The Importance of Genetic and Epigenetic Research in the Brassica Vegetables in the Face of Climate Change

Abstract

The genus Brassica includes many economically important crops providing nutrition as well as health-promoting substances. Most cultivars of the Brassica vegetables are F₁ hybrids, and breeding system was successfully established by effectively applying the phenomenon of heterosis or self-incompatibility. However, their production is constantly threatened by abiotic and biotic stresses such as the increasing numbers of races and isolates of pathogens, inappropriate cropping systems, and changing climate. Traditional methods of control are often costly and environmentally damaging, while the ideal way is to mine and use the abiotic or biotic resistance from the crop hosts. Fortunately, genomics and molecular genetics enables the rapid discover and application of plant breeding to improve adaptation to environmental conditions and abiotic or biotic resistance. Herein, we have summarized the important characteristics for breeding of the Brassica vegetables, including the trials for understanding the molecular mechanisms with genetic and epigenetic approaches. Some future perspectives are also given concerning how to efficiently use these genes and overcome global climate change.

3.1 Introduction

Brassicaceae is a diverse family of angiosperms containing 338 genera and 3,709 species, including the model plant Arabidopsis thaliana (Warwick et al. 2006). Three diploid species, Brassica rapa L. (AA, 2n = 20), Brassica nigra L. (BB, 2n = 16), and Brassica oleracea L. (CC, 2n = 18), and three allotetraploid species, Brassica juncea L. (AABB, 2n = 36), Brassica napus L. (AACC, 2n = 38), and Brassica carinata L. (BBCC, 2n = 34), are all involved in the genus Brassica, and the relationships of the genome of these six species are known as the triangle of U (Fig. 3.1) (U 1935).

Fig. 3.1

Genetic relationship in the genus Brassica known as the triangle of U. Diagram illustrating the genetic relationship between the diploids, B. rapa (AA genome), B. nigra (BB genome), and B. oleracea (CC genome) and the allotetraploids, B. juncea (AABB genome), B. carinata (BBCC genome), and B. napus (AACC genome)

B. rapa and B. oleracea show extreme morphological divergence (termed morphotype), which is due to selection by the plant breeders. With this effort, B. rapa comprises commercially important vegetable crops consumed worldwide such as leafy vegetables including Chinese cabbage (var. pekinensis), pak choi (var. chinensis), and komatsuna (var. perviridis), root vegetables including turnip (var. rapa), and oilseed (var. oleifera) (Fig. 3.2) (Cheng et al. 2014, 2016). The heading vegetable, Chinese cabbage, forms a head with large pale green-colored leaves and wide white midribs and is an important vegetable in Asia. The non-heading vegetables, pak choi and komatsuna, are also important vegetables in Asia. Turnip develops enlarged hypocotyls, and there are variations of both shape and color. There are morphotypes of oilseed in B. rapa, and seeds are used for oil extraction. B. oleracea comprises cabbage (var. capitata) from which the leafy heads are harvested, broccoli (var. italica) for cluster of flower buds, cauliflower (var. botrytis) for enlarged mass of the young, terminal inflorescence (described as the curd), and kohlrabi (var. gongylodes) for enlarged stems (Fig. 3.2) (Cheng et al. 2014, 2016). B. napus comprises the important oilseed crops such as canola or rapeseed (Fig. 3.2).

Fig. 3.2

Variations of the Brassica vegetables. B. rapa vegetables include Chinese cabbage (var. pekinensis) (a), pak choi (var. chinensis) (b), komatsuna (var. perviridis) (c), and turnip (var. rapa (d), mizuna (var. japonica) (e), chijimina (var. narinosa) (f), and sendai-yukina (var. chinensis) (g). B. oleracea vegetables include cabbage (var. capitata) (h), broccoli (var. italica) (i), and romanesco (var. botrytis) (j). B. napus crop includes canola (k)

Most commercial cultivars of B. rapa vegetables such as Chinese cabbage, komatsuna, and turnip or B. oleracea such as cabbage, broccoli, and cauliflower, are F₁ hybrids due to their agronomic benefits such as high yield, abiotic stress tolerance, disease resistance, and uniform phenotype (Fujimoto et al. 2018). Hybrid breeding came from the discovery of heterosis (or hybrid vigor), which is defined as the superior performance of hybrid plants over the parents (Crow 1998). In B. napus, F₁ hybrid production systems were introduced to replace open-pollinated cultivars leading to increased production. When breeding F₁ hybrid cultivars, breeders developed pure elite lines (inbred lines) as parents for hybrid production. About five to seven generations of selfing and selection based on traits concerned with the breeding objective such as disease resistance are required for developing inbred lines as parental candidates. The level of heterosis of crosses of all possible combinations of the inbred lines is used to identify suitable parents for F₁ hybrid generation. Self-incompatibility or cytoplasmic male sterility is successfully used for the production of F₁ hybrid seeds in B. rapa or B. oleracea vegetables to avoid contamination by non-hybrid seeds (Fujimoto and Nishio 2007; Yamagishi and Bhat 2014) (Fig. 3.3). The strength of self-incompatibility and stability of male sterility are important for harvesting highly pure F₁ seeds.

Fig. 3.3

The strategy for production of F₁ hybrid seeds. Breeders develop two elite parental lines to produce F₁ hybrid seeds. To avoid contamination by non-hybrid seeds, self-incompatibility (preventing self-pollination) or cytoplasmic male sterility is successfully used for the production of F₁ hybrid seeds in B. rapa or B. oleracea vegetables. Fertility needs to be restored to produce seeds of F₁ hybrid crops such as canola

Plants are highly perceptive toward extant environmental conditions. Temperature, water availability and salinity, soil pH and porosity, nutrient availability, and the amount of available photosynthetic active radiation (PAR) in a given geographic region have resulted in evolutionary adaptations to ecological niches that help to ensure successful flowering and germination (Franks and Weis 2009). Adaptation to the seasonal temperature variability experienced in more temperate climates has resulted in vernalization requirements for members of the Brassicaceae family (Shea et al. 2018a). This aims to ensure flowering does not occur until after a plant has perceived a prolonged period of cold and/or the shorter days experienced during the winter season, and promotes flowering during the more amenable spring season (Yan et al. 2003; Huijser and Schmid 2011).

Unlike other organisms, plants are sessile and thus incapable of migratory behavior within a generation. Therefore, migration due to climatic changes can require multiple generations and natural adaptations to environmental changes require evolutionary timescales to develop into viable strategies. The rapidity of current environmental change due to anthropogenic climate change is unprecedented in the geological record (Kemp et al. 2015) and presents a challenge to the increasing demands placed upon agricultural production (Howden et al. 2007; Namazkar et al. 2015) and very clear and present danger to the ecological stability of flora, and by extension, the fauna that rely on them as a resource for both food and habitat (Montoya and Raffaelli 2010). Along with the aforementioned abiotic factors affected by changes to climate, biotic factors such as disease and insects are of concern. For example, the anticipated increase to humidity and soil temperatures in some regions poses an increased danger from some soilborne pathogens (Das et al. 2016) and insects (DeLucia et al. 2012) and threatens both agricultural and wild cultivation (Newbery et al. 2016). With respect to the Brassica vegetables, Fusarium oxysporum (responsible for Fusarium wilt and root rot in Brassica) and Plasmodiophora brassicae (commonly known as clubroot) are of particular concern.

B. rapa is the first species within the genus Brassica that has been sequenced, and a doubled haploid (DH) line of Chinese cabbage, chiifu-401-42, was used for sequencing (Table 3.1). Genome sequence information in B. rapa and A. thaliana revealed that many orthologous genes are conserved (Wang et al. 2011). In addition, B. rapa genome has undergone a whole-genome triplication (WGT) after speciation between the genus Brassica and Arabidopsis (Fig. 3.4) (Wang et al. 2011). This WGT results in multiple copies of paralogous genes. Three subgenomes, the least fractioned subgenome (LF) and two more fractionated subgenomes (MF1 and MF2), were found within the B. rapa genome (Cheng et al. 2012). Whole-genome sequence of the other diploid species, B. oleracea and B. nigra, have been determined (Table 3.1) (Liu et al. 2014; Parkin et al. 2014; Yang et al. 2016). Furthermore, more complicated genomes of allotetraploid species, B. napus and B. juncea, have also been sequenced (Table 3.1) (Chalhoub et al. 2014; Yang et al. 2016). Recently pangenomes, which refers to a full genomic (genic) makeup of a species, and resequence of the other lines of reference genome were constructed in Brassica vegetables using more than one hundred lines within a species (Chen et al. 2015; Golicz et al. 2016; Bayer et al. 2018).

Table 3.1 Published representative genomes of the genus Brassica

Fig. 3.4

Timing of whole-genome triplication (WGT). There are two or three paralogs by WGT in B. rapa and B. oleracea, and some paralogs are deleted in B. rapa or B. oleracea genome after WGT

In this chapter, we introduce the important agronomical traits in the genus Brassica such as heterosis/hybrid vigor, self-incompatibility, disease resistance (biotic stress), vernalization, and abiotic stress tolerance from the concern of the global climate change.

3.2 What Is Epigenetics

Variation in DNA sequence can cause diverse gene expression changes that influences quantitative phenotypic variation such as morphotypes in the Brassica vegetables, which is an important factor determining plant value (Cheng et al. 2016). Gene expression regulatory networks are comprised of cis- and trans-acting factors, and differences in gene expression are attributable to genetic variation. In eukaryotes, the genome is compacted into chromatin, and the chromatin structure plays an important role in gene expression: gene expression can be controlled by changes in the structure of chromatin that does not involve changes in DNA sequence, and this phenomenon is termed “epigenetic” control (Fujimoto et al. 2012a). Accumulated evidence from researchers has demonstrated that epigenetic change plays an important role in the plant phenotype, and it is also involved in Brassica vegetables, such as heterosis, dominance relationship of the pollen determinant of self-incompatibility gene, or vernalization (Fujimoto et al. 2018; Itabashi et al. 2018). DNA methylation and histone modifications are well-known epigenetic modifications that can influence plant phenotype (Fig. 3.5).

Fig. 3.5

Epigenetic modification. DNA methylation and histone modifications such as methylation or acetylation regulate the structure of chromatin and controls gene expression

3.2.1 DNA Methylation

DNA methylation refers to an addition of a methyl group at the fifth carbon position of a cytosine ring (Fig. 3.5), and in plants, it is observed not only in the symmetric CG context but also in sequence contexts of CHG and CHH (where H is A, C, or T) (Cokus et al. 2008; Lister et al. 2008; Law and Jacobsen 2010; Osabe et al. 2012). DNA methylation is enriched in heterochromatic regions, such as in centromeric and pericentromeric regions, predominantly consisting of transposons (Zhang et al. 2006; Zilberman et al. 2007; Law and Jacobsen 2010). DNA methylation is also observed in euchromatic regions such as gene-coding regions, and it is widely seen in eukaryotes (Feng et al. 2010; Zemach et al. 2010; Vidalis et al. 2016). Genes having DNA methylation at only CG sites of transcribed regions are termed gene body methylation (gbM) (Vidalis et al. 2016).

Spontaneous epimutation is defined as heritable stochastic changes in the methylation states at CG, CHG, and CHH sites, and the rate of epimutation is overwhelmingly higher than the rate of genetic mutations in A. thaliana (Becker et al. 2011; Schmitz et al. 2011). Epimutation can sometimes act as the driving force of phenotypic variation (Fujimoto et al. 2012a; Quadrana and Colot 2016). DNA methylation has an important role in the regulation of gene expression, silencing of repeat sequences and transposons, and genome imprinting (Fujimoto et al. 2008a, 2011a; Osabe et al. 2012; Quadrana and Colot 2016). Most transposons are silenced via DNA methylation, are also immobile to protect genome integrity, and are silenced via DNA methylation (Miura et al. 2001; Singer et al. 2001; Fujimoto et al. 2008b; Tsukahara et al. 2009; Law and Jacobsen 2010; Sasaki et al. 2011).

DNA methylation in CG contexts is largely maintained by METHYLTRANSFERASE I (MET1), and those in CHG contexts are largely maintained by CHROMOMETHYLASE 3 (CMT3)-associated with di-methylation of the 9th lysine of H3 (H3K9me2) (Du et al. 2015; Quadrana and Colot 2016). CHH site methylation is maintained by CMT2 or DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) (Fig. 3.6) (Zemach et al. 2013; Stroud et al. 2014). The de novo methylation in all sequence contexts is catalyzed by DRM2 and is triggered by 24 nucleotide small interfering RNAs (24 nt-siRNAs) produced by the RNA interference (RNAi) pathway, termed RNA-directed DNA methylation (RdDM). Two plant-specific RNA polymerases, Polymerase IV (Pol IV) and Pol V, together with RNA-dependent RNA polymerase 2 (RDR2), dicer-like 3 (DCL3), and argonaute 4 (AGO4) proteins function in this RNAi pathway (Fig. 3.7) (Matzke and Mosher 2014; Quadrana and Colot 2016).

Fig. 3.6

Maintenance of DNA methylation

Fig. 3.7

Schematic representation of RNA-directed DNA methylation (RdDM)

Genome-wide profiles of epigenetic information define the epigenome, and recent advances in sequencing technology allow us to investigate the epigenome. DNA methylation states at the whole-genome levels have been examined using the methods such as whole-genome bisulfite sequencing (WGBS), methyl-CpG-binding domain sequencing (MBD-seq), epi-restriction-site associated DNA sequencing (EpiRAD-seq), and methylated DNA immunoprecipitation sequencing (MeDIP-seq) (Harris et al. 2010; Laird 2010; Schield et al. 2016). MeDIP-seq is a method to investigate the genome-wide methylation states by high-throughput sequencing enriched for methylated DNA fragments by immunoprecipitation using antibodies raised against methylcytosine (Laird 2010). WGBS directly sequences bisulfite converted DNA, and the methylation level at each cytosine position is calculated by dividing the number of methylated cytosines (mC) reads by the total number of reads (Fig. 3.8) (Laird 2010).

Fig. 3.8

Process of whole-genome bisulfite sequence (WGBS). Unmethylated cytosines (C) are converted to uracil (U) by bisulfite treatments. After bisulfite treatment, whole-genome sequences are determined and the number of C or T is summed for calculation of DNA methylation level (%). WGBS gives the methylated levels of all cytosine sites of the genome

Several types of hypomethylated Brassica vegetables have been analyzed. Treatment of B. rapa with 5-azaC, a cytidine analog that can inhibit DNA methylation, demonstrated male sterility, reduced seed size, and a late flowering phenotype, suggesting a strong relationship between DNA methylation and these traits (Amoah et al. 2012). Hypomethylated transgenic plants in B. rapa have been developed by the suppression of Decrease in DNA methylation 1 (DDM1) genes, by RNAi (Fujimoto et al. 2008b). DDM1 encodes a chromatin-remodeling factor, SWI2/SNF2, and plays an important role in the maintenance of DNA methylation (Vongs et al. 1993; Jeddeloh et al. 1999). B. rapa ddm1-RNAi transgenic plants showed reduced levels of DNA methylation and transcriptional reactivation of transposable elements, but they did not show any developmental abnormalities (Fujimoto et al. 2008b; Sasaki et al. 2011). Three mutants, braA.nrpd1, braA.rdr2, and braA.nrpe1, having dysfunction of genes involved in the RdDM pathway have been characterized (Grover et al. 2018). Nuclear RNA polymerase IV, subunit 1 (NRPD1), and Nuclear RNA polymerase V, subunit 1 (NRPE1), are components of the largest subunit of Pol IV and Pol V, respectively. braA.nrpd1 and braA.rdr2 reduced the accumulation of 24nt-siRNAs, while braA.nrpe1 did not show any change. There was no obvious vegetative defect in these three mutants, but silique and seed sizes in all three mutants are smaller than those in wild type (WT). As seed abortion occurs after fertilization, RdDM function is required in maternal sporophytic tissue (Grover et al. 2018).

Whole-genome DNA methylation states have been examined in the Brassica vegetables. In B. rapa, DNA methylation states have been examined by MeDIP-seq and DNA methylation states were compared between two inbred lines of Chinese cabbage. Most genes having difference of DNA methylation levels between the two lines showed similar gene expression levels, and about 30% of these genes were not expressed (Takahashi et al. 2018a). Using the same lines, tissues, and developmental stages that were harvested independently, WGBS was performed (Takahashi et al. 2018b). Between the MeDIP-seq and WGBS, the WGBS can assess different DNA methylation sequence contexts and was more sensitive (Takahashi et al. 2018a, b). WGBS has also been performed using B. rapa by several research groups, and the average methylation levels for CG, CHG, and CHH sites were 52.4%, 31.8%, and 8.3%, respectively (Chen et al. 2015), 37.2%, 17.3%, and 4.4%, respectively (Niederhuth et al. 2016), and 36.5%, 13.4%, and 5.3%, respectively (Takahashi et al. 2018b). This difference could be due to the variation of DNA methylation between lines or tissues. DNA methylation in the upstream and downstream regions of genes is negatively associated with expression levels, especially DNA methylation in the 200-bp upstream and downstream regions (Takahashi et al. 2018b). CHG and CHH methylation in exon or intron regions result in lower expression levels, indicating that CHG and CHH methylation in exon or intron regions are associated with gene silencing (Takahashi et al. 2018b). In contrast, there is no negative association between CG methylation in exons (except for the first exon) and expression levels, and genes having only CG methylation in the exon (gbM) show a moderate expression level, indicating that genes having gbM showed higher expression levels (Takahashi et al. 2018b), which is consistent with gbM genes in other plant species (Vidalis et al. 2016). There is a significant correlation in gbM between orthologous genes in B. rapa and A. thaliana (Niederhuth et al. 2016; Takahashi et al. 2018b). Significant correlation in gbM between paralogous genes is also found in B. rapa (Takahashi et al. 2018b), while the levels of methylation were inversely related to gene expression for each subgenome (DNA methylation: MF1 > MF2 > LF; Gene expression: LF > MF2 > MF1) (Cheng et al. 2015). The WGBS was also performed in B. oleracea, and the average methylation levels for CG, CHG, and CHH sites were 54.9%, 9.4%, and 2.4%, respectively (Parkin et al. 2014). An association between higher expression level and lower DNA methylation level was observed, and gbM related to higher gene expression level. At the subgenome level, lower methylation levels were found in the LF in B. oleracea (Parkin et al. 2014).

The 24 nt-siRNA levels are more associated with CHH methylation than CG and CHG methylation in B. rapa, suggesting that this CHH methylation was via RdDM (Takahashi et al. 2018b). Furthermore, the average methylation levels for CG, CHG, and CHH sites in the regions overlapping 24 nt-siRNA clusters were quite high even in the non-interspersed repeat regions (IRRs), indicating that 24 nt-siRNA clusters are strongly associated with DNA methylation (Takahashi et al. 2018b).

3.2.2 Histone Modification

Nucleosomes are formed by a histone octamer containing two of each of the core histones H2A, H2B, H3, and H4, and 147 bp of DNA is wrapped around this core. Alteration of chromatin structure, which causes changes in transcription, is regulated by various post-translational modifications of the N-terminal regions of histone proteins, such as methylation or acetylation (Fuchs et al. 2006). Histone lysine residues are able to be mono-, di-, or tri-methylated and each methylation state can be associated with different functions (Fuchs et al. 2006; He et al. 2011). In plants, histone deacetylation, H3K9me2, and H3K27me3 are associated with gene repression, and histone acetylation, H3K4me3, and H3K36me3 are associated with gene activation (Fuchs et al. 2006; He et al. 2011).

Different histone marks can be controlled by different histone lysine methyltransferase and can lead to different effects on gene regulation (Fuchs et al. 2006; Xiao et al. 2016). Histone lysine methyltransferases have a SET domain, which is evolutionally conserved, and SET domains have been identified in Drosophila melanogaster; SUPPRESSOR OF VARIEGATION 3-9 (SU(VAR)3-9), enhancer of zeste E(z), trithorax (TRX), and absent, small or homeotic disks 1 (ASH1). In A. thaliana, some members of ARABIDOPSIS TRITHORAX (ATX), ARABIDOPSIS TRITHORAX-RELATED (ATXR), and ASH1 HOMOLOG proteins (e.g., ATX1, ATX2, and ASHH2) are involved in H3K4me3 and/or H3K36me3. Histone lysine methyltransferases, KRYPTONITE (KYP)/SU(VAR)3-9 HOMOLOG 4 (SUVH4), SUVH5, and SUVH6, catalyze addition of H3K9me2 (Du et al. 2015). H3K27me3 addition is catalyzed by POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), which is composed of a subset of the Polycomb group (PcG) proteins (Zheng and Chen 2011).

Genome-wide profiles of histone modification are determined by a combination of chromatin immunoprecipitation (ChIP) and genomic tiling arrays (ChIP on chip) or ChIP and high-throughput sequencing (ChIP-seq) (Fig. 3.9), especially to detect methylation and acetylation of lysine residues on histone H3 because histone H3 undergoes the most extensive modification (Xiao et al. 2016). Using these technologies, the genome-wide distribution patterns of histone modifications such as H3K4me3, H3K9me2, H3K27me3, and H3K36me3 have been examined in some plants (Turck et al. 2007; Zhang et al. 2007, 2009; Bernatavichute et al. 2008; Oh et al. 2008; He et al. 2010; Roudier et al. 2011; Makarevitch et al. 2013).

Fig. 3.9

A schematic diagram representing the workflow of chromatin immunoprecipitation sequencing (ChIP-seq). DNA-bound histones are subjected for analyses and antibody against a specific histone modification is used for immunoprecipitation. ChIP DNA is purified and sequenced. The number of mapped reads to reference genome determines the level of histone modification

Information about histone modifications is limited in the Brassica vegetables. However, positive and negative control primer sets for H3K4me3, H3K9me2, H3K27me3, and H3K36me3 were developed in B. rapa (Kawanabe et al. 2016a), and these primer sets will be helpful for future ChIP analyses in B. rapa. Several suggestions were obtained during the process of making these primer sets. (1) H3K4me3 and H3K36me3 are enriched in transcriptionally active genes in B. rapa. (2) H3K9me2 is associated with TEs. (3) H3K27me3-targeted genes are conserved between A. thaliana and B. rapa. However, this has not been confirmed at the whole-genome level except for H3K9me2; a high resolution of the H3K9me2 states was examined in B. rapa (Takahashi et al. 2018b). From this ChIP-seq data, H3K9me2 tends to be overrepresented in TEs, but this overrepresentation is lower than DNA methylation and shows a more moderate association with TEs relative to DNA methylation, in B. rapa. The average expression level of genes having H3K9me2 in the exon and intron regions are lower than average of total genes. In addition, the level of H3K9me2 associates with DNA methylation levels but not with 24nt-siRNA levels (Takahashi et al. 2018b).

3.3 Heterosis or Hybrid Vigor

Heterosis or hybrid vigor is a phenomenon where hybrid progeny has superior performance compared to their parental inbred lines. The term ‘‘heterosis’’ was replaced to the more cumbersome word ‘‘heterozygosis’’, which did not express the superior performance of the hybrids (Shull 1948). Heterosis is observed in the agronomically important traits such as biomass, yield, and abiotic and biotic stress tolerance. Breeding of F₁ hybrid cultivars based on heterosis is used in many Brassica vegetables as well as many other crops (Fig. 3.10).

Fig. 3.10

A strategy of F₁ hybrid seed production system in Brassica vegetables. Breeders prepare the female and male lines and harvest the F₁ hybrid seeds using self-incompatibility or cytoplasmic male sterility. F₁ hybrid seeds are commercially sold and they have advantage such as high yield due to heterosis

Historically, F₁ hybrid cultivars were successfully introduced in maize production from 1940s (Crow 1998; Duvick 2001), and interpretation of genetic basis of heterosis began in the 1990s. The famous models such as dominance, overdominance, and epistasis have been suggested to explain the increased biomass and yield (Fig. 3.11) (Schnable and Springer 2013; Fujimoto et al. 2018). These hypotheses have been fundamental to heterosis research, but it is not clear if any one model can explain the molecular mechanism of heterosis. Quantitative trait locus (QTL) analysis is one of the popular approaches for elucidating the genetic bases of agriculturally important traits (Fig. 3.12). QTL analysis has been performed in maize, rice, sorghum, tomato, rapeseed, and cotton in attempts to understand the genetic basis of heterosis (Lippman and Zamir 2007). Most heterosis QTL studies focus on yield-related traits in biparental populations (Lippman and Zamir 2007). Other researchers tried to identify a QTL for general or specific combining ability in hybrids using multiparental populations (Giraud et al. 2017; Zhen et al. 2017). Single-nucleotide polymorphism (SNP) data of large populations have enabled comparisons of genetic architecture in a number of lines. In addition, genome-wide association studies (GWAS) using large SNP data have been incorporated into a genetic approach for heterosis (Fig. 3.13) (Yang et al. 2017a). Recent molecular analyses of transcriptomes, proteomes, and metabolomes, together with reference to the epigenome of the parents and hybrids, have begun to uncover some new facts about the generation of heterosis (Groszmann et al. 2011, 2013; Baranwal et al. 2012; Schnable and Springer 2013; Fujimoto et al. 2018; Miyaji and Fujimoto 2018).

Fig. 3.11

Three hypotheses to explain the genetic mechanism of heterosis

Fig. 3.12

The process of quantitative trait locus (QTL) analysis. The F₂-segregated populations derived from F₁ hybrid are produced. Using phenotype values (plant height, fresh weight, or leaf size, etc.) and genetic information determined by DNA markers, QTL analysis is performed. The genetically linked region with phenotype values is identified as a highest likelihood ratio (LOD) score. The horizontal line in QTL graph shows the significance threshold; therefore, the region between two flanking markers with the LOD peaks above the line is considered as the trait-related locus

Fig. 3.13

A schematic representation of genome-wide association study (GWAS). GWAS can find associations between DNA mutations and a certain phenotype (plant size in this figure). Manhattan plot makes it easier to visually locate the associations between SNPs and phenotypes. This enables researchers to estimate that the target gene is close to the DNA variants

In the dominance model, dominant alleles (A and B) suppress or complement the recessive alleles (a and b). In the overdominance model, heterozygosity (B/B’) at the key locus contributes to heterosis leading to superior performance. In the epistasis model, nonallelic genes (A and B) inherited from the parental lines interacts and contributes to heterosis.

3.3.1 Relationship Between Genetic Distance and Heterosis

For the crossability test for candidate of parental lines, all possible combinations of the inbred lines are used to identify suitable parents for F₁ hybrid generation. This is expensive, time-consuming, and labor-intensive. Thus, an efficient method for predicting hybrid performance in the parental generations is desired. One of the possible methods candidates the genetic distance between parental lines because it is believed that there is positive correlation between genetic distance and heterosis; crosses between more genetically divergent parental lines lead to greater heterosis in maize (Moll et al. 1965). However, positive correlation is not always observed between genetic distance and heterosis in plants (Barth et al. 2003; Girke et al. 2012; Yang et al. 2017a).

There are various types of DNA markers used for the analysis and identification of varietal difference in agricultural cultivars. These markers include cleaved amplified polymorphic sequences (CAPS)/restriction fragment length polymorphism (RFLP), amplified fragment length polymorphisms (AFLP), randomly amplified polymorphic DNA (RAPD), simple sequence repeats (SSRs), SNPs, and insertion/deletion polymorphism (InDel) markers (Fig. 3.14). SSR markers have been widely used because of high polymorphism, reproducibility, codominant inheritance, and genome-wide coverage. In addition, SSR markers require only small amounts of DNA for PCR and can be used for high-throughput analysis. SSR markers have been widely used for detecting genetic diversity and making genetic linkage maps, and many SSR markers are available for the genus Brassica (Suwabe et al. 2002, 2006; Lowe et al. 2004; Hatakeyama et al. 2010; Pino Del Carpio et al. 2011; Ramchiary et al. 2011; Guo et al. 2014). Sequencing technology enables us to identify SNPs easily, and SNPs are widespread in the B. rapa genome (Rafalski 2002; Metzker 2010). SNPs detected by RNA-sequencing (RNA-seq) in coding regions are used for developing gene-based markers (Fig. 3.15) (Paritosh et al. 2013). Restriction-site associated DNA sequencing (RAD-seq), where the flanking region is sequenced from a specific restriction site, is useful for developing DNA markers and high-throughput genotyping (Fig. 3.16) (Baird et al. 2008).

Fig. 3.14

Types of DNA markers. a InDel marker can detect the difference of the length of the PCR product when there is an insert/deletion. b CAPS marker (RFLP marker). PCR products are digested with a restriction enzyme, and the variation in the recognition site results in different number of bands. c SSR marker detects the variation in the number of repeating units of 2–6 base pairs of DNA. After PCR, the fragments can be separated by gel electrophoresis

Fig. 3.15

An illustration of the RNA-sequencing (RNA-seq). For making libraries for RNA-seq, the first-strand cDNA is synthesized by random hexamer primers using the short fragmented mRNA, which was purified by beads containing oligo (dT) from the total RNA. The second-strand cDNA is synthesized and sequenced on the high-throughput sequencer. Expression level in each gene is calculated by sequence read number

Fig. 3.16

The process of restriction-site associated DNA sequencing (RAD-seq). a Genomic DNA is digested with two different restriction enzymes. b Adapters (shown as green or orange square) are attached with the ends of restriction enzyme site. c Only fragments attached with the different adapters on either end are sequenced, and SNPs are detected from sequence information. RAD-seq targets a subset of the genome, thus providing advantages over whole-genome sequencing including low-cost discovery and genotyping and sequencing of greater numbers of samples

Using 32 F₁ hybrids of Chinese cabbage, genetic distance between parental lines and heterosis levels at three developmental stages was examined. For calculation of genetic distance, three types of DNA markers, SSR (multiallelic markers), CAPS (biallelic markers in exon regions based on SNP information of RNA-seq), and RAD-seq (biallelic markers on SNPs), were used because there is a concern of the ascertainment bias of DNA marker types (Kawamura et al. 2016). The genetic distance measured using the three types of DNA markers showed a high correlation. Of three developmental stages, cotyledon area at 6 days after sowing (DAS), leaf length x width of largest leaf at 21 DAS, and harvested biomass were examined (Fig. 3.17) (Kawamura et al. 2016). The intensity of heterosis is described by means of two indices, the mid-parent heterosis (MPH), and the best-parent heterosis (BPH). MPH is the performance of a hybrid relative to the mean value of its parental lines, whereas BPH is the performance of hybrids relative to the parent having the best value for the trait (Fig. 3.18) (Springer and Stupar 2007). The MPH and BPH were calculated by the phenotypic data in 32 F₁ hybrids and their parental lines, and the relationship between MPH or BPH and genetic distance of the parental lines was examined. Correlations were not observed between genetic distance and MPH or BPH of the parameter examined (Fig. 3.19) (Kawamura et al. 2016), indicating that the hybrid performance in Chinese cabbage cannot be predicted from the genetic distance of parental lines.

Fig. 3.17

The stages for examination of phenotype in F₁ hybrids and their parental lines in Chinese cabbage

Fig. 3.18

A graphical presentation of mid-parent heterosis (MPH) and best-parent heterosis (BPH). Y-axis indicates the phenotypic values such as biomass in parental lines and their F₁. MPH, heterosis over the mid-parent. BPH, heterosis over the better parent

Fig. 3.19

The relationship between genetic distance (GD) and mid-parent heterosis (relative ratio of plant size or biomass between F₁ and mid-parent values (rMPV)) in Chinese cabbage

3.3.2 Early Developmental and Yield Heterosis

The level of heterosis is trait-dependent, and heterosis in yield-related traits is important for F₁ hybrid cultivars (Springer and Stupar 2007; Flint-Garcia et al. 2009; Shi et al. 2011). In the commercial cultivar of Chinese cabbage, “W39”, a heterosis phenotype is seen at 4 DAS with hybrids having increased cotyledon size, while there is no difference in cotyledon size at 2 DAS between F₁ hybrids and best parent. The cell number per unit area of the cotyledon was greater for the female parent than the male parent or the hybrid (Fig. 3.20). In the first and second leaves of this F₁ hybrids, leaf size in F₁ hybrids was larger than that in best-parent, and the larger leaf size is associated with increased size and number of the photosynthetic palisade mesophyll cells (Fig. 3.20). Growth speed evaluated by counting leaf number in F₁ hybrids was not faster than parental lines (Saeki et al. 2016). Similar results were observed in the F₁ hybrids of the model plant A. thaliana and developed cotyledon with an increased size from a few days after sowing and greater leaf size in the first and second leaves (Fujimoto et al. 2012b; Meyer et al. 2012; Groszmann et al. 2014). Yield heterosis (25% greater than the better parent) was observed in “W39” of Chinese cabbage (Fig. 3.21). The prediction of yield heterosis from the early developmental stages could be useful to save time and labor because commercial F₁ hybrid of Chinese cabbage showed both early developmental and yield heterosis (Saeki et al. 2016). There was a moderate correlation in MPH between leaf size at 21DAS and yield but not in BPH (Kawamura et al. 2016). These results suggest that it is difficult to precisely predict the yield heterosis from the early developmental heterosis, though assessment of heterosis level in early developmental stages may be applied as the first screening of parental combinations of F₁ hybrid cultivars.

Fig. 3.20

Schematic representation of the size of seed, cotyledon, or leaf and cell size in F₁ hybrid and its parental lines, S27 (female line) and R29 (male line)

Fig. 3.21

Greater yield in commercial F₁ hybrid

Hormone signaling has been suggested to be important in heterotic hybrids of A. thaliana (Shen et al. 2012), and a model of hermetic modulation of hybrid vigor (concentration of salicylic acid (SA) in F₁ hybrids is in appropriate range for growth vigor performance) was suggested in A. thaliana (Zhang et al. 2016a). However, hormone profiles of 43 derivatives in 2-day cotyledons and 10-day first and second leaves were similar in parental lines and the F₁ hybrid of Chinese cabbage (Saeki et al. 2016).

3.3.3 Transcriptome Analysis in Heterosis

The underlying hypothesis for a transcriptomic approach is that genes whose expression changes in F₁ hybrids may be involved in heterosis. Transcriptome analyses initially used microarray technology and later, RNA-seq have been used to compare parental lines with their F₁ hybrids to identify genes potentially involved in heterosis (Fig. 3.15). Gene expression levels in F₁ hybrids are classified as additive or nonadditive. Additive gene expression level is defined as the expected changes in gene expression in F₁ hybrids where gene expression levels in F₁ hybrids are equal to the average level of parental gene expression (termed mid-parent value; MPV) (Fig. 3.22). Nonadditive gene expression level is unexpected changes in gene expression in the F₁ hybrids where gene expression levels in F₁ hybrids are either higher or lower than MPV (Fig. 3.22) (Fujimoto et al. 2018). RNA-seq enables us to not only compare the expression level of genes between the F₁ and parental lines but also to examine the parental allelic contributions to gene expression in F₁ hybrids at the whole-genome level (Chodavarapu et al. 2012).

Fig. 3.22

Classification of the mode of gene action in F₁ hybrid (F₁) compared with parental gene expression level (P₁ or P₂). LP; low-parent, HP; high-parent, MPV; mid-parent value

Upregulation of chloroplast-targeted genes occurs in the heterotic intraspecific hybrids of A. thaliana and rice, and the heterotic interspecific hybrids of A. thaliana and related species (Ni et al. 2009; Song et al. 2010; Fujimoto et al. 2011b, 2012b; Tonosaki et al. 2016). In F₁ hybrids of Chinese cabbage, “W39”, gene expression levels of eight chloroplast-targeted genes were examined by quantitative RT-PCR (RT-qPCR). Most genes showed higher expression levels in F₁ hybrids than in parental lines at 2 DAS, though expression level per se is low. At 3DAS, the expression levels of these genes increase in both F₁ hybrids and parental lines, but there was no difference in expression levels between F₁ hybrids and parental lines. From 4 to 6 DAS, there was no difference in expression levels between F₁ hybrids and parental lines. These results indicate that upregulation of chloroplast-targeted genes occurs at a specific developmental stage (Saeki et al. 2016). RNA-seq using 2-day cotyledons in F₁ hybrid and its parental lines of Chinese cabbage showed genes categorized into “Photosynthesis” and “Chloroplast part” tended to be upregulated, suggesting that chloroplast-targeted genes are upregulated at the whole-genome level. Stress-related genes tended to be downregulated in F₁ hybrids compared with in parental lines (Saeki et al. 2016).

As RNA-seq enables us to distinguish the parental alleles of transcripts in F₁ hybrids using SNP information, the parental alleles expressed in the F₁ hybrid of Chinese cabbage were examined. Most genes showed that differences in the expression levels between parental lines are maintained in the allelic bias of transcripts in F₁ hybrids (Saeki et al. 2016). Some genes showed allele-specific expression, and these genes tended to be categorized into “Translation” and “Ribosome” (Saeki et al. 2016).

3.3.4 Resequencing and SNP Analysis of the Parental Lines of a Commercial F₁ Hybrid Cultivar

SNP identification through the whole-genome resequencing of cultivar varieties has identified allelic mutations. Comparative variome analysis in a B. rapa collection has been reported and identified millions of high-quality SNPs (Cheng et al. 2016). The application of SNP markers has been used to identify seed coat color, hairiness, leaf morphology, and flowering time in B. rapa (Rahman et al. 2007; Zhang et al. 2008; Li et al. 2009). The functional loss of genes caused by SNPs and the distribution of high impact SNPs in comparison to the B. rapa reference genome sequence is desirable for trait analyses and breeding programs.

SNPs, genome structure, and composition between parental lines of the F₁ hybrid cultivar of Chinese cabbage, “W77”, were examined especially in protein-coding genes, by resequencing the genomes of the parental lines (Shea et al. 2018b). Not only moderate impact SNPs, nonsynonymous mutations without changing the framework of amino acid sequence but also high impact variants causing frameshifts, nonsense mutations, or other mutations that could possibly result in the loss of gene function were identified in both parental lines (Shea et al. 2018b). These putative nonfunctional genes that occurred specifically in each parent were distributed throughout the chromosome with high density. Functional markers derived from polymorphisms within genes that affect phenotypic variation are especially valuable in plant breeding, and thus these SNPs leading to nonfunctional genes will be applied to make functional markers that can assist future functional gene studies. If the dominance hypothesis (superior performance of hybrids results in the suppression/complementation of deleterious recessive alleles from one parent by beneficial or superior dominant alleles from the other (Crow 1998; Jones 1917)) applies to heterosis in Chinese cabbage, these putative loss-of-function genes in one parent could be the best candidate genes for heterosis of yield in Chinese cabbage.

Furthermore, the parental line-specific mutations in EcoRI sites by genome-wide comparative analysis were identified, and CAPS markers were developed. These CAPS markers can distinguish parental genotypes with codominance using agarose gel electrophoresis (Fig. 3.23), providing an easy and low-cost method of genotyping, suggesting that they can be applied for genetic analysis such as QTL analysis.

Fig. 3.23

Example of the electrophoresis of the genotyping. 96 F₂ plants derived from F₁ hybrid cultivar “W77” and CAPS marker developed were used. One band is in S11, two bands are in R09, and three bands are in F₁

3.3.5 Epigenetic Regulation and Heterosis

Recent study has revealed the possibility of an epigenetic contribution to heterosis. Enhanced growth similar to heterosis was observed in several of the hybrids between WT and specific epigenetic recombinant inbred lines (epiRIL) in A. thaliana (Dapp et al. 2015; Lauss et al. 2018). epiRILs differ only in DNA methylation levels, and their genetic backgrounds are almost the same (Johannes et al. 2009; Reinders et al. 2009; Teixeira et al. 2009). These two researches using hybrids between WT and epiRILs suggest that heterosis results in the difference of DNA methylation states between parental lines (Dapp et al. 2015; Lauss et al. 2018). In addition, two groups showed that DDM1 is a major regulator of heterosis in A. thaliana (Kawanabe et al. 2016b; Zhang et al. 2016a). The F₁ hybrids having homozygous mutations in ddm1 had reduced vegetative heterosis (Fig. 3.24). As DDM1 is involved in maintenance of DNA methylation, alterations in DNA methylation affect the level of heterosis (Kawanabe et al. 2016b; Zhang et al. 2016a).

Fig. 3.24

The F₁ hybrid between ddm1 mutant in Columbia (Col) and ddm1 mutant in C24 showed reduced level of heterosis. This result indicates that DDM1 plays a role in increasing leaf area of F₁ hybrids

There are few reports studying heterosis from the aspects of epigenetic regulation in Brassica vegetables. The hybrid broccoli, which showed larger curds, bigger leaves, and greater roots, was used for transcriptome and methylome analysis (Li et al. 2018b). Methylation-dependent restriction-site associated DNA (MethylRAD) method was used for methylome analysis. The DNA methylation levels were slightly higher in F₁ hybrids than MPV, and most of differentially methylated regions were intergenic. In addition, difference of DNA methylation in genes did not result in their difference of gene expression level (Li et al. 2018b). Although not so large, increased DNA methylation levels were observed in the heterotic hybrids of other plant species (Greaves et al. 2012; Shen et al. 2012, 2017). However, there is little evidence that the difference of DNA methylation between F₁ hybrids and parental lines directly affected differential gene expression between them. As mentioned above, the possible involvement of DNA methylation on heterosis has been proposed (Dapp et al. 2015; Kawanabe et al. 2016b; Zhang et al. 2016a; Lauss et al. 2018; Miyaji and Fujimoto 2018), and change of DNA methylation states in heterotic F₁ hybrids was revealed (He et al. 2010; Chodavarapu et al. 2012; Greaves et al. 2012; Shen et al. 2012, 2017). However, direct evidence is not yet obtained, and further study will be required.

3.3.6 Perspective

The F₁ hybrid cultivars have contributed to increasing crop yields during the last century. However, we still cannot predict the intensity of heterosis before the F₁ hybrids have been produced. This is because breeding of F₁ hybrid cultivars is still laborious, time-consuming, and costly. Most heterosis research has focused on growth vigor or increased yield, but there are a few reports showing the heterosis in biotic or abiotic stress tolerance (Rohde et al. 2004; Miller et al. 2015; Yang et al. 2015). Stable production will be more important for F₁ hybrid cultivars of Brassica vegetables by global climate change, and combining heterosis in different characters such as yield heterosis and stress tolerance heterosis could lead to producing better cultivars.

3.4 Self-incompatibility

Many species in the genus Brassica have a self‐incompatibility system, which is controlled by a single S locus with multiple alleles (Bateman 1955). The determinants of self‐recognition specificity in the stigma and the pollen have been identified; the female and male determinants are named S receptor kinase (SRK) and SP11/SCR (S‐locus protein 11/S‐locus cysteine rich) (SP11 hereafter), respectively (Stein et al. 1991; Schopfer et al. 1999; Suzuki et al. 1999; Takasaki et al. 2000). SRK is a membrane‐spanning serine–threonine kinase and has an extracellular domain (S domain), a transmembrane domain, and an intracellular domain (kinase domain) (Stein et al. 1991). SP11 is a small cysteine‐rich protein (Schopfer et al. 1999; Suzuki et al. 1999). These two determinants interact with each other in an allele‐specific manner (Kachroo et al. 2001; Takayama et al. 2001), and the interaction of these two factors induces reactions of self‐pollen rejection (Fig. 3.25). Many cultivars of Brassica vegetables are F₁ hybrids that are produced using the self‐incompatibility system. Now, DNA-based methods can examine whether two lines are compatible without performing a crossing test.

Fig. 3.25

Schematic representation of the self-incompatibility triggered by allele-specific interaction between SRK and SP11. SRK-m and SP11-m can interact with each other and this S haplotype-specific interaction leads to self-incompatibility. SRK-m and SP11-n cannot interact with each other, and thus S-n is compatible to S-m

3.4.1 Sequence Diversity of Multiple Alleles Located on S Locus

S determinants, SRK and SP11, are closely linked to each other in the S locus, and the alleles of these two genes are transmitted to the progeny together as a set. Therefore, this set of alleles is termed “S haplotype” (Fujimoto and Nishio 2007). The first candidate protein identified as the female S determinant, S‐locus glycoprotein (SLG), is also located in the S locus and segregates with SRK and SP11. There is a high degree of sequence similarity between SLG and the S domain of SRK (Stein et al. 1991). About 50 and 30 S haplotypes have been identified in B. oleracea and B. rapa, respectively (Nou et al. 1993; Ockendon 2000).

Nucleotide sequences of SLG, S domain of SRK, and SP11 of many S haplotypes have been determined in B. rapa and B. oleracea. There are sequence variations in SLG, S domain of SRK, and SP11 among the S haplotypes of B. rapa or B. oleracea (Kusaba et al. 1997; Sato et al. 2002). Deduced amino acid sequences of SP11 are more variable than SRK or SLG in B. rapa and B. oleracea (Watanabe et al. 2000; Sato et al. 2002). On the basis of nucleotide sequences in these genes, S haplotypes are classified into two groups, class-I and class-II (Fujimoto and Nishio 2007). The sequence variations of S domain of SRK and class-II SP11 between S haplotypes have relatively less nucleotide sequence variation compared with class-I S domain of SRK and SP11, respectively (Shiba et al. 2002), suggesting that class‐II S haplotype diversification occurred more recently than that of the class‐I S haplotypes.

3.4.2 Conservation of the Recognition Specificity After Speciation Between B. rapa and B. oleracea

From the sequence information of SLG, S domain of SRK, and SP11 in B. rapa and B. oleracea, interspecific pairs of S haplotypes, which have a high‐sequence similarity of both female and male S determinants between species, are identified (Kusaba et al. 1997; Sato et al. 2002, 2003). The same recognition specificity between interspecific pairs has been proved by pollination tests using interspecific hybrids, transgenic plants, and bioassay of recombinant SP11 proteins (Kimura et al. 2002; Sato et al. 2003, 2006), indicating that interspecific pairs between B. rapa and B. oleracea have the same recognition specificities.

The important regions for the recognition specificities of SRK and SP11 have been investigated by comparing amino acid sequences of SRK and SP11 in interspecific pairs. There were few amino acid substitutions in hypervariable regions (HVRs) of SRK between interspecific pairs, although the HVRs are highly variable among different S haplotypes, suggesting that the HVRs in SRK are important regions for recognition specificities (Sato et al. 2003). In SP11, the important regions for the recognition of the same haplotype of S domain of SRK have been identified by domain swapping or alanine‐scanning mutagenesis (Chookajorn et al. 2004; Sato et al. 2004).

3.4.3 The Diversification of the Genome Structure of S Locus

The genome structure of the S locus has been investigated in some S haplotypes of B. rapa and B. oleracea (Fujimoto et al. 2006a). In the center of the S locus of B. rapa, gene placement, distance between SP11, SRK, and SLG, and the orientation of these genes are different between S haplotypes, while sequence polymorphism in the flanking sequence is lower (Fig. 3.26) (Fujimoto et al. 2006a; Takuno et al. 2007). Recombination between SRK and SP11, which results in the breakdown of self‐incompatibility, seldom occurs, and recombination suppression is considered to be mainly due to the heteromorphism of the S locus. Between BrS‐8 and BrS‐46, which has a highly homologous region in SLG and the third to seventh exons of SRK, and recombination is detected in a part of SLG and part of SRK identified by the comparison of the whole-genome sequence of the S locus regions between these S haplotypes (Fig. 3.27) (Kusaba and Nishio 1999; Takuno et al. 2007). In this case, recombination within the S locus was identified, but this recombination did not result in the self-incompatibility recognition, suggesting it was not selected out. Comparison between class‐I and class‐II S haplotypes of B. rapa showed that genome structure of the S locus of a class‐II S haplotype is similar to that of class‐I S haplotypes, but that the order of SRK and SLG in the class‐II S haplotype is reversed compared to the class‐I S haplotypes (Fukai et al. 2003).

Fig. 3.26

Schematic representation of structural polymorphism of the S locus. In the center of S locus region covering SRK (blue), SP11 (red), and SLG (green) is diverged, while in the flaking region having high sequence homology

Fig. 3.27

Comparison of the genome structure of S locus regions between BrS-8 and BrS-46. Regions having high sequence homology are due to the recombination between these two S haplotypes

Interspecific pairs of S haplotypes are useful for the comparison of the S locus genome structure between species because S locus structure diverges within species and the ancestral S locus is common between interspecific pairs. Comparison of the structure of the S locus in three interspecific pairs demonstrated that the B. oleracea S locus is larger than the B. rapa S locus and revealed more retrotransposon‐like sequences, termed S‐locus retrotransposon families (STFs), in the S locus of B. oleracea than in that of B. rapa (Fujimoto et al. 2006a, 2008c). Most STFs are considered to have been inserted after speciation of B. rapa and B. oleracea (Fig. 3.28) (Fujimoto et al. 2006a, b). This transposable insertion into the S locus in B. oleracea may not be due to a specific event in the S locus because in most of the synthetic regions between B. rapa and B. oleracea, the region in B. oleracea is larger and contains many transposable elements than in B. rapa (Liu et al. 2014).

Fig. 3.28

Difference of the S locus regions between B. oleracea and B. rapa. S locus region in B. oleracea is larger than those in B. rapa, and this is due to the insertion of transposable elements in B. oleracea after speciation

3.4.4 Self-compatibility Results in the S Determinant Genes But also in the Downstream Genes of S Haplotype-Specific Interactions

Most plants in B. rapa and B. oleracea are self‐incompatible, and there are a few self‐compatible lines obtained by spontaneous mutations, suggesting an advantage of self‐incompatibility in these species. There is a self‐compatible line of Chinese kale, B. oleracea var. alboglabra. This line has the deletion of both the S domain and the transmembrane domain of SRK and SP11 (Nasrallah et al. 1994; Fujimoto et al. 2006b).

“Yellow sarson” is a self‐compatible oilseed cultivar (B. rapa var. oleifera) in India. The self‐compatibility of “Yellow sarson” is controlled by two loci, S and M, and the M locus is independent of the S locus (Hinata et al. 1983). “Yellow sarson” does not express SRK nor SP11, which is due to an insertion of a retrotransposon in SRK and deletion of the promoter region of SP11 (Nasrallah et al. 1994; Watanabe et al. 1997; Fujimoto et al. 2006b). M‐locus protein kinase (MLPK) has been isolated as a candidate gene of M by map‐based cloning (Fig. 3.29). MLPK belongs to a subfamily of receptor‐like cytoplasmic kinase (RLCK). MLPK of “Yellow sarson” has one amino acid substitution by a single-nucleotide change that leads to the loss of the autophosphorylation activity (Murase et al. 2004). Direct interaction between MLPK and SRK and phosphorylation of MLPK by SRK in vitro has been confirmed (Kakita et al. 2007a, b). There are two isoforms of MLPK by alternative transcriptional initiation sites; one localizes to the papillae cell membrane by myristoylation dependency and the other localizes to the plasma membrane by N-terminal hydrophobic region. Each MLPK isoform can complement the mlpk mutation (Kakita et al. 2007a). These results suggest that MLPK is involved in the downstream process of the S-allele-specific interaction through direct interaction with SRK.

Fig. 3.29

Schematic representation of map-based cloning. Map-based cloning is a method to identify the location of a candidate gene using the genetic and phenotypic information. In this case, genotype was determined using DNA markers in segregating population. Phenotype, SI (self-incompatibility) or SC (self-compatibility), is also determined by pollination test. From genotype and phenotype data, the location of M gene is identified in the region between marker-C and marker-E

Self‐compatible plants from a self‐pollinated population of an F₁ hybrid cultivar, “CR-Seiga 65” in Chinese cabbage having heterozygosity of BrS‐46 and BrS‐54 were identified. Pollination tests indicated that this self‐compatibility is linked to BrS‐54 and that the recognition function of the stigma is lost. The SRK allele of this self‐compatible plant, named BrSRK‐54f, is normally transcribed and translated, but gene conversion from SLG to SRK occurred resulting in the loss of the recognition specificity of BrSRK‐54 (Fig. 3.30) (Fujimoto et al. 2006c).

Fig. 3.30

Evidence of the gene conversion between SLG and S domain of SRK genes within the same S haplotype. This gene conversion results in the loss of recognition specificity of SRK

3.4.5 Dominance Relationship of S Haplotypes

Because self-incompatibility in Brassica vegetables is sporophytically controlled, there are dominance relationships of S haplotypes in the stigma and pollen (Thompson and Taylor 1966). Codominance is common and observed more frequently in the stigma than that in the pollen. The dominance relationships are different between the stigma and the pollen, and the dominance order of S haplotypes is nonlinear except for dominance relationship between class-II S haplotypes in the pollen (Thompson and Taylor 1966; Hatakeyama et al. 1998; Kakizaki et al. 2003; Yasuda et al. 2016).

The dominance relationship in the stigma is considered to be determined by the SRK protein itself; two models, competition of SRK-mediated signaling pathway and post-transcriptional modification of SRK, are proposed (Hatakeyama et al. 2001). The former model suggests the importance of the kinase domain in determining the dominance relationships of the SRK alleles; however, it is not clear which of the S domain or the kinase domain is important for determining the dominance relationship of SRK.

In pollen, class-I S haplotypes are dominant over class-II S haplotypes in the class-I/class-II S heterozygote plants of pollen (Nasrallah et al. 1991). In class-I/class-II S heterozygotes, expression of class-II SP11 is suppressed and the promoter region of class-II SP11 is DNA methylated (Fig. 3.31) (Shiba et al. 2002, 2006; Tarutani et al. 2010). The class-I S haplotypes have the SP11-methylation-inducing region (SMI) located in the S locus, and its sequence has homology to the promoter region of class-II S haplotypes (Fig. 3.31). The 24nt-small RNAs, Smi, are expressed from SMI during early stages of anther development, and these small RNAs can trigger de novo DNA methylation of the promoter region of class-II SP11 (Fig. 3.31). This indicates that class-I derived Smi induces silencing of the recessive SP11 allele by trans-acting de novo DNA methylation in the class-I/class-II S heterozygote plants (Tarutani et al. 2010). Between the class-II S haplotypes, there is a linear dominance order in pollen (BrS-44 > BrS-60 > BrS-40 > BrS-29), and DNA methylation is observed in the promoter region of recessive class-II SP11 allele in the heterozygotes of class-II S haplotypes (Kakizaki et al. 2003; Yasuda et al. 2016). Like class-I/class-II heterozygotes, 24nt-small RNAs termed SP11 methylation inducer 2 (Smi2) with sequence similarity to the promoter region of class-II S haplotypes are expressed, but they are expressed in all class-II S haplotypes. A linear dominance order in pollen is due to the sequence diversity within Smi among class-II S haplotypes; Smi2 derived from dominant class-II S haplotype can bind to the promoter region of recessive class-II S haplotypes but Smi2 derived from recessive class-II S haplotype cannot bind to the promoter region of dominant class-II S haplotypes because of nucleotide sequence difference (Fig. 3.32) (Yasuda et al. 2016).

Fig. 3.31

Dominance relationship in pollen. Smi derived from class-I S locus can induce the de novo DNA methylation in the promoter region of class-II SP11. De novo DNA methylation silences the expression of class-II SP11

Fig. 3.32

Mode of action of the dominance relationships via trans-acting small RNA. The single Smi2 regulates dominance hierarchy via a homology-dependent manner. In all dominant-recessive interactions, Smi2 variants derived from dominant SMI2 region exhibited high similarity to the all-recessive SP11 promoters and can induce the de novo DNA methylation leading to silencing of gene expression. By contrast, Smi2 variants derived from recessive SMI2 region cannot induce the DNA methylation in the dominant SP11 promoter because of low sequence similarity

3.4.6 S Haplotype Identification

Most cultivars of Brassica vegetables utilized for F₁ hybrid seed production system use self-incompatibility or cytoplasmic male sterility. As for the yield of F₁ hybrid seeds, F₁ hybrid breeding using the self-incompatibility system is much superior to that using male sterility. When self-incompatibility is used for harvesting F₁ hybrid seeds, identification of S haplotypes of breeding stocks is important for selecting parental combinations, thus avoiding the need for test crosses that are time-consuming. The method of S haplotype identification by DNA markers has been established by CAPS analysis using specific primer sets for amplification of SLG, or dot-blot analysis using high polymorphism of SP11 alleles among S haplotypes (Nishio et al. 1996; Fujimoto and Nishio 2003; Oikawa et al. 2011). CAPS analysis using class-I and class-II SLG-specific primer pairs, PS5/15 and PS3/21, respectively, is well established to identify S haplotypes in the Brassica vegetables (Nishio et al. 1996), and S haplotypes in many cultivars in B. rapa and B. oleracea have been identified using this method (Sakamoto et al. 2000; Sakamoto and Nishio 2001; Park et al. 2001). This method is fully useful in the B. rapa vegetables; however, PCR products were not amplified in some S haplotype of B. oleracea vegetables (Kawamura et al. 2015, 2017). Similar problems are also observed in radish (Haseyama et al. 2018). It has been shown that some class-I SLG alleles could not be amplified using the primer set, PS5/15 (Nishio et al. 1996), and deletion of the SLG gene has been found in B. oleracea (Okazaki et al. 1999). Thus, it is necessary to use other strategies to distinguish the parental S haplotype. Another primer set, PK1/PK4, is also used for the identification of class-I S haplotype (Nishio et al. 1997), although no major improvement was seen for B. oleracea vegetables (Kawamura et al. 2017). Other primer sets, PSA/PSB (class-I SLG/S domain of SRK), HVR2-F/R (class-I SLG/S domain of SRK), and 60-F/40-R (class-II SP11), or combination of these primer sets may improve the identification of S haplotypes in B. oleracea vegetables. In the non-PCR-amplified S haplotypes by PS5/PS15 or PS3/PS21 primer set, there are sequence differences in the regions covering the primer; thus identification of the nucleotide sequence of SLG in these S haplotype is required for designing new primer sets suitable for B. oleracea vegetables as well as radish.

In an F₁ hybrid seed production system, high seed purity is essential. To confirm the purity of F₁ hybrid seeds, a field grow-out trial can be performed but it is time-consuming and laborious. Therefore, a DNA-marker-based purity test is useful, and identification of the S haplotype can be applied to a purity test (Fujimoto and Nishio 2007). Furthermore, SSR markers, which can distinguish the parental alleles of F₁ hybrid cultivars, could be applied for purity testing of F₁ hybrid seeds, and SSR markers have the advantage of being able to assess multiple markers, increasing the accuracy. Highly polymorphic SSR markers have been identified in B. rapa and B. oleracea (Kawamura et al. 2015, 2017).

3.4.7 Stability of Self-incompatibility

As above mentioned, self-incompatibility is used for harvesting F₁ hybrid seeds in Brassica vegetables, and high seed purity of F₁ hybrid seeds is essential for commercial use. However, instability of self-incompatibility influenced by environmental factors such as high temperature sometimes leads to production of low-quality seeds containing high percentage of selfed seeds. Given the future global climate change, stable and strong self-incompatibility is required for F₁ hybrid breeding. If there is an S haplotype showing stable or strong self-incompatibility, this S haplotype is useful as maternal line of F₁ hybrid cultivar. Though there are a few reports examining the strength of self-incompatibility, this strength is controlled by genetic background (Ruffio-Châble et al. 1997; Hatakeyama et al. 2010). Further study will be required for identification of the factors involved in the stability of self-incompatibility in the Brassica vegetables.

Strength of self-incompatibility is important for harvesting highly pure F₁ hybrid seeds. However, weakening or overcoming self-incompatibility is also important for development of inbred lines because inbred lines are commonly obtained by bud pollination, which is laborious. Therefore, various methods have been developed for overcoming self-incompatibility, and carbon dioxide treatment to self-pollinated flowers in a greenhouse or a plastic house is effective to reduce labors for seed production of the inbred lines (Nakanishi et al. 1969; Nakanishi and Hinata 1973). Yield of selfed seeds by bud pollination is generally low in the lines having strong self-incompatibility. Effect of genotype on response to CO₂ gas is also known (Nakanishi and Hinata 1973; Niikura and Matsuura 2000). By the genetic analysis, two major QTLs overcoming self-incompatibility during CO₂ gas treatments were identified and they did not link with the S locus (Lao et al. 2014).

The molecular basis of how self-pollen hydration, germination, or pollen tube elongation is inhibited is not fully understood. In addition, identification of factors involved in the stability of self-incompatibility will be required for the high purity of F₁ hybrid seeds, especially in the near future facing global climate changes. It is possible that factors involved in inhibition of self-pollen hydration, germination, or pollen tube elongation might be involved in the stability of self-incompatibility. Further progress of research in this filed is desired.

3.5 Genetic and Epigenetic Regulation of Flowering in the Brassica Vegetables

The changes from vegetative to reproductive growth mark a major developmental transition in flowering plants. Controlling the time of transition is important in the Brassica vegetables, because once the transition starts it cannot be reversed. Correct timing can maximize the reproduction success and seed production through ensuring the flowering time under optimal conditions. The late flowering or late bolting is especially important for leafy Brassica vegetables, because premature bolting causes a decrease in productivity and market value. Therefore, much effort has been made in breeding programs to develop late bolting Brassica vegetable cultivars.

3.5.1 Environmental Factors of the Regulation of Flowering Time

Floral transition is highly responsive to environmental cues, and photoperiod and temperature play major roles (Srikanth and Schmid 2011). The regulation of flowering time, including its associated network, has been extensively studied in the model plant species A. thaliana (Putterill et al. 2004; Bäurle and Dean 2006; Fornara et al. 2010; Andrés and Coupland 2012; Song et al. 2013). More than 180 A. thaliana genes are recognized in flowering time control based on characterization of loss-of-function mutants or analysis of transgenic plants (Fornara et al. 2010). We know in A. thaliana, six major pathways control flowering time: the photoperiod/circadian clock pathway, vernalization pathway, ambient temperature pathway, age pathway, autonomous pathway, and gibberellin pathway (Fig. 3.33) (Kim et al. 2009; Fornara et al. 2010). Among them, the photoperiod response to changes in day length and the vernalization response to low temperatures are two major pathways that regulate flowering time in A. thaliana (Song et al. 2013). Other pathways are able to modulate the flowering response like the ambient temperature pathway, the age pathway, the sugar signaling pathway, and the stress pathway (Srikanth and Schmid 2011; Blümel et al. 2015).

Fig. 3.33

A simplified schematic showing FLOWERING LOCUS C (FLC), involving a complex network pathway for flowering in A. thaliana

Various numbers of genes and micro-RNAs (miRNAs) are involved in the regulation of flowering time, which help us to understand the involvement of these factors at the molecular level. Mainly, the photoreceptor proteins (phytochrome and/or cryptochrome) are controlling the photoperiodism, which is responsible for sensing red/far-red and blue light, respectively (Más et al. 2000). Photoperiod requirements are defined as either long day (LD) or short day (SD) with respect to the length of time of daylight. This photoperiod signal plays vital role in the floral development of several plant species, which is related to the annual cyclical seasonal changes, LD, coinciding with the spring and summer seasons, and SD, associated with the autumn and winter seasons, respectively (Corbesier and Coupland 2005).

Vernalization is defined as “the acquisition or acceleration of the ability to flower by a chilling treatment.” In A. thaliana, the prolonged exposure to cold will decrease the FLOWERING LOCUS C (FLC) expression, which acts as a floral repressor by inhibiting the activation of a set of genes required for transition of the apical meristem to a reproductive state (Kardailsky et al. 1999; Kobayashi et al. 1999; Michaels and Amasino 1999; Sheldon et al. 1999; Lee et al. 2000; Samach et al. 2000; Hepworth et al. 2002). Vernalization is an example of temperature-accelerated flowering (Song et al. 2012). When other specific conditions are met, including the presence of certain photoperiods and ambient temperatures, and vernalization, flowering only takes place many weeks or even months later (Kim et al. 2009).

B. rapa and B. oleracea show different responses to vernalization; B. rapa responds to seed vernalization, whereas B. oleracea requires plant vernalization (Lin et al. 2005). In seed-vernalization-responsive type, plants can sense low temperatures during seed germination. On the other hand, in plant-vernalization-responsive type, plants need to reach a certain developmental stage before they become sensitive to low temperatures (Friend 1985). In the plant-vernalization-responsive type, plants grow vegetative in the first year and flower in the following year after winter. B. napus is an important oilseed crop; natural variation in flowering time in response to vernalization was characterized into three groups (spring, winter, and semi-winter type) (Raman et al. 2016). Spring-type varieties are annual type generally seeded in spring and complete their life cycle in a single growing season without vernalization; winter (biennial) types have an obligate requirement usually seeded in the fall and complete development in the following spring under prolonged period of cold temperature. Semi-winter types are sown before winter, which gives flower after winter.

3.5.2 Photoperiod and Circadian Clock Mechanism in the Brassicaceae

The circadian clock mechanism controls the flowering time in concert with the photoperiodic flowering pathway (Jung and Müller 2009; Imaizumi 2010; Song et al. 2013, 2015). By the circadian clock mechanisms in LD condition, A. thaliana perceives LD light in the leaves, which involve the CONSTANS (CO), GIGANTEA (GI), and FLAVIN KELCH F BOX 1 (FKF1) genes. The interaction of upstream genes of CO such as GI and FKF1 releases repression of CO transcription by inducing degradation of the transcriptional repressor CYCLING DOF FACTOR1 (CDF1) (Srikanth and Schmid 2011). The transcription and protein function of CO tightly controlled by the light and circadian clock genes controls floral activator FLOWERING LOCUS T (FT) expression to induce flowering via the photoperiod pathway (Corbesier et al. 2007). FT expresses within the distal part of the leaf and moves through the phloem to the meristem acting as a long-distance systemic signal between leaves and the shoot meristem (Kardailsky et al. 1999; Weigel et al. 2000; Turck et al. 2008). FT interacts with the bZIP transcription factor (TF) FLOWERING LOCUS D (FD) to form a FT/FD heterodimer complex in the shoot apical meristem (SAM) (Abe et al. 2005; Wigge et al. 2005), which activates expression of the floral meristem identity genes, APETALA 1 (AP1) and FRUITFUL (FUL), thus initiating the development of flower buds (Abe et al. 2005; Wigge et al. 2005; Corbesier et al. 2007; Turck et al. 2008; Turnbull 2011).

As a main component of the clock, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a MYB-related TF, which plays an important role in the phytochrome-dependent induction of photosynthetic genes (Wang et al. 1997; Green and Tobin 2002), controls the circadian clock (Green and Tobin 2002; McClung 2014), stress response (Dong et al. 2011; Lai et al. 2012; Seo et al. 2012), and maintenance of photoperiodic flowering (Niwa et al. 2007; Fujiwara et al. 2008). Another core component of the circadian clock is LATE ELONGATED HYPOCOTYL (LHY) in A. thaliana (Wang et al. 1997; Wang and Tobin 1998), which is involved in the regulation of photoperiodic flowering (Fujiwara et al. 2008; Imaizumi 2010; Li et al. 2011; Lu et al. 2012).

In B. rapa, preferential retention is more important for CCA1 gene like CCA1/LHY/RVE and PRR gene families, but not ZTL/FKF1/LKP2 families because they are not retained in B. rapa genome (Lou et al. 2012). B. rapa has two copies of Bra.FT and three copies of Bra.CO (Zhang et al. 2015). In contrast, B. oleracea seems to carry four copies of Bol.FT and three copies of Bol.CO (Razi et al. 2008). Bra.FT.A07, often referred to as BrFT2, has a transposon insertion in the mapping parent R-o-18 and underlie a strong QTL for flowering time (Zhang et al. 2015). In a DH population derived from a Chinese cabbage and a rapid cycling line, a CO-like copy on A02 co-localized with a flowering QTL (Li et al. 2013b).

3.5.3 Vernalization Requirement and Responses in the Brassica Vegetables

In A. thaliana, mainly two key genes, FRIGIDA (FRI) and FLC, have been identified; FLC blocks flowering by binding to genes that promote flowering and repressing their transcription. Mainly FLC targeted three flowering time genes, FT, SOC1, and FD, with FLC binding to the promoters of SOC1 and FD and to the first intron of FT (Helliwell et al. 2006; Searle et al. 2006). Later, at the whole-genome level, more putative FLC targeted genes were identified by ChIP-seq. Five-hundred FLC binding sites were found, mostly located in the promoter region of genes containing one CArG box (the known target of MADS-box proteins) (Deng et al. 2011). In the photoperiod pathway, two genes (FT and SOC1) act downstream of the flowering activator CO that is being negatively regulated by FLC (Kim et al. 2009; Andrés and Coupland 2012).

Plant homeodomain (PHD) finger protein (VERNALIZATION INSENSITIVE 3, VIN3) induces during the exposure to cold, which acts to establish the initial repression of FLC (Sung and Amaniso 2004). Moreover, VIN3, VRN5, and VIN3/VRN5-like 1 (VEL1) interact with VRN2 protein and form PHD-PRC2 complex (Sung and Amaniso 2004; Wood et al. 2006; De Lucia et al. 2008). Vernalization reduces the FLC repression, which is associated with the enrichment of H3K27me3 mediated by the PHD-PRC2 mechanism (De Lucia et al. 2008). During exposure to cold, H3K27me3 is enriched in chromatin at the transcription start sites of FLC, and later H3K27me3 modification extends across the FLC gene due to warm temperature (Finnegan and Dennis 2007). A stable maintenance of repression requires PRC2, although the initial transcriptional repression of FLC is PRC2-independent (Gendall et al. 2001). After cold exposure, the maintenance of FLC silencing under warm conditions is therefore mediated by PHD-PRC2 spreading H3K27me3 over the FLC locus. Additionally, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), associated with H3K27me3, and VRN1 are also required for the maintenance of stable FLC repression (Levy et al. 2002; Mylne et al. 2006; Sung et al. 2006).

In B. rapa, several QTLs were identified for flowering time (VFR1, 2, and 3 in non-vernalized condition and FR1, 2, and 3 in vernalized condition) from a cross between an annual and a biennial oilseed cultivar (Teutonico and Osborn 1994; Osborn et al. 1997), which covers the region of BrFLC1 and BrFLC2 (Kole et al. 2001; Schranz et al. 2002). Eight QTLs for flowering with one major QTL, which co-localized with BrFLC2, were detected using a multi-population derived from several parental lines (rapid cycling, Chinese cabbage, yellow sarson, pak choi, and a Japanese vegetable turnip variety) (Lou et al. 2007). QTL analyses also showed the co-localization of a major QTL with BrFLC2 using other parental combinations between pak choi and yellow sarson (Zhao et al. 2010; Xiao et al. 2013). Over many years’ QTL analysis has shown a major QTL of flowering time co-localized with BrFLC2. QTL analysis was performed in two different conditions, greenhouse and open field using an F₂ population derived from a cross between an extremely late bolting line (Nou 6 gou and PL6) and early bolting line (A9709) of Chinese cabbage. Five QTLs were detected, but within two condition QTLs did not map in the same position. Among the five QTLs, three QTLs were co-localized with BrFTa (greenhouse), BrFLC1 (open field), and BrFLC5 (open field) (Kakizaki et al. 2011). In Chinese cabbage, an F₂ population was derived from the cross of an early bolting commercial F₁ varieties, “Early”, and an extremely late bolting line, “Tsukena No. 2”, where QTLs for bolting time after vernalization co-localized with the late bolting alleles of BrFLC2 and BrFLC3. In the extremely late bolting of “Tsukena No. 2”, large insertions were found in the first intron of BrFLC2 and BrFLC3, suggesting that weak repression of BrFLC2 and BrFLC3 transcripts by vernalization results in these insertions (Kitamoto et al. 2014). In addition, this group successfully developed new F₁ hybrids of Chinese cabbage by introducing these two FLC alleles from Tsukena No. 2 (Kitamoto et al. 2017).

In B. oleracea, QTL analysis identified a major QTL covering BoFLC2, while BoFLC1, BoFLC3, and BoFLC5 were not linked, using a population derived from a DH line of broccoli, Green Comet, and a DH line of cabbage, Reiho (Okazaki et al. 2007). Due to deletion of a single base in exon 4 leading to a frameshift mutation, suggesting that BoFLC2 contributes to the control of flowering time in Green Comet (non-vernalization type) (Okazaki et al. 2007). Another group conducted QTL analysis using the population derived from a rapid cycling line of B. oleracea var. alboglabra (A12DHd) and the broccoli variety, Green Duke. BoFLC2 is not responsible for the flowering time difference between the two lines because these two lines bear nonfunctional copies of BoFLC2; there is a single base deletion in exon 4 and deletion in the A12DH in Green Duke (Razi et al. 2008). The association between flowering time (under both glasshouse and field conditions) and a QTL at BoFLC2 has been shown using the population of two purple sprouting broccoli lines (E5 and E9); E9 requires longer cold periods than E5 to head (Irwin et al. 2016). Through allelic variation and sequence polymorphisms, BoFLC2 was shown to be a major determinant of heading date variation and vernalization response and alters the sensitivity and silencing dynamics of its expression (Irwin et al. 2016). In B. rapa, hybridized introgression of BoFLC2 from a plant-vernalized B. oleracea cultivar did not alter the vernalization phenotype in the derived BC₃F₂ offspring; however, the duration of cold required for successful vernalization leading to flowering was increased, suggesting that the duration of cold experienced is altered by allelic variation, while the difference in the developmental stage at which vernalization will occur between the two species is possibly controlled by another mechanism (Shea et al. 2017, 2018c).

In B. rapa, after vernalization, the expression of BrFLC genes was reduced and is stably maintained after returning to ambient temperatures (Fig. 3.34). During normal growth, three of the BrFLC paralogs (BrFLC2, BrFLC3, and BrFLC5) showed H3K4me3 modification, while only BrFLC1 showed accumulation of both H3K4me3 and H3K36me3. After 4 weeks of vernalization, the accumulation of H3K27me3 was observed in BrFLC1, BrFLC2, and BrFLC3, and maintained after returning to a warm temperature (Kawanabe et al. 2016a); the repression of BrFLC expression by prolonged cold treatment was associated with the histone modification. Previous studies of A. thaliana, long noncoding RNAs (lncRNAs) such as COLD-INDUCED LONG ANTISENSE INTRAGENIC RNA (COOLAIR), COLD-ASSISTED INTRONIC NONCODING RNA (COLDAIR), and COLD OF WINTER-INDUCED NONCODING RNA FROM THE PROMOTER (COLDWRAP) are involved in vernalization (Swiezewski et al. 2009; Heo and Sung 2011; Kim and Sung 2017). Cold-induced lncRNA COLDAIR is expressed from the mid-region of the first intron (Tsai et al. 2010; Heo and Sung 2011). In A. thaliana, the first intron, the promoter region, and exon 1 are important for the regulation of FLC expression by prolonged cold treatments (Sheldon et al. 2002). Although in B. rapa long insertions in the first intron cause a weak repression of BrFLC2 and BrFLC3 transcripts by vernalization, sequence similarity to the vernalization response element (VRE) in the first intron or to the COLDAIR of A. thaliana was not detected in the first intron of any of the B. rapa paralogs (Kitamoto et al. 2014). COLDAIR-like transcripts were not detected, but COOLAIR-like transcripts were detected only from BrFLC2, and these transcripts were induced by cold treatment in B. rapa (Li et al. 2016e).

Fig. 3.34

Schematic illustration of gene expression levels of genes involved in response to vernalization in B. rapa

High bolting resistance is an important trait for cultivation mainly in leafy vegetables such as Chinese cabbage or cabbage, which requires a deep understanding of the molecular mechanism to control the vernalization requirement. COOLAIR-like transcripts were detected only from BrFLC2, which regulated the suppression of BrFLC2 and maybe other BrFLCs (Li et al. 2016e), but in B. rapa, there is no report found about the transcripts of COLDAIR or COLDWRAP, and regions sharing sequence similarity to the COLDAIR (Kitamoto et al. 2014). Therefore, there is a possibility that lncRNAs may be involved in the regulation of repression of FLC, which will need to be assessed by RNA-seq. Thus, in the genus Brassica, it is important to identify the sequences required for vernalization, termed VREs, and to examine any sequence polymorphisms that may help to identify important regions and develop their relationship to sensitivity of vernalization; this will be helpful for marker-assisted selection (MAS) and serve as important tools for breeding in the genus Brassica.

3.5.4 Perspective

Future climatic shifts will affect the flowering time over the coming decades. Therefore, future research into flowering time and the various interconnected regulatory pathways continues to remain an invaluable source of information for the application of MAS and the continued development of hardy crop breeds. The current understanding of vernalization response and heading time, and its connection to the FLC haplotype is therefore an important consideration in the breeding of cole crops as climates begin to respond to an increased mean global temperature. In stable climatic regions, the breeding of such crops is well understood. The additional challenges of a changing climate, however, increase the demands placed upon both breeders and agricultural researchers.

3.6 Disease Resistance Genes in the Brassica Vegetables

Climate change, pathogen variations, and inappropriate farming methods are posing threat to current production of Brassica vegetables. Various pathogens, including virus, bacteria, fungi, and oomycete, can infect Brassica vegetables leading to production loss. Among the diseases, turnip mosaic virus, black rot, Fusarium wilt, and clubroot have been focused due to their impact on farming.

Traditional methods of disease prevention include physical, chemical, and biological control. Physical methods are often complicated and are not effective compared to other methods. Besides, chemical and biological controls may have effects under certain conditions but are costly and/or environmentally damaging. In contrast, natural resistance from Brassica hosts is the most desirable method of disease prevention.

There are two types of plant immunity: (1) pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) triggered immunity (PTI) activated through recognition of PAMPs/MAMPs by cell surface pattern recognition receptors (PRRs), and (2) effector-triggered immunity (ETI) activated through the recognition of pathogen-specific effector molecules by host resistance (R) genes, which reflects the “gene-for-gene” theory (Flor 1971; Chisholm et al. 2006). Most R genes encode nucleotide-binding leucine-rich repeat (NB-LRR) proteins, including coiled-coil NB-LRRs (CC-NB-LRRs) and toll interleukin-1 receptor-NB-LRRs (TIR-NB-LRRs). Some R genes also encode transmembrane receptor-like proteins (RLPs), transmembrane receptor-like kinases (RLKs), cytoplasmic kinases (CKs), and proteins with atypical molecular motifs (Jones and Dangl 2006; Liu et al. 2007; Neik et al. 2017). In recent years, a variety of R genes has been identified and successfully applied to improve the resistance against various diseases in the Brassica vegetables.

3.6.1 Turnip Mosaic Virus (TuMV)

The Brassica infecting viruses mainly include turnip mosaic virus (TuMV), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), and cauliflower mosaic virus (CaMV) with TuMV being the most prevalent and causing the greatest loss for Brassica crops. TuMV is a member of the virus genus Potyvirus. The disease was first described in 1921 in USA in B. rapa and then in B. oleracea in the UK (Smith 1935). Now, TuMV is threatening Brassica vegetables around the world, especially in Europe, Asia, and North America, resulting in a yield loss up to 30% (Tomlinson 1987; Walsh and Jenner 2002). The diseased plants show symptoms including slight leaf stunting, mottle, and chlorosis, or spot in the primary infection stage, and severe stunting, chlorosis, necrosis, and even withering of the whole plant in the late infection stage; during subsequent cold storage of the leaf head in Brassica, internal necrosis further develops and makes the leaf heads unmarketable. TuMV is difficult to control, because of its wide host range and high pathotype diversity, and has caused serious losses in almost all Brassica vegetables, as well as many non-Brassica vegetables such as radish, pea, and lettuce (Provvidenti 1980). Based on differential hosts or molecular variations, TuMV strains or pathotypes have been defined to strains C1-4 (Provvidenti 1980), C1-5 (Green and Deng 1985), pathotypes 1-12 (Walsh 1989; Jenner and Walsh 1996), four phylogenetic groups including basal-B, world-B, basal-BR, and Asian-BR (Ohshima et al. 2002, 2007), or four host types including B(B), BR, and B(R) (Tomimura et al. 2004; Tomitaka and Ohshima 2006; Nguyen et al. 2013). Another reason for the difficulty of control is the nonpersistent mode of transmission by at least 89 aphid species (Hamlyn 1953; Walsh and Jenner 2002). Traditional methods like chemical control are not effective and environmentally damaging, while natural resistance from Brassica hosts is the most desirable way of preventing TuMV. In recent years, a variety of R genes/loci has been identified and successfully applied to improve the resistance against TuMV in the Brassica vegetables.

TuMV resistance has been identified in most Brassica species (Doucet et al. 1990; Fjellstrom and Williams 1997; Walsh et al. 2002; Nyalugwe et al. 2014, 2015a, b). Extensive studies have revealed various inheritance types of TuMV resistance, which indicated a gene-for-gene system. Generally, the resistance genes are mostly found on the A genome, being dominant or recessive and displaying a qualitative genetic control by one or two genes, while the resistance on the C genome is mostly inherited in polygenic manner. In B. rapa, there are both dominant and recessive inheritance types. Suh et al. (1995) showed that the resistance to single strain was conferred by one or two dominant genes, while resistance to mix strains was controlled by more than two major effect genes. In recent years, new single dominant genes were identified by genetic analysis using different DH, F₂, or BC segregation populations (Chung et al. 2014; Jin et al. 2014; Li et al. 2015c). Recessive inheritance type of the resistance was also discovered. Yoon et al. (1993) reported that Chinese cabbage 0–2 line’s resistance to strain C4 and C5 was regulated by two recessive genes. Several other single recessive genes have also been identified, including recessive Turnip mosaic virus resistance 01(retr01) (Rusholme et al. 2007), resistance and necrosis to tumv 1(rnt1) (Fujiwara et al. 2011), retr02 (Qian et al. 2013), trs (Kim et al. 2013), and retr03 (Shopan et al. 2017). In B. oleracea, most studies have revealed dominant genetic structure. In Brussel sprouts, at least four genes control the resistance to TuMV (Pink et al. 1986). Pink and Walkey (1990) further analyzed the inheritance of disease resistance using new cabbage materials, and the estimated heritability of resistance ranged from 41 to 48%.

More than ten R genes to TuMV have been identified in the genus Brassica (Table 3.2). Turnip mosaic virus RESISTANCE IN BRASSICA 01 (TuRB01), a single dominant resistance gene to pathotype 1, was first mapped by Walsh et al. (1999) to a 7.2 cM interval on chromosome N6 of B. napus. TuRB01b was identified on a 2.9 Mb region of chromosome A06 from B. rapa, and comparative analysis indicated that TuRB01 and TuRB01b represent similar or identical alleles at the same A genome resistance locus (Lydiate et al. 2014). TuRB02, identified on the B. napus C genome linkage group (LG) N14, controls the degree of susceptibility to isolate CHN1 (Walsh et al. 1999). TuRB03, a single dominant gene conferring resistance to pathotype 4, was mapped to a 7.9 cM interval on chromosome N6 in B. napus (Hughes et al. 2003). retr01 represents the first mapped recessive gene in Brassica species and was mapped on chromosome R4 (Rusholme et al. 2007); another recessive gene rnt1 from B. rapa was mapped on chromosome R6 (Fujiwara et al. 2011). Besides, Li et al. (2015c) mapped a novel B. rapa resistance gene TuRBCS01 to a 1.98 Mb region on chromosome A04 using SSR and InDel markers. Using bulked segregation analysis (BSA), Shopan et al. (2017) identified another single recessive gene retr03. The previous mapping work opened the gate for isolation and analysis of the candidate R genes. The dominant gene ConTR01 and the recessive genes retr01, retr02, and retr03 were all supposed to be eIF4E or eIF(iso)4E encoding genes (Rusholme et al. 2007; Qian et al. 2013; Shopan et al. 2017). BcTuR3, isolated from non-heading Chinese cabbage, was a TIR-NB-LRR type R gene related to TuMV resistance (Ma et al. 2010). TuMV-R from B. rapa was mapped to a 0.34 Mb region on chromosome A06, with containing six candidate genes (Chung et al. 2014). TuRB07, a single dominant gene from B. rapa, was mapped to chromosome A06, and the candidate gene is Bra018863 (encoding CC-NB-LRR) (Jin et al. 2014).

Table 3.2 Mapped or cloned TuMV resistance genes in the genus Brassica

The protein–protein interaction in the Brassica–TuMV system has also received much attention. A variety of protein interactions has been characterized till now, using techniques such as yeast two-hybrid (Y2H), bimolecular fluorescence complement (BiFC) and co-immunoprecipitation (CoIP). Researchers have identified the cytoplasmic inclusion (CI) protein as the interactor and viral avirulence determinant for TuRB01, TuRB01b, and TuRB04, while P3 is the viral avirulence determinant for TuRB03 and TuRB05 (Jenner et al. 2000, 2002, 2003; Walsh et al. 2002). Another example is the plant eukaryotic initiation factor 4E (eIF4E) family, a well-known host factor that plays a critical role in the infection of several potyviruses. The interaction between viral protein genome-linked (VPg) of potyviruses and eIF4E or eIF(iso)4E of the host determines the virulence (Wittmann et al. 1997; Robaglia and Caranta 2006; Beauchemin et al. 2007). This eIF4E-mediated resistance often confers strong and broad-spectrum resistance (Yeam et al. 2007; Rodríguez-Hernández et al. 2012). In the genus Brassica, the eIF(iso)4E-encoding gene has been shown to be strongly linked to the recessive resistance genes retr01, retr02, and trs (Rusholme et al. 2007; Qian et al. 2013; Kim et al. 2013), and transgenic plants overexpressing eIF(iso)4E variants show broad-spectrum TuMV resistance (Kim et al. 2014). Except for the direct application of the identified resistance genes, the genes from TuMV have also been used in resistance breeding by host-induced gene silencing (HIGS), especially the coat protein (CP) gene derived from the virus. The viral CP can accumulate in the host cells and inhibit the virus replication, and thus confers resistance. Successful resistance enhancement by CP gene strategy has been reported on Brassica crops including oilseed rape and Chinese cabbage (Jan et al. 2000; Lehmann et al. 2003).

Although great progress has been made in terms of genetic mapping of the Brassica–TuMV resistance genes, these candidate genes still require further functional analysis, and the results from TuMV-Arabidopsis systems could provide evidence (Martín et al. 1999; Liu et al. 2015). Till now, there are few studies concerning other Brassica-affecting viruses including CMV, TMV, CaMV, etc. However, the progress made in TuMV in Brassica crops and CMV and TMV in other crops may open the gate for these future studies.

3.6.2 Black Rot (BR)

Bacterial diseases for the genus Brassica include black rot (BR) caused by Xanthomonas campestris pv. campestris (Xcc), soft rot caused by Erwinia carotovora subsp. carotovora, and leaf black spot caused by Alternaria oleracea, among which BR brought about the greatest loss and has been studied most extensively. Xcc belongs to the genus Xanthomonas, including many economically important pathogenic bacteria associated with plants. The disease has been identified in all Brassica growing continents, especially in Asia, Europe, and North America (Jensen et al. 2010; Singh et al. 2016). The pathogen usually invades through hydathodes or wounds, and spreads into the leaf and the whole plant through the vascular system (Vicente and Holub 2013). The disease starts as chlorotic lesions in the leaf margins, and the pathogen reproduces and its secretion blocks the vessels and water transport, resulting in V-shaped lesions and dark veins, and finally, the whole plant wilts leading to death (Schaad 1982; Alvarez et al. 1994). Xcc has a wide host range and can cause serious damage on B. oleracea, as well as other Brassicaceae crops, radish, ornamental crucifers, related weed species, and even A. thaliana (Bradbury 1986). Xcc has a high diversity and 11 races have been identified with race 1 and 4 being prevalent (Vicente et al. 2001; Fargier and Manceau 2007; Cruz et al. 2017). BR is a seed-borne disease, and the pathogen is mainly transmitted through contaminated seeds and transplants (Vicente and Holub 2013). BR can easily become epidemic under some favorable conditions like high humidity and warm temperature (Staub and Williams 1972; Vicente and Holub 2013). Countermeasures including seed treatment, soil disinfection, crop rotation, and biocontrol agents have some effects (Massomo et al. 2004). The development and use of resistant cultivars have long been considered as important methods of disease control (Taylor et al. 2002; Lee et al. 2015). In recent years, the inheritance of BR resistance has been studied in several Brassica species and some QTLs have been mapped using molecular markers (Vicente et al. 2001; Taylor et al. 2002).

Many studies focused on Brassica crops. However, only few resistance sources have been identified, including two extensively utilized cabbage accessions “Early Fuji” from Japan and PI436606 (cultivar Heh Yeh da Ping Tou) from China (Hunter et al. 1987; Camargo et al. 1995). Most studies for BR resistance were performed on B. oleracea crops and revealed complex genetic structures. Using F₂ and BC populations, Jamwal and Sharma (1986) showed that the BR resistance in the cauliflower cultivar SN445 was controlled by a dominant gene. Badger Inbred 16, a line derived from “Early Fuji”, contains QTLs for BR resistance (Camargo et al. 1995; Vicente et al. 2002). Ignatov et al. (1998) found in progenies of cabbage line PI436606, Portuguese kale ISA454, and Chinese kale SR1, that high resistance to race 1 was controlled by a dominant gene named R1, when a recessive gene r5 was responsible for the resistance to race 5. Vicente et al. (2002) found resistance to race 3 in the cabbage DH line BOH 85c and in PI436606 was controlled by a single dominant locus (Xca3). Recently, Saha et al. (2014, 2016) found that resistance to race 1 in cauliflower accession BR-207 was governed by a single dominant gene. In view that there are few highly resistant resources in C genome of B. oleracea, especially to the prevalent race 1 and 4, researchers tend to screen for useful sources from the A and B genomes. The gene conferring resistance to race 1 is present in the B genome of B. carinata, B. juncea, and B. nigra, while the gene conferring resistance to race 4 is present in the A genome of B. rapa, B. napus, and B. juncea (Taylor et al. 2002). Vicente et al. (2002) found that strong resistance to races 1 and 4 was controlled by a single dominant locus Xca1 in the B. carinata line PI199947, while resistance to race 4 in three B. napus lines was controlled by a single dominant locus (Xca4). Major dominant inheritance type in B. carinata was also proved in other studies (Guo et al. 1991; Sharma et al. 2016). Griffiths et al. (2009) identified five B. rapa accessions with variable resistance to race 1 and uniformly resistance to race 4, all of them having the oilseed plant growth type.

Most BR resistance research focused on QTL analysis or preliminary mapping. The first QTL analysis of BR resistance in Badger Inbred 16 using RFLP markers has revealed two major QTLs on LGs 1 and 9 (Camargo et al. 1995). Vicente et al. (2002) used B. napus DH populations and positioned the locus Xca4 on LG N5 of the A genome. Soengas et al. (2007) reported the genetics of broad-spectrum resistance in the Chinese cabbage accession B162, and resistance to both race 1 and 4 correlated and a cluster of highly significant QTL that explained 24–64% of the phenotypic variance was located on chromosome A06. To analyze resistance in cabbage line “Reiho”, Doullah et al. (2011) adopted sequence-related amplified polymorphism (SRAP) and CAPS markers and performed QTL analysis with F_2:3 families, and revealed QTLs on LG2 accounting for up to 10% of the phenotypic variation and another one on LG9 explaining 16% phenotypic variation. The high-throughput markers allow more accurate mapping. Kifuji et al. (2013) used expressed sequence tag (EST)-based SNP markers to map the resistance gene in “Early Fuji” to race 1, and three QTLs, i.e., QTL-1 (the major one), QTL-2, and QTL-3, were detected. Tonu et al. (2013) analyzed BR resistance QTLs, and the major QTL XccBo(Reiho)2 was detected on chromosome C8. Saha et al. (2014) mapped Xcc race 1 resistance gene Xca1bo in cauliflower line BR-161, with the 1.6 cM interval being flanked by one RAPD marker and one inter-simple sequence repeat (ISSR) marker. Kalia et al. (2017) further converted these markers to sequence characterized amplified region (SCAR) markers and proved that these markers were useful in MAS in cauliflower breeding. Sharma et al. (2016) firstly developed B. carinata F₂ mapping population and intron length polymorphic markers, to map the BR race 1 resistance locus Xca1bc in a 6.6 cM interval. Lee et al. (2015) firstly developed genome-wide SNP markers based on resequencing data and identified one major QTL on chromosome C03 in cabbage. In total, more than 20 QTLs with major or minor effects have been mapped on eight different Brassica chromosomes, suggesting that resistance to BR disease is complex and quantitatively controlled by multiple genes (Table 3.3).

Table 3.3 BR genes/QTLs identified in the genus Brassica

Current molecular and omics methods provide new opportunities for quick disease-related gene mining. Jiang et al. (2011) investigated the molecular resistance mechanisms to find the genes related to BR resistance in cauliflower, using suppression subtractive hybridization (SSH) technique. The results imply that some upregulated genes might be involved in cauliflower resistance responses, such as plant defensin PDF1.2, lipid transfer protein, and thioredoxin h. Tortosa et al. (2018) firstly investigated the dynamic changes in the metabolic profile of B. oleracea plants during an Xcc infection from leaves and found specific metabolic pathways such as alkaloids, coumarins, or sphingolipids are postulated as promising key role candidates in the infection response. Using RNA-seq, Afrin et al. (2018) revealed that six NB-encoding genes were highly expressed in resistant cabbage lines compared to susceptible cabbage lines, which were possibly related to BR resistance.

Although some R genes display single inheritance pattern, none has been cloned and functionally analyzed. Thus, more efforts will be needed on these directions to give a clear genetic structure for BR resistance and apply in resistance breeding. Furthermore, with increasingly more races and variabilities being discovered around the world (Singh et al. 2011; Rouhrazi and Khodakaramian 2014; Burlakoti et al. 2018), more resistance resources are becoming an urgent need.

3.6.3 Fusarium Wilt (FW)

Fusarium yellows or Fusarium wilt (FW) is one of the important diseases in the world and was first found in the USA and then in Japan and China (Xing et al. 2016). FW is caused by soilborne fungus, F. oxysporum, and contains many varieties and infects major crops and vegetables such as tomato, cotton, melon, and banana (Ploetz 2006; Ulloa et al. 2006; Charoenporn et al. 2010; Oumouloud et al. 2013). In Brassicaceae, leaf vegetables including cabbage (B. oleracea), Chinese cabbage, pak choi, komatsuna, and turnip (B. rapa) are usually infected by F. oxysporum. These Brassica vegetables are major a food source in Asia, especially in Japan, China, and Korea, which is threatened by the FW disease. Two forma specialis of F. oxysporum can inoculate Brassicaceae; F. oxysporum f. sp. conglutinans (Foc) inoculates B. oleracea and B. rapa, and has higher virulence on B. oleracea than on B. rapa, while F. oxysporum f. sp. rapae can inoculate only B. rapa (Daly and Tomkins 1995; Enya et al. 2008). When Brassicaceae is inoculated by F. oxysporum, the parenchyma tissue between the veins of their leaves becomes yellow and yellowing spots spread to the whole leaves (Walker 1930; Sherf and MacNab 1986; Daly and Tomkins 1995). The plants also show defoliation and stunted growth. Simultaneously or after the loss of the normal green color in the infected plants, the vascular elements in the diseased tissue become brown and the plants will finally die.

The pathogen usually invades the plants through young root, but can also invade through wounds in older roots (Sherf and MacNab 1986; Daly and Tomkins 1995). They move via water-conducting xylem tissue to root, stem, and leaves. In the susceptible cultivar, conidia attach to the root hair and the emergence site of the lateral roots, and grow into the central root surfaces between 1 to 3 days post-inoculation (dpi) (Pu et al. 2016). From 4 to 6 dpi, the mycelia spread from the epidermis into the cortical tissues, enter the xylem vessels, and move upward. After 7 dpi, hyphae are observed not only in the root, but also in the neighboring parenchymal tissues and surrounding cortical tissues. Microscopic analysis compared between the root of the resistant and susceptible cabbage cultivars after Foc inoculation showed that the infected points were observed in the susceptible cultivar at 1 dpi but not in the resistant cultivar (Pu et al. 2016). From 3 to 12 dpi, the infected points increase further in the susceptible cultivar. In the resistant cultivar, the infected points were observed from 3 dpi, but the number of points did not increase further and is less than the susceptible cultivar. Li et al. (2015b) also performed microscopic analysis using the root of the resistant and susceptible cabbage cultivars. In this report, there are few differences between resistant and susceptible cultivars from 1 to 3 dpi, while from 4 to 6 dpi, colonization was observed in the susceptible cultivar, but not in the resistant cultivar. They also compared the colonization pattern in root, stem base, upper stem, and petiole between the resistant and susceptible cultivars. In the susceptible cultivar, the colonization was observed in root, stem base, upper stem, and petiole, while in the resistant cultivar, few fungi were observed in root and stem base, and not in the upper stem and petiole. In summary, the fungus developmental speed is slower in the resistant cultivar than in the susceptible cultivar, and the resistant cultivar restricts the fungus development and spreading.

Only two types of Foc  resistance have been reported: type A and B. Type A resistance, which is stable under high or low temperature, followed a single dominant inheritance pattern and has been proven to be very effective in resistance breeding and has been introduced into various cabbage cultivars (Walker 1930, 1933; Blank 1937), generating the first series of resistant cabbage cultivars, distributed in 1920s, including “Wisconsin Hollander” (winter/storage type), “Wisconsin All Seasons” (mid-season type), “Copenhagen Market” (mid-season type), “All Head Early” (flat-head type), etc. (Walker et al. 1927; Walker and Blank 1934). At the same time, type B resistance is unstable under high temperature (above 24 °C) and follows a polygenic inheritance pattern, limiting its use in breeding (Walker et al. 1927; Walker 1930; Farnham et al. 2001). Foc race 1 has been found worldwide, while race 2 has only been reported in USA and Russia (Bosland et al. 1988; Morrison et al. 1994). While type A resistance is very effective to race 1, the cultivars of type A resistance have successfully controlled FW for decades. However, type A major gene resistance can be overcome by Foc race 2 and the genetic control of host resistance to race 2 remains unclear. Thus, efforts are needed to clarify the genetic structure of cabbage resistance to race 2. Foc favors hot climate and plentiful rainfall and can survive for more than 10 years even without a host, making it difficult to control through traditional methods like seed treatment, rotation, and fungicide (Tisdale 1923; Bosland et al. 1988; Fravel et al. 2003). Once this disease is present in the field, using FW resistant cultivar is the only successful method to maintain the yield.

Resistance genes to FW have been isolated in A. thaliana, B. rapa, and B. oleracea. In A. thaliana, six dominant RESISTANCE TO FUSARIUM OXYSPORUM loci (RFO1-6) contribute to the resistance to F. oxysporum f. sp. matthioli (Diener and Ausubel 2005). The strongest locus encodes RESISTANCE TO FUSARIUM OXYSPORUM 1 (RFO1), identical to WALL-ASSOCIATED KINASE-LIKE KINASE 22 (WAKL22), which encodes for receptor-like kinase. RFO1 does not have LRR domain, making RFO1 an atypical type resistance gene. RFO1 interacts with RFO2, RFO4, and RFO6, and RFO2 is identified as the receptor-like protein gene, which is a homologue to the PSY1 peptide receptor gene, PSY1R (Shen and Diener 2013). RFO3 and RFO5 are independent of RFO1, and RFO3 encodes a receptor-like kinase (Cole and Diener 2013; Diener 2013). To Foc race 1, RFO7 is associated with the resistance in A. thaliana (Diener 2013). In B. rapa, inoculation test using F₂ population showed that a single dominant gene regulates the resistance to Foc (Shimizu et al. 2014). The neighbor genes, Bra012688 and Bra012689, were identified in FW resistance inbred line of Chinese cabbage by transcriptome analysis (Shimizu et al. 2014). These two genes have TIR, NB, and LRR domains. In the susceptible line, these two genes are completely deleted. Shimizu et al. (2014) did not conclude whether Bra012688 or Bra012689 is the resistance gene to FW because all FW resistance lines contained these two genes and all susceptible lines lacked these two genes. In B. oleracea, the type A single dominant resistance gene FOC1 has been studied extensively in recent years, which is favored greatly by the release of the reference genome (Liu et al. 2014). Pu et al. (2012) mapped FW resistance gene FocBo1 to LG seven (O7) using both BSA and QTL analyses in cabbage. Lv et al. (2013) constructed a linkage map based on a cabbage DH population. Lv et al. (2014a) mapped the gene to the interval between two InDel markers, M10 and A1, flanking the resistance gene at 1.2 and 0.6 cM, respectively, and used these markers to breed resistant hybrids. Lv et al. (2014b) ultimately mapped the candidate resistance gene FOC1 using an enlarged cabbage F₂ population to a re-predicted Bol037156, which encodes a putative TIR-NB-LRR type R protein. Shimizu et al. (2015) further mapped the resistance locus FocBo1 by using 139 recombinant F₂ plants derived from resistant cabbage AnjuP01 and susceptible broccoli GCP04, and identified an orthologous gene of Bra012688 as a candidate gene. The genetic region including the FW resistance genes is conserved between B. rapa and B. oleracea. Most of the exons of Bra012688, one of the candidates to FW resistance gene in B. rapa, are conserved in FocBo1, but Bra012689 is not conserved in B. oleracea, indicating that Bra012688 may be the resistance gene to FW in B. rapa (Fig. 3.35).

Fig. 3.35

Genetic region containing resistance genes to Fusarium wilt (FW) conserved in B. rapa and B. oleracea. Black boxes and bars show exons and introns, respectively. Numbers under boxes show each length

Plant pathogens are categorized into biotrophs and necrotrophs by their lifestyles (Glazebrook 2005). Biotrophic pathogens get nutrients from living host tissues, while necrotrophic pathogens kill host tissue and gain nutrients from dead tissues. SA, jasmonic acid (JA), and ethylene (ET) are the phytohormones related to disease resistance. SA-dependent defenses act against biotrophic pathogens, and JA- and ET-depending defenses act against necrotrophic pathogens. F. oxysporum is considered a hemibiotrophic disease, because it begins its infection cycle as a biotroph, but change to a necrotroph at the later stage (Lyons et al. 2015). Transcriptome analysis using RNA-seq gives expression levels of all genes, allele-specific expression, and splicing variants (Mortazavi et al. 2008). Fusarium-inoculated and mock-treated plants were compared with each other using transcriptome analysis in A. thaliana, B. rapa, and B. oleracea. In B. rapa, using FW resistant and susceptible inbred lines of Chinese cabbage, the differentially expressed genes (DEGs) were identified by comparison between with and without Foc inoculation (Miyaji et al. 2017). Gene Ontology (GO) analysis using upregulated DEGs at 24 h after inoculation suggested that the resistant lines activated systemic acquired resistance, and that the susceptible lines activated tryptophan biosynthetic process and responses to chitin and ET (Fig. 3.36). At 72 h after inoculation, GO analysis indicated that the genes related to response to biotic stimulus and response to stress were expressed in the susceptible lines, but there are no overrepresentation in the resistant lines (Fig. 3.36). The transcriptome analysis in A. thaliana after F. oxysporum infection was also reported (Zhu et al. 2013); DEGs were compared between B. rapa and A. thaliana at the same time point, 24 h after inoculation (Miyaji et al. 2017). Genes encoding the peroxidase superfamily protein, chitinase, glutathione S-transferase, ACC OXIDASE 1, CYTOCHROME P450, and some TFs including WRKY51 and WRKY53 were common with between A. thaliana Columbia-0 (medium susceptible), and the resistant and susceptible inbred lines of Chinese cabbage (Fig. 3.36). In B. oleracea, RNA-seq was performed in the resistant cabbage variety and DEGs were identified by comparison between the samples inoculated with Foc or distilled water (Xing et al. 2016). They performed GO, clusters of orthologous groups (KOG), and Kyoto encyclopedia of genes and genomes pathway database (KEGG) analysis. From these analysis, calcium signaling, mitogen-activated protein kinase (MAPK) signaling, SA-mediated hypersensitive response, SA-dependent systemic acquired resistance, JA- and ET-mediated pathways, and the lignin biosynthesis pathway were activated at the early time point after Foc inoculation, indicating that their signaling and pathways are important for Foc resistance in cabbage (Fig. 3.37).

Fig. 3.36

Transcriptional change after Foc inoculation in the resistant and susceptible lines in B. rapa. HAI; hours after inoculation

Fig. 3.37

Transcriptional change and protein concentration after Foc inoculation in the resistant and susceptible lines in B. oleracea. SA; salicylic acid, HR; hypersensitive resistance, SAR; systemic acquired resistance, JA; jasmonic acid, ET; ethylene

Xylem sap proteome of the non-inoculated and Foc-inoculated root was also performed using liquid chromatography–mass spectrometry (LC-MS/MS) in the resistant and susceptible cultivars in B. oleracea (Pu et al. 2016). A large portion of up- and downregulated proteins was categorized into the protein acting on carbohydrates in the resistant and susceptible cultivars, suggesting that these proteins may have a role for Foc resistance. Both up- and downregulated oxidoreductases were induced in the susceptible cultivar, while there were only a few inductions of oxidoreductases in the resistant cultivar, indicating that the induced oxidoreductases are related to symptoms development in the susceptible cultivar. To note, they identified ten Foc cysteine-containing secreted small proteins as candidate effectors. Proteome was also performed using two races of Foc that differ in pathogenicity, race 1 and 2 (Li et al. 2015a). Race 2 has stronger pathogenicity compared with race 1. The high abundance proteins contained carbohydrate, amino acid, and ion metabolism in race 2, indicating that these proteins may be involved in the race 2’s stronger pathogenicity. Foc has four isoforms of the homolog of secreted-in-xylem 1 (SIX1) protein, and a SIX1 homolog is required for the full level of virulence on cabbage (Li et al. 2016a). They also analyzed whether SIX1 works as an avirulence gene in Foc by inoculation test using the FW resistance cabbage variety. Cabbage showed no disease symptoms by the inoculation of both Foc with WT and mutational SIX1, indicating that SIX1 is not an avirulence gene, but a virulence gene in Foc.

3.6.4 Clubroot (CR)

Clubroot (CR) disease, caused by the soilborne pathogen P. brassicae (Pb), is now threatening almost all the Brassica crops worldwide. CR was firstly reported in Russia in 1878, and the disease rapidly expanded to Europe, Asia, and USA during nineteenth and early twentieth century, becoming one of the most serious problems in almost every Brassica production area around the world. Pb infection is a two-phase process. The primary phase occurs in root hairs, and the secondary phase occurs in cells of the cortex and stele of the root. During the latter phase, multinucleate plasmodia induce clubs on roots, inhibiting the nutrient and water transport, causing abnormal cell enlargement, and uncontrolled cell division of infected roots, thus deforming them with characteristic clubs. Thus, the quality and commercial value of the crop products are seriously compromised (Piao et al. 2009). Pb has a wide host range and can affect cruciferous plants including all Brassica crops, common weeds like charlock and A. thaliana, as well as some non-cruciferous plant species such as Tropaeolum majus and Reseda alba (Dixon 1980; Ludwig-Müller et al. 1999). Pb has a complex pathotype differentiation and has been extensively studied (Ayers 1957). Currently, there are two main systems used for classification: the Williams system (four differential hosts) and the European clubroot differential (ECD) set (15 differential hosts), proposed by Buczacki et al. (1975), both of which are widely used in pathotype identifications all over the world (Donald et al. 2006). Pb is favored at low pH, and wet and warm weather (Diederichsen et al. 2009). The pathogen variation and its ability to survive in soil as resting spores for up to 15 years make it difficult to control by cultural practices or chemical treatments (Dixon 1980; Voorrips 1995; Kageyama and Asano 2009). Thus, breeding of resistant cultivars is a desirable means of utilizing the host resistance and reducing pollution to the environment. Currently, several important CR resistance genes/QTLs have been mapped or cloned, and MAS and introgression breeding have been widely used in improving the resistance in the Brassica crops (Manzanares-Dauleux et al. 2000; Nomura et al. 2005; Ueno et al. 2012; Lee et al. 2016; Li et al. 2016d; Hatakeyama et al. 2017).

Extensive studies for inheritance analysis have been performed in Brassica crops including B. rapa, B. oleracea, and B. napus. In B. rapa, many studies have indicated major dominant genes, which conferred resistance to specific Pb pathotypes (Wit and Van De Weg 1964; Toxopeus and Janssen 1975). A few important turnip resistance sources have been used for genetic analysis, resistance gene mapping, and B. rapa breeding. The major resistance from European fodder turnip cultivar “Siloga” was proved and widely used in turnip and Chinese cabbage breeding (Kuginuki et al. 1997; Suwabe et al. 2003, 2006; Hatakeyama et al. 2013). Other turnips possessing CR resistance genes include inbred line N-WMR-3 carrying major gene Crr3, “Gelria R” carrying major dominant resistance to race 4, European fodder turnip “Debra” carrying major genes CRk and CRc, and inbred line ECD04 with quantitative resistance to a series of Pb isolates, which was revealed by genetic analysis using different F₂, F₃, and BC segregation populations (Piao et al. 2004; Hirai et al. 2004; Saito et al. 2006; Sakamoto et al. 2008; Chen et al. 2013). Other major dominant genes were identified in Chinese cabbage accessions including T136-8 with CRa to race 2 (Matsumoto et al. 1998; Ueno et al. 2012), “Akiriso” and “CR Shinki” with CRb to race 3 and 4 (Kato et al. 2012, 2013; Zhang et al. 2014), “Jazz” with resistance gene Rcr2 to multi pathotypes (Huang et al. 2017), and 85-74 with race 4 resistance CRd (Pang et al. 2018). Quantitative-inherited resistance to a few pathotypes was found in T19 (Yu et al. 2017). Also, a pak choi cultivar “Flower Nabana” was found with pathotype 3 major dominant resistance gene Rcr1 (Chu et al. 2014; Yu et al. 2016).

In B. oleracea, most of these studies concluded that inheritance of this trait was polygenic (Piao et al. 2009). Using cabbage segregation populations, the resistance was shown to be recessive and controlled by two genes with additive effects (Chiang and Crête 1976). Also, dominant or incomplete dominant inheritance was found in cabbage and kale (Hansen 1989; Laurens and Thomas 1993). Further, based on qualitative and quantitative analyses, Voorrips and Kanne (1997) suggested four types of inheritance, one of which was controlled by two complementary genes. The polygenic inheritance of CR resistance in B. oleracea was further validated in broccoli accession CR7 (Figdore et al. 1993), cabbage resources Bindsachsener, Anju, C1220, and GZ87 (Voorrips et al. 1997; Nagaoka et al. 2010; Lee et al. 2016; Peng et al. 2018), and kale cultivars C10 and K269 (Grandclément and Thomas 1996; Moriguchi et al. 1999; Rocherieux et al. 2004; Nomura et al. 2005).

In B. rapa, several CR genes/QTLs conferring complete resistant accessions against specific pathogen isolates were found, and more than ten loci have been identified (Table 3.4). The mapping and cloning of the first loci CRb/CRa took over 20 years. Matsumoto et al. (1998) firstly mapped the dominant major gene CRa in ECD02 on LG3. Ueno et al. (2012) fine mapped the CRa locus using synteny to the A. thaliana genome and revealed a candidate gene encoding a TIR-NBS-LRR protein. This was the first report on the molecular characterization of a CR resistance gene in the genus Brassica. Then, an R locus to pathotype 4, CRb, was mapped by Piao et al. (2004) to an interval of 3 cM from the Chinese cabbage cultivar “CR Shinki”. Kato et al. (2012) identified of a CR resistance locus CRb^Kato to pathotype group 3 in Chinese cabbage “Akiriso”, and the markers were also linked to CRb. To fine map CRb, Kato et al. (2013) further developed 28 markers and located CRb in the 140-kb genomic region and found candidate resistance genes. Zhang et al. (2014) narrowed CRb locus to a region of 83.5 kb on a BAC clone, with several candidates. CRb was tightly linked to CRa and CRb^Kato. To identify the relationship, Hatakeyama et al. (2017) determined the sequence of an approximately 64-kb region, and CRb^Kato and CRa were determined to be the same TIR-NB-LRR gene, while CRb might be a different but closely linked locus. Another example is Crr1-4. At first, Kuginuki et al. (1997) employed RAPD marked to study CR resistance gene Crr1 in turnip cultivar “Siloga” using a DH population. Suwabe et al. (2003) identified Crr1 and Crr2 from G004 (Siloga derived) and concluded that these two loci were complementary. Besides, a weak QTL Crr4 was detected (Suwabe et al. 2006). Hirai et al. (2004) identified and mapped a novel locus Crr3 using RAPD markers, which originated from the turnip cultivar “Milan White”. Saito et al. (2006) used Chinese cabbage progenies and mapped the Crr3 gene in a 0.35 cM segment. Sakamoto et al. (2008) developed populations derived from resistant turnip cultivar “Debra” and identified two CR loci, CRk and CRc. CRk was located close to Crr3. Through fine mapping, Hatakeyama et al. (2013) revealed that Crr1 comprises two loci: Crr1a and Crr1b. Crr1a was cloned from the resistant line G004, encoding TIR-NB-LRR, and was functionally confirmed in susceptible A. thaliana and B. rapa. With the development of genomic and molecular genetics, especially the release of the reference genome sequence of B. rapa, more R loci were discovered. Chen et al. (2013) used SSR markers to map the resistance in ECD04, and six QTLs were identified. Chu et al. (2014) mapped a CR gene from pak choi cultivar “Flower Nabana” to the region between 24.26 Mb and 24.50 Mb on LG A03. Yu et al. (2016) applied bulked segregant analysis sequencing (BSA-seq) and identified a novel resistance gene Rcr1, and Bra019409 and Bra019410 encoding TIR-NB-LRRs were probable candidates. Yu et al. (2017) performed genotyping-by-sequencing (GBS) and revealed three QTLs for CR resistance to six pathotypes. A single co-localized QTL, designated as Rcr4, was on chromosome A03. Two QTLs for resistance to a novel pathotype 5x, designated Rcr8 and Rcr9, were detected, respectively. Huang et al. (2017) adopted SNP-based competitive allele-specific PCR (KASP) markers and bulked segregant RNA-sequencing (BSR-seq) strategies to identify the locus Rcr2 in CR-resistant Chinese cabbage “Jazz”, and Rcr2 was fine mapped to a 0.4 cM interval, with two TIR-NBS-LRRs as the likely candidates. Nguyen et al. (2018) found a dominant monogenic resistance locus CrrA5 in a Chinese cabbage inbred line 20-2ccl on the LG 5. Using BSA-seq, Pang et al. (2018) identified a new locus CRd to a 60 kb region on chromosome A03, which was located upstream of Crr3, using an F₂ segregation population derived from the resistant line 85-74.

Table 3.4 Mapped CR genes/QTLs in B. rapa

Using omics techniques such as RNA-seq and proteomics, significantly related genes were found to be involved in plant–pathogen interaction, calcium ion influx, pathogenesis-related (PR) pathway, chitin metabolism, hormone signaling, cell-wall modifications, antioxidant protein expression, glucosinolate biosynthesis, and glycolysis metabolism (Cao et al. 2008; Verma et al. 2014; Chen et al. 2016; Song et al. 2016a; Xu et al. 2016). Also, plant hormones, especially SA and JA, were all believed to be important in the interactions (Lovelock et al. 2013; Chu et al. 2014; Zhang et al. 2016b; Manoharan et al. 2016; Jia et al. 2017; Luo et al. 2018). Based on these data, some important gene families were further studied for their possible roles during Brassica–Pb interaction. MAPK cascades play key roles in responses to various biotic stresses. Piao et al. (2018) found 5 BraMKK and 16 BraMPK genes that exhibited a significantly different expression pattern between a pair of CR-resistant and susceptible near-isogenic lines (NILs). SWEET genes have been demonstrated as the targets of extracellular pathogens. Li et al. (2018a) identified several BrSWEETs that were significantly upregulated, especially in CR susceptible NIL upon Pb infection. Chitinases are believed to function as a guardian against chitin-containing pathogens. Chen et al. (2018) revealed that 14 chitinase genes were expressed differentially in response to Pb between CR resistance and susceptible NILs. Furthermore, reduced pathogen DNA content and CR symptoms were observed in the CR susceptible NILs after their treatment with chitin oligosaccharides 24 h prior to inoculation with Pb. The findings indicate that chitinases play a crucial role in pathogen resistance of the host plants.

Resistance in B. oleracea appears to be determined by quantitative genes (Piao et al. 2009). So far, a few CR QTLs were identified in cabbage, kale, and broccoli. Figdore et al. (1993) first identified three QTLs showing resistance to race 7 using broccoli. In resistant kale line C10, Grandclément and Thomas (1996) performed QTL detection with RAPD markers, suggesting the existence of at least two genetic mechanisms in the resistance; Rocherieux et al. (2004) further found two to five QTLs depending on the five pathotype used. Of the nine QTLs fully identified, PbBo1 was detected in all isolates and explained 20.7–80.7% of the phenotypic variation. Using another resistant kale line K269, Moriguchi et al. (1999) constructed a genetic map and identified two QTLs for resistance; similarly, Nomura et al. (2005) identified three QTLs. In cabbage, Voorrips et al. (1997) firstly reported two QTLs, pb-3 and pb-4, and a minor QTL contained in landrace Bindsachsener. Nagaoka et al. (2010) identified a major QTL, PbBo(Anju)1 on LG 2, from cabbage accession Anju with a maximum LOD score of 13.7. Tomita et al. (2013) examined the major locus PbBo(Anju)1 and other QTLs and found that a single major locus was not enough to confer sufficient resistance. Lee et al. (2016) employed the GBS technique to construct a high-resolution genetic map. QTLs survey using F_2:3 progenies revealed two and single major QTLs for race 2 and race 9, respectively. The QTLs showed similar locations to the previously reported CR loci for race 4 in B. oleracea, but were in different positions from any of the CR loci found in B. rapa, indicating the divergence of resistance genes in A and C genome. Peng et al. (2018) performed QTL analysis with SNP microarray and identified 23 QTLs for disease incidence and the other two correlated traits, individually explaining 6.1–17.8% of the phenotypic variation. In summary, over 30 QTLs have been found in B. oleracea so far (Table 3.5), indicating the complex genetic basis of CR resistance. It is difficult to compare these QTLs, due to the use of different CR sources and isolates.

Table 3.5 Mapped CR genes/QTLs in B. oleracea

3.6.5 Marker-Assisted Selection (MAS)

Molecular markers are specific inheritable and detectable DNA segments, which can be used for linkage map construction, gene mapping, and MAS. The marker types and mapping methods have been improved greatly from 1990s till now in the genomic era. In 1990s, low-efficiency RAPD, AFLP, CAPS/RFLP markers were mainly used. Since 2000s, convenient and easily detectable SSR, microsatellite, and InDel markers were applied in identification of the resistance genes. From 2010 onward, high-throughput-based methods of mapping have become popular, such as SNP-based markers like KASP markers, microarray, BSA, and GWAS. For example, Huang et al. (2017) adopted KASP markers and BSA-seq strategies to rapidly identify the locus Rcr2 in CR-resistant Chinese cabbage cultivar “Jazz”, and Rcr2 was fine mapped to a 0.4 cM interval, with two TIR-NB-LRRs as the candidates. Of special note, KASP technology possesses high levels of assay robustness and accuracy with notable savings in cost and time. For example, Li et al. (2016b) developed a KASP marker based on the TuMV resistance gene retr02, which could accurately genotype the allele in Chinese cabbage accessions.

MAS is a useful method to predict the phenotype at early developmental stages without field trials. Nowadays, there are some DNA markers against FW or CR resistance genes in the Brassica vegetables (Kawamura et al. 2015, 2017). The genomic era is also symbolized with high-efficiency integrated breeding (HIB), in which multi-MAS methods such as foreground and background analysis are combined with traditional methods such as microspore culture and backcrossing. For example, in the study of Liu et al. (2017b), the resistance-specific markers as well as genome background markers were used in cabbage resistance breeding to FW. Combined with microspore culture and backcrossing, the authors presented a rapid and effective way of generating FW resistance introgression lines in BC₂ generation. During HIB, the genomic background analysis is of great help in eliminating the undesirable linkage drags and rapidly finding the desirable individual.

New tools like CRISPR/Cas9-based genome editing provide new approach of molecular design breeding (MDB) (Cong et al. 2013; Li et al. 2013a). Compared with traditional genetic modification technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the RNA-guided Cas9 system is highly efficient and flexible (Nekrasov et al. 2013; Shan et al. 2013a). This technique has been widely used in field crops including rice, wheat, maize, and cotton, and model plants such as A. thaliana and tobacco (Altpeter et al. 2016; Scheben et al. 2017). Lawrenson et al. (2015) firstly employed CRISPR/Cas9 system on B. oleracea by targeting multicopy genes BolC.GA4.a, leading to Cas9-induced mutations and an expected dwarf phenotype associated with knockout of the target genes. Also, in B. napus, successful editing of target genes including CLAVATA and FAD2 has resulted in inheritable and stable mutations and desirable phenotypes (Yang et al. 2017b; Okuzaki et al. 2018), displaying great potential in its application. With increasingly more genome and transcriptome sequences being available, CRISPR/Cas9 technique will, with no doubt, bring revolution in crop breeding as a fast and accurate method.

3.6.6 Perspective

For certain Brassica species, the resistance resources to some diseases such as BR and CR are very limited. Generally, A genome is rich in TuMV and CR resistance and B genome possesses BR resistances. The inter-species crossing within Brassica genus are widely adopted, using embryo rescue, reciprocal crossing, and MAS, and inter-species hybridizations have been used to transfer and utilize the resistance. Fortunately, the six basic species and others such as B. incana, B. cretica, and B. fruticulosa in the Brassica genus, as well as its close Brassicaceae relatives such as Erucastrum cardaminoides, Raphanus raphanistrum, and Sinapsis arvensis could be used for crossing to facilitate resistance gene exchanges in breeding programs.

A sole resistance gene is easily broken down, which is caused by the pathogen variations as well as global climate changes. For example, a few B. rapa, B. oleracea, and B. napus varieties have been successfully cultivated, with resistance to the specific CR pathogen Pb races (Rocherieux et al. 2004; Werner et al. 2008; Chen et al. 2013). However, they all exhibited loss of resistance within a few years (Kuginuki et al. 1999). At the same time, the vast genetic variability of the CR pathogen Pb and infection by multiple races have been reported (Buczacki et al. 1975; Kuginuki et al. 1999). For BR pathogen Xcc, pathogen variations were frequently discovered and at least 11 races have been reported (Singh et al. 2011; Rouhrazi and Khodakaramian 2014; Burlakoti et al. 2018). Undoubtedly, more durable resistance is in urgent need to secure the Brassica crops production. Durable resistance was first defined by Johnson (1984) as resistance that remains effective during its prolonged and widespread use in an environment favorable to the disease. Complete race-specific R gene is highly effective but is easily broken down; polygenic quantitative resistance is considered to be more durable than qualitative resistance, but its effectiveness varies between cropping seasons due to environmental conditions (Lindhout 2002). Thus, the pyramiding of qualitative resistance with a high level of quantitative resistance in cultivars is an ideal way to maximize the effectiveness and durability of the resistance. This pyramiding model is supported and used in resistance breeding to BR (Vicente et al. 2002) and CR (Piao et al. 2009; Tomita et al. 2013). Thus, combining quantitative resistances with sole R genes is a promising strategy in resistance breeding.

3.7 Abiotic Stress

Plants are sessile organisms. Therefore, the environmental conditions where a plant is cultivated must be tolerable to ensure their successful growth. Abiotic conditions such as temperature, photoperiodicity, moisture, salinity, and soil conditions (nutritional content, pH, and physical characteristics such as porosity) all impose selective pressures upon plants. The adaptations of plant species to various abiotic stressors are thus dependent upon the climatic conditions present within their habitat.

Vernalization is an adaptation that ensures the plant flowers during the spring, when seasonal conditions are amenable to reproductive success (Shea et al. 2018a). Other abiotic stresses such as high ambient temperatures (those found in tropical and subtropical climates) and salt tolerance are also additional limiting factors of agronomic importance in the successful cultivation of the Brassica vegetables. As such, researches examining the molecular mechanisms involved in drought, heat, and salt response have been conducted to identify the regulatory pathways and genes involved.

Many genes induced by abiotic stress are upregulated irrespective of the type of abiotic stress, suggesting a shared regulatory pathway in stress response of plants. As such, the genes associated with abiotic stress response are classified into three groups. The first group of genes encodes for proteins that act to protect plant cells against stresses, e.g., late-embryogenesis abundant proteins (LEAs) (Olvera-Carrillo et al. 2011) and heat shock protein (HSPs)/chaperones (Zhu 2016; Jacob et al. 2017). The second group consists of genes that are involved in signaling cascades, e.g., calcium-dependent protein kinase (CDPK) (Ludwig et al. 2004; Asano et al. 2012) and MAPK (Danquah et al. 2014), or TFs, i.e., genes that regulate another gene’s transcription (Shinozaki and Yamaguchi-Shinozaki 2000). The third group consists of genes involved cellular homeostasis, e.g., aquaporins and ion transporters (Shah et al. 2017).

3.7.1 Late-Embryogenesis-Abundant (LEA) Proteins Can Confer Abiotic Stress Tolerance in the Genus Brassica

LEA proteins are a family of hydrophilic proteins associated with seed desiccation tolerance and play a protective role under salt, cold, and osmotic stresses in the genus Brassica. The expression analysis of LEA4-1, derived from B. napus, revealed that abscisic acid (ABA), salt, cold, and osmotic stresses all induce expression of LEA4-1 gene in leaf tissues, whereas the reproductive tissues such as flowers and developing seeds showed a constitutive expression of LEA4 that was upregulated in flowers placed under salt stress (Dalal et al. 2009). Such findings are consistent with studies examining the role of other LEAs in abiotic stress tolerance. Of the nine classified LEA groups, Groups 1, 2, and 3 have been shown to play a role in tolerance to abiotic stresses in other plants. A Group 1 LEA wheat protein PMA1959 was shown to increase the drought and salinity tolerances of transgenic rice (Cheng et al. 2002). Group 2 LEA wheat protein PMA80 conferred drought and salinity tolerances in transgenic rice (Cheng et al. 2002); chimeric double constructs overexpressing either RAB18 and COR47 or LTI29 and LTI30 conferred freezing stress tolerance to A. thaliana (Puhakainen et al. 2004); cold tolerance was conferred to transgenic cucumber seedlings expressing a pGT::Dhn24 gene fusion (which encodes a SK₃-type DHN24 dehydrin) derived from Solanum sogarandinum—a wild potato species native to central to northern Peru (Yin et al. 2006).

3.7.2 Calcium-Dependent Protein Kinases (CDPKs) Can Confer Abiotic Stress Tolerances in the Genus Brassica

Second messengers are molecules that relay signals received at receptors located on the cell membrane to target molecules in the cytosol and/or nucleus of the cell. The arrival of protein hormones, growth factors, or other signals is relayed to the cytosol in what is referred to as a signal cascade. Calcium is a universal second messenger and plays a key role in the signal transduction pathways in plants (Hetherington and Brownlee 2004). Ca²⁺ signaling systems are composed of a receptor located at the cellular membrane that responds to protein, hormone, or other external cellular signals. In turn, a system for propagation of the signal increases the concentration of Ca²⁺ in the cytosol, and downstream mechanisms then react to the increased concentration of Ca²⁺ in the cytosol. Other cellular systems are then responsible for attenuating the signal by returning Ca²⁺ cytosol concentration back to the pre-stimulus level (Sanders et al. 1999). CDPK is one of the four classes of Ca²⁺ receptors or binding proteins known to exist, with the other three classes comprised of calmodulins (CaM), calmodulin-like proteins, and calcineurin B-like proteins (Zielinski 1998; Hrabak et al. 2003; McCormack and Braam 2003; Kolukisaoglu et al. 2004). CDPKs are unique to the other three classes, functioning without requiring an independent calmodulin, because they contain both a protein kinase domain and a calmodulin-like domain in a single polypeptide, acting in both the direct Ca²⁺-binding and Ca²⁺-stimulated kinase activities (Roberts and Harmon 1992; Hamel et al. 2014).

In the genus Brassica, a genome-wide survey of B. rapa var. rapa identified 55 BrCDPK genes clustered into four subfamilies by phylogenetic analysis. RT-qPCR expression analyses confirmed that all of the identified BrCDPK genes responded to several of the tested abiotic stresses (cold, salt, drought, ABA, pst DC3000, 1-aminocyclopropane-1-carboxylic acid (ACC; the precursor of ET), JA, and SA) with transcriptional upregulation (Wu et al. 2017). To examine tolerance to the phytotoxic effects of SO₂ and salt stress, Tseng et al. (2007) introduced the maize Cu/ZnSOD and/or CAT genes into the chloroplasts of Chinese cabbage cultivar (B. rapa var. pekinensis cv. Tropical Pride), with the resultant SOD + CAT plants exhibiting an increased tolerance to SO₂ (up to 400 ppb) and visible damage one-sixth that of the WT Chinese cabbage plants. The SOD + CAT plants also showed increased tolerance to salinity after exposure to a high salt treatment of 200 mM NaCl for 4 weeks, with the photosynthetic activity of the SOD + CAT plants decreasing by 6% in comparison to a 72% reduction in WT Chinese cabbage plants (Tseng et al. 2007). Taken together these results suggest that CDPKs are involved in the stress responses of B. rapa to various abiotic stressors, with different CDPKs responding to multiple, albeit different abiotic stresses.

3.7.3 Abscisic Acid (ABA) Signaling and ABA-Dependent and Independent Transcription Factors

The ABA pathway is an evolutionarily conserved central regulator of abiotic stress response in plants and acts to mediate many of the responses in abiotic stress signaling (Wasilewska et al. 2008; Danquah et al. 2014). A regulon is a group of genes that are all regulated by the same regulatory protein. Two such abiotic stress-responsive regulons are controlled by ABA. The first contains the ABA-responsive element-binding proteins (AREB) and the ABA-binding factors (ABF), and the second is composed of the myelocytomatosis oncogene (MYC) and myeloblastosis oncogene (MYB) regulon. TFs are proteins with a DNA domain that binds to a recognition site located in the promoter region of a target gene, acting as either activators or repressors by regulating the transcriptional activity of the target gene, thereby regulating the target gene’s expression. The targets of ABA are TFs in the abiotic stress response that activate genes containing ABA-responsive elements (ABRE) or MYC-responsive (MYCR)/MYB-responsive (MYBR) regions within their promoters (Shinozaki and Yamaguchi-Shinozaki 2007; Fujita et al. 2013).

In B. rapa, genes involved in ABA signaling have been identified. Using transgenic A. thaliana with overexpression of one of the two AtHAB2-like proteins in B. rapa, BrHAB2a (Bra025964), was shown to be a putative negative regulator of ABA signaling conferring ABA insensitivity, suggesting that BrHAB2a functions as a protein phosphatase type 2C (PP2C-A), a key component of ABA signaling (Li et al. 2018a). During times of drought, plants reduce water loss via transpiration through the closure of stoma in a process known as stomatal closure. Each stoma is bordered by a pair of guard cells that shrink in response to ABA that is produced in response to drought stress, causing them to become flaccid and the stomatal opening to close. A metabolomic study of drought-stressed B. napus, utilizing gas chromatography–mass spectrometry (GC-MS/MS) and LC-MS/MS, identified metabolic signatures in response to ABA in guard cell protoplasts, suggesting that ABA comprises part of the complex signaling pathway of drought response in B. napus (Zhu and Assmann 2017). The previously mentioned Group 4 LEA genes studied in B. napus, involved in both drought and salt tolerance, are also ABA-induced, further supporting the role of ABA as one of the central signaling pathways in abiotic stress responses (Dalal et al. 2009).

3.7.4 Aquaporins and Ion Transporters, and Their Role in the Abiotic Stress Response of the Genus Brassica

Aquaporins are strongly conserved in both prokaryotes and eukaryotes, and are integral membrane proteins that function as channels in the transfer of water, small solutes, gasses, and ions across the cellular membrane (Takata et al. 2004; Afzal et al. 2016). Aquaporins are part of the highly conserved major intrinsic protein (MIP) superfamily of membrane proteins and are grouped by their localization within the cell. Aquaporins localized to the plasma membrane are further classified into three subgroups, nodulin-26 like intrinsic proteins (NIPs), plasma membrane intrinsic proteins (PIPs), and the uncategorized X intrinsic proteins (XIPs), and are prevalently found on the entirety of the cell surface. Small basic intrinsic proteins (SIPs) are localized to the endoplasmic reticulum (ER). Aquaporins localized to the membrane of vacuole, i.e., the tonoplast, are tonoplast intrinsic proteins (TIPs).

In plants, aquaporins are involved in the abiotic stress responses of drought, salinity, cold, and osmotic stress, functioning to provide osmotic and nutrient homeostasis. To that end, the transgenic overexpression of various aquaporin genes derived from several plants has generally conferred improved drought tolerance to transfected host plants, e.g., overexpression of a tomato SlTIP2;2 in transgenic tomato plants (Sade et al. 2009), wheat TaAQP7 (PIP2) overexpressed in tobacco (Zhou et al. 2012), and the overexpression of Vicia faba PIP1 (VfPIP1) in A. thaliana by preventing water loss through transpiration due to the induction of stomatal closure (Cui et al. 2008). Similarly, the B. napus aquaporin BnPIP1 conferred drought tolerance to transgenic tobacco plants, whereas the BnPIP1 antisense construct caused developmental abnormalities, altered leaf morphology, and decreased drought tolerance (Yu et al. 2005). Likewise, the overexpression of Panax ginseng aquaporin, PgTIP1, improved both salt and drought tolerances (Peng et al. 2007) and cold tolerance in transgenic A. thaliana plants overexpressing AtPIP1;4 or AtPIP2;5 with the latter study noting that, converse to other studies regarding drought tolerance, reduced drought tolerance was observed due to rapid water loss under drought conditions and most likely explained by an increase in hydraulic conductivity (Aharon et al. 2003). Lastly, a tolerance to borate toxicity in A. thaliana plants overexpressing AtTIP5;1 was observed, suggesting that TIPs may be involved in the vacuolar compartmentation of borate (Pang et al. 2010).

A cDNA-AFLP analysis following cadmium (Cd) treatment in B. juncea showed the transcription of drought- and ABA-responsive genes in response to exposure to Cd (Fusco et al. 2005). The aquaporins PIP1 and PIP2, denoted as BjCdR51 and BjCdR49 in B. juncea, were found to be transcribed in response to Cd stress for a day. This observation coupled with expression of the ABA and drought-responsive genes, aldehyde dehydrogenase BjCdR39 and RNA-binding BjCdR55, suggesting that Cd stress imposes water stress, triggering the ABA stress response pathway.

3.7.5 Heat Stress Response

Heat stress responses in plants have been studied for decades, but most of these studies examine the HSP accumulation, signal transduction, and TFs (Kotak et al. 2007; Nakashima et al. 2014; Dong et al. 2015). HSPs and chaperones are ubiquitous among the prokaryotes and eukaryotes, where they primarily act to ensure proper protein conformation after translation and resolve protein aggregates (Jacob et al. 2017). Based on the number of complete plant genomes and EST sequences currently available, there are 30 known heat stress transcription factors (HSF) encoding genes in Chinese cabbage (Huang et al. 2015).

Using two Chinese cabbage inbred lines, “Chiifu” and “Kenshin”, 51 genes (from 130,000 Brassica rapa ESTs) were selected to examine the differences in heat stress responses using RT-PCR. In both lines given heat stress treatment, six, eleven, and three genes were induced, stimulated, and reduced, respectively (Lee et al. 2010). Using the same Chinese cabbage inbred lines, different thermo-tolerances were profiled by transcriptome analysis to examine the transcriptional changes brought about by heat stress. Leaf disks (1 cm in diameter) incubated at 45 °C for 0.5, 1, 2, 3, or 4 h by floating on a water bath showed enrichment for the GO terms “response to heat,” “response to reactive oxygen species (ROS),” “response to temperature stimulus,” “response to abiotic stimulus,” and “MAPKKK cascade.” Most upregulated genes in response to heat stress were HSFs in both lines. Expression of the TF genes Bra024224 (MYB41) and Bra021735 (a bZIP/AIR1 (Anthocyanin-Impaired-Response-1)) were specific in the more heat-tolerant Kenshin lines, suggesting that HSFs and specific TF genes may be responsible for conferring heat tolerance in B. rapa (Dong et al. 2015). In Indian mustard, several-fold upregulation of the HSP101 was observed under heat stress (Bhardwaj et al. 2015).

DNA methylation has a significant effect on the genetic expression of plants in response to different abiotic stresses (Dowen et al. 2012; Karan et al. 2012; Shan et al. 2013b; Garg et al. 2015). DNA methylation patterns are altered under heat stress (Gao et al. 2014; Parkin et al. 2014; Li et al. 2016c; Liu et al. 2017a). Liu et al. (2018) analyzed differential methylation and gene expression in non-heading Chinese cabbage under heat stress and revealed the involvement of the different sets of differentially methylated genes at the early and late stages of heat stress. Changes to DNA methylation occurred by heat stress, affecting a large number and diverse set of genes in B. napus (Gao et al. 2014).

Tissue-specific changes in the expression of the B. rapa noncoding RNA fragments were found, and the most significant changes were observed in tRNA^Glu and tRNA^Asp under heat stress (Byeon et al. 2018a). Their analysis of tRNA fragments (tRFs) also confirmed that three isoacceptors (tRF5´^Asp(GUC), tRF^Gly(UCC), and tRF^Pseudo(UCC)) were severely underrepresented in heat-stressed tissues. The size of the tRF reads was changed significantly in the heat-stressed progeny, while tRFs mapping significantly increased to tRNA^Asp and decreased to tRNA^Ala, tRNA^Arg, and tRNA^Tyr (Byeon et al. 2018b). On the other hand, their enrichment analysis resulted in the significant difference in tRFs processing from tRNA^Ala(AGC), tRNA^Ala(UGC), tRNA^Arg(UGC), tRNA^Thr(UGU), tRNA^Pseudo(UCC), and tRNA^Val(CAC) isoacceptors. The expression of tRFs and snoRNA fragments (snoRFs) is changed by heat stress in B. rapa plant progenies but neither of small nuclear RNA fragments (snRFs) and ribosomal RNA fragments (rRFs) (Byeon et al. 2018b). Recently, it has been found that various types of ncRNAs like miRNAs, siRNAs, lncRNAs, and circular RNAs (circRNAs) play a vital role in heat response (de Lima et al. 2012; Khraiwesh et al. 2012). In B. rapa, miR398 and its target CSDs (i.e., miR398-CSD/CCS pathway) were found in the involvement of the heat stress responses, whereas miR156h and miR156g were found to be upregulated and BracSPL2 were downregulated (Yu et al. 2012). Stief et al. (2014) reported that miR156 can sustainably express the heat stress-responsive genes through SPL genes, especially SPL2 and SPL11 in A. thaliana. Furthermore, 34 specifically expressed lncRNAs and 192 lncRNAs-regulated target genes were identified in B. rapa under heat stress (Song et al. 2016b). In cabbage, heat stress-tolerant lines have stronger expression levels for a transcript of BoHsp70 and TF BoGRAS (SCL13) than that of the heat stress-sensitive lines when under heat stress but the expression levels is much lower at young stages (Park et al. 2013). The expression pattern of BolSGT1 genes in B. oleracea was analyzed under heat stress and found that BolSGT1a is highly upregulated until 1 h of heat stress treatments, and then subsequently decreased (Shanmugam et al. 2016).

Conversely to the increased heat tolerance conferred by HSFs, a transgenic study overexpressing SlHsfA3, derived from Solanum lycopersicum (tomato), in A. thaliana showed overexpression resulted in an increased heat tolerance and a late flowering phenotype, and sensitivity to salinity in germinating A. thaliana plants was increased, suggesting that HSFs are involved in other biological processes related to abiotic stress response (Li et al. 2013c). In B. napus, 6-week-old plants were treated with drought via no watering, and leaves were harvested at 3, 5, 7, 10, 12, and 14 days; the subsequent RT-qPCR transcriptional and LC-MS/MS proteomics analyses showed both differentially expressed HSP transcription levels and protein concentrations at the early stages of drought, with decreased transcription during prolonged drought conditions, suggesting that HSPs are initially upregulated in response to drought stress, most likely as a defensive response to maintain cellular homeostasis (Koh et al. 2015). Further supporting the idea that HSPs are involved in several abiotic stress responses, the expression of an HSP gene (HSP17.4) was found to be upregulated during drought stress in B. juncea, with transcripts present only in the drought-stressed plants. Upon rehydration, transcriptional levels of HSP17.4 were undetectable. Furthermore, the drought-tolerant variety showed a higher transcript accumulation in comparison to the sensitive variety, with drought-induced changes in gene expression in two contrasting genotypes correlating to the physiological responses of each cultivar (Aneja et al. 2015). HSP17.4 is a member of the class-I small heat shock protein (sHSP) family and encoded by At3g46230 in A. thaliana (Yu et al. 2013). These results in B. juncea coupled with similar results in B. rapa suggest that it is the small molecular weight HSPs and HSFs that likely comprise the abiotic stress response in the genus Brassica, and that the response of these small molecular weight proteins is not limited to only heat stress.

3.8 Perspective

The anticipated climatic changes due to increases in mean global temperature pose a challenge to agricultural production of Brassica vegetables. Improved knowledge of the genes and regulatory pathways involved with response to drought, salinity, nutrient deficiency, and temperature (both heat and cold) are fundamental to the success of directed breeding programs aimed at mitigating the impacts of climate change. The efforts that have successfully identified key regulatory genes and beneficial alleles for use in MAS in staple crops such as wheat, rice, and maize may prove useful in Brassica vegetables. However, a careful and thorough evaluation of potential markers should be carried out to confirm their usefulness in the context of a Brassica vegetable breeding program. In addition, a multidisciplinary approach would be beneficial, allowing for a more proactive setting of the goals for future breeding programs by utilizing projected climate changes within a given crop’s region of cultivation.