Keywords

1 Introduction

Banana is one of the commercially important crops that belongs to the family Musaceae and essential for food security in many tropical and subtropical countries (D’Hont et al. 2012). Due to its nutritional value, it has become one of the vital foodstuffs in our daily life (Biswas et al. 2015). As a consequence, the banana-producing area has increased, the processing industry has strengthened, and globally banana trade has expanded. A special emphasis has now been given to its improvements in many countries according to the consumer demand. In recent years, several research groups around the globe have initiated banana improvement projects using conventional and advance breeding technology such as cross-breeding, marker-assisted selection, transgenic breeding, biotechnological approach (micropropagation, somaclonal variation, protoplast culture), radiation breeding, etc. There are lots of obstacles in banana breeding due to its complex taxonomy, ploidy nature, low level of seed fertility and seed viability, and genome complexity. Recently, two genomes of banana have been sequenced which has facilitated the understanding of its genomics. A deep insight into banana genetics provides an enormous opportunity to elucidate the function of genes of interest and also to detect variable (or polymorphic) regions in the genome that are associated with agronomic traits (Liu 1997). Therefore, the genome sequence information of banana is the valuable resource to identify genes of interest and develop markers for the tagging of important agronomical traits. Banana scientists around the world are concentrating their view on genomics of banana to pull out pieces of vital information for the development of a marker and to identify genes responsible for disease, stress, yield, quality of fruits, etc.

In this chapter, we illustrate the present scenario in banana breeding programs as well as banana research around the world and also try to address how to overcome the difficulties in banana breeding.

2 Gene

Gene is the basic molecular unit of heredity that contains a series of nucleotides in which instructions are contained for the synthesis of RNA that translates into protein. Understanding its gene structure, function, evolution, and mode of inheritance is the key for improvement of any crop species. Crop improvement generally involves the insertion of important genes from cultivated or wild species to a cultivar. Most of the wild banana cultivars rich with disease-resistant, stress-tolerant, and pest- and nematode-resistant genes carry many other undesirable traits such as low yield, seedy fruits, nonedible fruits, etc., while edible banana cultivars carry only good fruit qualities. Therefore, important traits such as disease resistance need to be transferred to the cultivars for its improvement. There are two different strategies commonly used in banana crop improvements viz., conventional cross-breeding and transgenic breeding approach.

2.1 Identification and Characterization of Agronomical Important Genes in Banana

Genes are the functional parts of the genome and are directly linked to the agronomical traits of the crops. Identification and functional characterization of agronomically important genes is really a big challenge. It is essential to know the information on its molecular structure, position on the genome, evolution, segregation patterns, etc., for understanding how genes control traits. Recently, two banana genomes (A and B genomes) have been sequenced that speeds up the identification of important genes for subsequent application in the improvement of traits of the banana. Annotation of these genomes identified 36542 and 36638 protein-coding gene models in A and B genomes, respectively (D’Hont et al. 2012; Davey et al. 2013). This protein-coding gene model data set is the valuable resource for further identification and characterization of agronomically important genes in banana. In banana, disease-resistant genes are considered to be more important agronomical traits because several diseases such as black sigatoka, Fusarium wilt and bunchy top greatly hamper banana production around the world. Furthermore, most of the cultivated bananas are highly susceptible to the disease and they are vegetatively propagated. So, there is a high possibility of destruction of banana plantation in any particular region once the disease breaks out. Banana breeders have hence given more importance to disease resistance breeding. Several disease-resistant genes have been identified and characterized in banana; among them, NBS-RGCs, Pto-RGCs, and NPR1 are notable. Zhao et al. (2009) isolated and characterized NPR1-like gene from a local banana cultivar Dongguan Dajiao (Musa spp. ABB); this cultivar is well known to be resistant to Fusarium oxysporum f.sp. cubense (FOC) race 4. In this study, the author noted that this gene is induced by exogenous application of salicylic acid in cv. Dongguan Dajiao but not in cv. Fenjiao which is an FOC race 4 susceptible variety (Zhao et al. 2009). Therefore, this gene has a significant role in the combat FOC race 4 in banana, and it might be a promising candidate for a resistant variety breeding in banana through a transgenic approach. Carotenoids’ biosynthesis gene (PSY) was isolated from the banana cv. Asupina and cv. Cavendish for understanding the mechanism of pro-vitamin A carotenoid (pVAC ) accumulation in banana cultivars. This study showed that banana PSY gene is encoded by two paralogs (PSY1 and PSY2) where in PSY2 is highly expressed in fruit pulp than in the leaf. The enzymatic activity of Asupina PSYs is double to that of Cavendish PSYs; that is why Asupina and Cavendish accumulate different levels of fruit pVAC content (Mlalazi et al. 2012). Consequently, A-PSY2a is a good candidate for the genetic improvement of fruit pVAC content in bananas. In addition, gene discovery in banana is in progress using cDNA library sequencing and functional annotation.

2.2 Introduce or Transfer Important Genes for Improvement of Banana

In order to introduce/transfer new genes (or traits) into cultivars from their wild relatives of banana, two different strategies have been widely used, conventional breeding and transgenic breeding. Improvement of commercial variety of banana with important agronomic traits through cross-breeding approach is inefficient due to its triploidy and sterility. So, transgenic breeding approach overcomes the limitation of commercial banana variety improvement. A well-characterized gene could be cloned from the wild species of banana or other plant species and transferred to commercial cultivars. Furthermore, this approach is significantly effective only for the traits controlled by a single gene. Genetic transformation and transgenic regeneration method are well established for the banana plant. But to date, there is no significant progress of transgenic breeding in banana compared to other crop plants. In order to improve black sigatoka disease resistance in banana, several genes from different organisms (gene ThEn-42 from Trichoderma harzianum, StSy from grape, Cu, Zn-SOD from tomato) have been cloned and transferred to banana. After a 4-year field trial, some of the transgenic lines showed improved tolerance to black sigatoka disease (Vishnevetsky et al. 2011). The replication initiation protein-encoded gene (Rep) was transferred to the banana to develop a banana bunchy top virus-resistant cultivar (Tsao 2008). A plant gene that encoded the protein called cystatin is effective against nematodes. This gene which has been transferred to the Cavendish banana improved its tolerance to the nematode (Atkinson et al. 2004). Genes pflp and hrap were cloned from sweet potato and transferred to banana cultivars. In vitro assays showed that the transgenic lines were completely resistant to banana Xanthomonas wilt disease (Tripathi et al. 2009). A gene AMP1 encoding antimicrobial peptide was cloned from onion seeds and transformed in banana cultivar Rasthali (AAB, Silk gp). In this study, embryogenic cells of cv. “Rasthali” were transformed with Agrobacterium strain LB4404 harboring binary vector pCAMBIA2301 containing gene Ace-AMP1. Transgenic plants were then tested with their performance against Fusarium oxysporum f.sp. cubense race1 (Foc), and results suggest that Ace-AMP1 gene is able to improve resistance of banana against Fusarium wilt disease (Mohandas et al. 2013) under glass house conditions. To facilitate the improvement in nutritional content of banana with vitamin A, vitamin E, or iron, an Austrian scientist transferred vitamin A, vitamin E, or iron accumulation genes in banana cv. “Nakinyika,” “Mpologoma,” “Nakasabira,” and “Sukalindizi” (Pillay et al. 2012b).

3 Genetic Markers

A genetic marker is a visible phenotype or biochemical compound or gene or a segment of DNA that can be used to identify individuals or species. Genetic markers are inherited and segregated in Mendelian manner. Based on the principle or methodology, genetic markers are of three types: morphological markers, biochemical markers, and molecular markers. These genetic markers further classified into several subgroups according to the technology and principle.

3.1 Overview of Different Marker Technology Applied in Banana Research

3.1.1 Morphological Markers

A morphological marker is the visible, prominent phenotype that can be used to distinguish one or more individuals from a population. Agronomical traits such as plant height, leaf architecture, seed coat color, flower color, fruit flavor, leaf aroma, etc., are used as morphological markers to select individual in plant breeding. In banana leaf, architecture and texture are commonly used as morphological markers. For example, the leaf arrangement of diploid banana genotypes is upright (around 45° angle), triploid genotype is horizontal, and tetraploid genotype is downward. Parthenocarpy is one of the most important agronomic traits of banana. Most of the edible banana cultivars are parthenocarpic, they don’t produce seeds, and they are triploids. Consequently, this trait was also used to identify breeding materials in the banana breeding program. In 1955, Simmonds and Shepherd discovered banana hybrid identification system using 15 prominent traits, that are highly polymorphic between M. acuminata and M. balbisiana (Simmonds and Shepherd 1955). Among these, 13 traits were related to the reproductive organ, such as peduncle of inflorescence, pedicels of flower, male flower color, free tepal, stigma color, and arrangement of ovules, and the other seven were related to the bract of the male bud (shape, apex, shoulder, curling, color, color fading, and scars), while the other two were pseudostem color and petiolar canal of the leaves. Based on these phenotypic scoring systems, the M. acuminata × M. balbisiana hybrids could be characterized into different ploidy levels (diploid, triploid, and tetraploid) and five genetic groups (AA, AB, AAA, AAB, and ABB). Most of the morphological markers are polygenic and highly influenced by the environment. That is why the utility of morphological markers in plant breeding is limited. However, this type of marker is still used in banana breeding, only where other markers’ assay facilities are not available.

3.1.2 Biochemical Markers

Biochemical markers are the molecules or the products of genes such as proteins/enzymes which are polymorphic in their structure as well as their amino acid composition. For example, isozymes are encoded by the homologous gene located in the different loci in the genome, while allozymes are allelic variants of the same gene. Allozymes and isozymes move at different speeds through a gel because they differ from each other in size, structure, and charge. So, allelic variation can be detected among the individual in a population by using this type of markers. Bonner et al. (1974) first used peroxidase isoenzyme to distinguish banana clones and species. Unfortunately, they failed to distinguish banana clones and species. Thereafter, Rivera (1983) successfully distinguished ABB/BBB (Saba) and ABB (Bluggoe) genomic group using peroxidase and polyphenoloxiydase. After that several researchers around the globe used biochemical markers to characterize banana clones, hybrids, and species, and it was popular until the molecular markers were employed in banana (Rivera 1983; Jarret and Litz 1986a, b; Bhat et al. 1992a, b; Mandal et al. 2001; Megia et al. 2001; Dhanya et al. 2006). The biochemical markers are sensitive to the environment and have a reproducibility problem.

3.1.3 Molecular Markers

A molecular marker is a fragment of DNA that is located in a particular region of the genome and variable between two individuals. In the past decade, advances have been made in the molecular marker technology, and several marker technologies have been invented by a molecular biologist. The use of these marker technologies for crop improvement is now a routine work. Based on the principle of molecular marker technology, it can be categorized into several subgroups. The most useful and popular marker technologies that are commonly used in the banana crop improvement program are discussed herewith.

3.1.3.1 Restriction Fragment Length Polymorphism (RFLP )

RFLP marker is non-PCR-based molecular marker technique. The variation among the genotypes obtained from this technique is based on restriction site variation of the studied individuals. Restriction site is frequent in the genome, and it is variable between two individuals because the presence of restriction enzyme cleavage site in the genome of two different individuals may not be the same. The presence of restriction site variation between two sister individuals might be due to mutation, crossing over, genome duplication, insertion, or deletion of the DNA fragments. Restriction fragment length polymorphism (RFLP) is a robust, codominant in nature, highly reproducible, and able to characterize heterozygote and homozygote condition of the individual. RFLP markers successfully applied in banana genetics and breeding are useful in estimating variation in the chloroplast genome of the banana (Gawel and Jarret 1991; Baurens et al. 1997), phylogenetic relationship of the banana and its relatives (Gawel et al. 1992; Jarret et al. 1992; Wong et al. 2001), mapping (Miller et al. 2008), etc,. RFLP markers are often used in combination with other types of markers, such as RAPD, SSR, etc., in banana breeding (Bhat et al. 1995), although RFLP markers are not suitable for large-scale utilization in the breeding program because of their complicated assay technique, their expensive nature, and their requirement of sophisticated equipments.

3.1.3.2 Variable Number of Tandem Repeats (VNTR )

The VNTR marker also known as non-PCR-based marker technology is used in plant genetics and breeding research. This technique involves restriction digestion followed by the hybridization with the probe containing minisatellite sequences. In principle, the VNTR marker is the fragment of the genome composed of tandem repeat units of a 10–50-base motif (known as minisatellite) and flanked by the conserved DNA restriction site. Many reports showed that tandem repeats are highly abundant and randomly distributed throughout the plant genome (Biswas et al. 2015; Manzo-Sánchez 2008). Therefore tandem repeat containing fragments of the genome could be the vital source for development of a large number of VNTR markers. This marker is highly polymorphic and reproducible, but its assay technique is complex and costly. So this marker is not popular with banana researchers. Several VNTR markers have been developed in Musa as reported by Crouch et al. (1998, 1999a) and Kaemmer et al. (1997), and used in genotyping and characterization of Musa breeding populations. Recent advances in the genome sequence technology and availability of the whole genome sequence of Musa spp. provided the opportunity to develop Musa genome-specific VNTR marker. This marker can be useful in banana research for DNA fingerprinting, identification of varieties, cultivars, and individuals, and also at population-level study.

3.1.3.3 Random Amplified Polymorphic DNA (RAPD )

This marker is well known for its simple assay system. But a major drawback of this marker is reproducibility. Evidences showed that different laboratories achieved different results when tested under identical PCR parameters and conditions (Pillay et al. 2012a). The common uses of RAPD marker in banana research are to:

  • Detect diverse genotypes (Kaemmer et al. 1992; Howell et al. 1994; Bhat and Jarret 1995; Uma et al. 2006; Jain et al. 2007).

  • Identify duplicate accessions from the in vitro and ex vitro germplasm collection (Ray et al. 2006).

  • Identify somaclonal variation (Dhanapal et al. 2014).

  • Differentiate Musa genomic group (Howell et al. 1994; Rekha et al. 2001), genetic diversity study, etc. (Bhat and Jarret 1995).

The first RAPD marker was applied for fingerprinting of wild and cultivated species of banana in 1992 (Kaemmer et al. 1992). A and B genome-specific RAPD marker has been identified for Musa, and it is very useful to estimate genomic composition of banana cultivars (Pillay et al. 2000; Oselebe et al. 2006; Pillay et al. 2006). Uma et al. (2006) applied 80 RAPD primers to estimate intraspecific variation and relation of Indian wild Musa balbisiana Colla collection. In this study, only 4 primes produced 31 polymorphic bands, and all the 16 accessions were clustered into four as against seven clusters obtained through morphotaxonomic characterization. Some of the accessions failed to group in the same cluster as revealed by morphotaxonomy data. This result can be further improved using more RAPD markers and also using other types of markers such as ISSR, ITS, AFLP, etc. In recent years, RAPD has been used with other markers for the Musa genetics and breeding study (Howell et al. 1994; Lamare and Rao 2015). Bhat et al. (1995) reported that RAPD primer OPC-15 (5′-GACGGATCAG-3′) had the potential for distinguishing 55 of the cultivars by producing 24 bands but failed to characterize the clones of Gros Michel and Venkel.

3.1.3.4 Intersimple Sequence Repeats (ISSR )

ISSR is a PCR-based marker technique, and its assay system is similar to RAPD. This technique does not require any prior knowledge of genome sequences of the organism. Like RAPD, a versatile set of primers is used in the ISSR assay. Furthermore this marker technique is simple, fast, and cost effective. The main drawbacks of this technology are the dominant nature of the marker, that the homology of the bands is uncertain, and that they do not allow the identification of heterozygous from homozygous dominance. This marker involves amplification of the DNA fragments nearby at an amplifiable distance between two identical microsatellite repeat regions oriented in reverse orders (Spooner et al. 2005). ISSR has been widely used for varietal identification, genetic diversity analysis, genetic stability of the tissue culture-derived clone, etc. The use of ISSR in banana research has not received wide attention so far. Several studies using ISSR marker in banana research were mainly limited in small-scale germplasm characterization (Venkatachalam et al. 2007; HaiFei et al. 2010; Poerba and Ahmad 2010; QianJie et al. 2010; Khatri et al. 2011; Yao et al. 2012; Dhanapal et al. 2014; Lamare and Rao 2015) and in genetic fidelity testing of cultivated banana (Rout et al. 2009; Dhanapal et al. 2014). Choudhary et al. (2014) used 40 ISSRs with 60 RAPD markers to estimate molecular variability of 12 plantain ecotypes and found that ISSR is a better tool than RAPD for assessment of genetic diversity in plantain ecotypes. Another study reported the use of ISSR with RAPD to test genetic stability of three micropropagated banana (Musa spp.) cultivars and found that ISSR detected more polymorphism than RAPD (Ray et al. 2006). Similarly, ISSR were used for finding genetic uniformity of micropropagated banana plantlets (Rout et al. 2009), ​in vitro mutagenesis, and variance (Khatri et al. 2011). ISSRs were also employed to evaluate the genetic diversity and classification of 27 wild banana accessions collected in Guangxi, China (Qin et al. 2011). Padmesh et al. (2012) explored the pattern of genetic variation of 32 wild M. acuminata Colla collected from the Southwestern Ghats in the peninsula of India using ISSR markers.

3.1.3.5 Internal Transcribed Spacers (ITS)

The nuclear ribosomal genes (rDNA ) encoded three rRNA subunits, viz., 18S, 5.8S, and 26S. These subunits are present in the genome as clusters of tandemly repeated units of 250–20,000 copies (Rogers and Bendich 1987). rDNA is transcribed as a single unit along with the two spacers known as the internal transcribed spacers (ITS) that separate the 5.8S subunit from the 18S and the 26S subunits. The sequences of these ITS regions are variable among closely related clones or species. Therefore, this variation could be used as a marker, and it is a powerful tool for assessing phylogenetic relationships at the lower taxonomic levels. ITS markers are used for genetic diversity, population structure and clone identification. In Musa, ITS marker was used to distinguish A and B genomes. Nwakanma et al. (2003) amplified ITS region of seven M. acuminata (A genome) and five M. balbisiana (B genome) accessions. The result revealed that all the accessions produced 700-bp fragments; subsequently this fragment was digested with ten different restriction enzymes (AluI, CfoI, DdeI, HaeIII, HinfI, HpaII, MspI, RsaI, Sau3AI, and TaqI). Only RsaI produced a consistent polymorphic banding pattern between M. acuminata and M. balbisiana. The RsaI produces four fragments (350 bp, 180 bp, 120 bp, and 50 bp) in the M. balbisiana accessions and three fragments (530 bp, 120 bp, and 50 bp) in M. acuminata accessions. The fragment of 530 bp was unique to the A genome, while two fragments of 350 bp and 180 bp were specific to the B genome. Further 56 accessions from different genomic combinations of Musa spp. (viz., AA, AAA, AAB, AB, and ABB) were amplified with ITS primer and subsequently digested with RsaI. Results showed that only a 530-bp fragment was present in A genome-containing individuals but absent in B genome-containing individuals. On the other hand, a B genome-containing individual produced 350-bp and 180-bp fragments that were absent in A genome-containing individuals. This finding was a breakthrough for characterization of Musa spp. according to their genomic combination using the ITS marker.

3.1.3.6 Inter-retrotransposon Amplified Polymorphism (IRAP )

IRAP is a PCR-based dominant marker. This marker detects retrotransposon (RT) insertion-based polymorphism among the individuals. RTs are abundant in the plant genome, and they compose over 50 % of the total nuclear genome. RT elements act as a mutagenic agent by insertion or deletion in the genome thereby making them a good source of genomic variation (Heslop-Harrison 2000); thus RT can be used as a genetic marker for various purposes in plant genetics research. It has been used in fingerprinting, genetic diversity, and population structure studies in plants (Biswas et al. 2010a, b; Khadivi-Khub et al. 2015; Singh et al. 2015). IRAP markers are also used in Musa for classifying the genome constitution (Nair et al. 2005; Teo et al. 2005; Häkkinen et al. 2007; Pachuau et al. 2014), genetic diversity (Saraswathi et al. 2011), and identification of wild Musa spp. (Häkkinen et al. 2007; Häkkinen and Teo 2008).

Ty3-gypsy-like LTR sequence of banana (Musa acuminata monkey retrotransposon, AF 143332) was used to design IRAP primer, and it was used to identify the B genome in the banana cultivars. Nair et al. (2005) used this marker to classify 36 banana cultivars and observed multiple polymorphic bands. Among these bands, a specific band of 350 bp was observed in all the cultivars with the B genome. In ABB genomes, the band intensity was highly observed in AAB genomes. But there was an exceptional result found in the cultivar “Manoranjitham” (AAA), in which B-specific band was present with similar intensity observed in ABB (“Kosta bontha”). The author suggested that “Manoranjitham” was misidentified. Teo et al. (2005) applied IRAP methods to generate molecular markers for estimation of diversity, genome constitution, and relationships of Malaysian banana cultivars. IRAP is used with an RAPD marker to study intragroup diversity among Cavendish (AAA) clones of banana (Saraswathi et al. 2011). Singh et al. (2015) used IRAP and morphological markers to characterize Musa germplasm from Northeast India.

3.1.3.7 Amplified Fragment Length Polymorphism (AFLP )

AFLP is a PCR-based multilocus marker technology employed in many plant studies relating to genetic identity, phylogenetic relation, parentage identification of clones and cultivars, etc. It is a robust, highly reproducible, and dominant marker, but in a segregating population, AFLP can be detected as codominant. The AFLP marker is capable of generating 80–500-bp fragments from different genomic sites at the same time; therefore this marker detects high polymorphisms in different genomic regions simultaneously. There are many advantages of AFLP compared with other markers such as RAPD, RFLP, SSR, etc. AFLP has become particularly useful in the study of plant taxa, where genomic information is not available.

Thirty-nine accessions from four main sections of the genus Musa were evaluated using eight AFLP markers (Ude et al. 2002a). A wide range of variability was observed among the species within the sections of the genus Musa. This study proved that AFLP is a very useful tool in determining taxonomic relationships in the genus Musa and potentially useful to resolve some of the complicated taxonomic questions in the genus Musa. According to morphological data, Simmonds and Weatherup (1990) place M. peekeli ssp. peekeli as a close relative of M. peekeli ssp. angustigemma, while Argent (1976) suggests that M. angustigemma should rank under M. peekeli as a subspecies. AFLP data suggest that M. peekeli ssp. angustigemma is clearly distinct from M. peekeli ssp. peekeli, and therefore the specific rank of M. angustigemma should be retained.

Several studies suggested that the AFLP marker is effective for genetic diversity analysis in Musa and the level of polymorphism comparatively higher than other markers used in the Musa diversity analysis (Crouch et al. 1999; Loh et al. 2000; Wong et al. 2001, 2002). The relationship of cultivated banana with M. acuminata and M. balbisiana is precisely classified by AFLP markers (Wongniam et al. 2010). Youssef et al. (2011a) reported that AFLP is able to discriminate A, B, S, and T genomes within Musa species. In order to detect genetic stability of somaclonal variation in somatic embryo-derived plants of two banana cultivars, namely, “Grand Naine” and “Williams” (Musa acuminata Colla, AAA), AFLP marker technology was applied (Youssef et al. 2011b). Thirty primer combinations were used to detect polymorphism among somaclonal variants, and the result showed that 1.4 % and 1.6 % bands were polymorphic in “Grand Naine” and “Williams,” respectively. In addition, 8 and 16 bands were specific to the observed regenerated plants of “Grand Naine” and “Williams,” respectively, which were absent in their parents, while ten and five bands are completely absent in the regenerated plant of “Grand Naine” and “Williams,” respectively, which were exclusively present in their parents. AFLP markers were also used for the genetic analysis of Musa species in combination with other molecular markers (Opara et al. 2010; Youssef et al. 2011a) and also to identify somaclonal variation (James et al. 2004; Vroh-Bi et al. 2011; Youssef et al. 2011b).

3.1.3.8 Simple Sequence Repeat (SSR ) or Microsatellite Marker

An SSR marker is well known for its genome-wide distribution, codominant inheritance, reproducibility, multiallelic nature, and easy assay technique. Recent progress in DNA sequencing technology has provided an opportunity to routinely develop large sets of SSR markers. Transferability of a genic SSR marker is higher than genomic SSRs. This feature helps to design anchor markers for comparative mapping studies. Since they are often more conserved, genic SSRs may provide an insufficient degree of polymorphism to discriminate between closely related germplasm. Therefore genomic SSRs may be valuable complements. In the recent past decade, SSR markers are extensively used in a wide range of breeding applications. Several hundred EST-SSR markers were developed by in silico EST sequence mining of several Musa spp. (Li et al. 2012; Passos et al. 2012, 2013; Backiyarani et al. 2013; Ravishankar et al. 2015). SSR markers were developed from B genome of Musa (Buhariwalla et al. 2005; Ravishankar et al. 2013); 226 were developed from Musa GSS (survey of the genomic sequence) data (Ravishankar et al. 2012), and 41 were from Calcutta 4 using BAC sequences (Miller et al. 2010). Most of these markers are not freely accessible; some are redundant with alternate IDs or names, while their physical positions and functional natures are unknown. Consequently, the use of these markers in Musa spp. improvement is limited. In the whole genome sequences of two banana varieties, more than 0.1 million EST and several thousand GSS sequences were mined for SSR marker discovery, and 119,540 non-redundant SSR markers were developed (Biswas et al. 2015). Subsequently these markers were characterized, classified, and stored in a searchable database known as Musa marker database and it can be access through the following link http://www.agrogene.ac.cn:8088/mumdb/mumdb_home.html. A part of these markers was validated by wet lab assay and their potentiality was estimated for a genetic diversity study in Musa population.

3.1.3.9 DArT

Diversity arrays technology (DArT ) is DNA/DNA hybridization-based molecular marker technology, extensively used for a quick assessment of the structure of germplasm collections. This is the cost-effective genotyping technology that can detect all types of DNA variation including SNP, indel, CNV, and methylation. It also can detect simultaneously variation at numerous genomic loci without any sequence information of the organism. In recent years, this technology become popular and is regularly used in the crop improvement including Musa (Risterucci et al. 2009). A total of 836 DArT markers were developed and used for Musa spp. genotyping. Ten percent of these markers were A genome specific and able to target this genome portion in a related analysis, among diverse ploidy constitutions (Risterucci et al. 2009). Risterucci et al. (2009) in their study clearly demonstrated the usefulness of DArT markers for Musa spp. genotyping and genetic diversity analysis. They used four complexity reduction methods of DArT for Musa spp. and tested their performance on 48 Musa genotypes and finally invented two methods that produced more polymorphic information content than others. Subsequently, selected methods were used for large-scale Musa spp. genotyping.Musa accessions, around 168 in number that were collected from CIRAD (Neufchateau, Guadeloupe) and IITA (Ibadan, Nigeria) were used. All these accessions were derived from M. acuminata (A genome) and M. balbisiana (B genome). And the result reveals that DArT markers classify them according to their origin and genomic combinations. Kilian (2007) developed 1,500 DArT markers using a wide array of Musa accessions, which were used for Musa framework map. Further these markers with additional 380 markers have been used to construct a map at CIRAD.

3.1.3.10 Ecotilling

Ecotilling is a new class of molecular marker technology that was developed as a high-throughput and low-cost platform for the SNPs discovery and small indels. This technology was first used in Arabidopsis ecotypes and then adopted for many species including humans, switchgrass, poplar, melon, banana, etc. (Till et al. 2010). Since its discovery, ecotilling has been used for more than 20 plant species for genetic diversity, population structure, mapping, and QTL analysis. In principle, ecotilling is an enzymatic mismatch cleavage-based DNA hybridization technology. Around 700–1,600-bp genic regions are amplified by PCR using fluorescent labeled gene-specific primers. Subsequently, samples are denatured and annealed; then heteroduplexed molecules are produced through the hybridization of polymorphic amplicons. Mismatched regions or double-stranded duplexes are then cleaved using crude extract of celery juice containing the single-strand specific nuclease CEL I. Cleaved products are then resolved in denaturing polyacrylamide gel electrophoresis (PAGE) for observing banding pattern.

In Musa, ecotilling method was used for the discovery and characterization of nucleotide polymorphisms of diploid and polyploid accessions. Over 800 novel alleles in 80 accessions were identified as polymorphic using 14 gene-specific primers by Till et al. (2010). In this study, more than 6,000 polymorphisms were detected in over 800 alleles in 80 Musa accessions. Further, sequencing-based validation was performed for the detection of SNPs variation among the accessions. Consequently, sequencing and banding patterns reveal that ecotilling is the perfect platform for discovery of polymorphisms in homologous gene targets in Musa accessions.

3.2 Utility of Marker Technology in Banana Breeding

3.2.1 Genotype Identification

Genotype identification is known as genotyping . It is one of the routine works for plant breeders prior to selection of breeding material. Environment has great impact on phenotypic expression on gene or traits. Selection of breeding material based on the phenotypic traits from the population or germplasm collection maintained in a different environment may be misleading. Therefore, genotyping is important in a breeding programme. Genotyping is a DNA-based technology commonly used to characterize or identify any living organism. It is also known as fingerprinting or DNA fingerprinting. This methodology is extensively used in plant breeding for identification of individuals in a population, for discrimination between individuals in an inbred line, or for determination of genetic distance between genotypes in general. Different types of markers such as biochemical, RAPD, ISSR, RFLP, AFLP, SSR, etc., are commonly used for Musa spp. genotyping. In the early 1980s, biochemical markers are used for Musa spp. genotyping, for example, peroxidase and polyphenoloxidase used to identify “Saba” (ABB/BBB) and “Bluggoe” (ABB) types of banana (Rivera 1983). Dhanya et al. (2006) and Dhanya et al. (2006) used isozymes to identify banana cultivars resistant to banana bract mosiac virus (BBrMV). An RAPD marker has been developed for genotyping dwarf off-type Cavendish banana (Musa spp. AAA) cultivar. The primer OPJ-04 (5′-CCGAACACGG-3′) was found to amplify an approximately 1.5-kb band which is consistently present in all normal Cavendish cultivars but absent in dwarf Cavendish cultivars (Damasco et al. 1996). Bhat et al. (1995) and Bhat and Jarret (1995) used 60 RAPD markers for identification of 57 Musa cultivars; among these RAPD markers, 49 produced consistent results and were able to identify 55 cultivars but failed to characterize Gros Michel and Venkel clones. A total of 33 SSR markers were used to characterize 35 cultivated banana (Musa spp.) genotypes, including triploid cultivars and tetraploid hybrids (Creste et al. 2003). Christelova et al. (2011) used 19 sets of fluorescently labeled SSR primers for genotyping 70 diploid and 38 triploid banana accessions.

3.2.2 Germplasm Managements

Germplasm collection and its systematic management are essential for quick access of a right individual for the breeding program. In general, genebank collection consists of multiple copies of same genotypes; these duplicates may occur for various regions, for example, documentation error, the sampling of multiple individuals from genetically homogeneous collections, exchange of identical accessions between genebanks, etc. (Spooner et al. 2005). The redundant accessions of the genebank have no significant impact, but maintaining them in the genebank requires time, space, and resource. So the elimination of redundant accessions from a large collection of genebank is a big challenge. Correct classification and identification of unique accessions allows solving germplasm management problems. There is a high possibility of inclusion of redundant accessions in the vegetative propagated crop plant genebank such as banana, because the germplasms of this crop are collected based on its morphological traits and a similar clone is maintained in a different geographical region with a different name. Further, cultivars of the banana are evolved through human selection. Therefore efficient germplasm management is an important factor in banana breeding. In the last few decades, a significant number of studies have been conducted for the germplasm characterization of Musa spp. around the world using different kinds of molecular markers including RAPD, AFLP, SSR, etc. (Bhat and Jarret 1995; Creste et al. 2003; Christelová et al. 2011).

3.2.3 Genome Composition Determination

Most of the banana cultivars are triploid and derived from interspecies hybridization between M. acuminata (A genome) and M. balbisiana (B genome), while other banana and plantain genomes are derived from the combination of A, B, T, and S species genomes. The knowledge of genomic combination of the cultivars or a clone of banana and plantain is essential for its improvement. Several molecular markers have already been developed to identify genomic combination of banana and plantain cultivars. Three RAPD primers (A17, A18, D10) from OPERON Technologies (Alameda, CA, USA) were able to identify A and B genome of Musa spp. Primer A17 generated two fragments (600 bp, 100 bp) and primer D10 one fragment (320 bp) that was unique to M. acuminata (A genome), while primer A18 produced three fragments (200 bp, 250 bp, 300 bp) in M. balbisiana (B genome). The fragments B18250 and B18300 were always present in genotypes with at least one B genome; on the other hand, fragment A18200 was present in clones with two B genomes. These three RAPD primers are extensively used for the determination of banana genomic composition (Jones 2000; Pillay et al. 2000). A PCR-RFLP-based ITS marker is also applicable for banana A and B genome identification (Nwakanma et al. 2003). The ITS marker produced one fragment of 530 bp that is A genome specific and two B genome-specific fragments of 350 bp and 180 bp. An interspecific hybrid of A and B genome possessed all three fragments.

3.2.4 Genetic Diversity and Population Structure Estimation

Molecular marker technology is widely used for study genetic diversity, population structure, and phylogenetic relationships of the crop plants. Many studies have been attempted to estimate genetic diversity using different types of markers in banana and plantain wild and cultivar collections. For example, the genetic diversity of 100 Indonesian Musa cultivars from the different genomic groups of Musa (AA, AAA, AAB, ABB, and BB) was estimated using isozymes of malate dehydrogenase (MDH), peroxidase (PRX), and glutamate oxaloacetate transaminase (GOT) (Megia et al. 2001). The result reveals that MDH and PRX were more useful than GOT for genetic diversity study of Indonesian Musa cultivars.

AFLP marker technology was applied to estimate the genetic diversity of banana and plantain population collected from different geographical regions and also different cultivars and wild accessions (Ude et al. 2002a, b; Wong et al. 2002; Wang et al. 2007; Opara et al. 2010; Wongniam et al. 2010; Youssef et al. 2011a). Wong et al. (2001) used an AFPL marker to assess the genetic diversity of 32 Malaysian wild bananas Musa acuminata Colla, and results showed that AFLP efficiently classified Malaysian wild bananas based on genetic distance. Ude et al. (2002b) assessed the genetic diversity and phylogenetic relationships of M. acuminata and M. balbisiana and their natural hybrids and noted that AFLP markers were able to produce enough information about the genetic diversity of M. balbisiana accessions to further classify them into two subgroups.

Seven SSR markers were used to estimate genetic diversity and population structure of six M. ornate populations (Burgos-Hernández et al. 2013). A low level of genetic diversity was observed in the M. ornate populations. This low diversity may occur due to recent fragmentation of events, which meant that there was not enough time gap between populations to detect differences. It may also be the cause of self-pollination, clonal reproduction, bottleneck selection, or the decline in pollinator in population. This study was proposed to conserve and maintain all the remaining M. ornate population to maintain gene flow and increase the genetic diversity. The ex situ collection of 224 Musa spp. at Embrapa, Brazil, was analyzed by 16 SSR markers for the estimation of genetic diversity and population structure. The findings of this study showed that structure analysis might be useful in identification of ancestry of recently developed tetraploid hybrids and triploid cultivars by breeding programs.

3.2.5 Mapping and Marker-Assisted Breeding

Genome mapping sets up the roadmap of a genome that helps to locate important genes, manipulate them, identify the molecular environment of both coding and noncoding DNA sequences, etc. The advancements in the area of sequencing technology and molecular marker developments open the door for tagging agronomically important traits via mapping and marker-assisted breeding. Although several linkage maps have been developed in Musa spp., their quality and marker saturation remain poor; that is why their utility in banana breeding is still limited (Faure et al. 1993; Baurens et al. 1997; Vilarhinos 2004; Hippolyte et al. 2010). Faure et al. (1993) developed first partial molecular linkage map based on 77 markers, in which a significant number (36 %) of markers were deviated from Mendelian segregation. The second map was developed for M. acuminata cv “M53,” in which it exhibited 11 linkage groups with a significant number of markers distorted from the Mendelian segregation ratio. The linkage map developed by Vilarhinos (2004) is composed of 14 linkage groups; 59 % of markers are skewed from the Mendelian segregation ratio; the mapping population is derived from F1 and pseudo-testcross strategy used to construct the linkage group. The oversized linkage groups of this map may be the cause of distorted markers that are involved in the structural rearrangements of chromosomes. The high-density linkage map was developed in Musa sp. using DArT and SSR markers (Hippolyte et al. 2010). In this study, two parental maps and one reference map were constructed. The female parent map consists of 11 linkage groups with 261 markers (125 SSRs and 136 DArTs). The map spanned 920 cM, with one marker per 3.8 cM.; 59 and 9 markers were comprised in the largest and smallest linkage group, respectively. The male parent map is obtained at LOD 5; a total of 359 markers consisted the 9 linkage groups with a total map length of 1,081 cM., in which one marker is distributed per 2.9 cM., but the marker distribution in each linkage group was not uniform. Further a synthetic linkage reference map was developed using 489 markers (167 SSRs, 322 DArTs), among which 132 were anchored markers. This map contains 11 linkage groups covering 1,197 cM., at an average of 38 markers per linkage group and one marker per 2.8 cM.

Marker-assisted breeding is useful for identifying markers linked to important traits; these markers can be used for the direct screening under greenhouse condition at early stage of growth. This approach reduces time, cost, and gaps in the breeding program that dramatically speeds up the selection process. There are a few markers identified in Musa linked with some important agronomical trait that is related to disease resistance. Four methylation-sensitive amplification polymorphism (MSAP) markers were identified as linked markers for black sigatoka disease-resistant gene of banana (Gimenez et al. 2006). There are some RAPD markers used for selecting black sigatoka disease-resistant, nematode-resistant, and salt-tolerant banana clones (Miri et al. 2009). A banana SERK-related marker is associated with somatic embryogenic competence and disease-resistant response in Musa sp. (Huang et al. 2010; Cunha et al. 2015).

4 Conclusions

In the last few decades, gene discovery and marker technology have revolutionized plant breeding. Genes and their related functions were identified; they were cloned and utilized in bred crop improvement. Scientists around the globe are identifying agronomically important genes for banana varietal improvement. Molecular marker technology has made a great impact on banana breeding, including cultivar identification, parent selection for cross-breeding, identifying genomic group of wild species, germplasm management, characterizing somaclonal variation, etc.