Introduction

Brassica napus, an important worldwide edible oil crop, is often used within many biological research fields including plant pathology, crop science and biomass energy (Cardoza and Stewart 2007). Glucosinolate and erucic acid are both important agronomic traits within the oil quality of rapeseed, and achieving lower glucosinolate content (GSLC) and erucic acid content (EAC) is the important goal in rapeseed production and quality improvement due to the toxicity of glucosinolate after its enzymic breakdown (Mawson et al. 1994) and the antinutritional qualities of erucic acid (Badawy et al. 1994). However, gucosinolate can be used as biopesticides (in pest control and prevention or for the treatment of fungal diseases in plants), in the prevention of cancer in human or as flavor compounds (Halkier and Gershenzon 2006). Rapeseed oil with high erucic acid content is desired as raw material for these applications (Lühs and Friedt 1993).

Since GSLC and EAC are quantitative traits, they have complex genetic mechanisms and might be sensitive to environmental factors. To date many scientists have done numerous studies on these two traits in rapeseed based on the QTL mapping models developed by Lander and Bostein (1989), Zeng (1993), and Wang et al. (1999). For the QTL analysis of these two traits, the inheritance of seed glucosinolate accumulation in different Brassica species, some QTLs detected for GSLC have been described (Uzunova et al. 1995; Toroser et al. 1995; Howell et al. 2003; Sharpe and Lydiate 2003; Zhao and Meng 2003; Basunanda et al. 2007; Hasan et al. 2008; Feng et al. 2012). The genetic control of EAC is relatively simpler and only two alleles located separately in A and C genome chromosomes in B. napus were found. These correspond to homologous copies of the Arabidopsis fatty acid elongase gene FAE1 (Jönsson 1977; Anand and Downey 1981; Fourmann et al. 1998; Lemieux et al. 1990; James et al. 1995; Lassner et al. 1996). The results of the mentioned-above studies demonstrated that a number of QTLs are located on different chromosomes in allotetraploid rapeseed and all of the identified QTLs were in consideration of QTLs expressed only in the embryo nuclear genome. Although rapeseed is a new generation from its seed-producing plant which supplies assimilates for the seed development, there is currently a thought that glucosinolates are synthesized mainly in maternal vegetative organs such as young leaves and silique walls, and then transported actively to the embryo (Magrath and Mithen 1993). Recently, some models have been constructed to dissect the nuclear genetic effects for the seed quality traits on the different parts from maternal and offspring tissues (Foolad and Jones 1992; Zhu and Weir 1998; Wang et al. 1999; Cui et al. 2004, 2005). Previous studies have shown that the performance of most rapeseed quality traits were simultaneously affected by the expression of nuclear genes in the embryo and maternal plant genetic systems (Shi et al. 2003, 2006; Zhang et al. 2004a, b, 2011a, b; Wu et al. 2005, 2006; Variath et al. 2009, 2010; Wang et al. 2010; Chen et al. 2011a, b; Zhang et al. 2011a, b). Besides, some QTLs divided into different genetic systems across environments were identified for rice (Zheng et al. 2008; Shi et al. 2009a, b) and cotton (Liu et al. 2012, 2013). There is however, little work done on QTL identification simultaneously considering the effects of the maternal nuclear genome and their stability over different seasons in rapeseed which could further improve our understanding of the gene expression mechanism of quality traits in rapeseed.

In the present study, a DH population and newly-developed software named as QTL Network-CL-2.0-Seed, which can divide the total genetic effect of QTLs into embryo additive main effect, embryo dominant main effect, maternal additive main effect as well as their environmental interaction effects, were utilized to identify and dissect the QTLs for GSLC and EAC of rapeseed. A total of nine QTL loci for GSLC and three QTL loci for EAC with significant embryo additive main effects, embryo dominant main effects and/or maternal additive main effects were identified and could explain 83.8 and 89.7 % of their phenotypic variation, respectively. The results of this study could help to clarify further the nature of glucosinolate and erucic acid contents in rapeseed at the molecular level and provide a theoretical basis for the molecular marker-assisted selection (MAS) breeding for quality improvement under the influence of embryo and maternal plant genetic systems, and provide more reliable information for future cloning of the relevant genes.

Materials and methods

Materials

A doubled haploid (DH) segregating population of 202 lines (named as TN DH) was derived from a hybrid of Tapidor and Ningyou7. Tapidor is a kind of European winter cultivar with low GSLC and low EAC, whereas Ningyou7 is a Chinese semi-winter cultivar with higher GSLC and EAC (Qiu et al. 2006). The two parents Tapidor and Ningyou7 and 202 TN DHs which were kindly supplied by Huazhong Agricultural University, Wuhan, China, have different contents of rapeseed nutrition traits, especially GSLC and EAC.

Field experiments

The experiment was conducted on randomized block designed plots with two replications. The seeds of 204 materials including TN DHs and their parents were sown at the experimental farm of Zhejiang University in October of 2011 and 2012, respectively. After 40 days they were individually transplanted at a spacing of 25 cm × 25 cm, and each line contained 32 individual plants (8 rows with 4 plants per row). 177 BC1F1 1 (DHs × Tapidor) and 181 BC1F1 2 (DHs × Ningyou7) were derived from every TN DH by crossing each of the parents with hand emasculation in the spring of 2012 and 2013, respectively. Mature seeds of the parental lines, and of BC1F1 1 and BC1F1 2 materials were harvested for further analysis of quality traits.

Trait measurement

Spectral measurements and trait determination were performed using a Near Infrared Scanning Monochromator (Model 5000 NIRS Systems Inc, Silver Spring, MD, USA) and the WinISI II software (v1.5, FOSS NIRSystems, Silver Springs, MD, USA) to determine GSLC and EAC in rapeseed with about 3 g/sample in a small ring cup of 36-mm (inner diameter) (Wu et al. 2002).

Genetic linkage map for QTL mapping

The linkage map was constructed with different molecular markers, including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), methylation sensitive AFLP (MS-AFLP), simple sequence repeats (SSR), single nucleotide polymorphism (SNP), sequence-tagged site (STS), single-strand conformational polymorphism (SSCP) and cleaved amplified fragment length polymorphism (CAPS). The 786 molecular makers cover 19 linkage groups (A1-A10 and C1-C9), with a total genome size of 2117.2 cm and an average distance of 2.7 cm between marker pairs (Shi et al. 2009a, b).

Data analysis and QTL mapping

Statistics used for phenotypic analysis including mean, standard deviation, the minimum value, the maximum value, skewness and kurtosis of the target traits were calculated with the SAS 9.1 (SAS Institute, Cary, North Carolina, USA).

QTL analysis with environmental interaction effects was carried out on GSLC and EAC of two backcross populations in rapeseed by using the QTL Network-CL-2.0-Seed software and the mixed-model based composite interval mapping (MCIM) method with a 10 cM window size and a 1 cm walking speed (Yang et al. 2007). A logarithm of odds (LOD) threshold of 3 was used to indicate the existence of a putative QTL associated with a target trait. A maximum of 10 background makers were used to control genetic background and 1,000 permutations to estimate the LOD threshold used to declare significant QTL (Doerge and Churchill 1996). The confidence interval was set to 95 %. The QTL main effects including the embryo additive main effect, embryo dominance main effect and maternal additive main effect, QTL × environment interaction effects and corresponding P values were estimated by the Markov Chain Monte Carlo (MCMC) (Zheng et al. 2008; Liu et al. 2013). QTL were named based on the McCouch standard nomenclature (McCouch et al. 1997).

Result

Phenotypic variation for glucosionlate and erucic acid contents

Glucosinolate content (GSLC) and erucic acid content (EAC) of the two parents and two backcross populations (BC1F1 1 and BC1F1 2) are listed on Table 1. The results show that there were significant differences between parents in the performance of GSLC and EAC. In both years, GSLC and EAC in the oilseed of Ningyou7 were all significantly higher than those of Tapidor. Unlike GSLC, the average EAC of BC1F1 2 was greater than that of BC1F1 1. The influence of parents in backcross populations was relatively weak on GSLC, as could be observed in the results where the average GSLC in BC1F1 1 was only slightly higher than that in BC1F1 2 in both years. Transgressive segregation for GSLC and EAC was found in different backcross poupulations under two years, while GSLC was in bidirectional transgressive segregation and EAC was in unidirectional transgressive segregation (Fig. 1). The skewness and kurtosis of GSLC and EAC were all lower than 1, which showed that their distributions were normal. The results revealed that the distributions of these two quality traits were presented in an almost continuously variable manner (Fig. 1), showing that GSLC and EAC of rapeseed are quantitative traits, and confirming that they had complex genetic basis from the phenotype. The frequency distribution of GSLC showed one peak with continuous distribution, while the frequency distribution of EAC showed multiple peaks in continuous distribution. Furthermore, the significant correlation coefficient between GSLC and EAC (r = −0.232**) suggested that they were negatively correlated.

Table 1 The quality traits of Brassica napus L. including GSLC (μmol/g) and EAC (%) of parents and two backcross populations
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Fig. 1
figure 1

Frequency distributions of GSLC and EAC for BC1F1 1 and BC1F1 2 in 2012 and 2013. White and black arrows indicate the average values of the parents Tapidor and Ningyou7, respectively

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QTL mapping for glucosinolate and erucic acid contents

It was observed that twelve QTLs for GSLC and EAC were distributed in different linkage groups (Table 2; Fig. 2). All QTLs had extremely significant embryo and maternal additive main effects, in which three were found with slightly significant QTL × environment effects. The results showed that QTLs controlling the performance of GSLC and EAC were basically the genetic main effects and weakly influenced by environmental factors of the two seasons. QTLs associated with glucosinolate and erucic acid contents were, therefore, relatively stable over two years.

Table 2 QTL locations and the genetic effect components for GSLC and EAC of rapeseed under different years
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Fig. 2
figure 2

a, b Mapping of QTL with embryo and maternal effects for GSLC and EAC of rapeseed under different years

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Mapping QTL controlling glucosinolate content

Nine QTLs controlling GSLC in the present experiment were detected to locate in A3, A4, A8, A9, C1, C2, C2, C7 and C9 linkage group, namely qGSLC-3-1, qGSLC-4-2, qGSLC-8-3, qGSLC-9-4, qGSLC-11-5, qGSLC-12-6, qGSLC-12-7, qGSLC-17-8 and qGSLC-19-9, respectively (Table 2; Fig. 2). All QTLs could explain 83.8 % of phenotypic variation in GSLC (Table 2). The genetic effects from the allele of Ningyou7 could increase GSLC, while that from Tapidor could decrease the GSLC. Compared with other, qGSLC-19-9 had the larger genetic contribution (24.8 %) in Table 2 suggesting that it was the major QTL controlling the performance of GSLC in rapeseed and was very important for improving GSLC in oilseed breeding. qGSLC-3-1 and qGSLC-4-2 displayed significant embryo additive main effects, embryo dominant main effects and maternal additive main effects. While for qGSLC-3-1 the maternal additive main effect was larger than the embryo additive main effect, the opposite was true for qGSLC-4-2. qGSLC-12-6 and qGSLC-12-7 were located between the molecular markers HG-FT-C2 and HR-TP2-110 (63.9 cm), and between EM18ME6-220 and NA12C03 (124.3 cm) on the same linkage group C2, respectively. They both had significant embryo additive main effects, embryo dominant main effects and maternal additive main effects, but qGSLC-12-6 showed positive embryo dominant main effects and maternal additive main effects, while qGSLC-12-7 only had positive embryo main effects. qGSLC-8-3, qGSLC-11-5 and qGSLC-17-8 were all with the positive maternal additive main effects coming from the parent Ningyou7 and the negative embryo additive main effects from Tapidor. In contrast, the embryo additive main effect from qGSLC-17-8 was positive and the maternal additive main effect was negative. In addition, no environmental interaction effects of QTLs for GSLC were detected in this study except for qGSLC-9-4 in which the embryo and maternal additive interaction effects (AeE 2  = −1.010*, AmE 2  = 1.005*) in 2013 were both significant. Therefore, the QTL × environment interaction of qGSLC-9-4 could not be ignored when analyzing the performance of GSLC under the conditions of 2013.

Mapping QTL controlling erucic acid content

Three QTL loci for EAC, named as qEAC-7-1, qEAC-8-2 and qEAC-13-3, could explain 89.7 % of the phenotypic variation. The phenotypic contribution rate of single QTL ranged from 5.7 to 68.3 %. Among them, qEAC-7-1 located between maker HG-WRI1-A7 and CNU168 in the linkage group A7 had a higher genetic contribution (68.3 %), indicating that it was a major QTL controlling the EAC. Its embryo additive main effects (Ae = −62.123) and maternal additive main effects (Am = 63.380) of qEAC-7-1 were the largest among the three QTLs, while its embryo and maternal additive main effects were opposite, showing that its expression in the embryo and maternal nuclear genomes was different. Besides, qEAC-8-2 and qEAC-13-3 with significant embryo additive main effects, embryo dominant main effects, maternal additive main effects and slight QTL × year effects, were distributed on different linkage groups.qEAC-8-2 with the second largest genetic effects located on linkage group A8 had the narrowest confidence interval and a genetic contribution of 15.59 % in the phenotypic variation of EAC. The positive effects of its embryo additive main effects were mainly from the allele of Tapidor, while its negative maternal additive main effects were from Ningyou7. qEAC-13-3 was different for its position in the linkage group C3, and its negative maternal additive main effects (decreasing EAC) were also larger than positive embryo additive main effects (increasing EAC). It was found that the embryo dominant main effects of qEAC-8-2 and qEAC-13-3 were larger than that of qEAC-7-1, which could explain 0.02 and 0.01 % of the total phenotypic variation, respectively. The environmental interaction effects of qEAC-8-1 and qEAC-13-2 were similar, both having the significant embryo dominant interaction effects under different environmental conditions.

Discussion

Glucosinolate is a kind of sulfur-containing anionic hydrophilic secondary metabolite widely found in the roots, stems, leaves and seeds of cruciferous plants, especially in Brassica species (Barbara and Jonathan 2006). It is the main bioactive component in rapeseed and other cruciferous plants, which determines the flavor and nutritional quality of plants. Though it is the major secondary metabolite in Brassica plants, current knowledge on glucosinolate is mainly through the genetic studies of this metabolite in the model plant Arabidopsis. To a certain extent, it had revealed the mechanism of glucosinolate biosynthesis (Grubb and Abel 2006). Erucic acid is a kind of unique fatty acid in cruciferous plants (Ecke et al. 1995). It is of low nutritional value, because its carbon chain (C21:1) is longer than other fatty acids such as palmitic (C16:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3) and eicosenoic (C20:1) acids and cannot be easily decomposed and absorbed by the human body (Das et al. 2002). Erucic acid is also an important industrial raw material (Lühs and Friedt 1993). Therefore, the level of erucic acid in rapeseed oil will not only affect the nutritional value of edible rapeseed oil, but also influence the value of the use of rapeseed oil for industrial purposes.

For rapeseed, the current studies of glucosinolate and erucic acid contents are mainly focused on their accumulation, while their genetic research still in the developing stage. Previous QTL mapping results for GSLC and EAC were only focuses on the seed embryo genetic system (Toroser et al. 1995; Uzunova et al. 1995). However, the studies conducted by Kondra and Stefasom (1970) and Zhang et al. (1996) revealed that these quality traits could be controlled by the genetic effects from the genes of maternal plant, embryo or cytoplasm, while no environmental factors were considered. Shi et al. (2003) and Zhang et al. (2011a) found that the embryo and maternal main effects and their genotype × environmental interaction effects could simultaneously affect the performance of GSLC and EAC. In the present experiment, a newly-developed multi genetic system model for dicotyledonous seed quality traits was used to identify QTLs controlling the performance of GSLC and EAC from the different angle. As a result, twelve QTLs associated with GSLC and EAC were detected which could simultaneously express in embryo and maternal genetic linkage groups. Some of them with weak environment interaction effects indicated that GSLC and EAC were mainly controlled by genetic main effects from embryo and maternal plant genomes. The results showed that GSLC and EAC of rapeseed were mostly based on the embryo and maternal additive main effects in correspondence with the results got by Shi et al. (2003) and Zhang et al. (2011a, b). For GSLC, five QTLs (qGSLC-9-4, qGSLC-12-6, qGSLC-12-7, qGSLC-17-8 and qGSLC-19-9) corresponded to those mapped in previous researches on rapeseed (Toroser et al. 1995; Uzunova et al. 1995; Howell et al. 2003; Quijada et al. 2006; Feng et al. 2012), and qGSLC-9-4 might have been previously detected on A genome linkage groups of Brassica juncea L. (Ramchiary et al. 2007; Bisht et al. 2009). Besides, qGSLC-8-3, qGSLC-17-8 and qGSLC-19-9 had larger genetic contributive values and the GSLC of oilseed might be improved according to the molecular markers closely linked to these three QTL during early generations. For EAC, one major QTL, qEAC-7-1 was detected with the largest genetic contribution with embryo and maternal additive effects. This QTL might offer a new insight into cloning related genes for reducing EAC in rapeseed breeding. The other two QTLs (qEAC-8-2 and qEAC-13-3) located on linkage groups A8 and C3 were corresponded with the genes/QTLs had already been identified (Jourdren et al. 1996, Thormann et al. 1996; Fourmann et al. 1998; Qiu et al. 2006; Amar et al. 2008), which might be closely linked with a Brassica FAE1 homologue controlling erucic acid biosynthesis.

On the other hand, we compared the QTLs detected in this study and the previously mapped ones by using TN DH mapping population. It was found that there were some QTLs, qGSLC-9-4 and qGSLC-12-6 for GSLC, qEAC-8-2 and qEAC-13-3 for EAC being repeatedly detected for each detection (Qiu et al. 2006; Feng et al. 2012), while the others have not been reported. The interesting thing is that the maternal additive effects of these four QTLs were negative, which mean that alleles from Tapidor could decrease the content in seeds, and the maternal effects were higher than the embryo effects. More importantly, this study firstly verified that the QTLs of rapeseed quality traits were related to two different genetic systems and their environmental interaction effects at the same time. QTL × environment interaction effect was also an important part enjoying different influence on the phenotypic variation of glucosinolate and erucic acid contents. During the rapeseed growing season, rainfall in 2013 was more than that in 2012, while the temperature in 2012 higher than that in 2013. The weak environmental interaction would be a useful guide for its stable performance when selecting glucosinolate content across environments in rapeseed breeding, while the strong QTL × environment interaction effect for erucic acid contents showed that it could be influenced by the interaction of rainfall, temperature and soil water availability during seed development under different years. This could provide a new approach for the future breeding of rapeseed quality traits by molecular marker-assisted selection to improve the breeding efficiency, and also offer a new theoretical basis for cloning related genes in future.