Allele segregation analysis of F1 hybrids between independent Brassica allohexaploid lineages.

In the Brassica genus, we find both diploid species (one genome) and allotetraploid species (two different genomes) but no naturally occurring hexaploid species (three different genomes, AABBCC). Although hexaploids can be produced via human intervention, these neo-polyploids have quite unstable genomes and usually suffer from severe genome reshuffling. Whether these genome rearrangements continue in later generations and whether genomic arrangements follow similar, reproducible patterns between different lineages is still unknown. We crossed Brassica hexaploids resulting from different species combinations to produce five F1 hybrids and analyzed the karyotypes of the parents and the F1 hybrids, as well as allele segregation in a resulting test-cross population via molecular karyotyping using SNP array genotyping. Although some genomic regions were found to be more likely to be duplicated, deleted, or rearranged, a consensus pattern was not shared between genotypes. Brassica hexaploids had a high tolerance for fixed structural rearrangements, but which rearrangements occur and become fixed over many generations does not seem to show either strong reproducibility or to indicate selection for stability. On average, we observed 10 de novo chromosome rearrangements contributed almost equally from both parents to the F1 hybrids. At the same time, the F1 hybrid meiosis produced on average 8.6 new rearrangements. Hence, the increased heterozygosity in the F1 hybrid did not significantly improve genome stability in our hexaploid hybrids and might have had the opposite effect. However, hybridization between lineages was readily achieved and may be exploited for future genetics and breeding purposes.

Exploring the gene pool of Brassica napus by genomics-based approaches.

De novo allopolyploidization in Brassica provides a very successful model for reconstructing polyploid genomes using progenitor species and relatives to broaden crop gene pools and understand genome evolution after polyploidy, interspecific hybridization and exotic introgression. B. napus (AACC), the major cultivated rapeseed species and the third largest oilseed crop in the world, is a young Brassica species with a limited genetic base resulting from its short history of domestication, cultivation, and intensive selection during breeding for target economic traits. However, the gene pool of B. napus has been significantly enriched in recent decades that has been benefit from worldwide effects by the successful introduction of abundant subgenomic variation and novel genomic variation via intraspecific, interspecific and intergeneric crosses. An important question in this respect is how to utilize such variation to breed crops adapted to the changing global climate. Here, we review the genetic diversity, genome structure, and population-level differentiation of the B. napus gene pool in relation to known exotic introgressions from various species of the Brassicaceae, especially those elucidated by recent genome-sequencing projects. We also summarize progress in gene cloning, trait-marker associations, gene editing, molecular marker-assisted selection and genome-wide prediction, and describe the challenges and opportunities of these techniques as molecular platforms to exploit novel genomic variation and their value in the rapeseed gene pool. Future progress will accelerate the creation and manipulation of genetic diversity with genomic-based improvement, as well as provide novel insights into the neo-domestication of polyploid crops with novel genetic diversity from reconstructed genomes.

Genome-wide analysis of alternative splicing divergences between Brassica hexaploid and its parents.

MAIN CONCLUSION: Compared with its parents, Brassica hexaploid underwent significant AS changes, which may provide diversified gene expression regulation patterns and could enhance its adaptability during evolution Polyploidization is considered a significant evolution force that promotes species formation. Alternative splicing (AS) plays a crucial role in multiple biological processes during plant growth and development. To explore the effects of allopolyploidization on the AS patterns of genes, a genome-wide AS analysis was performed by RNA-seq in Brassica hexaploid and its parents. In total, we found 7913 (27540 AS events), 14447 (70179 AS events), and 13205 (60804 AS events) AS genes in Brassica rapa, Brassica carinata, and Brassica hexaploid, respectively. A total of 920 new AS genes were discovered in Brassica hexaploid. There were 56 differently spliced genes between Brassica hexaploid and its parents. In addition, most of the alternative 5' splice sites were located 4 bp upstream of the dominant 5' splice sites, and most of the alternative 3' splice sites were located 3 bp downstream of the dominant 3' splice sites in Brassica hexapliod, which was similar to B. carinata. Furthermore, we cloned and sequenced all amplicons from the RT-PCR products of GRP7/8, namely, Bol045859, Bol016025 and Bol02880. The three genes were found to produce AS transcripts in a new way. The AS patterns of genes were diverse between Brassica hexaploid and its parents, including the loss and gain of AS events. Allopolyploidization changed alternative splicing sites of pre-mRNAs in Brassica hexaploid, which brought about alterations in the sequences of transcripts. Our study provided novel insights into the AS patterns of genes in allopolyploid plants, which may provide a reference for the study of polyploidy adaptability.

Reconstituting the genome of a young allopolyploid crop, Brassica napus, with its related species.

Brassica napus (An An Cn Cn ) is an important worldwide oilseed crop, but it is a young allotetraploid with a short evolutionary history and limited genetic diversity. To significantly broaden its genetic diversity and create a novel heterotic population for sustainable rapeseed breeding, this study reconstituted the genome of B. napus by replacing it with the subgenomes from 122 accessions of Brassica rapa (Ar Ar ) and 74 accessions of Brassica carinata (Bc Bc Cc Cc ) and developing a novel gene pool of B. napus through five rounds of extensive recurrent selection. When compared with traditional B. napus using SSR markers and high-throughput SNP/Indel markers through genotyping by sequencing, the newly developed gene pool and its homozygous progenies exhibited a large genetic distance, rich allelic diversity, new alleles and exotic allelic introgression across all 19 AC chromosomes. In addition to the abundant genomic variation detected in the AC genome, we also detected considerable introgression from the eight chromosomes of the B genome. Extensive trait variation and some genetic improvements were present from the early recurrent selection to later generations. This novel gene pool produced equally rich phenotypic variation and should be valuable for rapeseed genetic improvement. By reconstituting the genome of B. napus by introducing subgenomic variation within and between the related species using intense selection and recombination, the whole genome could be substantially reorganized. These results serve as an example of the manipulation of the genome of a young allopolyploid and provide insights into its rapid genome evolution affected by interspecific and intraspecific crosses.

Genome-wide selection footprints and deleterious variations in young Asian allotetraploid rapeseed.

Brassica napus (AACC, 2n = 38) is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic groups, such as European and Asian rapeseed, and the impacts of historical large-scale introgression events on this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7, and its four progenitors and compared these genomes with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed but subsequently significantly diverged, with rapid genome differentiation after hybridization and intensive local selective breeding. The first historical introgression of B. rapa dramatically broadened the allelic pool but decreased the deleterious variations of Asian rapeseed. The second historical introgression of the double-low traits of European rapeseed (canola) has reshaped Asian rapeseed into two groups (double-low and double-high), accompanied by an increase in genetic load in the double-low group. This study demonstrates distinctive genomic footprints and deleterious SNP (single nucleotide polymorphism) variants for local adaptation by recent intra- and interspecies introgression events and provides novel insights for understanding the rapid genome evolution of a young allopolyploid crop.

The high-quality genome of Brassica napus cultivar 'ZS11' reveals the introgression history in semi-winter morphotype.

Allotetraploid oilseed rape (Brassica napus L.) is an agriculturally important crop. Cultivation and breeding of B. napus by humans has resulted in numerous genetically diverse morphotypes with optimized agronomic traits and ecophysiological adaptation. To further understand the genetic basis of diversification and adaptation, we report a draft genome of an Asian semi-winter oilseed rape cultivar 'ZS11' and its comprehensive genomic comparison with the genomes of the winter-type cultivar 'Darmor-bzh' as well as two progenitors. The integrated BAC-to-BAC and whole-genome shotgun sequencing strategies were effective in the assembly of repetitive regions (especially young long terminal repeats) and resulted in a high-quality genome assembly of B. napus 'ZS11'. Within a short evolutionary period (~6700 years ago), semi-winter-type 'ZS11' and the winter-type 'Darmor-bzh' maintained highly genomic collinearity. Even so, certain genetic differences were also detected in two morphotypes. Relative to 'Darmor-bzh', both two subgenomes of 'ZS11' are closely related to its progenitors, and the 'ZS11' genome harbored several specific segmental homoeologous exchanges (HEs). Furthermore, the semi-winter-type 'ZS11' underwent potential genomic introgressions with B. rapa (Ar ). Some of these genetic differences were associated with key agronomic traits. A key gene of A03.FLC3 regulating vernalization-responsive flowering time in 'ZS11' was first experienced HE, and then underwent genomic introgression event with Ar , which potentially has led to genetic differences in controlling vernalization in the semi-winter types. Our observations improved our understanding of the genetic diversity of different B. napus morphotypes and the cultivation history of semi-winter oilseed rape in Asia.

Genetic changes in a novel breeding population of Brassica napus synthesized from hundreds of crosses between B. rapa and B. carinata.

Introgression of genomic variation between and within related crop species is a significant evolutionary approach for population differentiation, genome reorganization and trait improvement. Using the Illumina Infinium Brassica 60K SNP array, we investigated genomic changes in a panel of advanced generation new-type Brassica napus breeding lines developed from hundreds of interspecific crosses between 122 Brassica rapa and 74 Brassica carinata accessions, and compared them with representative accessions of their three parental species. The new-type B. napus population presented rich genetic diversity and abundant novel genomic alterations, consisting of introgressions from B. rapa and B. carinata, novel allelic combinations, reconstructed linkage disequilibrium patterns and haplotype blocks, and frequent deletions and duplications (nonrandomly distributed), particularly in the C subgenome. After a much shorter, but very intensive, selection history compared to traditional B. napus, a total of 15 genomic regions with strong selective sweeps and 112 genomic regions with putative signals of selective sweeps were identified. Some of these regions were associated with important agronomic traits that were selected for during the breeding process, while others were potentially associated with restoration of genome stability and fertility after interspecific hybridization. Our results demonstrate how a novel method for population-based crop genetic improvement can lead to rapid adaptation, restoration of genome stability and positive responses to artificial selection.

Correction to: Incorporating pleiotropic quantitative trait loci in dissection of complex traits: seed yield in rapeseed as an example.

The article "Incorporating pleiotropic quantitative trait loci in dissection of complex traits: seed yield in rapeseed as an example", written by J. Zou et al, was originally published Online First without open access.

Assembly and comparison of two closely related Brassica napus genomes.

As an increasing number of plant genome sequences become available, it is clear that gene content varies between individuals, and the challenge arises to predict the gene content of a species. However, genome comparison is often confounded by variation in assembly and annotation. Differentiating between true gene absence and variation in assembly or annotation is essential for the accurate identification of conserved and variable genes in a species. Here, we present the de novo assembly of the B. napus cultivar Tapidor and comparison with an improved assembly of the Brassica napus cultivar Darmor-bzh. Both cultivars were annotated using the same method to allow comparison of gene content. We identified genes unique to each cultivar and differentiate these from artefacts due to variation in the assembly and annotation. We demonstrate that using a common annotation pipeline can result in different gene predictions, even for closely related cultivars, and repeat regions which collapse during assembly impact whole genome comparison. After accounting for differences in assembly and annotation, we demonstrate that the genome of Darmor-bzh contains a greater number of genes than the genome of Tapidor. Our results are the first step towards comparison of the true differences between B. napus genomes and highlight the potential sources of error in future production of a B. napus pangenome.

Incorporating pleiotropic quantitative trait loci in dissection of complex traits: seed yield in rapeseed as an example.

KEY MESSAGE: A comprehensive linkage atlas for seed yield in rapeseed. Most agronomic traits of interest for crop improvement (including seed yield) are highly complex quantitative traits controlled by numerous genetic loci, which brings challenges for comprehensively capturing associated markers/genes. We propose that multiple trait interactions underlie complex traits such as seed yield, and that considering these component traits and their interactions can dissect individual quantitative trait loci (QTL) effects more effectively and improve yield predictions. Using a segregating rapeseed (Brassica napus) population, we analyzed a large set of trait data generated in 19 independent experiments to investigate correlations between seed yield and other complex traits, and further identified QTL in this population with a SNP-based genetic bin map. A total of 1904 consensus QTL accounting for 22 traits, including 80 QTL directly affecting seed yield, were anchored to the B. napus reference sequence. Through trait association analysis and QTL meta-analysis, we identified a total of 525 indivisible QTL that either directly or indirectly contributed to seed yield, of which 295 QTL were detected across multiple environments. A majority (81.5%) of the 525 QTL were pleiotropic. By considering associations between traits, we identified 25 yield-related QTL previously ignored due to contrasting genetic effects, as well as 31 QTL with minor complementary effects. Implementation of the 525 QTL in genomic prediction models improved seed yield prediction accuracy. Dissecting the genetic and phenotypic interrelationships underlying complex quantitative traits using this method will provide valuable insights for genomics-based crop improvement.

Co-linearity and divergence of the A subgenome of Brassica juncea compared with other Brassica species carrying different A subgenomes.

BACKGROUND: There are three basic Brassica genomes (A, B, and C) and three parallel sets of subgenomes distinguished in the diploid Brassica (i.e.: B. rapa, A(r)A(r); B. nigra, B(ni)B(ni); B. oleracea, C(o)C(o)) and the derived allotetraploid species (i.e.: B. juncea, A(j)A(j)B(j)B(j); B. napus, A(n)A(n)C(n)C(n); B. carinata, B(c)B(c)C(c)C(c)). To understand subgenome differentiation in B. juncea in comparison to other A genome-carrying Brassica species (B. rapa and B. napus), we constructed a dense genetic linkage map of B. juncea, and conducted population genetic analysis on diverse lines of the three A-genome carrying Brassica species using a genotyping-by-sequencing approach (DArT-seq). RESULTS: A dense genetic linkage map of B. juncea was constructed using an F2 population derived from Sichuan Yellow/Purple Mustard. The map included 3329 DArT-seq markers on 18 linkage groups and covered 1579 cM with an average density of two markers per cM. Based on this map and the alignment of the marker sequences with the physical genome of Arabidopsis thaliana, we observed strong co-linearity of the ancestral blocks among the different A subgenomes but also considerable block variation. Comparative analyses at the level of genome sequences of B. rapa and B. napus, and marker sequence anchored on the genetic map of B. juncea, revealed a total of 30 potential inversion events across large segments and 20 potential translocation events among the three A subgenomes. Population genetic analysis on 26 accessions of the three A genome-carrying Brassica species showed that the highest genetic distance were estimated when comparing A(j)-A(n) than between A(n)-A(r) and A(j)-A(r) subgenome pairs. CONCLUSIONS: The development of the dense genetic linkage map of B. juncea with informative DArT-seq marker sequences and availability of the reference sequences of the A(r), and A(n)C(n) genomes allowed us to compare the A subgenome structure of B. juncea (A(j)) . Our results suggest that strong co-linearity exists among the three A Brassica genomes (A(r), A(n) and A(j)) but with apparent subgenomic variation. Population genetic analysis on three A-genome carrying Brassica species support the idea that B. juncea has distinct genomic diversity, and/or evolved from a different A genome progenitor of B. napus.

A high-density SNP genotyping array for Brassica napus and its ancestral diploid species based on optimised selection of single-locus markers in the allotetraploid genome.

KEY MESSAGE: The Brassica napus Illumina array provides genome-wide markers linked to the available genome sequence, a significant tool for genetic analyses of the allotetraploid B. napus and its progenitor diploid genomes. A high-density single nucleotide polymorphism (SNP) Illumina Infinium array, containing 52,157 markers, was developed for the allotetraploid Brassica napus. A stringent selection process employing the short probe sequence for each SNP assay was used to limit the majority of the selected markers to those represented a minimum number of times across the highly replicated genome. As a result approximately 60 % of the SNP assays display genome-specificity, resolving as three clearly separated clusters (AA, AB, and BB) when tested with a diverse range of B. napus material. This genome specificity was supported by the analysis of the diploid ancestors of B. napus, whereby 26,504 and 29,720 markers were scorable in B. oleracea and B. rapa, respectively. Forty-four percent of the assayed loci on the array were genetically mapped in a single doubled-haploid B. napus population allowing alignment of their physical and genetic coordinates. Although strong conservation of the two positions was shown, at least 3 % of the loci were genetically mapped to a homoeologous position compared to their presumed physical position in the respective genome, underlying the importance of genetic corroboration of locus identity. In addition, the alignments identified multiple rearrangements between the diploid and tetraploid Brassica genomes. Although mostly attributed to genome assembly errors, some are likely evidence of rearrangements that occurred since the hybridisation of the progenitor genomes in the B. napus nucleus. Based on estimates for linkage disequilibrium decay, the array is a valuable tool for genetic fine mapping and genome-wide association studies in B. napus and its progenitor genomes.

QTL mapping based on the embryo and maternal genetic systems for non-essential amino acids in rapeseed (Brassica napus L.) meal.

BACKGROUND: Non-essential amino acids are a good source of nitrogen and also very important contributors to the metabolic process. Analysis of quantitative trait locus (QTL) simultaneously located on the amphidiploid embryo and maternal plant nuclear genomes for non-essential amino acid contents in rapeseed meal across different environments was conducive to further clarify the genetic mechanism of seed quality traits. RESULTS: Twenty-eight QTLs associated with arginine (five QTLs), histidine (four QTLs), glutamic acid (three QTLs), glycine (three QTLs), proline (three QTLs), alanine (four QTLs) and aspartic acid (six QTLs) contents were identified in present study. All of these QTLs had significant additive main effects from embryo and maternal plant nuclear genomes with eight of them showing significant embryo dominance main effects and 12 showing notable QTL × environment interaction effects. Among them, 12 QTLs were major QTLs which could explain 13.27-35.71% of the phenotypic variation. Specially, five QTL clusters associated with several QTLs related to multiple traits were distributed on chromosomes A1, A4, A5, A7 and C2. CONCLUSION: Non-essential amino acids in rapeseed meal could be simultaneously controlled by the genetic effects from the QTLs which were located on the chromosomes both in the embryo and maternal plant genetic systems.

New insights into the genetic networks affecting seed fatty acid concentrations in Brassica napus.

BACKGROUND: Rapeseed (B. napus, AACC, 2n = 38) is one of the most important oil seed crops in the world, it is also one of the most common oil for production of biodiesel. Its oil is a mixture of various fatty acids and dissection of the genetic network for fatty acids biosynthesis is of great importance for improving seed quality. RESULTS: The genetic basis of fatty acid biosynthesis in B. napus was investigated via quantitative trail locus (QTL) analysis using a doubled haploid (DH) population with 202 lines. A total of 72 individual QTLs and a large number pairs of epistatic interactions associated with the content of 10 different fatty acids were detected. A total of 234 homologous genes of Arabidopsis thaliana that are involved in fatty acid metabolism were found within the confidence intervals (CIs) of 47 QTLs. Among them, 47 and 15 genes homologous to those of B. rapa and B. oleracea were detected, respectively. After the QTL mapping, the epistatic and the candidate gene interaction analysis, a potential regulatory pathway controlling fatty acid biosynthesis in B. napus was constructed, including 50 enzymes encoded genes and five regulatory factors (LEC1, LEC2, FUS3, WRI1 and ABI3). Subsequently, the interaction between these five regulatory factors and the genes involved in fatty acid metabolism were analyzed. CONCLUSIONS: In this study, a potential regulatory pathway controlling the fatty acid was constructed by QTL analysis and in silico mapping analysis. These results enriched our knowledge of QTLs for fatty acids metabolism and provided a new clue for genetic engineering fatty acids composition in B. napus.

Widespread and evolutionary analysis of a MITE family Monkey King in Brassicaceae.

BACKGROUND: Miniature inverted repeat transposable elements (MITEs) are important components of eukaryotic genomes, with hundreds of families and many copies, which may play important roles in gene regulation and genome evolution. However, few studies have investigated the molecular mechanisms involved. In our previous study, a Tourist-like MITE, Monkey King, was identified from the promoter region of a flowering time gene, BnFLC.A10, in Brassica napus. Based on this MITE, the characteristics and potential roles on gene regulation of the MITE family were analyzed in Brassicaceae. RESULTS: The characteristics of the Tourist-like MITE family Monkey King in Brassicaceae, including its distribution, copies and insertion sites in the genomes of major Brassicaceae species were analyzed in this study. Monkey King was actively amplified in Brassica after divergence from Arabidopsis, which was indicated by the prompt increase in copy number and by phylogenetic analysis. The genomic variations caused by Monkey King insertions, both intra- and inter-species in Brassica, were traced by PCR amplification. Genomic sequence analysis showed that most complete Monkey King elements are located in gene-rich regions, less than 3kb from genes, in both the B. rapa and A. thaliana genomes. Sixty-seven Brassica expressed sequence tags carrying Monkey King fragments were also identified from the NCBI database. Bisulfite sequencing identified specific DNA methylation of cytosine residues in the Monkey King sequence. A fragment containing putative TATA-box motifs in the MITE sequence could bind with nuclear protein(s) extracted from leaves of B. napus plants. A Monkey King-related microRNA, bna-miR6031, was identified in the microRNA database. In transgenic A. thaliana, when the Monkey King element was inserted upstream of 35S promoter, the promoter activity was weakened. CONCLUSION: Monkey King, a Brassicaceae Tourist-like MITE family, has amplified relatively recently and has induced intra- and inter-species genomic variations in Brassica. Monkey King elements are most abundant in the vicinity of genes and may have a substantial effect on genome-wide gene regulation in Brassicaceae. Monkey King insertions potentially regulate gene expression and genome evolution through epigenetic modification and new regulatory motif production.

Identification, evolution, and expression partitioning of miRNAs in allopolyploid Brassica napus.

The recently published genome of Brassica napus offers for the first time the opportunity to gain insights into the genomic organization and the evolution of miRNAs in oilseed rape. In this study, 12 small RNA libraries from two B. napus cultivars (Tapidor and Ningyou7) and their four double-haploid lines were sequenced, employing the newly sequenced B. napus genome, together with genomes of its progenitors Brassica rapa and Brassica oleracea. A total of 645 miRNAs including 280 conserved and 365 novel miRNAs were identified. Comparative analysis revealed a high level of genomic conservation of MIRNAs (75.9%) between the subgenomes of B. napus and its two progenitors' genomes, and MIRNA lost/gain events (133) occurred in B. napus after its speciation. Furthermore, significant partitioning of miRNA expressions between the two subgenomes in B. napus was detected. The data of degradome sequencing, miRNA-mediated cleavage, and expression analyses support specific interactions between miRNAs and their targets in the modulation of diverse physiological processes in roots and leaves, as well as in biosynthesis of, for example, glucosinolates and lipids in oilseed rape. These data provide a first genome-wide view on the origin, evolution, and genomic organization of B. napus MIRNAs.

High-resolution skim genotyping by sequencing reveals the distribution of crossovers and gene conversions in Cicer arietinum and Brassica napus.

KEY MESSAGE: We characterise the distribution of crossover and non-crossover recombination in Brassica napus and Cicer arietinum using a low-coverage genotyping by sequencing pipeline SkimGBS. The growth of next-generation DNA sequencing technologies has led to a rapid increase in sequence-based genotyping for applications including diversity assessment, genome structure validation and gene-trait association. We have established a skim-based genotyping by sequencing method for crop plants and applied this approach to genotype-segregating populations of Brassica napus and Cicer arietinum. Comparison of progeny genotypes with those of the parental individuals allowed the identification of crossover and non-crossover (gene conversion) events. Our results identify the positions of recombination events with high resolution, permitting the mapping and frequency assessment of recombination in segregating populations.

Characterization and expression patterns of small RNAs in synthesized Brassica hexaploids.

Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. We used high-throughput sequencing to compare miRNA expression profiles between Brassica hexaploid and its parents. A total of 613, 784 and 742 known miRNAs were identified in Brassica rapa, Brassica carinata, and Brassica hexaploid, respectively. We detected 618 miRNAs were differentially expressed (log(2)Ratio ≥ 1, P ≤ 0.05) between Brassica hexaploid and its parents, and 425 miRNAs were non-additively expressed in Brassica hexaploid, which suggest a trend of non-additive miRNA regulation following hybridization and polyploidization. Remarkably, majority of the non-additively expressed miRNAs in the Brassica hexaploid are repressed, and there was a bias toward repression of B. rapa miRNAs, which is consistent with the progenitor-biased gene repression in the synthetic allopolyploids. In addition, we identified 653 novel mature miRNAs in Brassica hexaploid and its parents. Finally, we found that almost all the non-additive accumulation of siRNA clusters exhibited a low-parent pattern in Brassica hexaploid. Non-additive small RNA regulation is involved in a range of biological pathways, probably providing a driving force for variation and adaptation in allopolyploids.

A high-throughput SNP array in the amphidiploid species Brassica napus shows diversity in resistance genes.

Single-nucleotide polymorphisms (SNPs)are molecular markers based on nucleotide variation and can be used for genotyping assays across populations and to track genomic inheritance. SNPs offer a comprehensive genotyping alternative to whole-genome sequencing for both agricultural and research purposes including molecular breeding and diagnostics, genome evolution and genetic diversity analyses, genetic mapping, and trait association studies. Here genomic SNPs were discovered between four cultivars of the important amphidiploid oilseed species Brassica napus and used to develop a B. napus Infinium™ array containing 5,306 SNPs randomly dispersed across the genome. Assay success was high, with >94 % of these producing a reproducible, polymorphic genotype in the 1,070 samples screened. Although the assay was designed to B. napus, successful SNP amplification was achieved in the B. napus progenitor species, Brassica rapa and Brassica oleracea, and to a lesser extent in the related species Brassica nigra. Phylogenetic analysis was consistent with the expected relationships between B. napus individuals. This study presents an efficient custom SNP assay development pipeline in the complex polyploid Brassica genome and demonstrates the utility of the array for high-throughput genotyping in a number of related Brassica species. It also demonstrates the utility of this assay in genotyping resistance genes on chromosome A7, which segregate amongst the 1,070 samples.

Quantitative trait loci that control the oil content variation of rapeseed (Brassica napus L.).

KEY MESSAGE: This report describes an integrative analysis of seed-oil-content quantitative trait loci (QTL) in Brassica napus , using a high-density genetic map to align QTL among different populations. Rapeseed (Brassica napus) is an important source of edible oil and sustainable energy. Given the challenge involved in using only a few genes to substantially increase the oil content of rapeseed without affecting the fatty acid composition, exploitation of a greater number of genetic loci that regulate the oil content variation among rapeseed germplasm is of fundamental importance. In this study, we investigated variation in the seed-oil content among two related genetic populations of Brassica napus, the TN double-haploid population and its derivative reconstructed-F2 population. Each population was grown in multiple experiments under different environmental conditions. Mapping of quantitative trait loci (QTL) identified 41 QTL in the TN populations. Furthermore, of the 20 pairs of epistatic interaction loci detected, approximately one-third were located within the QTL intervals. The use of common markers on different genetic maps and the TN genetic map as a reference enabled us to project QTL from an additional three genetic populations onto the TN genetic map. In summary, we used the TN genetic map of the B. napus genome to identify 46 distinct QTL regions that control seed-oil content on 16 of the 19 linkage groups of B. napus. Of these, 18 were each detected in multiple populations. The present results are of value for ongoing efforts to breed rapeseed with high oil content, and alignment of the QTL makes an important contribution to the development of an integrative system for genetic studies of rapeseed.