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Variation in patterns of recombination in plant genomes provides information about species evolution, genetic diversity and crop improvement. We investigated meiotic crossovers generated in biparental segregating and reciprocal backcross populations of the allopolyploid genome of rapeseed (Brassica napus) (AACC, 2n = 38). A structured set of 1445 intercrossed lines was derived from two homozygous de novo genome-assembled parents that represented the major genetic clusters of semi-winter Chinese and winter European rapeseeds, and was used to increase QTL resolution and achieve genomic reciprocal introgression. A high-density genetic map constructed with 6161 genetic bins and anchored centromere regions was used to establish the pattern of recombination variation in each chromosome. Around 93% of the genome contained crossovers at a mean rate of 3.8 cM Mb−1, with the remaining 7% attributed to centromeres or low marker density. Recombination hotspots predominated in the A genome, including two-thirds of those associated with breeding introgression from B. rapa. Genetic background might affect recombination variation. Introgression of genetic diversity from European winter to Chinese semi-winter rapeseed showed an increase in crossover rate under the semi-winter environment. Evidence for an elevated recombination rate having historically contributed to selective trait improvement includes accumulation of favorable alleles for seed oil content on hotspots of chromosome A10. Conversely, strong artificial selection may affect recombination rate variation, as appears to be the case with a coldspot resulting from strong selection for glucosinolate alleles on A09. But the cold region would be promptly reactivated by crossing design indicated by the pedigree analysis. Knowledge of recombination hotspots and coldspots associated with QTL for 22 traits can guide selection strategies for introgression breeding between the two gene pools. These results and rich genomic resources broaden our understanding of recombination behavior in allopolyploids and may advance rapeseed genetic improvement.

Abstract Selection breeding has played a key role in the improvement of seed yield and quality in oilseed rape (Brassica napus L.). We genotyped Tapidor (European), Ningyou7 (Chinese) and their progenitors with the Brassica 60 K Illumina Infinium SNP array and mapped a total of 29,347 SNP markers onto the reference genome of Darmor-bzh. Identity by descent (IBD) refers to a haplotype segment of a chromosome inherited from a shared common ancestor. IBDs identified on the C subgenome were larger than those on the A subgenome within both the Tapidor and Ningyou7 pedigrees. IBD number and length were greater in the Ningyou7 pedigree than in the Tapidor pedigree. Seventy nine QTLs for flowering time, seed quality and root morphology traits were identified in the IBDs of Tapidor and Ningyou7. Many more candidate genes had been selected within the Ningyou7 pedigree than within the Tapidor pedigree. These results highlight differences in the transfer of favorable gene clusters controlling key traits during selection breeding in Europe and China.

Improving the prediction accuracy of a complex trait of interest is key to performing genomic selection (GS) for crop breeding. For the complex trait measured in multiple environments, this paper proposes a two-stage method to solve a linear model that jointly models the genetic effects and the genotype × environment interaction (G × E) effects. In the first stage, the least absolute shrinkage and selection operator (LASSO) penalized method was utilized to identify quantitative trait loci (QTL). Then, the ordinary least squares (OLS) approach was used in the second stage to reestimate the QTL effects. As a case study, this approach was used to improve the prediction accuracies of flowering time (FT), oil content (OC), and seed yield per plant (SY) in Brassica napus (B. napus). The results showed that the G × E effects reduced the mean squared error (MSE) significantly. Numerous QTL were environment-specific and presented minor effects. On average, the two-stage method, named OLS post-LASSO, offers the highest prediction accuracies (correlations are 0.8789, 0.9045, and 0.5507 for FT, OC, and SY, respectively). It was followed by the marker × environment interaction (M × E) genomic best linear unbiased prediction (GBLUP) model (correlations are 0.8347, 0.8205, and 0.4005 for FT, OC, and SY, respectively), the LASSO method (correlations are 0.7583, 0.7755, and 0.2718 for FT, OC, and SY, respectively), and the stratified GBLUP model (correlations are 0.6789, 0.6361, and 0.2860 for FT, OC, and SY, respectively). The two-stage method showed an obvious improvement in the prediction accuracy, and this study will provide methods and reference to improve GS of breeding.

In recent years, much attention has been devoted to the development and applications of smart grid technologies, with special emphasis on flexible resources such as distributed generations (DGs), energy storages, active loads, and electric vehicles (EVs). Demand response (DR) is expected to be an effective means for accommodating the integration of renewable energy generations and mitigating their power output fluctuations. Despite their potential contributions to power system secure and economic operation, uncoordinated operations of these flexible resources may result in unexpected congestions in the distribution system concerned. In addition, the behaviors and impacts of flexible resources are normally highly uncertain and complex in deregulated electricity market environments. In this context, this paper aims to propose a DR based congestion management strategy for smart distribution systems. The general framework and procedures for distribution congestion management is first presented. A bi-level optimization model for the day-ahead congestion management based on the proposed framework is established. Subsequently, the robust optimization approach is introduced to alleviate negative impacts introduced by the uncertainties of DG power outputs and market prices. The economic efficiency and robustness of the proposed congestion management strategy is demonstrated by an actual 0.4 kV distribution system in Denmark.

In recent years, energy storage technology is widely used in distribution networks. It can significantly improve stability and power quality of the grid. An improved droop control strategy for energy power storage converter is proposed here, which based on complex filter, dividing the voltage into positive and negative sequence components in the αβ-axis. This method has small amount of calculation and easy realisation. This strategy can improve the output power quality of energy power storage converter. Finally, it proved that the improved voltage control strategy can effectively shorten the time of voltage recovery in the case of the power fluctuation of the contact line, and provide reference for other access storage power distribution network operation control through MATLAB/Simulink simulation.

A doubled haploid (DH) mapping population was obtained from microspore culture of an allohexaploid F1 from the cross between two recently-synthesized allohexaploid Brassica lines. We used single nucleotide polymorphism (SNP) genetic variation based on restriction-site associated DNA (RAD) sequencing to construct a high density genetic linkage map of the population. RAD libraries were constructed from the genomic DNA of both parents and 146 DH progenies. A total of 2.87 G reads with an average sequencing depth of 2.59 × were obtained in the parents and of 1.41 × in the progeny. A total of 290,422 SNPs were identified from clustering of RAD reads, from which we developed 7,950 high quality SNP markers that segregated normally (1:1) in the population. The linkage map contained all 27 chromosomes from the parental A, B and C genomes with a total genetic distance of 5725.19 cM and an average of 0.75 cM between adjacent markers. Genetic distance on non-integrated linkage groups was 1534.23 cM, or 21% of total genetic distance. Out of 146 DH progenies, 91 had a complete set of 27 chromosomes as expected of a hexaploid species, and 21 out of 27 chromosomes showed high collinearity between the physical and linkage maps. The loss of chromosome(s) or chromosome segment(s) in the DH population was associated with a reduction in pollen viability. Twenty-five additive QTL were associated with pollen viability and fertility-related traits (seed number, seed yield, pod length, plant height, 1000-seed weight). In addition, 44 intra-genomic and 18 inter-genomic epistatic QTL pairs were detected for 4 phenotypic traits. This provides confidence that the DH population may be selected for improved pollen viability and fertility in a future allohexaploid Brassica species.

Genomics-based prediction of hybrid performance promises to boost selection gain. The main goal of our study was to investigate the relevance of additive, dominance, and epistatic effects for determining hybrid seed yield in a biparental rapeseed population. We re-analyzed 60,000 SNP array and seed yield data points from an immortalized F2 population comprised of 318 hybrids and 180 parental lines by performing genome-wide QTL mapping and predictions in combination with five-fold cross-validation. Moreover, an additional set of 37 hybrids were genotyped and phenotyped in an independent environment. The decomposition of the phenotypic variance components and the cross-validated results of the QTL mapping and genome-wide predictions revealed that the hybrid performance in rapeseed was driven by a mix of additive, dominance, and epistatic effects. Interestingly, the genome-wide prediction accuracy in the additional 37 hybrids remained high when modeling exclusively additive effects but was severely reduced when dominance or epistatic effects were also included. This loss in accuracy was most likely caused by more pronounced interactions of environments with dominance and epistatic effects than with additive effects. Consequently, the development of robust hybrid prediction models, including dominance and epistatic effects, required much deeper phenotyping in multi-environmental trials.

Brassica carinata (BBCC) is an allotetraploid in Brassicas with unique alleles for agronomic traits and has huge potential as source for biodiesel production. To investigate the genome-wide molecular diversity, population structure and linkage disequilibrium (LD) pattern in this species, we genotyped a panel of 81 accessions of B. carinata with genotyping by sequencing approach DArTseq, generating a total of 54,510 polymorphic markers. Two subpopulations were exhibited in the B. carinata accessions. The average distance of LD decay (r2 = 0.1) in B subgenome (0.25 Mb) was shorter than that of C subgenome (0.40 Mb). Genome-wide association analysis (GWAS) identified a total of seven markers significantly associated with five seed quality traits in two experiments. To further identify the quantitative trait loci (QTL) for important agronomic and seed quality traits, we phenotyped a doubled haploid (DH) mapping population derived from the “YW” cross between two parents (Y-BcDH64 and W-BcDH76) representing from the two subpopulations. The YW DH population and its parents were grown in three contrasting environments; spring (Hezheng and Xining, China), semi-winter (Wuhan, China), and spring (Wagga Wagga, Australia) across 5 years for QTL mapping. Genetic bases of phenotypic variation in seed yield and its seven related traits, and six seed quality traits were determined. A total of 282 consensus QTL accounting for these traits were identified including nine major QTL for flowering time, oleic acid, linolenic acid, pod number of main inflorescence, and seed weight. Of these, 109 and 134 QTL were specific to spring and semi-winter environment, respectively, while 39 consensus QTL were identified in both contrasting environments. Two QTL identified for linolenic acid (B3) and erucic acid (C7) were validated in the diverse lines used for GWAS. A total of 25 QTL accounting for flowering time, erucic acid, and oleic acid were aligned to the homologous QTL or candidate gene regions in the C genome of B. napus. These results would not only provide insights for genetic improvement of this species, but will also identify useful genetic variation hidden in the Cc subgenome of B. carinata to improve canola cultivars.

Brassica juncea (AjAjBjBj), is an allotetraploid that arose from two diploid species, B. rapa (ArAr) and B. nigra (BnBn). It is an old oilseed crop with unique favorable traits, but the genetic improvement on this species is limited. We developed an approach to broaden its genetic base within several generations by intensive selection. The Ar subgenome from the Asian oil crop B. rapa (ArAr) and the Bc subgenome from the African oil crop B. carinata (BcBcCcCc) were combined in a synthesized allohexaploid (ArArBcBcCcCc), which was crossed with traditional B. juncea to generate pentaploid F1 hybrids (ArAjBcBjCc), with subsequent self-pollination to obtain newly synthesized B. juncea (Ar/jAr/jBc/jBc/j). After intensive cytological screening and phenotypic selection of fertility and agronomic traits, a population of new-type B. juncea was obtained and was found to be genetically stable at the F6 generation. The new-type B. juncea possesses good fertility and rich genetic diversity and is distinctly divergent but not isolated from traditional B. juncea, as revealed by population genetic analysis with molecular markers. More than half of its genome was modified, showing exotic introgression and novel variation. In addition to the improvement in some traits of the new-type B. juncea lines, a considerable potential for heterosis was observed in inter-subgenomic hybrids between new-type B. juncea lines and traditional B. juncea accessions. The new-type B. juncea exhibited a stable chromosome number and a novel genome composition through multiple generations, providing insight into how to significantly broaden the genetic base of crops with subgenome introgression from their related species and the potential of exploring inter-subgenomic heterosis for hybrid breeding.

Improving seed oil yield and quality are central targets in rapeseed (Brassica napus) breeding. The primary goal of our study was to examine and compare the potential and the limits of marker-assisted selection and genome-wide prediction of six important seed quality traits of B. napus. Our study is based on a bi-parental population comprising 202 doubled haploid lines and a diverse validation set including 117 B. napus inbred lines derived from interspecific crosses between B. rapa and B. carinata. We used phenotypic data for seed oil, protein, erucic acid, linolenic acid, stearic acid, and glucosinolate content. All lines were genotyped with a 60k SNP array. We performed five-fold cross-validations in combination with linkage mapping and four genome-wide prediction approaches in the bi-parental population. Quantitative trait loci (QTL) with large effects were detected for erucic acid, stearic acid, and glucosinolate content, blazing the trail for marker-assisted selection. Despite substantial differences in the complexity of the genetic architecture of the six traits, genome-wide prediction models had only minor impacts on the prediction accuracies. We evaluated the effects of training population size, marker density and phenotyping intensity on the prediction accuracy. The prediction accuracy in the independent and genetically very distinct validation set still amounted to 0.14 for protein content and 0.17 for oil content reflecting the utility of the developed calibration models even in very diverse backgrounds.

The prediction of the flowering time (FT) trait in Brassica napus based on genome-wide markers and the detection of underlying genetic factors is important not only for oilseed producers around the world but also for the other crop industry in the rotation system in China. In previous studies the low density and mixture of biomarkers used obstructed genomic selection in B. napus and comprehensive mapping of FT related loci. In this study, a high-density genome-wide SNP set was genotyped from a double-haploid population of B. napus. We first performed genomic prediction of FT traits in B. napus using SNPs across the genome under ten environments of three geographic regions via eight existing genomic predictive models. The results showed that all the models achieved comparably high accuracies, verifying the feasibility of genomic prediction in B. napus. Next, we performed a large-scale mapping of FT related loci among three regions, and found 437 associated SNPs, some of which represented known FT genes, such as AP1 and PHYE. The genes tagged by the associated SNPs were enriched in biological processes involved in the formation of flowers. Epistasis analysis showed that significant interactions were found between detected loci, even among some known FT related genes. All the results showed that our large scale and high-density genotype data are of great practical and scientific values for B. napus. To our best knowledge, this is the first evaluation of genomic selection models in B. napus based on a high-density SNP dataset and large-scale mapping of FT loci.

BACKGROUND: Oilseed rape (Brassica napus L.) is one of the most important oil crops. A primary limitation to the cultivation of this crop is the lack of available phosphorus (P) in soils. To elucidate the genetic control of P deficiency tolerance in Brassica napus, quantitative trait locus (QTL) for seed yield and yield related-traits in response to P deficiency were identified using a double haploid mapping population (TN DH) derived from a cross between a P-efficient cultivar, Ningyou 7 and a P-inefficient cultivar, Tapidor. RESULTS: Three field trials were conducted to determine seed yield (SY), plant height (PH), number of primary branches (BN), height to the first primary branch (FBH), relative first primary branch height (RBH), pod number per plant (PN), seed number per pod (SN) and seed weight of 1,000 seeds (SW) in 188 lines of TN DH population exposed to low P (LP) and optimal P (OP) conditions. P deficiency decreased PH, BN, SN, PN and SY, and increased FBH and RBH with no effect on SW. Three reproducible LP-specific QTL regions were identified on chromosomes A2, A3 and A5 that controlled SN, PN and SW respectively. In addition, six reproducible constitutive regions were also mapped with two each for SY-LP on A2, and FBH-LP on C6 and one each for PH-LP and SW-LP on A3. About 30 markers derived from 19 orthologous genes involved in Arabidopsis P homeostasis were mapped on 24 QTL regions by comparative mapping between Arabidopsis and Brassica napus. Among these genes, GPT1, MGD2 and SIZ1 were associated with two major loci regulating SY-LP and other yield-related traits on A2 between 77.1 and 95.0 cM. CONCLUSION: The stable QTLs detected under LP conditions and their candidate genes may provide useful information for marker-assisted selection in breeding high-yield B. napus varieties with improved P efficiency.

Polyploidization has played an important role in plant evolution and speciation, and newly formed allopolyploids have experienced rapid transcriptomic changes. Here, we compared the transcriptomic differences between a synthetic Brassica allohexaploid and its parents using a high-throughput RNA-Seq method. A total of 35,644,409 sequence reads were generated, and 32,642 genes were aligned from the data. Totals of 29,260, 29,060, and 29,697 genes were identified in Brassicarapa, Brassicacarinata, and Brassica allohexaploid, respectively. We compared 7,397 differentially expressed genes (DEGs) between Brassica hexaploid and its parents, as well as 2,545 nonadditive genes of Brassica hexaploid. We hypothesized that the higher ploidy level as well as secondary polyploidy might have influenced these changes. The majority of the 3,184 DEGs between Brassica hexaploid and its paternal parent, B. rapa, were involved in the biosynthesis of secondary metabolites, plant-pathogen interactions, photosynthesis, and circadian rhythm. Among the 2,233 DEGs between Brassica hexaploid and its maternal parent, B. carinata, several played roles in plant-pathogen interactions, plant hormone signal transduction, ribosomes, limonene and pinene degradation, photosynthesis, and biosynthesis of secondary metabolites. There were more significant differences in gene expression between the allohexaploid and its paternal parent than between it and its maternal parent, possibly partly because of cytoplasmic and maternal effects. Specific functional categories were enriched among the 2,545 nonadditive genes of Brassica hexaploid compared with the additive genes; the categories included response to stimulus, immune system process, cellular process, metabolic process, rhythmic process, and pigmentation. Many transcription factor genes, methyltransferases, and methylation genes showed differential expression between Brassica hexaploid and its parents. Our results demonstrate that the Brassica allohexaploid can generate extensive transcriptomic diversity compared with its parents. These changes may contribute to the normal growth and reproduction of allohexaploids.

BACKGROUND: Tocopherols are important antioxidants in vegetable oils; when present as vitamin E, tocopherols are an essential nutrient for humans and livestock. Rapeseed (Brassica napus L, AACC, 2 n = 38) is one of the most important oil crops and a major source of tocopherols. Although the tocopherol biosynthetic pathway has been well elucidated in the model photosynthetic organisms Arabidopsis thaliana and Synechocystis sp. PCC6803, knowledge about the genetic basis of tocopherol biosynthesis in seeds of rapeseed is scant. This project was carried out to dissect the genetic basis of seed tocopherol content and composition in rapeseed through quantitative trait loci (QTL) detection, genome-wide association analysis, and homologous gene mapping. METHODOLOGY/PRINCIPAL FINDINGS: We used a segregating Tapidor × Ningyou7 doubled haploid (TNDH) population, its reconstructed F(2) (RC-F(2)) population, and a panel of 142 rapeseed accessions (association panel). Genetic effects mainly contributed to phenotypic variations in tocopherol content and composition; environmental effects were also identified. Thirty-three unique QTL were detected for tocopherol content and composition in TNDH and RC-F(2) populations. Of these, seven QTL co-localized with candidate sequences associated with tocopherol biosynthesis through in silico and linkage mapping. Several near-isogenic lines carrying introgressions from the parent with higher tocopherol content showed highly increased tocopherol content compared with the recurrent parent. Genome-wide association analysis was performed with 142 B. napus accessions. Sixty-one loci were significantly associated with tocopherol content and composition, 11 of which were localized within the confidence intervals of tocopherol QTL. CONCLUSIONS/SIGNIFICANCE: This joint QTL, candidate gene, and association mapping study sheds light on the genetic basis of seed tocopherol biosynthesis in rapeseed. The sequences presented here may be used for marker-assisted selection of oilseed rape lines with superior tocopherol content and composition.

Brassica napus (AACC, 2n = 38), an oil crop of world-wide importance, originated from interspecific hybridization of B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18), and has six FLOWERING LOCUS T (FT) paralogues. Two located on the homeologous chromosomes A2 and C2 arose from a lineage distinct from four located on A7 and C6. A set of three conserved blocks A, B and C, which were found to be essential for FT activation by CONSTANS (CO) in Arabidopsis, was identified within the FT upstream region in B. napus and its progenitor diploids. However, on chromosome C2, insertion of a DNA transposable element (TE) and a retro-element in FT upstream blocks A and B contributed to significant structural divergence between the A and C genome orthologues. Phylogenetic analysis of upstream block A indicated the conserved evolutionary relationships of distinct FT genes within Brassicaceae. We conclude that the ancient At-α whole genome duplication contributed to distinct ancestral lineages for this key adaptive gene, which co-exist within the same genus. FT-A2 was found to be transcribed in all leaf samples from different developmental stages in both B. rapa and B. napus, whereas FT-C2 was not transcribed in either B. napus or B. oleracea. Silencing of FT-C2 appeared to result from TE insertion and consequent high levels of cytosine methylation in TE sequences within upstream block A. Interestingly, FT-A7/C6 paralogues were specifically silenced in winter type B. napus but abundantly expressed in spring type cultivars under vernalization-free conditions. Motif prediction indicated the presence of two CO protein binding sites within all Brassica block A and additional sites for FT activation in block C. We propose that the ancestral whole genome duplications have contributed to more complex mechanisms of floral regulation and niche adaptation in Brassica compared to Arabidopsis.

We identified nine FLOWERING LOCUS C homologues (BnFLC) in Brassica napus and found that the coding sequences of all BnFLCs were relatively conserved but the intronic and promoter regions were more divergent. The BnFLC homologues were mapped to six of 19 chromosomes. All of the BnFLC homologues were located in the collinear region of FLC in the Arabidopsis genome except BnFLC.A3b and BnFLC.C3b, which were mapped to noncollinear regions of chromosome A3 and C3, respectively. Four of the homologues were associated significantly with quantitative trait loci for flowering time in two mapping populations. The BnFLC homologues showed distinct expression patterns in vegetative and reproductive organs, and at different developmental stages. BnFLC.A3b was differentially expressed between the winter-type and semi-winter-type cultivars. Microsynteny analysis indicated that BnFLC.A3b might have been translocated to the present segment in a cluster with other flowering-time regulators, such as a homologue of FRIGIDA in Arabidopsis. This cluster of flowering-time genes might have conferred a selective advantage to Brassica species in terms of increased adaptability to diverse environments during their evolution and domestication process.

High yield is the most important goal in crop breeding, and boron (B) is an essential micronutrient for plants. However, B deficiency, leading to yield decreases, is an agricultural problem worldwide. Brassica napus is one of the most sensitive crops to B deficiency, and considerable genotypic variation exists among different cultivars in response to B deficiency. To dissect the genetic basis of tolerance to B deficiency in B. napus, we carried out QTL analysis for seed yield and yield-related traits under low and normal B conditions using the double haploid population (TNDH) by two-year and the BQDH population by three-year field trials. In total, 80 putative QTLs and 42 epistatic interactions for seed yield, plant height, branch number, pod number, seed number, seed weight and B efficiency coefficient (BEC) were identified under low and normal B conditions, singly explaining 4.15-23.16% and 0.53-14.38% of the phenotypic variation. An additive effect of putative QTLs was a more important controlling factor than the additive-additive effect of epistatic interactions. Four QTL-by-environment interactions and 7 interactions between epistatic interactions and the environment contributed to 1.27-4.95% and 1.17-3.68% of the phenotypic variation, respectively. The chromosome region on A2 of SYLB-A2 for seed yield under low B condition and BEC-A2 for BEC in the two populations was equivalent to the region of a reported major QTL, BE1. The B. napus homologous genes of Bra020592 and Bra020595 mapped to the A2 region and were speculated to be candidate genes for B efficiency. These findings reveal the complex genetic basis of B efficiency in B. napus. They provide a basis for the fine mapping and cloning of the B efficiency genes and for breeding B-efficient cultivars by marker-assisted selection (MAS).

Although much research has been conducted, the genetic architecture of heterosis remains ambiguous. To unravel the genetic architecture of heterosis, a reconstructed F(2) population was produced by random intercross among 202 lines of a double haploid population in rapeseed (Brassica napus L.). Both populations were planted in three environments and 15 yield-correlated traits were measured, and only seed yield and eight yield-correlated traits showed significant mid-parent heterosis, with the mean ranging from 8.7% (branch number) to 31.4% (seed yield). Hundreds of QTL and epistatic interactions were identified for the 15 yield-correlated traits, involving numerous variable loci with moderate effect, genome-wide distribution and obvious hotspots. All kinds of mode-of-inheritance of QTL (additive, A; partial-dominant, PD; full-dominant, D; over-dominant, OD) and epistatic interactions (additive × additive, AA; additive × dominant/dominant × additive, AD/DA; dominant × dominant, DD) were observed and epistasis, especially AA epistasis, seemed to be the major genetic basis of heterosis in rapeseed. Consistent with the low correlation between marker heterozygosity and mid-parent heterosis/hybrid performance, a considerable proportion of dominant and DD epistatic effects were negative, indicating heterozygosity was not always advantageous for heterosis/hybrid performance. The implications of our results on evolution and crop breeding are discussed.

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, ArAr; B. nigra, BniBni; B. oleracea, CoCo) and the derived allotetraploid species (i.e.: B. juncea, AjAjBjBj; B. napus, AnAnCnCn; B. carinata, BcBcCcCc). 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 Aj-An than between An-Ar and Aj-Ar 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 Ar, and AnCn genomes allowed us to compare the A subgenome structure of B. juncea (Aj). Our results suggest that strong co-linearity exists among the three A Brassica genomes (Ar, An and Aj) 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Dense consensus genetic maps based on high-throughput genotyping platforms are valuable for making genetic gains in Brassica napus through quantitative trait locus identification, efficient predictive molecular breeding, and map-based gene cloning. This report describes the construction of the first B. napus consensus map consisting of a 1,359 anchored array based genotyping platform; Diversity Arrays Technology (DArT), and non-DArT markers from six populations originating from Australia, Canada, China and Europe. We aligned the B. napus DArT sequences with genomic scaffolds from Brassica rapa and Brassica oleracea, and identified DArT loci that showed linkage with qualitative and quantitative loci associated with agronomic traits. Results: The integrated consensus map covered a total of 1,987.2 cM and represented all 19 chromosomes of the A and C genomes, with an average map density of one marker per 1.46 cM, corresponding to approximately 0.88 Mbp of the haploid genome. Through in silico physical mapping 2,457 out of 3,072 (80%) DArT clones were assigned to the genomic scaffolds of B. rapa (A genome) and B. oleracea (C genome). These were used to orientate the genetic consensus map with the chromosomal sequences. The DArT markers showed linkage with previously identified non-DArT markers associated with qualitative and quantitative trait loci for plant architecture, phenological components, seed and oil quality attributes, boron efficiency, sucrose transport, male sterility, and race-specific resistance to blackleg disease. Conclusions: The DArT markers provide increased marker density across the B. napus genome. Most of the DArT markers represented on the current array were sequenced and aligned with the B. rapa and B. oleracea genomes, providing insight into the Brassica A and C genomes. This information can be utilised for comparative genomics and genomic evolution studies. In summary, this consensus map can be used to (i) integrate new generation markers such as SNP arrays and next generation sequencing data; (ii) anchor physical maps to facilitate assembly of B. napus genome sequences; and (iii) identify candidate genes underlying natural genetic variation for traits of interest. [ABSTRACT FROM AUTHOR]

Rapeseed (Brassica napus L.) is the most important oil crop of temperate climates. Rapeseed oil contains tocopherols, also known as vitamin E, which is an indispensable nutrient for humans and animals due to its antioxidant and radical scavenging abilities. Moreover, tocopherols are also important for the oxidative stability of vegetable oils. Therefore, seed oil with increased tocopherol content or altered tocopherol composition is a target for breeding. We investigated the role of nucleotide variations within candidate genes from the tocopherol biosynthesis pathway. Field trials were carried out with 229 accessions from a worldwide B. napus collection which was divided into two panels of 96 and 133 accessions. Seed tocopherol content and composition were measured by HPLC. High heritabilities were found for both traits, ranging from 0.62 to 0.94. We identified polymorphisms by sequencing selected regions of the tocopherol genes from the 96 accession panel. Subsequently, we determined the population structure (Q) and relative kinship (K) as detected by genotyping with genome-wide distributed SSR markers. Association studies were performed using two models, the structure-based GLM?+?Q and the PK-mixed model. Between 26 and 12 polymorphisms within two genes (BnaX.VTE3.a, BnaA.PDS1.c) were significantly associated with tocopherol traits. The SNPs explained up to 16.93% of the genetic variance for tocopherol composition and up to 10.48% for total tocopherol content. Based on the sequence information we designed CAPS markers for genotyping the 133 accessions from the second panel. Significant associations with various tocopherol traits confirmed the results from the first experiment. We demonstrate that the polymorphisms within the tocopherol genes clearly impact tocopherol content and composition in B. napus seeds. We suggest that these nucleotide variations may be used as selectable markers for breeding rapeseed with enhanced tocopherol quality. [ABSTRACT FROM AUTHOR]

Background: Rapeseed (Brassica napus L.) has spring and winter genotypes adapted to different growing seasons. Winter genotypes do not flower before the onset of winter, thus leading to a longer vegetative growth period that promotes the accumulation and allocation of more resources to seed production. The development of winter genotypes enabled the rapeseed to spread rapidly from southern to northern Europe and other temperate regions of the world. The molecular basis underlying the evolutionary transition from spring- to winter- type rapeseed is not known, however, and needs to be elucidated. Results: We fine-mapped the spring environment specific quantitative trait locus (QTL) for flowering time, qFT10-4, in a doubled haploid (DH) mapping population of rapeseed derived from a cross between Tapidor (winter-type) and Ningyou7 (semi-winter) and delimited the qFT10-4 to an 80-kb region on chromosome A10 of B. napus. The BnFLC.A10 gene, an ortholog of FLOWERING LOCUS C (FLC) in Arabidopsis, was cloned from the QTL. We identified 12 polymorphic sites between BnFLC.A10 parental alleles of the TN-DH population in the upstream region and in intron 1. Expression of both BnFLC.A10 alleles decreased during vernalization, but decreased more slowly in the winter parent Tapidor. Haplotyping and association analysis showed that one of the polymorphic sites upstream of BnFLC.A10 is strongly associated with the vernalization requirement of rapeseed (r2 = 0.93, χ2 = 0.50). This polymorphic site is derived from a Tourist-like miniature inverted-repeat transposable element (MITE) insertion/ deletion in the upstream region of BnFLC.A10. The MITE sequence was not present in the BnFLC.A10 gene in spring-type rapeseed, nor in ancestral 'A' genome species B. rapa genotypes. Our results suggest that the insertion may have occurred in winter rapeseed after B. napus speciation. Conclusions: Our findings strongly suggest that (i) BnFLC.A10 is the gene underlying qFT10-4, the QTL for phenotypic diversity of flowering time in the TN-DH population, (ii) the allelic diversity caused by MITE insertion/ deletion upstream of BnFLC.A10 is one of the major causes of differentiation of winter and spring genotypes in rapeseed and (iii) winter rapeseed has evolved from spring genotypes through selection pressure at the BnFLC.A10 locus, enabling expanded cultivation of rapeseed along the route of Brassica domestication. [ABSTRACT FROM AUTHOR]

Background: Rapeseed (Brassica napus L.) has spring and winter genotypes adapted to different growing seasons. Winter genotypes do not flower before the onset of winter, thus leading to a longer vegetative growth period that promotes the accumulation and allocation of more resources to seed production. The development of winter genotypes enabled the rapeseed to spread rapidly from southern to northern Europe and other temperate regions of the world. The molecular basis underlying the evolutionary transition from spring- to winter- type rapeseed is not known, however, and needs to be elucidated. Results: We fine-mapped the spring environment specific quantitative trait locus (QTL) for flowering time, qFT10-4, in a doubled haploid (DH) mapping population of rapeseed derived from a cross between Tapidor (winter-type) and Ningyou7 (semi-winter) and delimited the qFT10-4 to an 80-kb region on chromosome A10 of B. napus. The BnFLC.A10 gene, an ortholog of FLOWERING LOCUS C (FLC) in Arabidopsis, was cloned from the QTL. We identified 12 polymorphic sites between BnFLC.A10 parental alleles of the TN-DH population in the upstream region and in intron 1. Expression of both BnFLC.A10 alleles decreased during vernalization, but decreased more slowly in the winter parent Tapidor. Haplotyping and association analysis showed that one of the polymorphic sites upstream of BnFLC.A10 is strongly associated with the vernalization requirement of rapeseed (r2 = 0.93, χ2 = 0.50). This polymorphic site is derived from a Tourist-like miniature inverted-repeat transposable element (MITE) insertion/ deletion in the upstream region of BnFLC.A10. The MITE sequence was not present in the BnFLC.A10 gene in spring-type rapeseed, nor in ancestral 'A' genome species B. rapa genotypes. Our results suggest that the insertion may have occurred in winter rapeseed after B. napus speciation. Conclusions: Our findings strongly suggest that (i) BnFLC.A10 is the gene underlying qFT10-4, the QTL for phenotypic diversity of flowering time in the TN-DH population, (ii) the allelic diversity caused by MITE insertion/deletion upstream of BnFLC.A10 is one of the major causes of differentiation of winter and spring genotypes in rapeseed and (iii) winter rapeseed has evolved from spring genotypes through selection pressure at the BnFLC.A10 locus, enabling expanded cultivation of rapeseed along the route of Brassica domestication. [ABSTRACT FROM AUTHOR]

Phosphorus (P) deficiency in soils is a major limiting factor for plant growth worldwide. Plants have developed adaptive strategies in response to P deficiency. The objective of this study was to map quantitative trait loci (QTL) for P efficiency using a recombinant inbred (RI) population consisting of 124 lines derived from a cross between Brassica napus P-inefficient cv. B104-2 and P-efficient cv. Eyou Changjia. Six traits (shoot dry weight, root dry weight, root/shoot ratio, P concentration, shoot P uptake and shoot P use efficiency) at vegetative stage were examined under high P (HP, 1 mM) and low P (LP, 5 μM) conditions during three separate experimental trial periods. Their relative values (i.e., the ratio of a trait value under the LP condition to that under the HP condition) of these six traits were also determined. Eyou Changjia produced more biomass and acquired more P under the LP condition and, thus, had a higher relative dry weight and relative P uptake than B104-2, indicating Eyou Changjia was high P efficiency. A total of 71 QTL were detected on 13 linkage groups, including 28 QTL under the LP condition, 22 QTL under the HP condition and 21 QTL for relative traits. Nineteen and nine QTL were specific for the LP and HP conditions, respectively, suggesting that different mechanisms existed under the two P condition. Twelve of the twenty-one QTL for relative traits co-localized with QTL identified under the two P conditions. In addition, 18 orthologous genes involved in the P metabolic pathway of Arabidopsis were in silico mapped to the QTL confidence intervals identified in B. napus by comparative genomic analysis. These QTL and their corresponding candidate genes should be further investigated to better understand P efficiency in B. napus. [ABSTRACT FROM AUTHOR]

llopolyploidy plays an important role in plant evolution and confers obvious advantages on crop growth and breeding compared to low ploidy levels. The present investigation was aimed at synthesising the first known chromosomally stable hexaploid Brassica with the genome constitution AABBCC. More than 2,000 putative hexaploid plants were obtained through large-scale hybridisation from various combinations of crosses between different cultivars of Brassica carinata (BBCC) and B. rapa (AA). The majority of plants after two generations of selfing within selected hexaploid plants (H) were aneuploid, and only 80 plants (4.6%) had the expected hexaploid chromosome number (2 n = 54). The hexaploid ratio increased to an average of 23.0 and 26.3% in the H and H generations, respectively, and was accompanied by an increase in pollen fertility. The appearance of aneuploid plants in each generation could be detected having various chromosomal abnormalities at meiosis. The frequency of hexaploid plants varied significantly among different cultivar combinations, from 0 to 56% in the H generation, and it showed a positive correlation with pollen fertility. The frequency of SSR allelic fragments lost or novel alleles gained was significantly lower in H than in H and H, which reflects increasing genome stability in H. The A and C genomes were significantly less stable than the B genome, which may mainly result from frequent homoeologous pairing and rearrangements between the A and C genomes. Methods to establish a stable hexaploid Brassica crop by intercrossing these lines followed by intensive selection are also discussed. [ABSTRACT FROM AUTHOR]

Intersubgenomic heterosis in rapeseed has been revealed in previous studies by using traditional Brassica napus (AACC) to cross partial new type B. napus with A/C introgression from the genomes of B. rapa and B. carinata, respectively. To further enlarge the genetic basis of B. napus and to facilitate a sustained heterosis breeding in rapeseed, it is crucial to create a population for substantial new type B. napus diversified at both A/C genomes. In this experiment, hundreds of artificial hexaploid plants (AABBCC) involving hundreds of B. carinata/ B. rapa combinations were first crossed with elite lines of partial new type B. napus. The pentaploid plants (AABCC) were open-pollinated in isolated conditions, and their offspring were successively self-pollinated and intensively selected for two generations. Thereafter, a population of substantial new type B. napus mainly with a genomic composition of AACC harbouring genetic diversity from 25 original cultivars of B. rapa and 72 accessions of B. carinata was constructed. The population was cytologically verified to have the correct chromosome constitution of AACC and differed genetically from traditional B. napus, in terms of the genome components of A/C and B as well as the novel genetic variations induced by the interspecific hybridisation process. Synchronously, rich phenotypic variation with plenty of novel valuable traits was observed in the population. The origin of the novel variations and the value of the population are discussed. [ABSTRACT FROM AUTHOR]

Background and Aims: Phosphorus (P) deficiency is one of the major limitations for crop production. A significant relationship exists between plant P uptake from soils and the accumulation of P and other mineral elements in seeds. The aims of this study were to identify and characterize genetic loci (QTLs) controlling the accumulation of mineral elements in seeds of Brassica napus grown with contrasting P availabilities. [ABSTRACT FROM PUBLISHER]

Phosphorus (P) deficiency in soils is a major limiting factor for crop growth worldwide. Changes in root morphology and architecture represent as an important mechanism of adaptation of plants to low P (LP) stress. To elucidate the genetic control of tolerance to P deficiency in Brassica napus, quantitative trait loci (QTL) for root morphology in response to LP were identified in three independent paper culture experiments, and dissected through QTL meta-analysis. In total, 62 significant QTL for total root length, root surface area, root volume, total dry weight, and plant P uptake under high and low P conditions were detected in the three experiments. Forty-five of these QTL were clustered within four linkage groups and were integrated into eight unique QTL by two rounds of QTL meta-analysis. Three of the unique QTL, uq.A1, uq.C3a and uq.C3b, were specific for LP condition. uq.C3a and uq.C3b were identified specifically for root traits and P uptake under LP stress, and may contribute to the adaptability of B. napus to P deficiency. Two functional markers, BnIPS2-C3 and BnGPT1-C3, which were developed from the genes AtIPS2 and AtGPT1 in Arabidopsis, were located in the confidence intervals of uq.C3a and uq.C3b, respectively. And AtGPT1 that corresponded to the interval of uq.C3b by in silico mapping was a possible candidate gene of uq.C3b. These results confirmed the importance of root traits for the adaptability of B. napus to LP and partially revealed the genetic basis of tolerance to P deficiency. These findings should be valuable for further study of the mechanism of P efficiency and the breeding of P-efficient cultivars by marker-assisted selection. [ABSTRACT FROM AUTHOR]

Background: FAE1 (fatty acid elongase1) is the key gene in the control of erucic acid synthesis in seeds of Brassica species. Due to oil with low erucic acid (LEA) content is essential for human health and not enough LEA resource could be available, thus new LEA genetic resources are being sought for Brassica breeding. EcoTILLING, a powerful genotyping method, can readily be used to identify polymorphisms in Brassica. Results: Seven B. rapa, nine B. oleracea and 101 B. napus accessions were collected for identification of FAE1 polymorphisms. Three polymorphisms were detected in the two FAE1 paralogues of B. napus using EcoTILLING and were found to be strongly associated with differences in the erucic acid contents of seeds. In genomic FAE1 sequences obtained from seven B. rapa accessions, one SNP in the coding region was deduced to cause loss of gene function. Molecular evolution analysis of FAE1 homologues showed that the relationship between the Brassica A and C genomes is closer than that between the A/C genomes and Arabidopsis genome. Alignment of the coding sequences of these FAE1 homologues indicated that 18 SNPs differed between the A and C genomes and could be used as genomespecific markers in Brassica. Conclusion: This study showed the applicability of EcoTILLING for detecting gene polymorphisms in Brassica. The association between B. napus FAE1 polymorphisms and the erucic acid contents of seeds may provide useful guidance for LEA breeding. The discovery of the LEA resource in B. rapa can be exploited in Brasscia cultivation. [ABSTRACT FROM AUTHOR]