Oilseed Rape Accessions Research Articles

Genome-wide association study exposed the pleiotropic genes for yield-related and oil quality traits in Brassica napus L.

Oilseed rape (Brassica napus L.) is an allotetraploid (AACC, 2n ​= ​38) crop, valued for its edible oil and protein content. seed yield and nutritional composition of rapeseed are influenced by its yield and oil quality traits. However, the genetic basis of yield-related and oil-quality traits remain ambiguous. A panel of 266 diversified oilseed rape accessions was genotyped using 223 simple sequence repeat (SSR) markers covering all 19 chromosomes to identify significant markers associated with yield and quality traits. Twelve yield-related and six quality traits were investigated in two consecutive years (2014 and 2015), with three replications in two environments (Changshun, CS; and Qinghe, QH). Using the model GLM with population structure and kinship (Q ​+ ​K), a total of 25 significant SSR markers (P ​< ​0.001) were detected to be associated with these twelve yield-related and six quality traits, explaining 4.56%–19.17% of the phenotypic variation for each trait. Based on these markers, BnaA03g23490D, BnaC09g46370D, BnaA07g37150D, BnaA01g32590D, and BnaC09g37280D were identified as pleiotropic genes controlling multiple traits. These candidate genes illustrated the potential for the genetic understanding of yield and oil quality traits. Most importantly, these significant markers can be used for marker-assisted breeding of oilseed rape in different environments.

Genome-wide and candidate gene association studies identify BnPAP17 as conferring the utilization of organic phosphorus in oilseed rape

Phosphorus (P) is essential for living plants, and P deficiency is one of the key factors limiting the yield in rapeseed production worldwide. As the most important organ for plants, root morphology traits (RMTs) play a key role in P absorption. To investigate the genetic variability of RMT under low P availability, we dissected the genetic structure of RMTs by genome-wide association studies (GWAS), linkage mapping and candidate gene association studies (CGAS). A total of 52 suggestive loci were associated with RMTs under P stress conditions in 405 oilseed rape accessions. The purple acid phosphatase gene BnPAP17 was found to control the lateral root number (LRN) and root dry weight (RDW) under low P stress. The expression of BnPAP17 was increased in shoot tissue in P-efficient cultivars compared to root tissue and P-inefficient cultivars in response to low P stress. Moreover, the haplotype of BnPAP17Hap3 was detected for the selective breeding of P efficiency in oilseed rape. Over-expression of the BnPAP17Hap3 could promote the shoot and root growth with enhanced tolerance to low P stress and organic phosphorus (Po) utilization in oilseed rape. Collectively, these findings increase our understanding of the mechanisms underlying BnPAP17-mediated low P stress tolerance in oilseed rape.

Genetic relationships among resynthesized, semi-resynthesized and natural Brassica napus L. genotypes

The allopolyploidization event that created cultivated oilseed rape Brassica napus L, followed by intense breeding, reduced its genetic diversity. Resynthesized (RS) B. napus L. obtained by interspecific hybridization between genotypes of B. rapa L. and B. oleracea L. can be a valuable source for broadening genetic diversity in cultivated oilseed rape. In this study, we determined the extent of DNA polymorphism among natural accessions of oilseed rape, resynthesized B. napus, their parental species and double-low quality semi-RS lines carrying the Rfo gene. Using 10 selected primer combinations, 522 polymorphic AFLP markers were scored in the complete set of 100 Brassica sp. To detect relationships between these genotypes, a cluster analysis was performed using the Jaccard’s distance. Resynthesized allopolyploids clustered directly between their diploid parents. Cultivated accessions of oilseed rape created a compact group away from resynthesized allopolyploids as well as semi-RS lines. The natural oilseed rape group, which consists of 49 cultivars and breeding lines of oilseed rape, is characterized by lower genetic diversity than the group of 33 accessions of resynthesized oilseed rape, and the analysis showed that the double-low quality semi-RS lines represent a specific genetic variation of B. napus. The de novo resynthesized B. napus lines and the semi-RS lines of double-low quality generated from them, provide a significant opportunity for enrichment the gene pool of oilseed rape.

Classification of oilseed rape accessions according to sulfur-related plant traits in short-term experiments reflects agronomic performance in field experiments

Abstract Sulfur (S) deficiency can negatively affect seed yield and nitrogen (N) use efficiency of winter oilseed rape. In this study, it was tested whether genotypic variation in sulfur-related plant traits exists and how this is related to agronomic performance of oilseed rape accessions at low and high N availability in the field. A range of highly diverse oilseed rape accessions was grown in short-term hydroponic experiments under N-limiting conditions. Accessions showed high variation in S uptake and S distribution within shoots, and particularly in leaf S concentration, malate:sulfate ratio and glucosinolate concentration, while variation in N-related plant traits was less pronounced. Accessions could be classified into four groups by cluster analysis using the measured plant traits in hydroponics. The same grouping of the accessions could also be achieved by using only the leaf S parameters. Apart from the variation in hydroponic experiments, groups also differed in agronomic parameters in the field. Within glucosinolate-containing accessions those characterized by high S uptake and S distribution in hydroponics showed superior yield both at low and high N supply. Low-glucosinolate (00) accessions with high S uptake in hydroponics had higher seed S concentrations than other 00-accessions in field experiments. It was concluded that assessing S-related plant traits under controlled conditions in an early growth stage is effective for identifying genotypes with superior S utilization.

Clubroot resistance QTL are modulated by nitrogen input in Brassica napus.

Nitrogen levels can modulate the effectiveness of clubroot resistance in an isolate- and host-specific manner. While the same QTL were detected under high and low nitrogen, their effects were altered. Clubroot, caused by Plasmodiophora brassicae, is one of the most damaging diseases of oilseed rape and is known to be affected by nitrogen fertilization. However, the genetic factors involved in clubroot resistance have not been characterized under nitrogen-limiting conditions. This study aimed to assess the variability of clubroot resistance under different nitrogen levels and to characterize the impact of nitrogen supply on genetic resistance factors. Linkage analyses and a genome-wide association study were conducted to detect QTL for clubroot resistance and evaluate their sensitivity to nitrogen. The clubroot response of a set of 92 diverse oilseed rape accessions and 108 lines derived from a cross between 'Darmor-bzh' (resistant) and 'Yudal' (susceptible) was studied in the greenhouse under high- and low-nitrogen conditions, following inoculation with the P. brassicae isolates eH and K92-16. Resistance to each isolate was controlled by a major QTL and a few small-effects QTL. While the same QTL were detected under both high and low nitrogen, their effects were altered. Clubroot resistance to isolate eH, but not K92-16, was greater under a low-N supply versus a high-N supply. New sources of resistance were found among the oilseed rape accessions under both low and high-N conditions. The results are discussed relative to the literature and from a crop improvement perspective.

Host Range of Plasmodiophora brassicae on Cruciferous Crops and Weeds in China.

Clubroot caused by Plasmodiophora brassicae is an increasingly important soilborne disease in China. The host range of P. brassicae was investigated with 30 cruciferous plants, including 16 crop species, 9 ornamentals, and 5 weeds in field and pot-cultured conditions. In the field, 17 species from five genera produced visible galls, and these included radish, Capsella bursa-pastoris, Orychophragmus violaceus, Sinapis alba, and 13 Brassica crops. In pot-cultured conditions, an additional 13 plant species (11 genera) were determined to be hosts of P. brassicae. Five common weeds were found to be hosts of P. brassicae, including C. bursa-pastoris, Lepidium apetalum, Descurainia sophia, S. alba, and Thellungiella salsuginea. The infection of these plants was confirmed via polymerase chain reaction (PCR) with primers specific to P. brassicae. No galls were found on Matthiola incana roots in the field or in pots and no resting spores of P. brassicae were observed in M. incana roots, although P. brassicae was detected in M. incana roots via PCR. Microscopic examination revealed infection only in the root hairs of M. incana roots. These results suggested that M. incana was highly resistant to P. brassicae in China and could be developed as a bait crop. In total, 297 accessions of oilseed rape were tested in the field, and 3 accessions of Brassica napus and 1 accession of B. juncea were found to be highly resistant to clubroot disease. These resistant resources provide options for managing clubroot in P. brassicae-infested fields.

Identification of Candidate Genes for Seed Glucosinolate Content Using Association Mapping in Brassica napus L.

Rapeseed contains glucosinolates, a toxic group of sulfur-containing glucosides, which play critical roles in defense against herbivores and microbes. However, the presence of glucosinolates in rapeseed reduces the value of the meal as feed for livestock. We performed association mapping of seed glucosinolate (GS) content using the 60K Brassica Infinium single nucleotide polymorphism (SNP) array in 520 oilseed rape accessions. A total of 11 peak SNPs significantly associated with GS content were detected in growing seasons of 2013 and 2014 and were located on B. napus chromosomes A08, A09, C03, and C09, respectively. Two associated regions of GS content covered by these markers were further verified, and three B. napus homologous genes involved in the biosynthesis and accumulation of GS were identified. These genes were multigene family members and were distributed on different chromosomes. Moreover, two genes (BnGRT2 and BnMYB28) associated with GS content were validated by the qRT-PCR analysis of their expression profiles. The further identification and functionalization of these genes will provide useful insight into the mechanism underlying GS biosynthesis and allocation in B. napus, and the associated SNPs markers could be helpful for molecular maker-assisted breeding for low seed GS in B. napus.

Complementary diversity for nitrogen uptake and utilisation efficiency reveals broad potential for increased sustainability of oilseed rape production

Nitrogen use efficiency (NUE) has become a major global megatrend for sustainable agricultural production, particularly in association with the reduced release of nitrogen-derived greenhouse gases from soils and nitrate contamination of waterways. Oilseed rape (Brassica napus L.), the second most important oilseed crop worldwide, requires relatively high N fertilisation but releases an N balance surplus after harvest. The use of genetic variation to breed more efficient varieties is a promising option to improve agricultural sustainability. We assessed 30 diverse winter oilseed rape accessions at contrasting nitrogen fertilisation levels for 33 physiological traits associated with N uptake and utilisation efficiency. Relationships among traits across the diversity panel were used to identify key complementary factors contributing to overall NUE. Trait correlations revealed considerable variation for NUE parameters, including positive effects of early flowering and high leaf N concentration on enhanced N utilisation under low N input. Furthermore, seed yield per se was found to be more important than the seed N concentration for simultaneously achieving both high N utilisation and reduced N balance surplus. Independent inheritance of N uptake and N utilisation traits suggests potential for further improvement of NUE in oilseed rape by targeted combination of contributing factors in new, high-yielding varieties.

Association mapping of seed quality traits in Brassica napus L. using GWAS and candidate QTL approaches

Single nucleotide polymorphisms (SNPs) have rapidly become the molecular marker of choice in plant and animal association mapping (AM) studies. In this work, a genome-wide association study (GWAS) and candidate quantitative trait loci (cQTL) approaches were used to identify SNP markers associated with seed quality traits, in a Brassica napus L. association panel composed of 89 adapted winter oilseed rape accessions. Six seed quality traits (oil and protein content, linolenic acid, total glucosinolates, hemicellulose and cellulose content) were evaluated in two different locations for two seasons. For GWAS, 4025 SNP markers evenly distributed along the B. napus genome were genotyped using a 6K Illumina array platform. For cQTL, 100 SNP markers previously discovered in genomic regions underlying seed quality QTL were genotyped using a competitive allele-specific PCR (KASPar). Analysis of the population structure revealed the presence of two weakly differentiated subpopulations (F ST = 0.037), with 82 % of the pairwise kinship comparisons ranging from 0 to 0.1. The GWAS approach resulted in the identification of 17 and 5 significant associations for seed glucosinolate content and seed hemicellulose content, respectively. The cQTL approach identified 4 significant associations for seed glucosinolate content and 6 significant associations for seed hemicellulose content. The associated SNPs were consistently identified across environments and were mapped to previously reported QTL. These results illustrate the suitability of AM to identify SNP markers associated with seed quality traits in B. napus.

Evaluation of yield and agronomic traits and their genetic variation in 488 global collections of Brassica napus L.

It is important to evaluate yield and agronomic traits when selecting for elite lines in Brassica napus L. In this study, 488 global collections of B. napus L. were evaluated for yield and agronomic traits during two consecutive years under growing conditions in central China. A series of phenotypic data for yield and agronomic traits were obtained. Large variations in yield and agronomic traits were found among these accessions, making selection possible for these characters. Chinese accessions, in general, tended to have higher plot yield (POY), higher seed yield per plant (PY), higher thousand-seed weight (TSW), more seeds per silique (SS), and fewer siliques per plant (PS) than foreign accessions. Comparing winter, semi-winter, and spring accessions, semi-winter accessions tended to have the highest POY, highest PY, highest TSW, most SS and fewest PS. Genotypic variation for TSW accounted for 71.23 % of the total variation, and it was the highest for all 12 of the phenotypic traits. Significant correlations were observed between different traits. Principal component analysis (PCA) revealed that significant variation among the traits with the first four principal components could explain 49.8 % of the total variation. Three major groups (winter, spring and semi-winter oilseed rape accessions) could be distinguished when projecting the accessions onto the first two PCAs. The information on variations in yield and agronomic traits detected in this study provided useful parents for rapeseed breeding. Moreover, the phenotypic data on yield and agronomic traits obtained may be used in our subsequent genome-wide association studies for B. napus.

Deciphering the structural organization of the oil bodies in the Brassica napus seed as a mean to improve the oil extraction yield

A population of 95 winter oilseed rape accessions displaying contrasted lipid and protein contents has been used to find relations between the oil extraction efficiency and the structural organization of seed oil bodies (OBs). Evaluation of the crushing ability using the micro-pressing technique, revealed a high positive correlation between compressibility index and oil extraction yield. OBs hydrodynamic diameter decreased with the increases of temperature or pH and was negatively correlated to content in oleosins, the main structural OB proteins. OB stability was significantly affected by (i) genotype, the accessions with the lowest oil extraction levels displaying the most stable oil bodies, (ii) lowering the pH leading to OBs aggregation at pH lower than 7 and, (iii) the addition of divalent cations able to neutralize the negative charges on the OB surface. As far as genotypes characterized by the lowest oil extractability levels presented the most stable OBs, a link between seed extractability and OB stability could be suggested. Temperature, pH medium, and salt presence appearing to affect OB microstructure and suspension stability, could also be considered towards improvement of oil extraction efficiency.

Molecular characterization of oilseed rape accessions collected from multi continents for exploitation of potential heterotic group through SSR markers

Evaluation of the genetic diversity in conventional and modern rapeseed cultivars is essential for conservation, management and utilization of these genetic resources for high yielding hybrid production. The objective of this research was to evaluate a collection of 86 oilseed rape cultivars with 188 simple sequence repeat (SSR) markers to assess the genetic variability, heterotic group identity and relationships within and between the groups identified among the genotypes. A total of 631 alleles at 188 SSR markers were detected including 53 and 84 unique and private alleles respectively, which indicated great richness and uniqueness of genetic variation in these selected cultivars. The mean number of alleles per locus was 3.3 and the average polymorphic information content was 0.35 for all microsatellite loci. Unweighted Pair Group Method with Arithmetic Mean clustering and principal component analysis consistently divided all the cultivars into four distinct groups (I, II, III and IV) which largely coincided with their geographical distributions. The Chinese origin cultivars are predominantly assembled in Group II and showed wide genetic base because of its high allelic abundance at SSR loci while most of the exotic cultivars grouped into Group I and were highly distinct owing to the abundant private and unique alleles. The highest genetic distance was found between Group I and IV, which mainly comprised of exotic and newly synthesized yellow seeded (1728-1 and G1087) breeding lines, respectively. Our study provides important insights into further utilization of exotic Brassica napus accessions in Chinese rapeseed breeding and vice versa.

Regional association analysis delineates a sequenced chromosome region influencing antinutritive seed meal compounds in oilseed rapeThis article is one of a selection of papers from the conference “Exploiting Genome-wide Association in Oilseed Brassicas: a model for genetic improvement of major OECD crops for sustainable farming”.

This study describes the use of regional association analyses to delineate a sequenced region of a Brassica napus chromosome with a significant effect on antinutritive seed meal compounds in oilseed rape. A major quantitative trait locus (QTL) influencing seed colour, fibre content, and phenolic compounds was mapped to the same position on B. napus chromosome A9 in biparental mapping populations from two different yellow-seeded × black-seeded B. napus crosses. Sequences of markers spanning the QTL region identified synteny to a sequence contig from the corresponding chromosome A9 in Brassica rapa. Remapping of sequence-derived markers originating from the B. rapa sequence contig confirmed their position within the QTL. One of these markers also mapped to a seed colour and fibre QTL on the same chromosome in a black-seeded × black-seeded B. napus cross. Consequently, regional association analysis was performed in a genetically diverse panel of dark-seeded, winter-type oilseed rape accessions. For this we used closely spaced simple sequence repeat (SSR) markers spanning the sequence contig covering the QTL region. Correction for population structure was performed using a set of genome-wide SSR markers. The identification of QTL-derived markers with significant associations to seed colour, fibre content, and phenolic compounds in the association panel enabled the identification of positional and functional candidate genes for B. napus seed meal quality within a small segment of the B. rapa genome sequence.

Identification of QTLs for phenolic compounds in oilseed rape (Brassica napus L.) by association mapping using SSR markers

Association mapping identifies quantitative trait loci (QTLs) by examining the marker-trait associations that can be attributed to the strength of linkage disequilibrium between markers and functional polymorphisms across a set of diverse germplasm. In this study, association mapping was performed to detect QTL-linked and genome wide SSR markers linked to phenolic compounds of extraction meal in a population of 49 genetically diverse oilseed rape cultivars of dark-seeded, winter-type oilseed rape accessions. Correction for population structure was performed using 559 genome wide SSR markers. Results showed that seed colour is an important contributor to seed meal quality. Totally, 52 SSR markers linked to phenolic compounds were detected, five of them being QTL linked markers. Some of these markers were already mapped on Brassica napus chromosomes that contain known QTL controlling oilseed rape meal quality traits. Our results demonstrate that association mapping is a useful approach to complement and enhance previous QTL information for marker-assisted selection.

RFLP and AFLP Analysis of Inter- and Intraspecific Variation of Brassica rapa and B. napus Shows that B. rapa Is an Important Genetic Resource for B. napus Improvement

Fingerprinting of 29 accessions of oilseed rape, including seven accessions of Brassica napus, and 22 accessions of B. rapa ( B. campestris) from Europe, North America, and China was analyzed using RFLP and AFLP markers. In total, 1 477 polymorphic RFLP bands and 183 polymorphic AFLP bands from 166 enzyme-probe combinations and two pairs of AFLP primers, respectively, were scored for the 29 accessions. On average, RFLP analysis showed that the Arabidopsis EST probe detected more polymorphic bands in Brassica than the random genomic probe performed. More polymorphic RFLP markers were detected with the digestion of EcoR I or BamH I than HindIII. According to the number of bands amplified from each accession, the copy numbers of each gene in the genomes of B. rapa and B. napus were estimated. The average copy numbers in B. rapa of China, B. rapa of Europe, and B. napus, were 3.2, 3.1, and 2.9, respectively. Genetic distance based on the AFLP data was well correlated with that based on the RFLP data ( r = 0.72, P<0.001), but 0.39 smaller on average. Genetic diversity analysis showed that Chinese B. rapa was more polymorphic than Chinese B. napus and European materials. Some European B. napus accessions were clustered into European B. rapa, which were distinctly different from Chinese B. napus. The larger variations of Chinese accessions of B. rapa suggest that they are valuable in oilseed rape breeding. Novel strategies to use intersubgenomic heterosis between genome of B. rapa (A rA r) and genome of B. napus (A nA nC nC n) were elucidated.

Analysis of MS2Bnap genomic DNA homologous to MS2 gene from Arabidopsis thaliana in two dominant digenic male sterile accessions of oilseed rape (Brassica napus L.)

PCR technique was employed to isolate gene homologous to the MS2Bnap (X99922.1) from two rapeseed (Brassica napus L.) dominant digenic male sterile lines, namely 220A (male sterile) and 220B (male fertile), 6A (male sterile) and 6C (male fertile). The isolated 2,581 bp sequences from 220A (named 220A-gDNA, GenBank accession number AY288778), 220B (220B-gDNA, AY257490), 6A (6A-gDNA, DQ060318) and 6C (6C-gDNA, DQ060319) all contained six introns. Forty-one single nucleotide polymorphism (SNP) sites were detected by alignment of these four sequences, seven of them dispersed in the exon regions. Two SNPs (1247, 1656) were detected between 220A-gDNA and 220B-gDNA, and the one at nucleotide 1247 of 220A-gDNA with A replaced by C was a missense mutation, which may be the putative male sterility site in 220A. All eight SNPs identified between 6A-gDNA and 6C-gDNA were located in the third intron, so the proteins encoded by them are the same. The one SNP between 6A-/6C-gDNA and 220A-/220B-gDNA at nucleotide 2474 of 220A-/220B-gDNA with C replaced by G was a missense mutation. Mutation site of BNMS2PROT (CAA68190.1) encoded by MS2Bnap in 220A(254) and 6A/6C(584) is different, which indicated dominant digenic male sterile line 220AB and 6CA have some difference in the molecular level. Comparison of structure of MS2Bnap in B. napus with that of MS2 in Arabidopsis thaliana revealed that the similarity of exons between these two genes is higher than that of introns.

Genetic diversity of oilseedBrassica napus germ plasm based on restriction fragment length polymorphisms

Oilseed rape (Brassica napus) is an important oilseed crop worldwide. Cultivars have been developed for many growing regions, however little is known about genetic diversity inB. napus germ plasm. The purpose of the research presented here was to study the genetic diversity and relationships ofB. napus accessions using restriction fragment length polymorphisms (RFLPs). Eighty threeB. napus accessions were screened using 43 genomic DNA clones which revealed 161 polymorphic fragments. Each accession was uniquely identified by the markers with the exception of the near-isogenic cvs 'Triton' and 'Tower'. The RFLP data were analyzed by cluster analysis of similarity coefficients and by principal component analysis. Overall, there were three major groups of cultivars. The first group included only spring accessions, the second mostly winter accessions and the third, rutabagas and oilseed rape accessions from China and Japan. These results indicate that withinB. napus, winter and spring cultivars represent genetically distinct groups. The grouping of accessions by cluster analysis was generally consistent with known pedigrees. This consistency included the grouping of lines derived both by backcrossing or self-pollination with their parents.

The response of oilseed rape (Brassica napus ssp. oleifera) accessions with different glucosinolate and erucic acid contents to four isolates of Peronospora parasitica (downy mildew) and the identification of new sources of resistance

A total of 101 Brassica napus ssp. oleifera accessions with seed differing in glucosinolate and erucic acid contents were screened for resistance to four isolates of Peronospora parasitica at the cotyledon stage. Two groups of accessions with different resistance factors were identified. Lines that were homogeneous for resistance were selected from seedling populations of accessions that exhibited a heterogeneous reaction to some isolates. The resistance of one group differs from that of cv, Cresor, the only oilseed rape cultivar reported to have an isolate‐specific gene for resistance to P. parasitica. The isolate specificity of the second group was identical to that of cv, Cresor, A comparison of the response of host accessions which expressed moderate to full susceptibility at the cotyledon stage, with no clear differential response to any of the four P. parasitica isolates, indicated that those with high glucosinolate and high erucic acid contents (12 accessions) were slightly but significantly less susceptible than those with high glucosinolate and low erucic acid (19 accessions), or low glucosinolate and low erucic acid contents (28 accessions). The mean differences between accessions with low erucic acid but differing in glucosinolate content were inconsistent. The last result was further confirmed by investigating the expression of resistance to three isolates of P. parasitica at three different seedling growth stages among 11 accessions of oilseed rape with seeds low in erucic acid but differing in glucosinolate content.