Detection of two homologous major QTLs and development of diagnostic molecular markers for sucrose content in peanut.

KEY MESSAGE: We identified two stable and homologous major QTLs for sucrose content in peanut, and developed breeder-friendly molecular markers for marker-assisted selection breeding. Sucrose content is a crucial quality trait for edible peanuts, and increasing sucrose content is a key breeding objective. However, the genetic basis of sucrose content in peanut remains unclear, and major quantitative trait loci (QTLs) for sucrose content have yet to be identified. In this study, a high-density genetic map was constructed based on whole-genome re-sequencing data from a peanut RIL population. This map consisted of 2,042 bins and 24,142 SNP markers, making it one of the most comprehensive maps to date in terms of marker density. Two major QTLs (qSCA06.2 and qSCB06.2) were identified, explaining 31.41% and 24.13% of the phenotypic variance, respectively. Notably, these two QTLs were located in homologous genomic regions between the A and B subgenomes. The elite allele of qSCA06.2 was exclusive to Valencia-type, while the elite allele of qSCB06.2 existed in other peanut types. Importantly, the distribution of alleles from two homologous QTLs in the RIL population and diverse germplasm accessions consistently demonstrated that only the combination of elite allelic genotypes from both QTLs/genes resulted in a significantly dominant phenotype, accompanied by a substantial increase in sucrose content. The newly developed diagnostic markers for these QTLs were confirmed to be reliable and could facilitate future breeding efforts to enhance sucrose content using marker-assisted selection techniques. Overall, this study highlights the co-regulation of sucrose content by two major homologous QTLs/genes and provides valuable insights into the genetic basis of sucrose in peanuts.

Functional genomics of Brassica napus: Progresses, challenges, and perspectives.

Brassica napus, commonly known as rapeseed or canola, is a major oil crop contributing over 13% to the stable supply of edible vegetable oil worldwide. Identification and understanding the gene functions in the B. napus genome is crucial for genomic breeding. A group of genes controlling agronomic traits have been successfully cloned through functional genomics studies in B. napus. In this review, we present an overview of the progress made in the functional genomics of B. napus, including the availability of germplasm resources, omics databases and cloned functional genes. Based on the current progress, we also highlight the main challenges and perspectives in this field. The advances in the functional genomics of B. napus contribute to a better understanding of the genetic basis underlying the complex agronomic traits in B. napus and will expedite the breeding of high quality, high resistance and high yield in B. napus varieties.

High-throughput phenotyping-based quantitative trait loci mapping reveals the genetic architecture of the salt stress tolerance of Brassica napus.

Salt stress is a major limiting factor that severely affects the survival and growth of crops. It is important to understand the salt stress tolerance ability of Brassica napus and explore the underlying related genetic resources. We used a high-throughput phenotyping platform to quantify 2111 image-based traits (i-traits) of a natural population under three different salt stress conditions and an intervarietal substitution line (ISL) population under nine different stress conditions to monitor and evaluate the salt stress tolerance of B. napus over time. We finally identified 928 high-quality i-traits associated with the salt stress tolerance of B. napus. Moreover, we mapped the salt stress-related loci in the natural population via a genome-wide association study and performed a linkage analysis associated with the ISL population, respectively. These results revealed 234 candidate genes associated with salt stress response, and two novel candidate genes, BnCKX5 and BnERF3, were experimentally verified to regulate the salt stress tolerance of B. napus. This study demonstrates the feasibility of using high-throughput phenotyping-based quantitative trait loci mapping to accurately and comprehensively quantify i-traits associated with B. napus. The mapped loci could be used for genomics-assisted breeding to genetically improve the salt stress tolerance of B. napus.

The mechanism of white flower formation in Brassica rapa is distinct from that in other Brassica species.

KEY MESSAGE: A single nucleotide (G) deletion in the third exon of BraA02.PES2-2 (Bra032957) leads to the conversion of flower color from yellow to white in B. rapa, and knockout mutants of its orthologous genes in B. napus showed white or pale yellow flowers. Brassica rapa (2n = 20, AA) is grown worldwide as an important crop for edible oil and vegetables. The bright yellow flower color and long-lasting flowering period give it aesthetic qualities appealing to countryside tourists. However, the mechanism controlling the accumulation of yellow pigments in B. rapa has not yet been completely revealed. In this study, we characterized the mechanism of white flower formation using a white-flowered natural B. rapa mutant W01. Compared to the petals of yellow-flowered P3246, the petals of W01 have significantly reduced content of yellowish carotenoids. Furthermore, the chromoplasts in white petals of W01 are abnormal with irregularly structured plastoglobules. Genetic analysis indicated that the white flower was controlled by a single recessive gene. By combining BSA-seq with fine mapping, we identified the target gene BraA02.PES2-2 (Bra032957) homologous to AtPES2, which has a single nucleotide (G) deletion in the third exon. Seven homologous PES2 genes including BnaA02.PES2-2 (BnaA02g28340D) and BnaC02.PES2-2 (BnaC02g36410D) were identified in B. napus (2n = 38, AACC), an allotetraploid derived from B. rapa and B. oleracea (2n = 18, CC). Knockout mutants of either one or two of BnaA02.PES2-2 and BnaC02.PES2-2 in the yellow-flowered B. napus cv. Westar by the CRISPR/Cas9 system showed pale-yellow or white flowers. The knock-out mutants of BnaA02.PES2-2 and BnaC02.PES2-2 had fewer esterified carotenoids. These results demonstrated that BraA02.PES2-2 in B. rapa, and BnaA02.PES2-2 and BnaC02.PES2-2 in B. napus play important roles in carotenoids esterification in chromoplasts that contributes to the accumulation of carotenoids in flower petals.

Comprehensive evaluation of Chinese peanut mini-mini core collection and QTL mapping for aflatoxin resistance.

BACKGROUND: Aflatoxin contamination caused by Aspergillus fungi has been a serious factor affecting food safety of peanut (Arachis hypogaea L.) because aflatoxins are highly harmful for human and animal health. As three mechanisms of resistance to aflatoxin in peanut including shell infection resistance, seed infection resistance and aflatoxin production resistance exist among naturally evolved germplasm stocks, it is highly crucial to pyramid these three resistances for promoting peanut industry development and protecting consumers' health. However, less research effort has been made yet to investigate the differentiation and genetic relationship among the three resistances in diversified peanut germplasm collections. RESULTS: In this study, the Chinese peanut mini-mini core collection selected from a large basic collection was systematically evaluated for the three resistances against A. flavus for the first time. The research revealed a wide variation among the diversified peanut accessions for all the three resistances. Totally, 14 resistant accessions were identified, including three with shell infection resistance, seven with seed infection resistance and five with aflatoxin production resistance. A special accession, Zh.h1312, was identified with both seed infection and aflatoxin production resistance. Among the five botanic types of A. hypogaea, the var. vulgaris (Spanish type) belonging to subspecies fastigiata is the only one which possessed all the three resistances. There was no close correlation between shell infection resistance and other two resistances, while there was a significant positive correlation between seed infection and toxin production resistance. All the three resistances had a significant negative correlation with pod or seed size. A total of 16 SNPs/InDels associated with the three resistances were identified through genome-wide association study (GWAS). Through comparative analysis, Zh.h1312 with seed infection resistance and aflatoxin production resistance was also revealed to possess all the resistance alleles of associated loci for seed infection index and aflatoxin content. CONCLUSIONS: This study provided the first comprehensive understanding of differentiation of aflatoxin resistance in diversified peanut germplasm collection, and would further contribute to the genetic enhancement for resistance to aflatoxin contamination.

Detection of a major QTL and development of KASP markers for seed weight by combining QTL-seq, QTL-mapping and RNA-seq in peanut.

KEY MESSAGE: Combining QTL-seq, QTL-mapping and RNA-seq identified a major QTL and candidate genes, which contributed to the development of KASP markers and understanding of molecular mechanisms associated with seed weight in peanut. Seed weight, as an important component of seed yield, is a significant target of peanut breeding. However, relatively little is known about the quantitative trait loci (QTLs) and candidate genes associated with seed weight in peanut. In this study, three major QTLs on chromosomes A05, B02, and B06 were determined by applying the QTL-seq approach in a recombinant inbred line (RIL) population. Based on conventional QTL-mapping, these three QTL regions were successfully narrowed down through newly developed single nucleotide polymorphism (SNP) and simple sequence repeat markers. Among these three QTL regions, qSWB06.3 exhibited stable expression, contributing mainly to phenotypic variance across environments. Furthermore, differentially expressed genes (DEGs) were identified at the three seed developmental stages between the two parents of the RIL population. It was found that the DEGs were widely distributed in the ubiquitin-proteasome pathway, the serine/threonine-protein pathway, signal transduction of hormones and transcription factors. Notably, DEGs at the early stage were mostly involved in regulating cell division, whereas DEGs at the middle and late stages were primarily involved in cell expansion during seed development. The expression patterns of candidate genes related to seed weight in qSWB06.3 were investigated using quantitative real-time PCR. In addition, the allelic diversity of qSWB06.3 was investigated in peanut germplasm accessions. The marker Ah011475 has higher efficiency for discriminating accessions with different seed weights, and it would be useful as a diagnostic marker in marker-assisted breeding. This study provided insights into the genetic and molecular mechanisms of seed weight in peanut.

DELLA proteins BnaA6.RGA and BnaC7.RGA negatively regulate fatty acid biosynthesis by interacting with BnaLEC1s in Brassica napus.

Seed oil content (SOC) and fatty acid (FA) composition determine the quality and economic value of rapeseed (Brassica napus). Little is known about the role of gibberellic acid (GA) in regulating FA biosynthesis in B. napus. Here, we discovered that four BnaRGAs (B. napus REPRESSOR OF GA), encoding negative regulators of GA signalling, were suppressed during seed development. Compared to the wild type, SOC was reduced in gain-of-function mutants bnaa6.rga-D and ds-3, which also showed reduced oleic acid and increased linoleic acid contents. By contrast, the loss-of-function quadruple mutant bnarga displayed higher SOC during early seed development than the wild type, with increased oleic acid and reduced linoleic acid contents. Notably, only BnaA6.RGA and BnaC7.RGA physically interacted with two BnaLEC1s, which function as essential transcription factors in FA biosynthesis. The FA composition did not significantly differ between bnarga bnalec1 sextuple mutants and bnalec1, suggesting that BnaLEC1s are epistatic to BnaRGAs in the regulation of FA composition. Furthermore, BnaLEC1-induced activation of BnaABI3 expression was repressed by BnaA6.RGA, indicating that GA triggers the degradation of BnaRGAs to relieve their repression of BnaLEC1s, thus promoting the transcription of downstream genes to facilitate oil biosynthesis. Therefore, we uncovered a developmental stage-specific role of GA in regulating oil biosynthesis via the GA-BnaRGA-BnaLEC1 signalling cascade, providing a novel mechanistic understanding of how phytohormones regulate FA biosynthesis in seeds. BnaRGAs represent promising targets for oil crop improvement.

Long-read sequencing reveals widespread intragenic structural variants in a recent allopolyploid crop plant.

Genome structural variation (SV) contributes strongly to trait variation in eukaryotic species and may have an even higher functional significance than single-nucleotide polymorphism (SNP). In recent years, there have been a number of studies associating large chromosomal scale SV ranging from hundreds of kilobases all the way up to a few megabases to key agronomic traits in plant genomes. However, there have been little or no efforts towards cataloguing small- (30-10 000 bp) to mid-scale (10 000-30 000 bp) SV and their impact on evolution and adaptation-related traits in plants. This might be attributed to complex and highly duplicated nature of plant genomes, which makes them difficult to assess using high-throughput genome screening methods. Here, we describe how long-read sequencing technologies can overcome this problem, revealing a surprisingly high level of widespread, small- to mid-scale SV in a major allopolyploid crop species, Brassica napus. We found that up to 10% of all genes were affected by small- to mid-scale SV events. Nearly half of these SV events ranged between 100 bp and 1000 bp, which makes them challenging to detect using short-read Illumina sequencing. Examples demonstrating the contribution of such SV towards eco-geographical adaptation and disease resistance in oilseed rape suggest that revisiting complex plant genomes using medium-coverage long-read sequencing might reveal unexpected levels of functional gene variation, with major implications for trait regulation and crop improvement.

A CACTA-like transposable element in the upstream region of BnaA9.CYP78A9 acts as an enhancer to increase silique length and seed weight in rapeseed.

Rapeseed (Brassica napus L.) is a model plant for polyploid crop research and the second-leading source of vegetable oil worldwide. Silique length (SL) and seed weight are two important yield-influencing traits in rapeseed. Using map-based cloning, we isolated qSLWA9, which encodes a P450 monooxygenase (BnaA9.CYP78A9) and functions as a positive regulator of SL. The expression level of BnaA9.CYP78A9 in silique valves of the long-silique variety is much higher than that in the regular-silique variety, which results in elongated cells and a prolonged phase of silique elongation. Plants of the long-silique variety and transgenic plants with high expression of BnaA9.CYP78A9 had a higher concentration of auxin in the developing silique; this induced a number of auxin-related genes but no genes in well-known auxin biosynthesis pathways, suggesting that BnaA9.CYP78A9 may influence auxin concentration by affecting auxin metabolism or an unknown auxin biosynthesis pathway. A 3.7-kb CACTA-like transposable element (TE) inserted in the 3.9-kb upstream regulatory sequence of BnaA9.CYP78A9 elevates the expression level, suggesting that the CACTA-like TE acts as an enhancer to stimulate high gene expression and silique elongation. Marker and sequence analysis revealed that the TE in B. napus had recently been introgressed from Brassica rapa by interspecific hybridization. The insertion of the TE is consistently associated with long siliques and large seeds in both B. napus and B. rapa collections. However, the frequency of the CACTA-like TE in rapeseed varieties is still very low, suggesting that this allele has not been widely used in rapeseed breeding programs and would be invaluable for yield improvement in rapeseed breeding.

High-throughput phenotyping accelerates the dissection of the dynamic genetic architecture of plant growth and yield improvement in rapeseed.

Rapeseed is the second most important oil crop species and is widely cultivated worldwide. However, overcoming the 'phenotyping bottleneck' has remained a significant challenge. A clear goal of high-throughput phenotyping is to bridge the gap between genomics and phenomics. In addition, it is important to explore the dynamic genetic architecture underlying rapeseed plant growth and its contribution to final yield. In this work, a high-throughput phenotyping facility was used to dynamically screen a rapeseed intervarietal substitution line population during two growing seasons. We developed an automatic image analysis pipeline to quantify 43 dynamic traits across multiple developmental stages, with 12 time points. The time-resolved i-traits could be extracted to reflect shoot growth and predict the final yield of rapeseed. Broad phenotypic variation and high heritability were observed for these i-traits across all developmental stages. A total of 337 and 599 QTLs were identified, with 33.5% and 36.1% consistent QTLs for each trait across all 12 time points in the two growing seasons, respectively. Moreover, the QTLs responsible for yield indicators colocalized with those of final yield, potentially providing a new mechanism of yield regulation. Our results indicate that high-throughput phenotyping can provide novel insights into the dynamic genetic architecture of rapeseed growth and final yield, which would be useful for future genetic improvements in rapeseed.

Genome-wide expression quantitative trait locus analysis in a recombinant inbred line population for trait dissection in peanut.

The transcriptome connects genome to the gene function and ultimate phenome in biology. So far, transcriptomic approach was not used in peanut for performing trait mapping in bi-parental populations. In this research, we sequenced the whole transcriptome in immature seeds in a peanut recombinant inbred line (RIL) population and explored thoroughly the landscape of transcriptomic variations and its genetic basis. The comprehensive analysis identified total 49 691 genes in RIL population, of which 92 genes followed a paramutation-like expression pattern. Expression quantitative trait locus (eQTL) analysis identified 1207 local eQTLs and 15 837 distant eQTLs contributing to the whole-genome transcriptomic variation in peanut. There were 94 eQTL hot spot regions detected across the genome with the dominance of distant eQTL. By integrating transcriptomic profile and annotation analyses, we unveiled a putative candidate gene and developed a linked marker InDel02 underlying a major QTL responsible for purple testa colour in peanut. Our result provided a first understanding of genetic basis of whole-genome transcriptomic variation in peanut and illustrates the potential of the transcriptome-aid approach in dissecting important traits in non-model plants.

Transposon insertions within alleles of BnaFLC.A10 and BnaFLC.A2 are associated with seasonal crop type in rapeseed.

In Brassicaceae, the requirement for vernalization is conferred by high expression of FLOWERING LOCUS C (FLC). The expression of FLC is known to be repressed by prolonged exposure to cold. Rapeseed (Brassica napus L.) cultivars can be classified into spring, winter, and semi-winter crop types, depending on their respective vernalization requirements. In addition to two known distinct transposon insertion events, here we identified a 4.422 kb hAT and a 5.625 kb long interspersed nuclear element transposon insertion within BnaFLC.A10, and a 810 bp miniature inverted-repeat transposable element (MITE) in BnaFLC.A2. Quantitative PCR demonstrated that these insertions lead to distinct gene expression patterns and contribute differentially to the vernalization response. Transgenic and haplotype analysis indicated that the known 621 bp MITE in the promoter region of BnaFLC.A10 is a transcriptional enhancer that appears to be the main determinant of rapeseed vernalization, and has contributed to the adaptation of rapeseed in winter cultivation environments. In the absence of this transposon insertion, the functional allele of BnaFLC.A2 is a major determinant of vernalization demand. Thus, the combination of BnaFLC.A10 carrying the 621 bp MITE insertion and a functional BnaFLC.A2 appears necessary to establish the winter rapeseed crop phenotype.

Genetic mapping of quantitative trait loci and a major locus for resistance to grey leaf spot in maize.

KEY MESSAGE: The genetic basis of GLS resistance was dissected using two DH populations sharing a common resistant parent. A major QTL repeatedly detected in multiple developmental stages and environments was fine mapped in a backcross population. Grey leaf spot (GLS), caused by Cercospora zeae-maydis or Cercospora zeina, is a highly destructive foliar disease worldwide. However, the mechanism of resistance against GLS is not well understood. To study the inheritance of this resistance, we developed two doubled haploid (DH) populations sharing a common resistant parent. The two DH populations were grown in two locations representing the typical maize-growing mountain area in Southwest China for 2 years. GLS disease severity was investigated 2 or 3 times until maturity in the 2 years, and the area under the disease progress curve was calculated. Two high-density linkage maps were constructed by genotyping-by-sequencing. A total of 22 quantitative trait loci (QTLs) were detected for GLS resistance, with most QTLs being repeatedly detected in different stages, locations and years. The confidence intervals of two major QTLs (qGLS_Y2-2 and qGLS_Z2-1) on chromosome 2 from the two DH populations overlapped with each other and were integrated into one consensus QTL (qGLS_YZ2-1). Using highly resistant and highly susceptible plants from a BC3 population, we fine mapped this genetic locus to a genomic region of 2.4 Mb. Using a panel of 255 inbred lines from breeding programmes, we detected associations between markers in the qGLS_YZ2-1 region and GLS resistance. The peak marker (ID-B1) will be very useful for marker-assisted breeding for GLS resistance.

Knock-out of TERMINAL FLOWER 1 genes altered flowering time and plant architecture in Brassica napus.

BACKGROUND: TERMINAL FLOWER 1 (TFL1) is a member of phosphatidylethanolamine-binding protein (PEBP) family, which plays an important role in the determination of floral meristem identity and regulates flowering time in higher plants. RESULTS: Five BnaTFL1 gene copies were identified in the genome of Brassica napus. The phylogenetic analysis indicated that all five BnaTFL1 gene copies were clustered with their corresponding homologous copies in the ancestral species, B. rapa and B. oleracea. The expression of the BnaTFL1s were confined to flower buds, flowers, seeds, siliques and stem tissues and displayed distinct expression profiles. Knockout mutants of BnaC03.TFL1 generated by CRISPR/Cas9 exhibited early flowering phenotype, while the knockout mutants of the other gene copies had similar flowering time as the wild type. Furthermore, knock-out mutants of individual BnaTFL1 gene copy displayed altered plant architecture. The plant height, branch initiation height, branch number, silique number, number of seeds per silique and number of siliques on the main inflorescence were significantly reduced in the BnaTFL1 mutants. CONCLUSIONS: Our results indicated that BnaC03.TFL1 negatively regulates flowering time in B. napus. BnaC03.TFL1 together with the other BnaTFL1 paralogues are essential for controlling the plant architecture.

An auxin signaling gene BnaA3.IAA7 contributes to improved plant architecture and yield heterosis in rapeseed.

Plant architecture is the key factor affecting overall yield in many crops. The genetic basis underlying plant architecture in rapeseed (Brassica napus), a key global oil crop, is elusive. We characterized an ethyl methanesulfonate (EMS)-mutagenized rapeseed mutant, sca, which had multiple phenotypic alterations, including crinkled leaves, semi-dwarf stature, narrow branch angles and upward-standing siliques. We identified the underlying gene, which encodes an Aux/IAA protein (BnaA3.IAA7). A G-to-A mutation changed the glycine at the 84th position to glutamic acid (G84E), disrupting the conserved degron motif GWPPV and reducing the affinity between BnaA3.IAA7 and TIR1 (TRANSPORT INHIBITOR RESPONSE 1) in an auxin dosage-dependent manner. This change repressed the degradation of BnaA3.IAA7 and therefore repressed auxin signaling at low levels of auxin that reduced the length of internodes. The G84E mutation reduced branch angles by enhancing the gravitropic response. The heterozygote +/sca closely resembled a proposed ideal plant architecture, displaying strong yield heterosis through single-locus overdominance by improving multiple component traits. Our findings demonstrate that a weak gain-of-function mutation in BnaA3.IAA7 contributes to yield heterosis by improving plant architecture and would be valuable for breeding superior rapeseed hybrid cultivars and such a mutation may increase the yield in other Brassica crops.

Identification and characterization of a new dwarf locus DS-4 encoding an Aux/IAA7 protein in Brassica napus.

KEY MESSAGE: A dominant dwarfing gene, ds - 4 , encodes an Aux/IAA protein that negatively regulates plant stature through an auxin signaling pathway. Dwarfism is an important agronomic trait affecting yield in many crop species. The molecular mechanisms underlying dwarfism in oilseed rape (Brassica napus) are poorly understood, restricting the progress of breeding dwarf varieties in this species. Here, we identified and characterized a new dwarf locus, DS-4, in B. napus. Next-generation sequencing-assisted genetic mapping and candidate gene analysis found that DS-4 encodes a nucleus-targeted auxin/indole-3-acetic acid (Aux/IAA) protein. A substitution (P87L) was found in the highly conserved degron motif of the Aux/IAA7 protein in the ds-4 mutant. This mutation co-segregated with the phenotype of individuals in the BC1F2 population. The P87L substitution was confirmed as the cause of the extreme dwarf phenotype by ectopic expression of the mutant allele BnaC05.iaa7 (equivalent to ds-4) in Arabidopsis. The P87L substitution blocked the interaction of BnaC05.iaa7 with TRANSPORT INHIBITOR RESPONSE 1 in the presence of auxin. The BnaC05.IAA7 gene is highly expressed in the cotyledons, hypocotyls, stems and leaves, but weakly in the roots and seeds of B. napus. Our findings provide new insights into the molecular mechanisms underlying dominant (gain-of-function) dwarfism in B. napus. Our identification of a distinct gene locus controlling plant height may help to improve lodging resistance in oilseed rape.

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.

Sequence variation and functional analysis of a FRIGIDA orthologue (BnaA3.FRI) in Brassica napus.

BACKGROUND: Allelic variation at the FRIGIDA (FRI) locus is a major contributor to natural variation of flowering time and vernalization requirement in Arabidopsis thaliana. Dominant FRI inhibits flowering by activating the expression of the MADS box transcriptional repressor FLOWERING LOCUS C (FLC), which represses flowering prior to vernalization. Four FRI orthologues had been identified in the domesticated amphidiploid Brassica napus. Linkage and association studies had revealed that one of the FRI orthologues, BnaA3.FRI, contributes to flowering time variation and crop type differentiation. RESULTS: Sequence analyses indicated that three out of the four BnaFRI paralogues, BnaA3.FRI, BnaA10.FRI and BnaC3.FRI, contained a large number of polymorphic sites. Haplotype analysis in a panel of 174 B. napus accessions using PCR markers showed that all the three paralogues had a biased distribution of haplotypes in winter type oilseed rape (P < 0.01). Association analysis indicated that only BnaA3.FRI contributes to flowering time variation in B. napus. In addition, transgenic functional complementation demonstrated that mutations in the coding sequence of BnaA3.FRI lead to weak alleles, and subsequently to flowering time variation. CONCLUSION: This study for the first time provides a molecular basis for flowering time control by BnaA3.FRI in B. napus, and will facilitate predictive oilseed rape breeding to select varieties with favorable flowering time and better adaption to latitude and seasonal shifts due to changing climate.

Dissection of the genetic architecture of three seed-quality traits and consequences for breeding in Brassica napus.

Genome-wide association studies (GWASs) combining high-throughput genome resequencing and phenotyping can accelerate the dissection of genetic architecture and identification of genes for plant complex traits. In this study, we developed a rapeseed genomic variation map consisting of 4 542 011 SNPs and 628 666 INDELs. GWAS was performed for three seed-quality traits, including erucic acid content (EAC), glucosinolate content (GSC) and seed oil content (SOC) using 3.82 million polymorphisms in an association panel. Six, 49 and 17 loci were detected to be associated with EAC, GSC and SOC in multiple environments, respectively. The mean total contribution of these loci in each environment was 94.1% for EAC and 87.9% for GSC, notably higher than that for SOC (40.1%). A high correlation was observed between phenotypic variance and number of favourable alleles for associated loci, which will contribute to breeding improvement by pyramiding these loci. Furthermore, candidate genes were detected underlying associated loci, based on functional polymorphisms in gene regions where sequence variation was found to correlate with phenotypic variation. Our approach was validated by detection of well-characterized FAE1 genes at each of two major loci for EAC on chromosomes A8 and C3, along with MYB28 genes at each of three major loci for GSC on chromosomes A9, C2 and C9. Four novel candidate genes were detected by correlation between GSC and SOC and observed sequence variation, respectively. This study provides insights into the genetic architecture of three seed-quality traits, which would be useful for genetic improvement of B. napus.

A comprehensive and precise set of intervarietal substitution lines to identify candidate genes and quantitative trait loci in oilseed rape (Brassica napus L.).

KEY MESSAGE: A set of intervarietal substitution lines were developed in rapeseed by recurrent backcrossing and marker-assisted selection and employed for mapping both qualitative and quantitative traits. Intervarietal substitution lines (ISLs) may be assembled into advanced secondary mapping populations that have remarkable potential for resolving trait loci and mapping candidate genes. To facilitate the identification of important genes in oilseed rape (canola, Brassica napus), we developed 89 ISLs using an elite cultivar 'Zhongyou 821' (ZY821) as the recipient and a re-synthesized line 'No.2127' as the donor. In the whole process of ISLs development, the target chromosome segments were selected based on the genotypes of 300 microsatellite markers evenly distributed across the genome. Eighty-nine ISLs fixed at BC5F4 were genotyped by sequencing using double digestion to survey the lengths of target substitution segments from the donor parent and the background segments from the recurrent parent. The total length of the substituted chromosome segments was 3030.27 Mb, representing 3.56 × of the Darmor-bzh reference genome sequence (version 4.1). Gene mapping was conducted for two qualitative traits, flower colour and seed-coat colour, and nine quantitative traits including yield- and quality-related traits, with 19 QTLs identified for the latter. Overlapping substitution segments were identified for flower colour and seed-coat colour loci, as well as for QTLs consistently detected in 2 or 3 years. These results demonstrate the value of these ISLs for locus resolution and subsequent cloning, targeted mutation or editing of genes controlling important traits in oilseed rape.