BnaC09.tfl1 controls determinate inflorescence trait in Brassica napus.

Determinate inflorescence is indeed a pivotal agricultural characteristic in crops, notably impacting the architecture modification of Brassica napus (AACC, 2n = 38). Previous study identified a crucial gene Bnsdt2 that encodes the transcription factor BnaC09.TFL1 (Terminal Flower 1). Here by two alleles were cloned and sequenced from indeterminate 2982 and determinate 4769, respectively, we found that BnaC09.TFL1 harbors two T/C and G/C non-synonymous mutations in exon 1, and contains sixty-six differences in a 1.9 Kb promoter sequence. Subsequently, BnaC09.TFL1 was introduced into B. napus 571 line by genetic complementation and overexpression, transgenic plants 571CTO lines and 571TClines were all restored to the indeterminate inflorescence. Interestingly, after BnaC09.TFL1 was knocked out in 'Westar', transgenic plants WestarTcr lines were mutated to determinate inflorescences. Additionally, a NIL-4769 line was constructed to evaluate the effect of BnaC09.TFL1 on agronomic traits of Brassica napus, the results demonstrated that BnaC09.tfl1 reduced the plant height and increased the branch number and branch thousand grain weight of Brassica napus. Finally, we performed RT-qPCR, GUS staining and subcellular localization experiments to analyze the expression pattern of BnaC09.TFL1, the results showed that the expression of BnaC09.TFL1 at shoot apex of NIL-4769 was higher than that of 4769, GUS activity was detected at apical of Arabidopsis thaliana and BnC09.TFL1-GFP was detected in cell membrane, nucleus and cytoplasm. Our findings provide a firm molecular foundation for the study of rapeseed's molecular mechanism of determinate inflorescence formation, as well as theoretical guidance for the application of determinate inflorescence in rapeseed breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01503-7.

Fine mapping and causal gene identification of a novel QTL for early flowering by QTL-seq, Target-seq and RNA-seq in spring oilseed rape.

KEY MESSAGE: A novel quantitative trait locus for early flowering in spring oilseed rape, BnaC08cqDTF, was mapped to an 86-kb region on chromosome C08, and its causal gene, CRY2, was uncovered. Days to flowering is a very important agronomic and adaptive trait of Brassica napus oilseed rape (AACC, 2n = 38). We previously identified BnaC08cqDTF as a novel candidate quantitative trait locus (QTL) for early flowering in spring oilseed rape. Here, we present fine mapping of the locus and a study of its causal gene. Initial mapping was performed by QTL sequencing of DNA pools of BC3F2 plants with extreme flowering times derived from crosses between the spring-type cv. No. 4512 (early flowering) and cv. No. 5246 (late flowering), along with fine mapping by target sequencing of the BC3F2 and BC4F2 populations. Fine mapping narrowed down BnaC08cqDTF to an 86-kb region on chromosome C08. The region harbored fifteen genes. After comparative analyses of the DNA sequences for mutation between A and C syntenic regions and detected by RNA-seq and qRT-PCR between the two parents, we found that BnaC08G0010400ZS harbors an A/G nonsynonymous mutation in exon 3. This single nucleotide polymorphism (SNP) haplotype was also correlated with early flowering in a 256 accession panel. BnaC08G0010400ZS is a homolog of the AT1G04400 gene (CRY2) in Arabidopsis. The analyses of transgenic Arabidopsis verified that BnaC08G0010400ZS is responsible for early flowering. Our results contribute to a better understanding of the genetic control mechanism of early flowering in spring Brassica napus and will promote the breeding for early mature varieties.

Multi-omics analysis reveals the mechanism of seed coat color formation in Brassica rapa L.

KEYMESSAGE: Multi-omics analysis of the transcriptome, metabolome and genome identified major and minor loci and candidate genes for seed coat color and explored the mechanism of flavonoid metabolites biosynthesis in Brassica rapa. Yellow seed trait is considered an agronomically desirable trait with great potential for improving seed quality of Brassica crops. Mechanisms of the yellow seed trait are complex and not well understood. In this study, we performed an integrated metabolome, transcriptome and genome-wide association study (GWAS) on different B. rapa varieties to explore the mechanisms underlying the seed coat color formation. A total of 2,499 differentially expressed genes and 116 differential metabolites between yellow and black seeds with strong association with the flavonoid biosynthesis pathway was identified. In addition, 330 hub genes involved in the seed coat color formation, and the most significantly differential flavonoids biosynthesis were detected based on weighted gene co-expression network analysis. Metabolite GWAS analysis using the contents of 42 flavonoids in developing seeds of 159 B. rapa lines resulted in the identification of 1,626 quantitative trait nucleotides (QTNs) and 37 chromosomal intervals, including one major locus on chromosome A09. A combination of QTNs detection, transcriptome and functional analyses led to the identification of 241 candidate genes that were associated with different flavonoid metabolites. The flavonoid biosynthesis pathway in B. rapa was assembled based on the identified flavonoid metabolites and candidate genes. Furthermore, BrMYB111 members (BraA09g004490.3C and BraA06g034790.3C) involved in the biosynthesis of taxifolin were functionally analyzed in vitro. Our findings lay a foundation and provide a reference for systematically investigating the mechanism of seed coat color in B. rapa and in the other plants.

A novel locus (Bnsdt2) in a TFL1 homologue sustaining determinate growth in Brassica napus.

BACKGROUND: The determinate growth habits is beneficial for plant architecture modification and the development of crops cultivars suited to mechanized production systems. Which play an important role in the genetic improvement of crops. In Brassica napus, a determinate inflorescence strain (4769) has been discovered among doubled haploid (DH) lines obtained from a spring B. napus × winter B. napus cross, but there are few reports on it. We fine mapped a determinate inflorescence locus, and evaluated the effect of the determinate growth habit on agronomic traits. RESULTS: In this study, we assessed the effect of the determinate growth habit on agronomic traits. The results showed that determinacy is beneficial for reducing plant height and flowering time, advancing maturity, enhancing lodging resistance, increasing plant branches and maintaining productivity. Genetic analysis in the determinate (4769) and indeterminate (2982) genotypes revealed that two independently inherited recessive genes (Bnsdt1, Bnsdt2) are responsible for this determinate growth trait. Bnsdt2 was subsequently mapped in BC2 and BC3 populations derived from the combination 2982 × 4769. Bnsdt2 could be delimited to an approximately 122.9 kb region between 68,586.2 kb and 68,709.1 kb on C09. BLAST analysis of these candidate intervals showed that chrC09g006434 (BnaC09.TFL1) is homologous to TFL1 of A. thaliana. Sequence analysis of two alleles identified two non-synonymous SNPs (T136C, G141C) in the first exon of BnaC09.TFL1, resulting in two amino acid substitutions (Phe46Leu, Leu47Phe). Subsequently, qRT-PCR revealed that BnaC09.TFL1 expression in shoot apexes was significantly higher in NIL-4769 than in 4769, suggesting its essential role in sustaining the indeterminate growth habit. CONCLUSIONS: In this study, the novel locus Bnsdt2, a recessive genes for determinate inflorescence in B. napus, was fine-mapped to a 68,586.2 kb - 68,709.1 kb interval on C09. The annotated genes chrC09g006434 (BnaC09.TFL1) that may be responsible for inflorescence traits were found.

Fine mapping of the QTL cqSPDA2 for chlorophyll content in Brassica napus L.

BACKGROUND: Chlorophyll is the most important factor enabling plants to absorb, transfer and transform light energy and plays an important role in yield formation. Brassica napus is one of the most important oil crops. Breeding Brassica napus for high light efficiency by improving photosynthetic efficiency has considerable social and economic value. In Brassica napus, there have been studies of the initial location of chlorophyll in seed embryos and pericarps, but there are few reports on the fine mapping of chlorophyll QTLs. We constructed near-isogenic lines (NIL), fine-mapped a chlorophyll locus, and evaluated the effect of this dominant locus on agronomic traits. RESULTS: The cqSPDA2 locus was mapped to an interval of 21.87-22.91 Mb on the chromosome A02 of Brassica napus using doubled haploid (DH) lines. To fine-map cqSPDA2, we built NIL and designed Indel primers covering the mapping interval. The 469 individuals in the BC3F2 population were analyzed using these indel primers. Among these indel primers, 15 could narrow the mapping interval to 188 kb between Indel3 and Indel15. Next, 16 indel primers and 19 SSR primers were designed within the new narrower mapping interval, and 5 of the primer-amplified fragments were found to be polymorphic and tightly linked to the cqSPDA2 locus in the BC4F2 population. The mapping interval was narrowed to 152 kb on A02 between SSR2 and Indel15. By gene expression analysis, we found three annotated genes in the mapping interval, including BnaA02g30260D, BnaA02g30290D and BnaA02g30310D, which may be responsible for chlorophyll synthesis. CONCLUSIONS: The locus cqSPDA2, a dominant QTL for chlorophyll content in Brassica napus, was fine-mapped to a 21.89-22.04 Mb interval on A02. Three annotated genes (BnaA02g30260D, BnaA02g30290D and BnaA02g30310D) that may be responsible for chlorophyll synthesis were found.

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.

Fine mapping of the Brassica napus Bnsdt1 gene associated with determinate growth habit.

KEY MESSAGE: The newly discovered determinate plant growth habit of Brassica napus is a potential trait that might contribute to the genetic improvement of rapeseed. Brassica napus is an important species of rapeseed and has an indeterminate growth habit. However, a determinate inflorescence strain (4769) has been discovered among doubled haploid (DH) lines obtained from a spring B. napus × winter B. napus cross. We assessed the effect of the determinate growth habit on agronomic traits. The results showed that determinacy is beneficial for reducing plant height and flowering time, advancing maturity and maintaining productivity. We also investigated the inheritance of determinacy. A genetic analysis revealed that the phenotype of the determinate trait is controlled by one recessive gene, Bnsdt1. Mapping of the Bnsdt1 gene was subsequently conducted in BC1 and BC3 populations derived from combination 2014 × 4769. The results showed that the Bnsdt1 gene could be delimited to a region of approximately 220 kb, between 16,627 and 16,847 kb on A10. Within the target region, whole-genome re-sequencing identified two candidate regions (16,628-16,641 and 16,739-16,794 kb) of approximately 68 kb. A Blast analysis of the two candidate intervals found that BnaA10g26300D/GSBRNA2T00136426001 (BnTFL1) is homologous to the TFL1 gene of A. thaliana. Subsequently, quantitative reverse transcription (qRT)-PCR revealed that BnTFL1 was specifically expressed in the shoot apex. Collectively, the results of expression analysis provide preliminary evidence that BnTFL1 is a candidate gene for the inflorescence trait in 4769.

Constructing a dense genetic linkage map and mapping QTL for the traits of flower development in Brassica carinata.

KEY MESSAGE: An integrated dense genetic linkage map was constructed in a B. carinata population and used for comparative genome analysis and QTL identification for flowering time. An integrated dense linkage map of Brassica carinata (BBCC) was constructed in a doubled haploid population based on DArT-Seq(TM) markers. A total of 4,031 markers corresponding to 1,366 unique loci were mapped including 639 bins, covering a genetic distance of 2,048 cM. We identified 136 blocks and islands conserved in Brassicaceae, which showed a feature of hexaploidisation representing the suggested ancestral crucifer karyotype. The B and C genome of B. carinata shared 85 % of commonly conserved blocks with the B genome of B. nigra/B. juncea and 80 % of commonly conserved blocks with the C genome of B. napus, and shown frequent structural rearrangements such as insertions and inversions. Up to 24 quantitative trait loci (QTL) for flowering and budding time were identified in the DH population. Of these QTL, one consistent QTL (qFT.B4-2) for flowering time was identified in all of the environments in the J block of the B4 linkage group, where a group of genes for flowering time were aligned in A. thaliana. Another major QTL for flowering time under a winter-cropped environment was detected in the E block of C6, where the BnFT-C6 gene was previously localised in B. napus. This high-density map would be useful not only to reveal the genetic variation in the species with QTL analysis and genome sequencing, but also for other applications such as marker-assisted selection and genomic selection, for the African mustard improvement.

Identification, evolution and expression analyses of the whole genome-wide PEBP gene family in Brassica napus L.

BACKGROUND: With the release of genomic data for B.rapa, B.oleracea, and B.napus, research on the genetic and molecular functions of Brassica spp. has entered a new stage. PEBP genes in plants play an important role in the transition to flowering as well as seed development and germination. Molecular evolutionary and functional analyses of the PEBP gene family in B.napus based on molecular biology methods can provide a theoretical basis for subsequent investigations of related regulators. RESULTS: In this paper, we identified a total of 29 PEBP genes from B.napus that were located on 14 chromosomes and 3 random locations. Most members contained 4 exons and 3 introns; motif 1 and motif 2 were the characteristic motifs of PEBP members. On the basis of intraspecific and interspecific collinearity analyses, it is speculated that fragment replication and genomic replication are the main drivers of for the amplification and evolution of the PEBP gene in the B.napus genome. The results of promoter cis-elements prediction suggest that BnPEBP family genes are inducible promoters, which may directly or indirectly participate in multiple regulatory pathways of plant growth cycle. Furthermore, the tissue-specific expression results show that the expression levels of BnPEBP family genes in different tissues were quite different, but the gene expression organization and patterns of the same subgroup were basically the same. qRT‒PCR revealed certain spatiotemporal patterns in the expression of the PEBP subgroups in roots, stems, leaves, buds, and siliques, was tissue-specific, and related to function. CONCLUSIONS: A systematic comparative analysis of the B.napus PEBP gene family was carried out at here. The results of gene identification, phylogenetic tree construction, structural analysis, gene duplication analysis, prediction of promoter cis-elements and interacting proteins, and expression analysis provide a reference for exploring the molecular mechanisms of BnPEBP family genes in future research.

QTL analysis and candidate gene prediction for seed density per silique by QTL-seq and RNA-seq in spring Brassica napus L.

Seed density per silique (SD) is an important agricultural trait and plays an important role in the yield performance of Brassica napus L. (B. napus). In this study, a genetic linkage map was constructed using a double haploid (DH) population with 213 lines derived from a cross between a low SD line No. 935 and a high SD line No. 3641, and a total of 1,098,259 SNP (single-nucleotide polymorphisms) markers and 2,102 bins were mapped to 19 linkage groups. Twenty-eight QTLs for SD were detected on chromosomes A02, A04, A05, A09, C02, C03, C06, and C09 of B. napus, of which eight QTLs were on chromosome A09 and explained 5.89%-13.24% of the phenotypic variation. Furthermore, a consistent QTL for SD on chromosome A09, cqSD-A9a, was identified in four environments by QTL meta-analysis, explaining 10.68% of the phenotypic variation. In addition, four pairs of epistatic interactions were detected in the DH population via QTL epistasis analysis, indicating that SD is controlled not only by additive effects but also by epistatic effects that play an important role in spring B. napus., but with little environmental effect. Moreover, 18 closely linked SSR markers for cqSD-A9a were developed, as a result, it was mapped to a 1.86Mb (7.80-9.66 Mb) region on chromosome A09. A total of 13 differentially expressed genes (DEGs) were screened in the candidate interval by RNA-seq analysis, which were differentially expressed in buds, leaves and siliques both between and siliques both between two parents and two pools of extremely high-SD and low-SD lines in the DH population. Three of 13 DEGs were possible candidate genes that might control SD: BnaA09g14070D, which encodes a callose synthase that plays an important role in development and stress responses; BnaA09g14800D, a plant synaptic protein that encodes a membrane component; and BnaA09g18250D, which is responsible for DNA binding, transcriptional regulation, and sequence-specific DNA binding and is involved in the response to growth hormone stimulation. Overall, these results lay a foundation for fine mapping and gene cloning for SD in B. napus.

Genetic characterization and fine mapping of a yellow-seeded gene in Dahuang (a Brassica rapa landrace).

The development of yellow-seeded cultivars in Brassica rapa (B. rapa) would improve the quality and quantity of available oil. The identification and mapping of the seed coat color gene may aid in the development of yellow-seeded cultivars and facilitate introgression of the yellow-seeded gene into desirable Brassica napus (B. napus) lines through marker-assisted selection. In the current study, we investigated the inheritance of a yellow-seeded landrace in B. rapa, "Dahuang", originating from the Qinghai-Tibetan plateau. Genetic analysis revealed that the phenotype of the yellow-seeded trait in Dahuang is controlled by one recessive gene, termed Brsc1. Mapping of the Brsc1 gene was subsequently conducted in a BC(1) population comprised 456 individuals, derived from (Dahuang × 09A-126) × Dahuang. From a survey of 256 amplified fragment length polymorphism (AFLP) primer combinations, 10 tightly linked AFLP markers were obtained. The closest AFLP markers flanking Brsc1, Y10 and Y06, were 0.2 and 0.4 cM away, respectively. Subsequently, using simple sequence repeat (SSR) markers in the reference map, the Brsc1 gene was mapped on A09 in B. rapa. Blast analysis revealed that seven AFLP markers showed sequence homology to A09 of B. rapa, wherein six AFLP markers in our map were in the same order as those in A09 of B. rapa. The two closest markers, Y10 and Y06, delimited the Brsc1 gene within a 2.8 Mb interval. Furthermore, Y05 and Y06, the two closest AFLP markers on one side linked to Brsc1, were located in scaffold000059 on A09 of B. rapa, whereas the closet AFLP marker on the opposite side of Brsc1, Y10, was located in scaffold000081 on A09 of B. rapa. Molecular markers developed from these studies may facilitate marker-assisted selection (MAS) of yellow-seeded lines in B. rapa and B. napus and expedite the process of map-based cloning of Brsc1.

Genome-Wide Association Study of Phenylalanine Derived Glucosinolates in Brassica rapa.

Glucosinolates (GSLs) are sulfur-containing bioactive compounds usually present in Brassicaceae plants and are usually responsible for a pungent flavor and reduction of the nutritional values of seeds. Therefore, breeding rapeseed varieties with low GSL levels is an important breeding objective. Most GSLs in Brassica rapa are derived from methionine or tryptophan, but two are derived from phenylalanine, one directly (benzylGSL) and one after a round of chain elongation (phenethylGSL). In the present study, two phenylalanine (Phe)-derived GSLs (benzylGSL and phenethylGSL) were identified and quantified in seeds by liquid chromatography and mass spectrometry (LC-MS) analysis. Levels of benzylGSL were low but differed among investigated low and high GSL genotypes. Levels of phenethylGSL (also known as 2-phenylethylGSL) were high but did not differ among GSL genotypes. Subsequently, a genome-wide association study (GWAS) was conducted using 159 B. rapa accessions to demarcate candidate regions underlying 43 and 59 QTNs associated with benzylGSL and phenethylGSL that were distributed on 10 chromosomes and 9 scaffolds, explaining 0.56% to 70.86% of phenotypic variations, respectively. Furthermore, we find that 15 and 18 known or novel candidate genes were identified for the biosynthesis of benzylGSL and phenethylGSL, including known regulators of GSL biosynthesis, such as BrMYB34, BrMYB51, BrMYB28, BrMYB29 and BrMYB122, and novel regulators or structural genes, such as BrMYB44/BrMYB77 and BrMYB60 for benzylGSL and BrCYP79B2 for phenethylGSL. Finally, we investigate the expression profiles of the biosynthetic genes for two Phe-derived GSLs by transcriptomic analysis. Our findings provide new insight into the complex machinery of Phe-derived GSLs in seeds of B. rapa and help to improve the quality of Brassicaceae plant breeding.

Comparative Mapping Combined With Map-Based Cloning of the Brassica juncea Genome Reveals a Candidate Gene for Multilocular Rapeseed.

Multilocular traits exist in a variety of plants and exert important effects on plant yield. Previous genetic studies have shown that multilocular trait of the Brassica juncea cultivar Duoshi is controlled by two recessive genes, Bjln1 and Bjln2. In previous studies, the Bjln1 gene is located on chromosome A07, and the Bjln1 candidate gene is BjuA07.CLV1. In this study, a BC4 mapping population for the Bjln2 gene was generated. This population was used to construct genetic linkage maps of the Bjln2 gene using amplified fragment length polymorphism (AFLP), intron length polymorphism (IP) and simple sequence repeat (SSR) methodology. The results showed that the Bjln2 gene was restricted to a 0.63 cM interval. BLAST alignment with B. juncea revealed the Bjln2 gene was located within a 11.81-16.65 Mb region on chromosome B07. Moreover, the candidate gene BjuB07.CLV1 (equivalent to Bjln2) was cloned by comparing mapping and map-based cloning, and BjuB07.CLV1 gene was shown to have the ability to restore the bilocular traits in a genetic complementation experiment. The sequencing revealed that a 4961 bp insertion interrupted the coding sequence of the BjuB07.CLV1 gene, resulting in an increase in locule number. Expression analysis revealed that BjuB07.CLV1 was expressed in all tissues and the expression level in bilocular plants was significantly higher than that in multilocular plants. In addition, markers closely linked to the Bjln2 gene were developed and used for molecular marker-assisted breeding of multilocular traits.

Mutations in the CDS and promoter of BjuA07.CLV1 cause a multilocular trait in Brassica juncea.

Multilocular trait has recently attracted considerable attention for its potential to increase yield. Our previous studies indicated that two genes (Bjln1 and Bjln2) are responsible for multilocular siliques in Brassica juncea and the Bjln1 gene has been delimited to a 208-kb region. In present study, the Bjln1 gene was successfully isolated using the map-based cloning method. Complementation test indicated that the BjuA07.CLV1 (equivalent to BjLn1) could rescue the multilocular phenotype and generate bilocular siliques. Two amino acids changes at positions 28 and 63 in BjuA07.clv1 as well as a 702-bp deletion in its promoter have been proved to affect the carpel numbers. Microscopic analyses suggested that BjuA07.CLV1 is involved in the maintenance of shoot and floral meristem size. The expression level of BjuA07.clv1 was significantly reduced in the SAM. Furthermore, WUS, CLV2, CLV3, RPK2 and POL, key genes in the CLV/WUS signal pathway, showed lower expression level in the multilocular plants. These data suggest that the mutations in the CDS and promoter of BjuA07.clv1 reduced its function and expression level, which disturbed CLV/WUS signal pathway, thereby leading to the enlargement of the shoot and floral meristem and resulting in the multilocular siliques.

Mapping and Identifying a Candidate Gene (Bnmfs) for Female-Male Sterility through Whole-Genome Resequencing and RNA-Seq in Rapeseed (Brassica napus L.).

In oilseed crops, carpel and stamen development play vital roles in pollination and rapeseed yield, but the genetic mechanisms underlying carpel and stamen development remain unclear. Herein, a male- and female-sterile mutant was obtained in offspring of a (Brassica napus cv. Qingyou 14) × (Qingyou 14 × B. rapa landrace Dahuang) cross. Subsequently, F2-F9 populations were generated through selfing of the heterozygote plants among the progeny of each generation. The male- and female-sterility exhibited stable inheritance in successive generations and was controlled by a recessive gene. The mutant kept the same chromosome number (2n = 38) as B. napus parent but showed abnormal meiosis for male and female. One candidate gene for the sterility was identified by simple sequence repeat (SSR) and insertion deletion length polymorphism (InDel) markers in F7-F9 plants, and whole-genome resequencing with F8 pools and RNA sequencing with F9 pools. Whole-genome resequencing found three candidate intervals (35.40-35.68, 35.74-35.75, and 45.34-46.45 Mb) on chromosome C3 in B. napus and candidate region for Bnmfs was narrowed to approximately 1.11-Mb (45.34-46.45 M) by combining SSR and InDel marker analyses with whole-genome resequencing. From transcriptome profiling in 0-2 mm buds, all of the genes in the candidate interval were detected, and only two genes with significant differences (BnaC03g56670D and BnaC03g56870D) were revealed. BnaC03g56870D was a candidate gene that shared homology with the CYP86C4 gene of Arabidopsis thaliana. Quantitative reverse transcription (qRT)-PCR analysis showed that Bnmfs primarily functioned in flower buds. Thus, sequencing and expression analyses provided evidence that BnaC03g56870D was the candidate gene for male and female sterility in the B. napus mutant.

Fine Mapping and Whole-Genome Resequencing Identify the Seed Coat Color Gene in Brassica rapa.

A yellow seed coat is a desirable agronomic trait in the seeds of oilseed-type Brassica crops. In this study, we identified a candidate gene for seed coat color in Dahuang, a landrace of Brassica rapa. A previous study of Dahuang mapped the seed coat color gene Brsc1 to a 2.8-Mb interval on chromosome A9 of B. rapa. In the present study, the density of the linkage map for Brsc1 was increased by adding simple sequence repeat (SSR) markers, and the candidate region for Brsc1 was narrowed to 1.04 Mb. In addition, whole-genome resequencing with bulked segregant analysis (BSA) was conducted to identify candidate intervals for Brsc1. A genome-wide comparison of SNP profiles was performed between yellow-seeded and brown-seeded bulk samples. SNP index analyses identified a major candidate interval on chromosome A9 (A09:18,255,838-18,934,000, 678 kb) containing a long overlap with the target region recovered from the fine mapping results. According to gene annotation, Bra028067 (BrTT1) is an important candidate gene for Brsc1 in the overlapping region. Quantitative reverse transcription (qRT)-PCR revealed that BrTT1 mainly functions in the seed. Point mutations and small deletions in BrTT1 were found between yellow- and brown-seeded Dahuang plants. Collectively, the expression and sequence analysis results provide preliminary evidence that BrTT1 is a candidate gene for the seed coat color trait in Dahuang.

The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes.

Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the molecular evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asymmetrical gene loss in duplicated genomic blocks, asymmetrical amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large number of paralogous and orthologous genes. Genes related to the production of anticancer phytochemicals and morphological variations illustrate consequences of genome duplication and gene divergence, imparting biochemical and morphological variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus.