Genome-wide characterization of LEA gene family reveals a positive role of BnaA.LEA6.a in freezing tolerance in rapeseed (Brassica napus L.).

BACKGROUND: Freezing stress is one of the major abiotic stresses that causes extensive damage to plants. LEA (Late embryogenesis abundant) proteins play a crucial role in plant growth, development, and abiotic stress. However, there is limited research on the function of LEA genes in low-temperature stress in Brassica napus (rapeseed). RESULTS: Total 306 potential LEA genes were identified in B. rapa (79), B. oleracea (79) and B. napus (148) and divided into eight subgroups. LEA genes of the same subgroup had similar gene structures and predicted subcellular locations. Cis-regulatory elements analysis showed that the promoters of BnaLEA genes rich in cis-regulatory elements related to various abiotic stresses. Additionally, RNA-seq and real-time PCR results indicated that the majority of BnaLEA family members were highly expressed in senescent tissues of rapeseed, especially during late stages of seed maturation, and most BnaLEA genes can be induced by salt and osmotic stress. Interestingly, the BnaA.LEA6.a and BnaC.LEA6.a genes were highly expressed across different vegetative and reproductive organs during different development stages, and showed strong responses to salt, osmotic, and cold stress, particularly freezing stress. Further analysis showed that overexpression of BnaA.LEA6.a increased the freezing tolerance in rapeseed, as evidenced by lower relative electrical leakage and higher survival rates compared to the wild-type (WT) under freezing treatment. CONCLUSION: This study is of great significance for understanding the functions of BnaLEA genes in freezing tolerance in rapeseed and offers an ideal candidate gene (BnaA.LEA6.a) for molecular breeding of freezing-tolerant rapeseed cultivars.

Identification of environment-insensitive genes for oil content by combination of transcriptome and genome-wide association analysis in rapeseed.

BACKGROUND: The primary objective of rapeseed breeding is to enhance oil content, which is predominantly influenced by environmental factors. However, the molecular mechanisms underlying the impact of these environmental factors on oil accumulation remain inadequately elucidated. In this study, we used transcriptome data from two higher (HOC) and two lower oil content (LOC) inbred lines at 35 days after pollination (DAP) to investigate genes exhibiting stable expression across three different environments. Meanwhile, a genome-wide association study (GWAS) was utilized to detect candidate genes exhibiting significant associations with seed oil content across three distinct environments. RESULTS: The study found a total of 405 stable differentially expressed genes (DEGs), including 25 involved in lipid/fatty acid metabolism and 14 classified as transcription factors. Among these genes, BnBZIP10-A09, BnMYB61-A06, BnAPA1-A08, BnPAS2-A10, BnLCAT3-C05 and BnKASIII-C09 were also found to exhibit significant associations with oil content across multiple different environments based on GWAS of 50 re-sequenced semi-winter rapeseed inbred lines and previously reported intervals. Otherwise, we revealed the presence of additive effects among BnBZIP10-A09, BnKASIII-C09, BnPAS2-A10 and BnAPA1-A08, resulting in a significant increase in seed oil content. Meanwhile, the majority of these stable DEGs are interconnected either directly or indirectly through co-expression network analysis, thereby giving rise to an elaborate molecular network implicated in the potential regulation of seed oil accumulation and stability. CONCLUSIONS: The combination of transcription and GWAS revealed that natural variation in six environment-insensitive gene regions exhibited significant correlations with seed oil content phenotypes. These results provide important molecular marker information for us to further improve oil content accumulation and stability in rapeseed.

QTL Mapping and Transcriptome Analysis Reveal Candidate Genes Regulating Seed Color in Brassica napus.

Yellow seeds are desirable in rapeseed breeding because of their higher oil content and better nutritional quality than black seeds. However, the underlying genes and formation mechanism of yellow seeds remain unclear. Here, a novel yellow-seeded rapeseed line (Huangaizao, HAZ) was crossed with a black-seeded rapeseed line (Zhongshuang11, ZS11) to construct a mapping population of 196 F2 individuals, based on which, a high-density genetic linkage map was constructed. This map, comprising 4174 bin markers, was 1618.33 cM in length and had an average distance of 0.39 cM between its adjacent markers. To assess the seed color of the F2 population, three methods (imaging, spectrophotometry, and visual scoring) were used and a common major quantitative trait locus (QTL) on chromosome A09, explaining 10.91-21.83% of the phenotypic variance, was detected. Another minor QTL, accounting for 6.19-6.69% of the phenotypic variance, was detected on chromosome C03, only by means of imaging and spectrophotometry. Furthermore, a dynamic analysis of the differential expressions between the parental lines showed that flavonoid biosynthesis-related genes were down-regulated in the yellow seed coats at 25 and 35 days after flowering. A coexpression network between the differentially expressed genes identified 17 candidate genes for the QTL intervals, including a flavonoid structure gene, novel4557 (BnaC03.TT4), and two transcription factor genes, namely, BnaA09G0616800ZS (BnaA09.NFYA8) and BnaC03G0060200ZS (BnaC03.NAC083), that may regulate flavonoid biosynthesis. Our study lays a foundation for further identifying the genes responsible for and understanding the regulatory mechanism of yellow seed formation in Brassica napus.

Transcriptome and Regional Association Analyses Reveal the Effects of Oleosin Genes on the Accumulation of Oil Content in Brassica napus.

Rapeseed stores lipids in the form of oil bodies. Oil bodies in the seeds of higher plants are surrounded by oleosins. Adjusting oleosin protein levels can prevent the fusion of oil bodies and maintain oil body size during seed development. However, oil contents are affected by many factors, and studies on the complex molecular regulatory mechanisms underlying the variations in seed oil contents of B. napus are limited. In this study, a total of 53 BnOLEO (B. napus oleosin) genes were identified in the genome of B. napus through a genome-wide analysis. The promoter sequences of oleosin genes consisted of various light-, hormone-, and stress-related cis-acting elements, along with transcription factor (TF) binding sites, for 25 TF families in 53 BnOLEO genes. The differentially expressed oleosin genes between two high- and two low-oil-content accessions were explored. BnOLEO3-C09, BnOLEO4-A02, BnOLEO4-A09, BnOLEO2-C04, BnOLEO1-C01, and BnOLEO7-A03 showed higher expressions in the high-oil-content accessions than in low-oil-content accessions, at 25, 35, and 45 days after pollination (DAP) in two different environments. A regional association analysis of 50 re-sequenced rapeseed accessions was used to further analyze these six BnOLEO genes, and it revealed that the nucleotide variations in the BnOLEO1-C01 and BnOLEO7-A03 gene regions were related to the phenotypic variations in seed oil content. Moreover, a co-expression network analysis revealed that the BnOLEO genes were directly linked to lipid/fatty acid metabolism, TF, lipid transport, and carbohydrate genes, thus forming a molecular network involved in seed oil accumulation. These favorable haplotypes can be utilized in molecular marker-assisted selection in order to further improve seed oil contents in rapeseed.

Functional study of CYP90A1 and ALDH3F1 gene obtained by transcriptome sequencing analysis of Brassica napus seedlings treated with brassinolide.

Sclerotinia disease and weeds of Brassica napus greatly reduce crop yields. However, brassinolides can improve the resistance of plants to sclerotinia diseases and herbicides. In this study, we investigated the effects of brassinolide on the occurrence, physiological indices, yield, and gene expression of Fanming No. 1 seeds under sclerotinia and glufosinate stress. The results showed that soaking of the seeds in 0.015% brassinolide for 6 h reduced the incidence of sclerotinia by 10%. Additionally, in response to glufosinate stress at the seedling stage, the enzyme activities of catalase and superoxide dismutase increased by 9.6 and 19.0 U/gFW/min, respectively, and the soluble sugar content increased by 9.4 mg/g, increasing the stress resistance of plants and yield by 2.4%. LHCB1, fabF, psbW, CYP90A1, ALDH3F1, ACOX1, petF, and ACSL were screened by transcriptome analysis. ALDH3F1 and CYP90A1 were identified as key genes. Following glufosinate treatment, transgenic plants overexpressing ALDH3F1 and CYP90A1 were found to be resistant to glufosinate, and the expression levels of the ALDH3F1 and CYP90A1 were 1.03-2.37-fold as high as those in the control. The expression level of ATG3, which is an antibacterial gene related to sclerotinia disease, in transgenic plants was 2.40-2.37-fold as high as that in the control. Our results indicate that these two key genes promote plant resistance to sclerotinia and glufosinate. Our study provides a foundation for further studies on the molecular mechanisms of rapeseed resistance breeding and selection of new resistant varieties.

Regional association and transcriptome analysis revealed candidate genes controlling plant height in Brassica napus.

Plant height is a key morphological trait in rapeseed, which not only plays an important role in determining plant architecture, but is also an important characteristic related to yield. Presently, the improvement of plant architecture is a major challenge in rapeseed breeding. This work was carried out to identify genetic loci related to plant height in rapeseed. In this study, a genome-wide association study (GWAS) of plant height was performed using a Brassica 60 K Illumina Infinium SNP array and 203 Brassica napus accessions. Eleven haplotypes containing important candidate genes were detected and significantly associated with plant height on chromosomes A02, A03, A05, A07, A08, C03, C06, and C09. Moreover, regional association analysis of 50 resequenced rapeseed inbred lines was used to further analyze these eleven haplotypes and revealed nucleotide variation in the BnFBR12-A08 and BnCCR1-C03 gene regions related to the phenotypic variation in plant height. Furthermore, coexpression network analysis showed that BnFBR12-A08 and BnCCR1-C03 were directly connected with hormone genes and transcription factors and formed a potential network regulating the plant height of rapeseed. Our results will aid in the development of haplotype functional markers to further improve plant height in rapeseed. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01337-1.

Whole-Genome Identification and Comparative Expression Analysis of Anthocyanin Biosynthetic Genes in Brassica napus.

Anthocyanins contribute to most colors of plants and play protective roles in response to abiotic stresses. Brassica napus is widely cultivated worldwide as both an oilseed and a vegetable. However, only several high anthocyanin-containing cultivars have been reported, and the mechanisms of anthocyanin accumulation have not been well-elucidated in B. napus. Here, the phenotype, comparative whole-genome identification, and gene expression analysis were performed to investigate the dynamic change of the anthocyanin content and the gene expression patterns of anthocyanin biosynthetic genes (ABGs) in B. napus. A total of 152 ABGs were identified in the B. napus reference genome. To screen out the critical genes involved in anthocyanin biosynthesis and accumulation, the RNA-seq of young leaves of two B. napus lines with purple leaves (PL) or green leaves (GL), and their F1 progeny at 41, 91, and 101 days were performed to identify the differentially expressed genes. The comparative expression analysis of these ABGs indicated that the upregulation of TT8 together with its target genes (such as DFR, ANS, UFGT, and TT19) might promote the anthocyanin accumulation in PL at the early developmental stage (41-91 days). While the downregulation of those ABGs and anthocyanin degradation at the late developmental stage (91-101 days) might result in the decrease in anthocyanin accumulation. Our results would enhance the understanding of the regulatory network of anthocyanin dynamic accumulation in B. napus.

Genome-wide identification and expression profiling of the COBRA-like genes reveal likely roles in stem strength in rapeseed (Brassica napus L.).

The COBRA-like (COBL) genes play key roles in cell anisotropic expansion and the orientation of microfibrils. Mutations in these genes cause the brittle stem and induce pathogen responsive phenotypes in Arabidopsis and several crop plants. In this study, an in silico genome-wide analysis was performed to identify the COBL family members in Brassica. We identified 44, 20 and 23 COBL genes in B. napus and its diploid progenitor species B. rapa and B. oleracea, respectively. All the predicted COBL genes were phylogenetically clustered into two groups: the AtCOB group and the AtCOBL7 group. The conserved chromosome locations of COBLs in Arabidopsis and Brassica, together with clustering, indicated that the expansion of the COBL gene family in B. napus was primarily attributable to whole-genome triplication. Among the BnaCOBLs, 22 contained all the conserved motifs and derived from 9 of 12 subgroups. RNA-seq analysis was used to determine the tissue preferential expression patterns of various subgroups. BnaCOBL9, BnaCOBL35 and BnaCOBL41 were highly expressed in stem with high-breaking resistance, which implies these AtCOB subgroup members may be involved in stem development and stem breaking resistance of rapeseed. Our results of this study may help to elucidate the molecular properties of the COBRA gene family and provide informative clues for high stem-breaking resistance studies.

Genomic insights into the origin, domestication and diversification of Brassica juncea.

Despite early domestication around 3000 BC, the evolutionary history of the ancient allotetraploid species Brassica juncea (L.) Czern & Coss remains uncertain. Here, we report a chromosome-scale de novo assembly of a yellow-seeded B. juncea genome by integrating long-read and short-read sequencing, optical mapping and Hi-C technologies. Nuclear and organelle phylogenies of 480 accessions worldwide supported that B. juncea is most likely a single origin in West Asia, 8,000-14,000 years ago, via natural interspecific hybridization. Subsequently, new crop types evolved through spontaneous gene mutations and introgressions along three independent routes of eastward expansion. Selective sweeps, genome-wide trait associations and tissue-specific RNA-sequencing analysis shed light on the domestication history of flowering time and seed weight, and on human selection for morphological diversification in this versatile species. Our data provide a comprehensive insight into the origin and domestication and a foundation for genomics-based breeding of B. juncea.

Use of Comparative Transcriptomics Combined With Physiological Analyses to Identify Key Factors Underlying Cadmium Accumulation in Brassica juncea L.

The contamination of soils with cadmium (Cd) has become a serious environmental issue that needs to be addressed. Elucidating the mechanisms underlying Cd accumulation may facilitate the development of plants that accumulate both high and low amounts of Cd. In this study, a combination of phenotypic, physiological, and comparative transcriptomic analyses was performed to investigate the effects of different Cd concentrations (0, 5, 10, 30, 50 mg/kg) on Brassica juncea L. Our results suggest that B. juncea L. seedlings had a degree of tolerance to the 5 mg/kg Cd treatment, whereas higher Cd stress (10-50 mg/kg) could suppress the growth of B. juncea L. seedlings. The contents of soluble protein, as well as MDA (malondialdehyde), were increased, but the activities of CAT (catalase) enzymes and the contents of soluble sugar and chlorophyll were decreased, when B. juncea L. was under 30 and 50 mg/kg Cd treatment. Comparative transcriptomic analysis indicated that XTH18 (xyloglucan endotransglucosylase/hydrolase enzymes), XTH22, and XTH23 were down-regulated, but PME17 (pectin methylesterases) and PME14 were up-regulated, which might contribute to cell wall integrity maintenance. Moreover, the down-regulation of HMA3 (heavy metal ATPase 3) and up-regulation of Nramp3 (natural resistance associated macrophage proteins 3), HMA2 (heavy metal ATPase 2), and Nramp1 (natural resistance associated macrophage proteins 1) might also play roles in reducing Cd toxicity in roots. Taken together, the results of our study may help to elucidate the mechanisms underlying the response of B. juncea L. to various concentrations of Cd.

Genome-wide identification and expression analysis of CaM/CML genes in Brassica napus under abiotic stress.

Calmodulin (CaM) and calmodulin-like (CML) proteins are primary calcium (Ca2+) sensors and are involved in the regulation of plant development and stress responses by converting calcium signals into transcriptional responses, protein phosphorylation, or metabolic changes. However, the characterization and expression profiling of CaM/CML genes in Brassica napus remain limited. The present study reports that 25 BnaCaM and 168 BnaCML genes were identified in B. napus. The phylogenetics, gene structures, gene motifs, gene chromosomal locations, syntenic and Ka/Ks analysis, promoter cis-acting elements, and expression characteristics in various organs and under abiotic stress were evaluated. The phylogenetic results revealed a total of 11 subgroups, including one unique clade of CaMs distinct from CMLs. Most of group I (CaM), II, III, and X members are intron rich, while members from the other seven groups are intron-less. The majority of CaM/CML proteins have four EF-hands. Syntenic analysis showed that 91.3 % orthologous CaM/CML gene pairs between B. rapa and B. oleracea were retained as homologous gene pairs in B. napus. Ka/Ks analysis indicated that the majority of BnaCaM/CML experienced purifying selection. Expression analysis showed that BnaCaMs genes are highly and ubiquitously expressed in all of the organs and tissues examined, while distinct BnaCMLs are expressed specifically in particular organs and tissues. In total, 129 BnaCaM/CML were induced by abiotic stress and phytohormones. BnaCMLs from group IV, VI, VIII, and X were strongly induced by freezing treatment, but were not or just slightly induced by chilling treatment. The present study is the first to analyze the CaM/CML gene family in B. napus, which is useful for understanding the functions of the BnaCaM/CML in modulating plant responses to abiotic stress, especially freezing stress.

Genome-wide identification and functional analysis of the TIFY gene family in the response to multiple stresses in Brassica napus L.

BACKGROUND: TIFY is a plant-specific protein family with a diversity of functions in plant development and responses to stress and hormones, which contains JASMONATE ZIM-domain (JAZ), TIFY, PPD and ZML subfamilies. Despite extensive studies of TIFY family in many other species, TIFY has not yet been characterized in Brassica napus. RESULTS: In this study, we identified 77, 36 and 39 TIFY family genes in the genome of B. napus, B. rapa and B. oleracea, respectively. Results of the phylogenetic analysis indicated the 170 TIFY proteins from Arabidopsis, B. napus, B. rapa and B. oleracea could be divided into 11 groups: seven JAZ groups, one PPD group, one TIFY group, and two ZIM/ZML groups. The molecular evolutionary analysis showed that TIFY genes were conserved in Brassicaceae species. Gene expression profiling and qRT-PCR revealed that different groups of BnaTIFY members have distinct spatiotemporal expression patterns in normal conditions or following treatment with different abiotic/biotic stresses and hormones. The BnaJAZ subfamily genes were predominantly expressed in roots and up-regulated by NaCl, PEG, freezing, methyl jasmonate (MeJA), salicylic acid (SA) and Sclerotinia sclerotiorum in leaves, suggesting that they have a vital role in hormone signaling to regulate multiple stress tolerance in B. napus. CONCLUSIONS: The extensive annotation and expression analysis of the BnaTIFY genes contributes to our understanding of the functions of these genes in multiple stress responses and phytohormone crosstalk in B. napus.

GWAS and co-expression network combination uncovers multigenes with close linkage effects on the oleic acid content accumulation in Brassica napus.

BACKGROUND: Strong artificial and natural selection causes the formation of highly conserved haplotypes that harbor agronomically important genes. GWAS combination with haplotype analysis has evolved as an effective method to dissect the genetic architecture of complex traits in crop species. RESULTS: We used the 60 K Brassica Infinium SNP array to perform a genome-wide analysis of haplotype blocks associated with oleic acid (C18:1) in rapeseed. Six haplotype regions were identified as significantly associated with oleic acid (C18:1) that mapped to chromosomes A02, A07, A08, C01, C02, and C03. Additionally, whole-genome sequencing of 50 rapeseed accessions revealed three genes (BnmtACP2-A02, BnABCI13-A02 and BnECI1-A02) in the A02 chromosome haplotype region and two genes (BnFAD8-C02 and BnSDP1-C02) in the C02 chromosome haplotype region that were closely linked to oleic acid content phenotypic variation. Moreover, the co-expression network analysis uncovered candidate genes from these two different haplotype regions with potential regulatory interrelationships with oleic acid content accumulation. CONCLUSIONS: Our results suggest that several candidate genes are closely linked, which provides us with an opportunity to develop functional haplotype markers for the improvement of the oleic acid content in rapeseed.

Comprehensive analyses of the annexin (ANN) gene family in Brassica rapa, Brassica oleracea and Brassica napus reveals their roles in stress response.

Annexins (ANN) are a multigene, evolutionarily conserved family of calcium-dependent and phospholipid-binding proteins that play important roles in plant development and stress resistance. However, a systematic comprehensive analysis of ANN genes of Brassicaceae species (Brassica rapa, Brassica oleracea, and Brassica napus) has not yet been reported. In this study, we identified 13, 12, and 26 ANN genes in B. rapa, B. oleracea, and B. napus, respectively. About half of these genes were clustered on various chromosomes. Molecular evolutionary analysis showed that the ANN genes were highly conserved in Brassicaceae species. Transcriptome analysis showed that different group ANN members exhibited varied expression patterns in different tissues and under different (abiotic stress and hormones) treatments. Meanwhile, same group members from Arabidopsis thaliana, B. rapa, B. oleracea, and B. napus demonstrated conserved expression patterns in different tissues. The weighted gene coexpression network analysis (WGCNA) showed that BnaANN genes were induced by methyl jasmonate (MeJA) treatment and played important roles in jasmonate (JA) signaling and multiple stress response in B. napus.

Trilocular phenotype in Brassica juncea L. resulted from interruption of CLAVATA1 gene homologue (BjMc1) transcription.

As a desirable agricultural trait, multilocular trait of rapeseed (Brassica rapa; Brassica napus; Brassica juncea), always represents higher yield per plant compared with bilocular plants. We previously isolated a trilocular gene locus, Bjmc1, and identified a set of molecular markers linked to the trilocular gene. With a map-based cloning, we identified that the BjMc1 was located in B genome of Brassica juncea, and it was a CLAVATA1 (CLV1) gene homologue. The insertion of a copia-LTR retrotransposable element 1 (RTE1) into the coding region of BjMc1 interrupted its transcription in rapeseed, leading to the trilocular phenotype. Phylogenetic analysis showed that Mc1 genes were conserved and widespread in land plants. Two amino acid sites had undergone positive selection in the ancestor of Mc1 genes, and then purifying selection was the dominant force after the divergence of dicots and monocots from their common ancestor in the evolutionary process, indicating that Mc1 genes are conserved in modern land plants. Our results provided new insights in molecular regulatory mechanism of multilocularity in rapeseed, and better understanding of molecular mechanism in crop yield improvement.

Genome-Wide Identification, Localization, and Expression Analysis of Proanthocyanidin-Associated Genes in Brassica.

Proanthocyanidins (PA) is a type of prominent flavonoid compound deposited in seed coats which controls the pigmentation in all Brassica species. Annotation of Brassica juncea genome survey sequences showed 72 PA genes; however, a functional description of these genes, especially how their interactions regulate seed pigmentation, remains elusive. In the present study, we designed 19 primer pairs to screen a bacterial artificial chromosome (BAC) library of B. juncea. A total of 284 BAC clones were identified and sequenced. Alignment of the sequences confirmed that 55 genes were cloned, with every Arabidopsis PA gene having 2-7 homologs in B. juncea. BLAST analysis using the recently released B. rapa or B. napus genome database identified 31 and 58 homologous genes, respectively. Mapping and phylogenetic analysis indicated that 30 B. juncea PA genes are located in the A-genome chromosomes except A04, whereas the remaining 25 genes are mapped to the B-genome chromosomes except B05 and B07. RNA-seq data and Fragments Per Kilobase of a transcript per Million mapped reads (FPKM) analysis showed that most of the PA genes were expressed in the seed coat of B. juncea and B. napus, and that BjuTT3, BjuTT18, BjuANR, BjuTT4-2, BjuTT4-3, BjuTT19-1, and BjuTT19-3 are transcriptionally regulated, and not expressed or downregulated in yellow-seeded testa. Importantly, our study facilitates in better understanding of the molecular mechanism underlying Brassica PA profiles and accumulation, as well as in further characterization of PA genes.

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

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

Cloning of TTG1 gene and PCR identification of genomes A, B and C in Brassica species.

Arabidopsis Transparent Testa Glabra 1 (TTG1) genes were cloned from three diploid Brassica species (B. rapa, B. nigra and B. oleracea) and two amphidiploids species (B. juncea and B. carinata) by homology cloning. TTG1 homologues identified in all the accessions of the investigated species had a coding sequence of 1,014 bp. One copy was obtained from each diploid species and two copies from each amphidiploid species. Combined analysis of the TTG1 sequences cloned in this study with those obtained from public databases demonstrated that three, forty-five and seven nucleotides were specific variations in TTG1 genes from genomes A, B and C, respectively. Primers designed with genome-specific nucleotide variations were able to distinguish among TTG1 genes originating from genomes A, B and C in Brassica. Therefore, the TTG1 gene could serve as a candidate marker gene to detect the pollen flow of Brassica and provide an alternative method for the detection of pollen drift and risk assessment of gene flow in Brassica species.

De novo transcriptome of Brassica juncea seed coat and identification of genes for the biosynthesis of flavonoids.

Brassica juncea, a worldwide cultivated crop plant, produces seeds of different colors. Seed pigmentation is due to the deposition in endothelial cells of proanthocyanidins (PAs), end products from a branch of flavonoid biosynthetic pathway. To elucidate the gene regulatory network of seed pigmentation in B. juncea, transcriptomes in seed coat of a yellow-seeded inbred line and its brown-seeded near- isogenic line were sequenced using the next-generation sequencing platform Illumina/Solexa and de novo assembled. Over 116 million high-quality reads were assembled into 69,605 unigenes, of which about 71.5% (49,758 unigenes) were aligned to Nr protein database with a cut-off E-value of 10(-5). RPKM analysis showed that the brown-seeded testa up-regulated 802 unigenes and down-regulated 502 unigenes as compared to the yellow-seeded one. Biological pathway analysis revealed the involvement of forty six unigenes in flavonoid biosynthesis. The unigenes encoding dihydroflavonol reductase (DFR), leucoantho-cyanidin dioxygenase (LDOX) and anthocyanidin reductase (ANR) for late flavonoid biosynthesis were not expressed at all or at a very low level in the yellow-seeded testa, which implied that these genes for PAs biosynthesis be associated with seed color of B. juncea, as confirmed by qRT-PCR analysis of these genes. To our knowledge, it is the first time to sequence the transcriptome of seed coat in Brassica juncea. The unigene sequences obtained in this study will not only lay the foundations for insight into the molecular mechanisms underlying seed pigmentation in B.juncea, but also provide the basis for further genomics research on this species or its allies.