Flowering time adaptation is a major breeding goal in the allopolyploid species Brassica napus. To investigate the genetic architecture of flowering time, a genome-wide association study (GWAS) of flowering time was conducted with a diversity panel comprising 523 B. napus cultivars and inbred lines grown in eight different environments. Genotyping was performed with a Brassica 60K Illumina Infinium SNP array. A total of 41 single-nucleotide polymorphisms (SNPs) distributed on 14 chromosomes were found to be associated with flowering time, and 12 SNPs located in the confidence intervals of quantitative trait loci (QTL) identified in previous researches based on linkage analyses. Twenty-five candidate genes were orthologous to Arabidopsis thaliana flowering genes. To further our understanding of the genetic factors influencing flowering time in different environments, GWAS was performed on two derived traits, environment sensitivity and temperature sensitivity. The most significant SNPs were found near Bn-scaff_16362_1-p380982, just 13 kb away from BnaC09g41990D, which is orthologous to A. thaliana CONSTANS (CO), an important gene in the photoperiod flowering pathway. These results provide new insights into the genetic control of flowering time in B. napus and indicate that GWAS is an effective method by which to reveal natural variations of complex traits in B. napus.

Yellow seed is a desirable quality trait of the Brassica oilseed species. Previously, several seed coat color genes have been mapped in the Brassica species, but the molecular mechanism is still unknown. In the present investigation, map-based cloning method was used to identify a seed coat color gene, located on A9 in B. rapa. Blast analysis with the Arabidopsis genome showed that there were 22 Arabidopsis genes in this region including at4g09820 to at4g10620. Functional complementation test exhibited a phenotype reversion in the Arabidopsis thaliana tt8-1 mutant and yellow-seeded plant. These results suggested that the candidate gene was a homolog of TRANSPARENT TESTA8 (TT8) locus. BrTT8 regulated the accumulation of proanthocyanidins (PAs) in the seed coat. Sequence analysis of two alleles revealed a large insertion of a new class of transposable elements, Helitron in yellow sarson. In addition, no mRNA expression of BrTT8 was detected in the yellow-seeded line. It indicated that the natural transposon might have caused the loss in function of BrTT8. BrTT8 encodes a basic/helix-loop-helix (bHLH) protein that shares a high degree of similarity with other bHLH proteins in the Brassica. Further expression analysis also revealed that BrTT8 was involved in controlling the late biosynthetic genes (LBGs) of the flavonoid pathway. Our present findings provided with further studies could assist in understanding the molecular mechanism involved in seed coat color formation in Brassica species, which is an important oil yielding quality trait.

SUMMARYS45A, a double recessive mutant at both the BnMs1 and BnMs2 loci in Brassica napus, produces no pollen in mature anthers and no seeds by self-fertilization. The BnMs1 and BnMs2 genes, which have redundant functions in the control of male fertility, are positioned on linkage groups N7 and N16, respectively, and are located at the same locus on Arabidopsis chromosome 1 based on collinearity between Arabidopsis and Brassica. Complementation tests indicated that one candidate gene, BnCYP704B1, a member of the cytochrome P450 family, can rescue male sterility. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of the developing anther showed that pollen-wall formation in the mutant was severely compromised, with a lack of sporopollenin or exine. The phenotype was first evident at the tetrad stage (stage 7) of anther development, coinciding with the maximum BnCYP704B1 mRNA accumulation observed in tapetal cells at stages 7-8 (haploid stage). TEM also suggested that development of the tapetum was seriously defective due to the disturbed lipid metabolism in the S45A mutant. A TUNEL assay indicated that the pattern of programmed cell death in the tapetum of the S45A mutant was defective. Lipid analysis showed that the total fatty acid content was reduced in the S45A mutant, indicating that BnCYP704B1 is involved in lipid metabolism. These data suggest that BnCYP704B1 participates in a vital tapetum-specific metabolic pathway that is not only involved in exine formation but is also required for basic tapetal cell development and function.

Yellow seeds are a favorable trait for Brassica crops breeding due to better quality than their black-seeded counterparts. Here, we compared the Brassica napus seed coat transcriptomes between yellow- and brown-seeded near-isogenic lines (Y-NIL and B-NIL) that were developed from the resynthesized yellow-seeded line No. 2127-17. A total of 4,974 differentially expressed genes (DEG) were identified during seed development, involving 3,128 up-regulated and 1,835 down-regulated genes in yellow seed coats. Phenylpropanoid and flavonoid biosynthesis pathways were enriched in down-regulated genes, whereas the top two pathways for up-regulated genes were plant–pathogen interaction and plant hormone signal transduction. Twelve biosynthetic genes and three regulatory genes involved in the flavonoid pathway exhibited similar expression patterns in seed coats during seed development, of which the down-regulation mainly contributed to the reduction of proanthocyanidins (PAs) in yellow seed coats, indicating that these genes associated with PA biosynthesis may be regulated by an unreported common regulator, possibly corresponding to the candidate for the dominant black-seeded gene D in the NILs. Three transcription factor (TF) genes, including one bHLH gene and two MYB-related genes that are located within the previous seed coat color quantitative trait locus (QTL) region on chromosome A09, also showed similar developmental expression patterns to the key PA biosynthetic genes and they might thus potentially involved participate in flavonoid biosynthesis regulation. Our study identified novel potential TFs involved in PAs accumulation and will provide pivotal information for identifying the candidate genes for seed coat color in B. napus.

SUMMARYHere, we describe the characteristics of a Brassica napus male sterile mutant 7365A with loss of the BnMs3 gene, which exhibits abnormal enlargement of the tapetal cells during meiosis. Later in development, the absence of the BnMs3 gene in the mutant results in a loss of the secretory function of the tapetum, as suggested by abortive callose dissolution and retarded tapetal degradation. The BnaC.Tic40 gene (equivalent to BnMs3) was isolated by a map-based cloning approach and was confirmed by genetic complementation. Sequence analyses suggested that BnaC.Tic40 originated from BolC.Tic40 on the Brassica oleracea linkage group C9, whereas its allele Bnms3 was derived from BraA.Tic40 on the Brassica rapa linkage group A10. The BnaC.Tic40 gene is highly expressed in the tapetum and encodes a putative plastid inner envelope membrane translocon, Tic40, which is localized into the chloroplast. Transmission electron microscopy (TEM) and lipid staining analyses suggested that BnaC.Tic40 is a key factor in controlling lipid accumulation in the tapetal plastids. These data indicate that BnaC.Tic40 participates in specific protein translocation across the inner envelope membrane in the tapetal plastid, which is required for tapetal development and function.