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Identification of genomic regions associated with multi-silique trait in Brassica napus.

ABSTRACT: BACKGROUND:Although rapeseed (Brassica napus L.) mutant forming multiple siliques was morphologically described and considered to increase the silique number per plant, an important agronomic trait in this crop, the molecular mechanism underlying this beneficial trait remains unclear. Here, we combined bulked-segregant analysis (BSA) and whole genome re-sequencing (WGR) to map the genomic regions responsible for the multi-silique trait using two pools of DNA from the near-isogenic lines (NILs) zws-ms (multi-silique) and zws-217 (single-silique). We used the Euclidean Distance (ED) to identify genomic regions associated with this trait based on both SNPs and InDels. We also conducted transcriptome sequencing to identify differentially expressed genes (DEGs) between zws-ms and zws-217. RESULTS:Genetic analysis using the ED algorithm identified three SNP- and two InDel-associated regions for the multi-silique trait. Two highly overlapped parts of the SNP- and InDel-associated regions were identified as important intersecting regions, which are located on chromosomes A09 and C08, respectively, including 2044 genes in 10.20-MB length totally. Transcriptome sequencing revealed 129 DEGs between zws-ms and zws-217 in buds, including 39 DEGs located in the two abovementioned associated regions. We identified candidate genes involved in multi-silique formation in rapeseed based on the results of functional annotation. CONCLUSIONS:This study identified the genomic regions and candidate genes related to the multi-silique trait in rapeseed.

SUBMITTER: Chai L

PROVIDER: S-EPMC6480887 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Project description:BackgroundFlower and fruit development are vital stages of the angiosperm lifecycle. We previously investigated the multi-silique trait in the rapeseed (Brassica napus) line zws-ms on a genomic and transcriptomic level, leading to the identification of two genomic regions and several candidate genes associated with this trait. However, some events on the transcriptome level, like alternative splicing, were poorly understood.MethodsPlants from zws-ms and its near-isogenic line (NIL) zws-217 were both grown in Xindu with normal conditions and a colder area Ma'erkang. Buds from the two lines were sampled and RNA was isolated to perform the transcriptomic sequencing. The numbers and types of alternative splicing (AS) events from the two lines were counted and classified. Genes with AS events and expressed differentially between the two lines, as well as genes with AS events which occurred in only one line were emphasized. Their annotations were further studied.ResultsFrom the plants in Xindu District, an average of 205,496 AS events, which could be sorted into five AS types, were identified. zws-ms and zws-217 shared highly similar ratios of each AS type: The alternative 5' and 3' splice site types were the most common, while the exon skipping type was observed least often. Eleven differentially expressed AS genes were identified, of which four were upregulated and seven were downregulated in zws-ms. Their annotations implied that five of these genes were directly associated with the multi-silique trait. While samples from colder area Ma'erkang generated generally reduced number of each type of AS events except for Intron Retention; but the number of differentially expressed AS genes increased significantly. Further analysis found that among the 11 differentially expressed AS genes from Xindu, three of them maintained the same expression models, while the other eight genes did not show significant difference between the two lines in expression level. Additionally, the 205 line-specific expressed AS genes were analyzed, of which 187 could be annotated, and two were considered to be important.DiscussionThis study provides new insights into the molecular mechanism of the agronomically important multi-silique trait in rapeseed on the transcriptome level and screens out some environment-responding candidate genes.

Project description:The defatted Brassica napus (rapeseed) meal can be high-protein feed for livestock as the protein value of rapeseed meal is higher than that of the majority of other vegetable proteins. Extensive work has already been carried out on developing canola rapeseed where the focus was on reducing erucic acid and glucosinolate content, with less consideration to other antinutritional factors such as tannin, phytate, sinapine, crude fiber, etc. The presence of these antinutrients limits the use and marketing of rapeseed meals and a significant amount of it goes unused and ends up as waste. We investigated the genetic architecture of crude protein, methionine, tryptophan, total phenols, β-carotene, glucosinolates (GLSs), phytate, tannins, sinapine, and crude fiber content of defatted seed meal samples by conducting a genome-wide association study (GWAS), using a diversity panel comprising 96 B. napus genotypes. Genotyping by sequencing was used to identify 77,889 SNPs, spread over 19 chromosomes. Genetic diversity and phenotypic variations were generally high for the studied traits. A total of eleven genotypes were identified which showed high-quality protein, high antioxidants, and lower amount of antinutrients. A significant negative correlation between protein and limiting amino acids and a significant positive correlation between GLS and phytic acid were observed. General and mixed linear models were used to estimate the association between the SNP markers and the seed quality traits and quantile-quantile (QQ) plots were generated to allow the best-fit algorithm. Annotation of genomic regions around associated SNPs helped to predict various trait-related candidates such as ASP2 and EMB1027 (amino acid biosynthesis); HEMA2, GLU1, and PGM (tryptophan biosynthesis); MS3, CYSD1, and MTO1 (methionine biosynthesis); LYC (β-carotene biosynthesis); HDR and ISPF (MEP pathway); COS1 (riboflavin synthesis); UGT (phenolics biosynthesis); NAC073 (cellulose and hemicellulose biosynthesis); CYT1 (cellulose biosynthesis); BGLU45 and BGLU46 (lignin biosynthesis); SOT12 and UGT88A1 (flavonoid pathway); and CYP79A2, DIN2, and GSTT2 (GLS metabolism), etc. The functional validation of these candidate genes could confirm key seed meal quality genes for germplasm enhancement programs directed at improving protein quality and reducing the antinutritional components in B. napus.

Project description:Seed number is a key character/trait tightly related to the plant fitness/evolution and crop domestication/improvement. The seed number per silique (SNPS) shows a huge variation from several to more than 30, however the underlying regulatory mechanisms are poorly known, which has hindered its improvement. To answer this question, several representative lines with extreme SNPS were previously subjected to systematic genetic and cytological analyses. The results showed that the natural variation of seed number per silique is mainly controlled by maternal and embryonic genotype, which are co-determined by ovule number per ovary, fertile ovule ratio, ovule fertilization rate, and fertilized ovule development rate. More importantly, we also mapped two repeatable quantitative trait loci (QTLs) for SNPS using the F2:3 population derived from Zhongshuang11 and No. 73290, of which the major QTL qSN.A6 has been fine-mapped. In the current study, the near-isogenic lines (NILs) of qSN.A7 were successfully developed by the successive backcross of F1 with Zhongshuang11. First, the effect of qSN.A7 was validated by evaluating the SNPS of two types of homozygous NILs from BC3F2 population, which showed a significant difference of 2.23 on average. Then, qSN.A7 was successfully fine-mapped from the original 4.237 to 1.389 Mb, using a BC4F2 segregating population of 2,551 individuals. To further clarify the regulatory mechanism of qSN.A7, the two types of homologous NILs were subjected to genetic and cytological analyses. The results showed that the difference in SNPS between the two homologous NILs was determined by the embryonic genotypic effect. Highly accordant with this, no significant difference was observed in ovule number per ovary, ovule fertility, fertilization rate, and pollen fertility between the two homologous NILs. Therefore, the regulatory mechanism of qSN.A7 is completely different from the cloned qSS.C9 and qSN.A6. These results will advance the understanding of SNPS and facilitate gene cloning and molecular breeding in Brassica napus.

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Identification of genomic regions associated with multi-silique trait in Brassica napus.

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