A high content of seed glucosinolates and their degradation products imposes anti-nutritional effects on livestock; therefore, persistent efforts are made to reduce the seed GSL content to increase the commercial value of rapeseed meal. Here, we dissected the genetic structure of SGC by genome-wide association studies (GWAS) combined with transcriptome-wide association studies (TWAS). Fifteen reliable quantitative trait loci (QTLs) were identified to be associated with the reduced SGC in modern B. napus cultivars by GWAS. Analysis of the selection strength and haplotypes at these QTLs revealed that low SGC was predominantly generated by the coselection of qGSL.A02.2, qGSL.C02.1, qGSL.A09.2, and qGSL.C09.1. Integration of the results from TWAS, comprehensive bioinformatics, and POCKET algorithm analyses indicated that BnaC02.GTR2 (BnaC02g42260D) is a candidate gene underlying qGSL.C02.1. Using CRISPR/ Cas9-derived Bna.gtr2s knockout mutants, we experimentally verified that both BnaC02.GTR2 and its three paralogs positively regulate seed GSL accumulation but negatively regulated vegetative tissue GSL contents. In addition, we observed smaller seeds with higher seed oil content in these Bna.gtr2 mutants. Furthermore, both RNA-seq and correlation analyses suggested that Bna.GTR2s might play a comprehensive role in seed development, such as amino acid accumulation, GSL synthesis, sugar assimilation, and oil accumulation. This study unravels the breeding selection history of low-SGC improvement and provides new insights into the molecular function of Bna.GTR2s in both seed GSL accumulation and seed development in B. napus.

Background Brassica napus is an important vegetable oil source worldwide. Seed coat content is a complex quantitative trait that negatively correlates with the seed oil content in B. napus. Results Here we provide insights into the genetic basis of natural variation of seed coat content by transcriptome-wide association studies (TWAS) and genome-wide association studies (GWAS) using 382 B. napus accessions. By population transcriptomic analysis, we identify more than 700 genes and four gene modules that are significantly associated with seed coat content. We also characterize three reliable quantitative trait loci (QTLs) controlling seed coat content by GWAS. Combining TWAS and correlation networks of seed coat content-related gene modules, we find that BnaC07.CCR-LIKE (CCRL) and BnaTT8s play key roles in the determination of the trait by modulating lignin biosynthesis. By expression GWAS analysis, we identify a regulatory hotspot on chromosome A09, which is involved in controlling seed coat content through BnaC07.CCRL and BnaTT8s. We then predict the downstream genes regulated by BnaTT8s using multi-omics datasets. We further experimentally validate that BnaCCRL and BnaTT8 positively regulate seed coat content and lignin content. BnaCCRL represents a novel identified gene involved in seed coat development. Furthermore, we also predict the key genes regulating carbon allocation between phenylpropane compounds and oil during seed development in B. napus. Conclusions This study helps us to better understand the complex machinery of seed coat development and provides a genetic resource for genetic improvement of seed coat content in B. napus breeding.

Most crops are sensitive to salt stress, but their degree of susceptibility varies among species and cultivars. In order to understand the salt stress adaptability of Brassica napus to salt stress, we collected the phenotypic data of 505 B. napus accessions at the germination stage under 150 or 215 mM sodium chloride (NaCl) and at the seedling stage under 215 mM NaCl. Genome-wide association studies (GWAS) of 16 salt tolerance coefficients (STCs) were applied to investigate the genetic basis of salt stress tolerance of B. napus. In this study, we mapped 31 salts stress-related QTLs and identified 177 and 228 candidate genes related to salt stress tolerance were detected at germination and seedling stages, respectively. Overexpression of two candidate genes, BnCKX5 and BnERF3 overexpression, were found to increase the sensitivity to salt and mannitol stresses at the germination stage. This study demonstrated that it is a feasible method to dissect the genetic basis of salt stress tolerance at germination and seedling stages in B. napus by GWAS, which provides valuable loci for improving the salt stress tolerance of B. napus. Moreover, these candidate genes are rich genetic resources for the following exploration of molecular mechanisms in adaptation to salt stress in B. napus.