Genetic dissection of Brassica napus seed vigor after aging.

KEY MESSAGE: Stable and novel QTLs that affect seed vigor under different storage durations were discovered, and BnaOLE4, located in the interval of cqSW-C2-3, increased seed vigor after aging. Seed vigor is an important trait in crop breeding; however, the underlying molecular regulatory mechanisms governing this trait in rapeseed remain largely unknown. In the present study, vigor-related traits were analyzed in seeds from a doubled haploid (DH) rapeseed (Brassica napus) population grown in 2 different environments using seeds stored for 7, 5, and 3 years under natural storage conditions. A total of 229 quantitative trait loci (QTLs) were identified and were found to explain 3.78%-17.22% of the phenotypic variance for seed vigor-related traits after aging. We further demonstrated that seed vigor-related traits were positively correlated with oil content (OC) but negatively correlated with unsaturated fatty acids (FAs). Some pleiotropic QTLs that collectively regulate OC, FAs, and seed vigor, such as uq.A8, uq.A3-2, uq.A9-2, and uq.C3-1, were identified. The transcriptomic results from extreme pools of DH lines with distinct seed vigor phenotypes during accelerated aging revealed that various biological pathways and metabolic processes (such as glutathione metabolism and reactive oxygen species) were involved in seed vigor. Through integration of QTL analysis and RNA-Seq, a regulatory network for the control of seed vigor was constructed. Importantly, a candidate (BnaOLE4) from cqSW-C2-3 was selected for functional analysis, and transgenic lines overexpressing BnaOLE4 showed increased seed vigor after artificial aging. Collectively, these results provide novel information on QTL and potential candidate genes for molecular breeding for improved seed storability.

New insight into the genetic basis of oil content based on noninvasive three-dimensional phenotyping and tissue-specific transcriptome in Brassica napus.

BACKGROUND: Increasing seed oil content is the most important breeding goal in Brassica napus, and phenotyping is crucial to dissect its genetic basis in crops. To date, QTL mapping for oil content has been based on whole seeds, and the lipid distribution is far from uniform in different tissues of seeds in B. napus. In this case, the phenotype based on whole seeds was unable to sufficiently reveal the complex genetic characteristics of seed oil content. RESULTS: Here, the three-dimensional (3D) distribution of lipid was determined for B. napus seeds by magnetic resonance imaging (MRI) and 3D quantitative analysis, and ten novel oil content-related traits were obtained by subdividing the seeds. Based on a high-density genetic linkage map, 35 QTLs were identified for 4 tissues, the outer cotyledon (OC), inner cotyledon (IC), radicle (R) and seed coat (SC), which explained up to 13.76% of the phenotypic variation. Notably, 14 tissue-specific QTLs were reported for the first time, 7 of which were novel. Moreover, haplotype analysis showed that the favorable alleles for different seed tissues exhibited cumulative effects on oil content. Furthermore, tissue-specific transcriptomes revealed that more active energy and pyruvate metabolism influenced carbon flow in the IC, OC and R than in the SC at the early and middle seed development stages, thus affecting the distribution difference in oil content. Combining tissue-specific QTL mapping and transcriptomics, 86 important candidate genes associated with lipid metabolism were identified that underlie 19 unique QTLs, including the fatty acid synthesis rate-limiting enzyme-related gene CAC2, in the QTLs for OC and IC. CONCLUSIONS: The present study provides further insight into the genetic basis of seed oil content at the tissue-specific level.

Integration of QTL Mapping and Gene Fishing Techniques to Dissect the Multi-Main Stem Trait in Rapeseed (Brassica napus L.).

Rapeseed is one of the most important oilseed crops in the world. Improving the production of rapeseed is beneficial to relieve the shortage of edible vegetable oil. As the organ of support and transport, the main stem of rapeseed controls the plant architecture, transports the water and nutrients, and determines the number of inflorescence. Increasing the number of main stems would be helpful for the yield improvement in Brassica napus (B. napus). This attractive multi-main stem (MMS) trait was observed in the KN DH population. We investigated not only the frequency of MMS traits but also dissected the genetic basis with QTL mapping analysis and Gene-Fishing technique. A total of 43 QTLs were identified for MMS based on high-density linkage map, which explained 2.95-14.9% of the phenotypic variation, among which two environmental stable QTLs (cqMMS.A3-2 and cqMMS.C3-5) were identified in winter and semi-winter environments. Epistatic interaction analysis indicated cqMMS.C3-5 was an important loci for MMS. According to the functional annotation, 159 candidate genes within QTL confidence intervals, corresponding to 148 Arabidopsis thaliana (A. thaliana) homologous genes, were identified, which regulated lateral bud development and tiller of stem, such as shoot meristemless (STM), WUSCHEL-regulated-related genes, cytokinin response factors (CRF5), cytokinin oxidase (CKX4), gibberellin-regulated (RDK1), auxin-regulated gene (ARL, IAR4), and auxin-mediated signaling gene (STV1). Based on Gene-Fishing analysis between the natural plants and the double-main stem (DMS) plant, 31 differentially expressed genes (DEGs) were also obtained, which were related to differentiation and formation of lateral buds, biotic stimulus, defense response, drought and salt-stress responses, as well as cold-response functional genes. In addition, by combining the candidate genes in QTL regions with the DEGs that were obtained by Gene-Fishing technique, six common candidate genes (RPT2A, HLR, CRK, LRR-RLK, AGL79, and TCTP) were identified, which might probably be related to the formation of MMS phenotype. The present results not only would give a new insight into the genetic basis underlying the regulation of MMS but also would provide clues for plant architecture breeding in rapeseed.

Genetic dissection of harvest index and related traits through genome-wide quantitative trait locus mapping in Brassica napus L.

The harvest index (HI) is the ratio of grain yield to the total biomass and represents the harvestable yield of crops. In Brassica napus, the HI is lower than that of other economically important crops, and limited relevant studies have been carried out regarding this issue. In this study, phenotypic analyses of 11 related traits showed the complexity of HI and the feasibility of cultivating desirable varieties with high HI. Quantitative trait loci (QTL) mapping based on a high-density genetic map identified 160 QTL, 163 epistatic loci pairs for HI and three closely related traits: seed yield (SY), biomass yield (BY) and plant height (PH), including two, five and three major QTL for HI, SY and PH, respectively. The related candidate genes underlying the QTL and epistatic loci with coding region variation were identified and investigated, including BnaA02g14010D, homologous to OsTB1, which functions as a negative regulator for lateral branching, and BnaA02g18890D, homologous to OsGW2, which controls grain width and weight. The complex correlation of HI with related traits, numerous QTL and epistatic loci and the candidate genes identified here provide new insights into the genetic architecture of HI, which might further enhance effective breeding strategies for yield improvement in rapeseed.

Integration of proteomic and genomic approaches to dissect seed germination vigor in Brassica napus seeds differing in oil content.

BACKGROUND: Rapeseed (Brassica napus, B. napus) is an important oil seed crop in the world. Previous studies showed that seed germination vigor might be correlated with seed oil content in B. napus, but the regulation mechanism for seed germination has not yet been explained clearly. Dissecting the regulation mechanism of seed germination and germination vigor is necessary. RESULTS: Here, proteomic and genomic approaches were used to analyze the germination process in B. napus seeds with different oil content. The identification of 165 differentially expressed proteins (DEPs) in the germinating seeds of B. napus with high and low oil content was accomplished by two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE). The comparative proteomic results revealed that seeds with high oil content had higher metabolic activity, especially for sulfur amino acid metabolism. Thirty-one unique genes were shown to be significantly changed during germination between the seeds with high and low oil content, and thirteen of these genes were located within the confidence interval of germination-related quantitative trait locus (QTLs), which might play an important role in regulating seed germination vigor. CONCLUSIONS: The present results are of importance for the understanding of the regulation mechanism for seed germination vigor in B. napus.