Cloning and characterization of the

Empirical threshold values for quantitative trait mapping.

A set of additive loci for seed oil content were identified using association mapping and one of the novel loci on the chromosome A5 was validated by linkage mapping.
 
Increasing seed oil content is one of the most important goals in the breeding of oilseed crops including Brassica napus, yet the genetic basis for variations in this important trait remains unclear. By genome-wide association study of seed oil content using 521 B. napus accessions genotyped with the Brassica 60K SNP array, we identified 50 loci significantly associated with seed oil content using three statistical models, the general linear model, the mixed linear model and the Anderson–Darling test. Together, the identified loci could explain approximately 80 % of the total phenotypic variance, and 29 of these loci have not been reported previously. Furthermore, a novel locus on the chromosome A5 that could increase 1.5–1.7 % of seed oil content was validated in an independent bi-parental linkage population. Haplotype analysis showed that the favorable alleles for seed oil content exhibit cumulative effects. Our results thus provide valuable information for understanding the genetic control of seed oil content in B. napus and may facilitate marker-based breeding for a higher seed oil content in this important oil crop.

A genome-wide association study reveals novel elite allelic variations in seed oil content of Brassica napus.

Genome- and transcriptome-wide association studies provide insights into the genetic basis of natural variation of seed oil content in Brassica napus.

High-density linkage maps can improve the precision of QTL localization. A high-density SNP-based linkage map containing 3207 markers covering 3072.7 cM of the Brassica napus genome was constructed in the KenC-8 × N53-2 (KNDH) population. A total of 67 and 38 QTLs for seed oil and protein content were identified with an average confidence interval of 5.26 and 4.38 cM, which could explain up to 22.24% and 27.48% of the phenotypic variation, respectively. Thirty-eight associated genomic regions from BSA overlapped with and/or narrowed the SOC-QTLs, further confirming the QTL mapping results based on the high-density linkage map. Potential candidates related to acyl-lipid and seed storage underlying SOC and SPC, respectively, were identified and analyzed, among which six were checked and showed expression differences between the two parents during different embryonic developmental periods. A large primary carbohydrate pathway based on potential candidates underlying SOC- and SPC-QTLs, and interaction networks based on potential candidates underlying SOC-QTLs, was constructed to dissect the complex mechanism based on metabolic and gene regulatory features, respectively. Accurate QTL mapping and potential candidates identified based on high-density linkage map and BSA analyses provide new insights into the complex genetic mechanism of oil and protein accumulation in the seeds of rapeseed.

/pdf/genetic-dissection-of-seed-oil-and-protein-content-and-2vn92n33my.pdf

Genetic dissection of seed oil and protein content and identification of networks associated with oil content in Brassica napus.

This report describes an integrative analysis of seed-oil-content quantitative trait loci (QTL) in
 Brassica napus
 , using a high-density genetic map to align QTL among different populations.
 Rapeseed (Brassica napus) is an important source of edible oil and sustainable energy. Given the challenge involved in using only a few genes to substantially increase the oil content of rapeseed without affecting the fatty acid composition, exploitation of a greater number of genetic loci that regulate the oil content variation among rapeseed germplasm is of fundamental importance. In this study, we investigated variation in the seed-oil content among two related genetic populations of Brassica napus, the TN double-haploid population and its derivative reconstructed-F2 population. Each population was grown in multiple experiments under different environmental conditions. Mapping of quantitative trait loci (QTL) identified 41 QTL in the TN populations. Furthermore, of the 20 pairs of epistatic interaction loci detected, approximately one-third were located within the QTL intervals. The use of common markers on different genetic maps and the TN genetic map as a reference enabled us to project QTL from an additional three genetic populations onto the TN genetic map. In summary, we used the TN genetic map of the B. napus genome to identify 46 distinct QTL regions that control seed-oil content on 16 of the 19 linkage groups of B. napus. Of these, 18 were each detected in multiple populations. The present results are of value for ongoing efforts to breed rapeseed with high oil content, and alignment of the QTL makes an important contribution to the development of an integrative system for genetic studies of rapeseed.

Quantitative trait loci that control the oil content variation of rapeseed (Brassica napus L.)

Quantitative Trait Loci (QTL) for oil content has been previously analyzed in a SG-DH population from a cross between a Chinese cultivar and a European cultivar of Brassica napus. Eight QTL with additive and epistatic effects, and with environmental interactions were evaluated. Here we present an integrated linkage map of this population predominantly based on informative markers derived from Brassica sequences, including 249 orthologous A. thaliana genes, where nearly half (112) are acyl lipid metabolism related genes. Comparative genomic analysis between B. napus and A. thaliana revealed 33 colinearity regions. Each of the conserved A. thaliana segments is present two to six times in the B. napus genome. Approximately half of the mapped lipid-related orthologous gene loci (76/137) were assigned in these conserved colinearity regions. QTL analysis for seed oil content was performed using the new map and phenotypic data from 11 different field trials. Nine significant QTL were identified on linkage groups A1, A5, A7, A9, C2, C3, C6 and C8, together explaining 57.79% of the total phenotypic variation. A total of 14 lipid related candidate gene loci were located in the confidence intervals of six of these QTL, of which ten were assigned in the conserved colinearity regions and felled in the most frequently overlapped QTL intervals. The information obtained from this study demonstrates the potential role of the suggested candidate genes in rapeseed kernel oil accumulation.

Molecular mapping of Arabidopsis thaliana lipid-related orthologous genes in Brassica napus

Brassica napus is an important oilseed crop. Dissection of the genetic architecture underlying oil-related biological processes will greatly facilitates the genetic improvement of rapeseed. The differential gene expression during pod development offers a snapshot on the genes responsible for oil accumulation in. To identify candidate genes in the linkage peaks reported previously, we used RNA sequencing (RNA-Seq) technology to analyze the pod transcriptomes of German cultivar Sollux and Chinese inbred line Gaoyou. The RNA samples were collected for RNA-Seq at 5-7, 15-17 and 25-27 days after flowering (DAF). Bioinformatics analysis was performed to investigate differentially expressed genes (DEGs). Gene annotation analysis was integrated with QTL mapping and Brassica napus pod transcriptome profiling to detect potential candidate genes in oilseed. Four hundred sixty five and two thousand, one hundred fourteen candidate DEGs were identified, respectively, between two varieties at the same stages and across different periods of each variety. Then, 33 DEGs between Sollux and Gaoyou were identified as the candidate genes affecting seed oil content by combining those DEGs with the quantitative trait locus (QTL) mapping results, of which, one was found to be homologous to Arabidopsis thaliana lipid-related genes. Intervarietal DEGs of lipid pathways in QTL regions represent important candidate genes for oil-related traits. Integrated analysis of transcriptome profiling, QTL mapping and comparative genomics with other relative species leads to efficient identification of most plausible functional genes underlying oil-content related characters, offering valuable resources for bettering breeding program of Brassica napus. This study provided a comprehensive overview on the pod transcriptomes of two varieties with different oil-contents at the three developmental stages.

/pdf/transcriptome-analysis-of-brassica-napus-pod-using-rna-seq-1oi0ip10vo.pdf

Transcriptome analysis of Brassica napus pod using RNA-Seq and identification of lipid-related candidate genes

The lobed-leaf character is a clearly recognizable morphologic trait in rapeseed (Brassica napus L.) that has potential advantages in hybrid production. The present study was carried out to analyze the effect of lobed-leaf trait on agronomic traits and to fine map the LOBED-LEAF 1 (BnLL1) gene in B. napus. The lobed-leaf line Yuye 87 and entire-leaf cultivar Zheyou 50 were used to produce four F2 and eight BC1 populations for genetic analysis. Multiple comparison analysis between lobed-leaf character and agronomic traits was carried out in two F2 populations of reciprocal crosses between Yuye 87 and Zheyou 50. Results show that the lobed-leaf phenotype is controlled by a single an incomplete dominant gene and has no significant adverse effects on 10 main agronomic traits. The location of BnLL1 gene was narrowed down to a 36.7-kb homologous region of B. rapa between genes Bra009506 and Bra009511. On the basis of the annotation of orthologous genes in Arabidopsis, Bra009510 is considered to be a promising candidate for BnLL1 gene. The AT5G03790 is the orthologous gene of Bra009510 in Arabidopsis thaliana, with known functions in the formation of simple serrated leaves and in the suppression of bract formation. It is suggested that previously described lobed-leaf gene BnMOSAICLEAF (BnML) and the BnLL1 are the same genes controlling the lobed-leaf phenotype in B. napus, whereas the lobed-leaf gene BcLEAFLOBED 1 (BcLL1) is their homologue in Brassica campestris ssp. chinensis. These findings will facilitate the cloning and functional identification of the BnLL1 gene in future studies.

Genetic analysis and fine mapping of the LOBED-LEAF 1 (BnLL1) gene in rapeseed (Brassica napus L.)

Increasing seed oil content has become one of the most important breeding criteria in rapeseed (Brassica napus). However, oil content is a complex quantitative trait. QTL mapping in a double haploid population (SG population) emerging from a cross between a German (Sollux) and Chinese (Gaoyou) cultivars revealed one QTL for oil content on linkage group A1 (OilA1), which was mapped to a 17 cM genetic interval. To further validate and characterize the OilA1, we constructed a high-resolution map using B. rapa sequence resources and developed a set of near-isogenic lines (NILs) by employing a DH line SG-DH267 as donor and Chinese parent Gaoyou as recurrent background. The results showed highly conserved synteny order between B. rapa and B. napus within the linkage group A1 and revealed a possible centromere region between two markers ZAASA1-38 and NTP3 (2.5 cM). OilA1 was firstly validated by 250 BC5F2 plants and was confirmed in a 10.6 cM interval between the markers ZAASA1-47 and ZAASA1-77. Further substitution mapping was conducted by using two generations of QTL-NILs, 283 lines from eight BC5F3:4 families and 428 plants from six BC5F4 sub-NILs and thus narrowed the OilA1 interval to 6.9 cM and 4.3 cM (1.4 Mb), respectively. Field investigations with two replications using homozygous BC5F3:4 sister sub-NILs indicated that NILs, which carry a Sollux chromosome segment across the target region showed significant higher oil content (1.26 %, p < 0.001) than their sister NILs containing Gaoyou chromosome. The OilA1 locus is of particular interest for breeding purpose in China because 80 % of Chinese cultivars do not carry this desirable allele.

Jian-Yi Zhao

Papers

Quantitative trait loci that control the oil content variation of rapeseed (Brassica napus L.)

Molecular mapping of Arabidopsis thaliana lipid-related orthologous genes in Brassica napus

Transcriptome analysis of Brassica napus pod using RNA-Seq and identification of lipid-related candidate genes

Genetic analysis and fine mapping of the LOBED-LEAF 1 (BnLL1) gene in rapeseed (Brassica napus L.)

Characterization of the quantitative trait locus OilA1 for oil content in Brassica napus