Stable, Environmental Specific and Novel QTL Identification as Well as Genetic Dissection of Fatty Acid Metabolism in Brassica napus

Abstract

Fatty acid (FA) composition is the typical quantitative trait in oil seed crops, of which study is not only closely related to oil content, but is also more critical for the quality improvement of seed oil. The double haploid (DH) population named KN with a high density SNP linkage map was applied for quantitative trait loci (QTL) analysis of FA composition in this study. A total of 406 identified QTL were detected for eight FA components with an average confidence interval (CI) of 2.92 cM, the explained phenotypic variation (PV) value ranged from 1.49 to 45.05%. Totally, 204 consensus and 91 unique QTL were further obtained via meta-analysis method for the purpose of detecting multiple environment expressed and pleiotropic QTL, respectively. Of which, 74 stable expressed and 22 environmental specific QTL were also revealed, respectively. In order to make clear the genetic mechanism of FA metabolism at individual QTL level, conditional QTL analysis was also conducted and more than two thousand conditional QTL which could not be detected under the unconditional mapping were detected, which indicated the complex interrelationship of the QTL controlling FA content in rapeseed. Through comparative genomic analysis and homologous gene annotation, 61 candidates related to acyl lipid metabolism were identified underlying the CI of FA QTL. To further visualize the genetic mechanism of FA metabolism, an intuitive and meticulous network about acyl lipid metabolism was constructed and some closely related candidates were positioned. This study provided a more accurate localization for stable and pleiotropic QTL, and a deeper dissection of the molecular regulatory mechanism of FA metabolism in rapeseed.

Keywords: Brassica napus; QTL mapping; acyl lipid metabolism; fatty acid; genetic dissection.

Figure 1

Distribution of FA concentrations of KN DH population in multiple environments. The unit of x-axis represents the percentage of each FA composition in the sum of all FAs. The unit of y-axis represents the number of lines. K represents the male parent of “KenC-8” and N represents the female parent of “N53-2” of the KN-DH population.

Figure 2

Distribution of QTL for each FA and oil content in each linkage group. The entire graph consists of 16 circles. From the inside to outside, the first 14 circles represent the 14 breeding microenvironments, it contains seven winter type of 08DL to 14YL, two spring type of 10GS and 11GS and five semi-winter type of 11WH to 11 HG, identified QTL for the nine traits located in each circle and indicated by different colors. The fifteenth circle with blue background represents the region includes consensus QTL for each trait and the gray spokes highlight the region of unique QTL. The outermost circle represents the 19 linkage groups of B. napus and they also distinguished by different colors. The abbreviation of PA, SA, OA, LA, ALA, EIA, EA, FAS, and OC represents the traits of C16:0, C18:0, C18:1, C18:2, C18:3, C20:1, C22:1, FAS, and oil content, respectively.

Figure 3

qPCR analysis for the five acyl lipid metabolism related candidates. The x-axis indicates the three different developmental stages of seed and y-axis indicates the mRNA expression level of each candidate gene. The green represents the material contains low C18:1 concentration and the purple represents the material contains high concentration of C18:1. Early indicates that the materials were collected at the stage of 15 days after flowering, middle indicates the stage of 30 days after flowering and late indicates the stage of 45 days after flowering. The expression of each gene were calculated based on three biological replicates. Bar represent the standard deviation calculated by ΔΔ Ct method. **Significant at p < 0.01, *** Significant at p < 0.001, n.s. No significance p > 0.05.

Figure 4

Gene interaction network analysis associated with the acyl lipid metabolism related candidates underlying the CI of unique QTL. The visualized interaction network of 54 candidates was constructed by String software and exhibited by Cytoscape V-3.5.0 software. The five different regions represent different metabolic pathway of acyl lipid metabolism. Nodes represent the potential candidates and edges represent the interaction of them. Node size represents “Degree” and edge size represents “Combined-score,” and the color for nodes and edges represents the “Betweenness certrality” and “Edge Betweenness”, respectively. All the value for these four parameters was calculated by Network Analyzer that included in Cytoscape V-3.5.0 software.

Figure 5

Potential regulatory network and some candidate genes associated with acyl lipid metabolism in B. napus. All the candidate genes used in the network originated from the CI of unique QTL of the seven FA compositions. Different colors with the genes indicated the candidates located in different consensus QTL associated with different breeding environment. The dashed line indicates that multiple reactions in this step might exist.