QTL Mapping And Candidate Gene Analysis For The Fatty Acidcomponents In Soybean Seeds

Soybean oil is the main source of the plant oils worldwide. Due to its contribution to human and plant defense systems, it has attracted broad attention. The comprehensive effect of genetic and environmental factors on fatty acid, therefore, QTL mapping and candidate gene cloning for major fatty acid components are more important on the molecular marker-assisted breeding for soybean fatty acid. Based on the qualitative and quantitative determination method for fatty acid components and molecular marker technology, we constructed a high density genetic linkage map by SSR and SNP markers using a LHD2 × NHZ F5: 7-8-RIL population. The QTLs underlying fatty acid components were also identified and analyzed across multiple environments. According to the bioinformatics analysis, the candidate genes involved in fatty acid biosynthesis and accumulation were discovered among these loci. The candidate genes associated with fatty acid were cloned according to the reference genome information, and their sequence alignments were analyzed to find the sequence differences between both parents. Moreover, their linkage analyses were also conducted using the dCAPs primers in the RIL population. Additionally, the spatial and temporal expression of candidate genes during soybean seed development stage was also detected to discover their relationship with the fatty acid accumulation by the real-time quantitative PCR method. The main results were as follows:1. Qualitative and quantitative determination method for soybean fatty acid compositions by gas chromatographyIn this study, the method of methyl heating extraction and gas chromatographic analysis were adopted. With five fatty acid methyl ester(methyl palmitate, methyl stearate, methyl oleate, methyl linoleic acid and linolenic acid methyl ester) as the standard samples, qualitative quantitative detection method of soybean fatty acid compositions was established on the base of the standard curves(R2 > 0.99) of fatty acid methyl ester and the regression equation. This method can qualitatively and quantitatively detect the absolute content of fatty acid composition accurately in soybean seeds. Through the comparative analysis for fatty acid and crude fat content in different soybean varieties, we found that this method can significantly improve the efficiency of extraction yield of fatty acid in the grain and the detected the content of total fatty acid were more than 94% of the total fat content. Besides, the absolute content of the fatty acid composition in grains can be accurately calculated with this method. It is great significant for the detection and the breeding of soybean fatty acids.2. QTL mapping for soybean fatty acid components based on SSR and SNP methodsBased on a genetic linkage map and fatty acid phenotypic data in three environments, 52 additive QTLs underlying fatty acid components on 19 LGs except chromosome 4 were identified with ICIM method of ICIMpping V3.3 software, explaining 5%-40% of the phenotypic variation. Among them, 15 QTLs were the novel loci. In addition, 17 QTLs were detected in multi-environment and multi-components with phenotypic contribution of 5%-24%.In order to achieve the fine mapping of soybean fatty acid, a high density genetic linkage map was established using the SLAF-seq method. This map consisted of 5,785 SNP markers on 20 LGs and spanned 2,255.18 cM in genomic size with an average distance of 0.39 cM between adjacent markers. 24 additive QTLs on 12 LGs were identified by ICIM method explaining phenotypic contribution of 7%-31%. Of them, three QTLs(qS-FA12-1, qS-OA16-1 and qS-LNA20-1) were detected in multiple environments associated with the same fatty acid composition and two QTLs(qS-FA7-1 and qS-FA12-2) were detected under the same environment for different fatty acid component. A QTL qLNA6-1 for LNA from the mapping based on SSR marker showed the stable performance across three environments. Therefore, it can be taken as the major QTL with the PVE up to 28.76%. Based on the fine mapping, in this interval of qLNA6-1 there were four QTLs detected, i.e. q S-LA6-1 for linoleic acid, qS-LNA6-1 for linolenic acid, qS-OA6-1 for stearic acid and qS-PA6-1 for palmitic acid. It provided the foundation for the candidate gene cloning to regulate fatty acid components in the future.3. Candidate gene cloning and sequence alignment for soybean fatty acid componentsTen candidate genes in the key loci were cloned and sequenced in both parents. We identified two transcription factors, i.e. a DOF transcription factor Gm08DOF-1 and a MYB transcription factor Gm20MYB-1, with one InDel and one SNP mutants between both parents. The sequence alignment results showed that an InDel(GGT) missing in Gm08DOF-1 caused an absence of Proline in the parent of NHZ; a SNP(T/C) mutant in Gm20MYB-1 caused the change of amino acid from Phenylalanine in LHD2 to Leucine in NHZ. The genotypes were identified using two primers and the associations between two loci and the fatty acid components were analyzed in RIL population. The results showed that the four kinds of fatty acid components were associated with both loci, except stearic acid.4. The spatial and temporal expression analysis of candidate genes for fatty acid componentsThe real-time PCR was used to detect the expression profiles of Gm08DOF-1, Gm20MYB-1 and the genes encoding key enzymes in the fatty acid biosynthesis pathway. The relative content of saturated fatty acids decreased obviously in the seed development of 50- 60 days. However, the expression levels of Gm08DOF-1and Gm20MYB-1 were increased gradually. The two genes may play the major role in the process of saturated fatty acids desaturation. Otherwise, we also analyzed the expression levels of the key enzyme genes in the fatty acid biosynthesis pathway. The expression levels of FAD3 B and FAD2 C were highly significant positive correlation with the two saturated fatty acid components and highly significant negative correlation with linoleic acid in NHZ.