Figure 3. An SSR based linkage map of 22 QTLs for seed isoflavone content in soybean. Note: marker names are labeled at the right side of each linkage group, and distance between makers labeled at the left side, the isoflavones-related QTLs are indicated by colorful labels..
Five loci associated with daidzin content were found in 4 linkage groups. The QTL qDaidzin-8-1, was positioned between Sat_406 and Sat_409 on the chromosome 8 (A2) linkage group, with a LOD value of 3.19 and accounted for 1.65% of the phenotypic variation explained (PVE). The additive effect value for this QTL was -0.34, with the negative value indicating that the allele associated with increased daidzin content derived from the low-isoflavone male parent Jinong 18. Four other QTLs with positive additive effects were located on chromosome 2 (D1b), chromosome 6 (C2) and chromosome 16 (J) were identified for daidzin explaining 1.65-2.06% of PVE with LOD scores ranging from 3.71 to 4.67 (Table 2).
For glycitin content, six loci were dispersed on 6 linkage groups. QTLs qGlycitin-7-1, qGlycitin-11-1, qGlycitin-16-1 and qGlycitin-17-1 had PVEs ranging from 0.35% to 1.76%, and LOD values ranging from 2.98 to 7.82 and had additive effects with negative values, indicating that the increased glycitin content originated from Jinong 18. Loci qGlycitin-2-1 and qGlycitin-3-1 located on chromosome 2 (D1b) and chromosome 3 (N), with PVE of 1.76% and 1.86% and additive effects of 0.05 and 0.18 (Table 2).
Genistin content was associated with seven loci in 5 linkage groups. The QTL qGenistin-9-1, found on chromosome 9 (K) linkage group between Satt260 and Sat_243, with a genetic distance from Sat_243 of 5.12 cM, had a PVE of 0.79% and an additive effect of -0.0033. However, the remaining QTL associated with genistin, which were located in chromosomes 8 (A2), 11 (B1), 14 (B2) and 19 (L), with PVEs ranging from 0.79-0.91% and LOD scores ranging from 24.43- 37.30 (Table 2), all had positive additive effect values, indicating the associated increases in genistein were derived from the female Jinong 17.
Four loci associated total isoflavone content were located in 4 linkage groups. The QTL qIsoflavone-3-1 was detected on chromo-some 3 (N) between Satt009 and Satt624, 3.0 cM from Satt009, with a LOD values of 11.41, and it had additive effect of -0.14. However, the remining three QTLs had positive additive effects values. These QTL, qIsoflavone-9-1, qIsoflavone-11-1, qIsoflavone-19-1, were located on chromosomes 9 (K), 11 (B1) and 19 (L), and had PVEs from 0.96% to 1.65% with LOD scores ranging from 10.51-11.17 (Table 2).
2.4. Identification of candidate genes in flavone or isoflavone-related QTL regions
According to the annotation from SoyBase database (https://www.soybase.org), fifty-eight candidate genes were identified (Table S1). Among them, many genes were annotated as transcript factors and enzymes. Interestingly, twenty of the candidate genes are involved in phenylpropanoid metabolism, which is a metabolic pathway associated with flavonoid metabolism and isoflavonoid metabolism [34](Table 3). Of the twenty genes, seven genes relates to lactase Laccase 2,3,5,6,7 and Laccase 15/TT10 were identified here whne compared with A. thaliana , and several studies have shown that laccase can regulate the synthesis of phenylpropanoid and lignin [35-37], as phenylpropanoid, lignin also associated with synthesis of flavonoid and isoflavonid metabolism [38]. Meanwhile, several transcript factors of A. thaliana homolog, e.g. bHLH42 , MYB20 and TTG1 , to directly regulate flavones or isoflavones synthesis in A. thaliana [39,44,52]. These results imply the accuracy of the presemted QTL mapping here. Finally, the RNA-Seq atlas of soybean different tissues the was obtained from Phytozome [40,41] to use select candidate genes. The expression dynamic variation of all these identified genes expressed in the seed were differed than other tissues (Figure 4). We found that nine of the fifty-six genes with expression data were highly expressed in seeds, Glyma.02G076300 ,Glyma.02G147800 , Glyma.06G136900 , Glyma.08G062000 ,Glyma.08G062100 , Glyma.09G020300 , Glyma.16G158400 ,Glyma.17G156000 and Glyma.19G102000 , respectively. Some genes were also expressed in seeds, although their expression was not higher than in other tissues, such as Glyma.02G125100 ,Glyma.06G16500 and Glyma.11G189100 . As we expected, some of phenylpronoid-related genes were also identified as seed-high or seed-available expression genes. The parental lines of the mapping population are not the same as the soybean used in the public RNA-seq altas, and the differences in expression of a gene among tissues are releavent candidate genes with expression in the seeds.
Table 3. Phenylpronoid-related candidate genes associated with QTL for soybean seed isoflavone content.