Construction of a high-density genetic map based on large-scale markers developed by specific length amplified fragment sequencing (SLAF-seq) and its application to QTL analysis for isoflavone content in Glycine max

Background: Quantitative trait locus (QTL) mapping is an efficient approach to discover the genetic architecture underlying complex quantitative traits. However, the low density of molecular markers in genetic maps has limited the efficiency and accuracy of QTL mapping. In this study, specific length amplified fragment sequencing (SLAF-seq), a new high-throughput strategy for large-scale SNP discovery and genotyping based on next generation sequencing (NGS), was employed to construct a high-density soybean genetic map using recombinant inbred lines (RILs, Luheidou2 x Nanhuizao, F-5:8). With this map, the consistent QTLs for isoflavone content across various environments were identified. Results: In total, 23 Gb of data containing 87,604,858 pair-end reads were obtained. The average coverage for each SLAF marker was 11.20-fold for the female parent, 12.51-fold for the male parent, and an average of 3.98-fold for individual RILs. Among the 116,216 high-quality SLAFs obtained, 9,948 were polymorphic. The final map consisted of 5,785 SLAFs on 20 linkage groups (LGs) and spanned 2,255.18 cM in genome size with an average distance of 0.43 cM between adjacent markers. Comparative genomic analysis revealed a relatively high collinearity of 20 LGs with the soybean reference genome. Based on this map, 41 QTLs were identified that contributed to the isoflavone content. The high efficiency and accuracy of this map were evidenced by the discovery of genes encoding isoflavone biosynthetic enzymes within these loci. Moreover, 11 of these 41 QTLs (including six novel loci) were associated with isoflavone content across multiple environments. One of them, qIF20-2, contributed to a majority of isoflavone components across various environments and explained a high amount of phenotypic variance (8.7% - 35.3%). This represents a novel major QTL underlying isoflavone content across various environments in soybean. Conclusions: Herein, we reported a high-density genetic map for soybean. This map exhibited high resolution and accuracy. It will facilitate the identification of genes and QTLs underlying essential agronomic traits in soybean. The novel major QTL for isoflavone content is useful not only for further study on the genetic basis of isoflavone accumulation, but also for marker-assisted selection (MAS) in soybean breeding in the future.