Integrated BSA-Seq and RNA-Seq reveals genes associated with salt tolerance at the germination stage in foxtail...

Salt stress has a significantly negative impact on growth, development, and yield of foxtail millet. Identifying and utilizing genes involved in salt tolerance for breeding is one of the effective ways to address this issue. However, no quantitative trait loci (QTLs) or genes have been identified in foxtail millet using a forward genetics approach. Therefore, it is essential to mine salt tolerance QTLs and candidate genes to further clarify the genetic basis of this complex trait in foxtail millet.

In this study, an F_8:9 recombinant inbred line (RIL) population derived from a cross between Hongmiaozhangu (HMZG, salt-tolerant) and Changnong 35 (CN35, salt-sensitive) was utilized to identify salt tolerance QTLs at the germination stage via bulk segregant analysis sequencing (BSA-seq) using 20 extreme salt-tolerant and 20 extreme salt-sensitive RILs. Two QTLs related to relative germination potential (RGP) under salt stress, qRGP6-1 (19,520,000-20,590,000) and qRGP6-2 (20,610,000-20,900,000), were detected on chromosome 6 (Chr. 6) by ∆SNP-index/∆InDel-index and Euclidean Distance (ED) algorithms. In the mapping region, 46 candidate genes were identified. Two of these genes, Seita.6G119500 and Seita.6G119700, were differentially expressed between the parental lines under salt stress, as revealed by RNA-sequencing (RNA-seq) and validated by quantitative real-time PCR (qRT-PCR). Among them, Seita.6G119500 encodes a BURP domain-containing protein that has been reported to be involved in abiotic stress responses. Furthermore, a G/A SNP was detected between HMZG and CN35, leading to a nonsynonymous amino acid substitution from arginine (R) to histidine (H) in the salt-sensitive parent CN35. Association analysis using the Kompetitive Allele-Specific PCR (KASP) marker developed from the SNP variation within Seita.6G119500 revealed that the marker genotype was associated with RGP across the RIL population.

This study identifies a candidate gene Seita.6G119500 associated with salt tolerance in foxtail millet and provides a molecular marker for breeding salt-tolerant cultivars via marker-assisted selection (MAS).