Mapping QTL Conferring Salt Tolerance At Seedling Stage And Cloning Of Candidate Genes In Soybean

Soil salinization and secondary salinization is an important factor affecting crop yields. At least 20% of the world’s arable land is salt-affected.In China, soil salinization is more and more serious, which becomes one of the main environmental factors restricting China’s agricultural development. Soybean is an important economic crop, and its production and quality are significantly constrained by salt stress. Therefore, breeding salt-tolerant varieties is very important for improving soybean production in China.In this study, a RIL population (NJRIKY) containing 427 lines was used for QTL analysis of salinity tolerance traits at soybean seedling stage. A high-density genetic map was used, which has 4737 bin markers developed by whole genome re-sequencing technology. Two salt tolerance candidate genes, GmRPL23 and GmAKR, were cloned and analyzed using bioinfomatic tools. The results are as follows:1. The phenotyping was conducted in the plant growht room under the following conditions:14-h of day length at 29℃ temprature, and 10-h of night at 23 ℃ temprature. Twelve plants of each RIL family were grown in hydroponic solutions until seedling stage, when two groups with six plants per group for each RIL family were transferred to 0 and 150mM NaCl solutions, respectively. After 6 days, the root length and dry weight of each plant under two conditions were measured. The whole experiment was repeated three times. Four traits were collected including root length under 0 and 150mM NaCl treatments (CKRL and STRL), the relative root length (RRL= STRL/CKRL) and relative dry weight (RDW= STDW/CKDW) were calculated respectively. The results show that heritability of CKRL, CKDW, RRL and RDW are 64.4%,86.2%; 58.7%,78.4%, respectively.2.1n this study, we used RRL and RDW as indicators of soybean salt tolerance and performed QTL analysis by QTL Network 2.1. We detected 16 salt tolerance additive QTL loci on chromosome 1,2,4,5,6,7,9,10,11,12,15,16 and 18, which explained 25.8% of total genetic variation, and the confidence intervals ranged from 1.1 cM to 2.6 cM. In addition, we detected 4 pairs of epistatic QTLs for RRL, and these epistatic QTLs were also detected as additive QTL loci. Comparing with previous QTL studies, we found the QTL on chromosome 18 (linkage group G) (qrrl18-1) is likely to be the same QTL with the locus (qpsdGl) found by Chen Huatao on linkage group G, with much narrower confidence interval in this study (1.7cM comparing with the previous interval of 21.5cM). Six out of seven salt tolerant QTL identified in this study harbored good candidate genes for salt tolerance (such as peroxidase, MYB, bZIP, NAC-like protein, etc.). The predicted functions of these salt tolerance genes largely support the mechanism of salt tolerance. By rating RRL、 RDW and their mean as salt tolerant indicators, we selected eight salt-tolerant RIL families, including WKY-133, WKY-166, WKY-348, WKY-351/WKY-397, WKY-465, WKY-481 and WKY-513.3. Previous studies have mapped a salt tolerant QTL on chromosome 3 between 40344151 to 41800718 bp. Combined with RNA-seq analysis of Nannong 1138-2 and Kefeng No.1 under different salt tolerance (Provided by Dr. Chen Shouyi from Chinese Academy of Science), we selected the region of 38-40Mb on chromosome 3 to screen for salt tolerance candidate genes. We predicted 81 genes within this region, among which 51 genes have SNP/Indel differences between Nannong 1138-2 and Kefeng No.l. By functional annotation and semi-quantitatively PCR experiments, we initially selected 6 genes as salt-tolerant candidate genes. We isolated mRNA from the roots, stems and leaves of Nannong 1138-2, and analyzed the gene expression of these 6 genes by RT-PCR.2 genes expressed in soybean roots, stems and leaves. Under 150mM NaCl treatment, the expression levels of them in roots and leaves were strongly induced. The expression level of one in the roots and leaves of Nannong 1138-2 increased after 12 hours of salt treatment, but is constantly expressed in the stems. The expression level of the other on in roots increased 6 hours after salt treatment, while its expression in leaves increased 24 hours after salt treatment. These results suggested these two genes might be related to salt tolerance. Named these 2 genes according to the function of them, one gene encodes a 60S ribosomal protein (Ribosomal protein L23), which is usually involved in protein synthesis, named GmRPL23. The other gene encodes an ankyrin repeat-containing protein(Ankyrin Repeat-Containing), OsAKR.5 gene was reported associated with stress tolerance, named GmAKR.