Mapping Salt Tolerance QTLs In Soybean And Functional Analysis Of A Novel Soybean Salt Tolerance Gene GmSKC1

Salinization and second salinization of soil are important factors affected agricultural production and ecological environment. To date, there are twenty percentage Cultivated Land affected by salinization and second salinization. The trend of Second salinization of soil is more serious in arid and semi-arid regions. Salinity affects 4 ×107 ha of agriculture land which limited agricultural development in china. Breeding salt-tolerant varieties are one of the effective ways to overcome the affect of agricultural production by soil salinization; therefore, enhancing the salt-tolerant capacity of crop has important practical significance for the expansion of arable land and improving agricultural production.Soybean [Glycine max （L.） Merr.] is one of the most important sources of vegetable protein and edible oil world-widely. As soybean varieties are rich and have a great difference of salt tolerance among them, we can cultivate salt tolerance soybean cultivars with pyramiding good traits. The purpose of this study are:1, SSR markers were used to map QTLs conferring tolerance to salt stress in a population of recombinant inbred lines （RILs） derived from a cross between Nannong1138-2 （salt tolerant） and Kefeng No.1 （salt sensitive）. We also examined the stability of QTL detected in two environments; field and greenhouse.2, cloning and functional analysis of a novel soybean salt tolerance gene GmSKCl.1、A RIL population was used to map salt tolerance QTL using SSR markers in soybean under salt stress. Under controlled conditions （greenhouse experiment）, plant survival days （PSD） and percentage of plant survival （PPS） responsive to salt stress were measured. Six QTLs were detected on six linkage groups, explaining 7.8% to 19.2% of the total phenotypic variation. In the field, visual salt tolerance ratings of the RILs ranged from 0 to 5. Three QTLs were identified on two linkage groups, explaining 7.1% to 19.7% of the total phenotypic variation. Seven QTLs related to the salt tolerance were identified in this study which have not previously been reported. The QTL qpsdG.l （greenhouse experiment） and qtrG.l （field experiment） mapped to the same genomic region （between markers Sat₁64 and Sat₃58） on linkage group G, suggesting a major QTL related to salt tolerance located on linkage group G. qppsK.1 and qpsdK.1 were also mapped on the same genomic region on linkage group K, suggesting that maybe another important salt tolerance QTL on linkage group K. A QTL （qppsN.l） associated with marker Satt237 on linkage group N was co-located with previously report. A 100-seed weight （SW） QTL （qswB2.1） was co-located with a salt tolerance QTL（qppsB2.1）, so the QTL （qppsB2.1） may be important to increase 100-seed weight of soybean under salt stress.2、A novel soybean gene encoding a HKT-like transporter （GmSKCl） was identified and function analyzed in transgenic plant. GmSKCl containing an open reading frame （ORF） of 1260 nucleotides. The ORF encoded a protein of 419 amino acids, with a potential molecular mass of 47.06 kDa and a predicted pI value of 8.59. Comparison of the genomic and cDNA sequences of GmSKCl identified no intron. The deduced amino acid sequence of GmSKCl was 38-49% identity to other plant HKT-like sequences. RT-PCR analysis showed that the expressions of GmSKCl were upregulated by salt stress （150 mM NaCl） in the root and leave but not in the stem. Subcellular localization of GmSKCl showed that was typically localized on the plasma membrane of the onion epidermal cell. We constructed a molecular phylogenetic tree of soybean GmSKCl and other fifteen HKT-type proteins from various organisms. Phylogenetic tree of publicly available full-length HKT coding sequences show that the gene family splits into three major branches. AtHKT1, GmSKC1, McHKT2, SmHKT1, SsHKTl, EcHKTl and EcHKT2 were classified in the first subfamily.To investigate the role of GmSKCl in K+ and Na+ transport, we compared K+ and Na+ accumulation in the root and shoot of WT tobacco and transgenic tobacco plants. Without salt stress, K+ and Na+ contents in transgenic tobacco plants shoots and roots were not substantially different from those in WT tobacco plants. Several days after treatment with 200mM NaCl, however, transgenic tobacco plants shoots and roots had a higher K+ content and lower Na+ content than non-transgenic plants （WT）. These results suggest that GmSKCl is a Na+ transport which affects Na+ transport in roots and shoots, and regulating the Na+/K+ balance in the roots and shoots. We found that overexpression of GmSKCl enhanced transgenic tobacco plants tolerance to salt stress, as compared with non-transgenic plants. Our findings suggest that GmSKC1 plays an important role response to salt stress and is useful in engineering crop plants with enhanced tolerance to salt stress.We constructed RNAi interference vector （pB7GWIWG2 （Ⅱ）-GmSKCl） and transformed soybean; Over-expression vector pMDC83-GmSKCl was also transformed soybean. Transformation of cultivar Nannong88-31 by infecting cotyledonary-node with A. tumefaciens strain EHA105. As a result, two RNAi regeneration soybean plants and four overexpression regeneration soybean plants harvested seeds.The novel soybean salt tolerant gene GmSKC1 was transformed into CAPS marker and integrated into the genetic map of NJRIKY RIL population using software Mapmaker/Exp3.0. As a result, GmSKC1 was mapped in linkage group D1a+Q. As the genome of soybean has been sequenced recently, we BLAST the sequence of GmSKC1 to soybean genome database and located chromosome Gm01; then two SSR markers （Sat₂01 and Satt267） associated with GmSKC1 on the genetic map of NJRIKY RIL population BLAST to soybean genome database and also located chromosome Gm01. Together above results, the novel salt tolerant gene GmSKC1 mapped in linkage group D1a+Q is accurate. Therefore, GmSKCl gene was transformed into a molecular marker （CAPS） and can be used for marker-assisted selection （MAS） breeding.3、 Salt tolerance identification of 405 M3 mutagenesis materials during seedling stage in pod which induced by 0.4% EMS from the control Nannong94-16. As a result, the salt tolerance phenotypes of two mutagenesis materials （165 and 405） were found better than Nannong94-16. SSR markers （619 pairs of SSR primers） were used to screen genomic difference among mutagenesis materials （165 and 405） and the control Nannong94-16. As a result, there were six SSR markers showed difference among mutagenesis materials （165 and 405） and the control Nannong94-16, therefore, using SSR markers detected difference in DNA level was 0.97 percent,99.03 percent for the consistency. The statistics result showed that genomic difference was very small among mutagenesis materials （165 and 405） and the control Nannong94-16. In addition to the agronomic trait of 100-seed weight, the other agronomic traits had no difference or no significant difference. Therefore, mutagenesis materials 165 and 405 were confirmed salt tolerant mutants from control Nannong94-16. Moreover, six different SSR markers are located among five linkage groups that showed the effectiveness and randomness of artificial mutagenesis. We compared locations of six different SSR markers with salt tolerance QTLs mapped previously in this study. The result showed that the different SSR marker Satt725 was associated with the salt tolerance QTL（qpsdK.1）, and the distance between markers Satt710 and Satt725 is 5.9 cM on linkage group K.