Hybrid Identification with SSR Markers,Analysis of Salt Tolerance, Cloning and Expression of GMSSOS1in Glycine Max,Glycine Soja and Their Hybrid
|School||Nanjing Agricultural College|
|Keywords||Glycine max Glycine soja SSR markers Agronomic traits Salttolerance SOS1gene Real-time fluorescent quantitative PCR|
In this study, the Glycine max cultivars Lee68, Nannong1138-2, Nannong8831were used as the female parents, and the hybrid strains(F5)4076,4111,3060and185selected for salt tolerance generation by generation from the cross combination of Glycine max and Glycine soja were used as the male parents,11backcrosses or three-way crosses (A-K)were designed and213single hybrids were harvested. The orthogonal experimental design was conducted to optimize the soybean SSR-PCR system, with which to analyze the SSR polymorphism of the above parents and to get the parental complementary SSR markers for the hybrid identification. Then the salt tolerance of the identified true hybrids of C(Lee68×3060), G(nannong1138-2×3060) cross combinations and their parents was evaluated with parameters including the salt tolerance coefficient, relative growth rate, dry matter accumulation, thiobarbituric acid reactive substances (TBARS) contents, relative electrolytic leakage and Pn. In addition, the agronomic traits (such as the plant height, branch No., node No. of stem, pod No. per plant, seed No. per plant, seed weight per plant,100-seed weight) of the partial experimental materials were analyzed and compared. Finally, using the Glyeine max N23674cultivar (the salt sensitive), Glyeine soja BB52population (the salt tolerant) and their hybrids trains (4076), the cloning of full-length ORF (open reading frame) of soybean plasma membrane Na+/H+antiporter gene (GmsSOS1), and its bioinformatics analysis and expression at mRNA level under NaCl stress were investigated. The results showed that:The effective polymorphic SSR markers could be found among the7parents, and30true hybrids were gained from213single hybrids by the parental complementary markers. Under salt stress, higher salt tolerance coefficient, relative growth rate and dry matter accumulation were displayed in salt-stressed seedlings of the true hybrid C2, C10, C14of C cross combination, and G3; G9strains of G cross combination, when compared with their female parent Nannong1138-2, Lee68and even be equal to their male parent3060. The TBARS contents and relative electrolytic leakage significantly increased in leaves of Lee68(C cross combination female parent), a slight increase was found in the other materials. TBARS contents significantly increased in roots of male parent3060strain, a slight increase was found in the other materials, all the changes were between the parents. The TBARS contents in leaves and roots of Nannong1138-2(G combination female parent) were significantly higher than those of other materials tested in either control or salt treatment, when compared with the control, the rise was greater than the other hybrid strains under salt treatment. The net photosynthetic rate (Pn) decline was also significantly lower than their parents. In addition, most of the agronomic traits (such as the plant height, branch No., node No. of stem, pod No. per plant, weight per100seeds) were between their parents. The above-mentioned provides not only a new effective method for hybrid identification between Glycine max and Glycine soja using SSR markers together with simple plant physiological parameters, but also the excellent materials for breeding new salt tolerant soybean germplasms.A plasma membrane Na+/H+antiporter gene (named as GmsSOSl, GenBank accession No. JQ287499) was isolated by RT-PCR and RACE in the Glycine max N23674cultivar, Glycine soja BB52population and their hybrid trains4076. The ORF sequences of GmsSOS1were exactly the same when amplified from the above three soybean materials. The GmsSOS1ORF has a length of3432bp encoding a protein of1143amino acids. By gene structure analysis it contained23exons and22introns, and by multiple sequence alignment analysis it showed72%,72%,69%,70%,69%and69%identities in amino acids to the plasma membrane Na+/H+antiporter genes from Vitis vinifera, Populus euphratica, Lycopersicon esculentum, Zygophyllum xanthoxylun, Thellungiella halophila, Arabidopsis thaliana, respectively. Moreover, it showed12transmembrane domains of GmsSOS1by TMHMM prediction. It indicates that monocots and dicots may exist opposite sex in the evolution by phylogenetic analysis. The results of real-time quantitative fluorescent PCR showed that GmsSOSl gene could maintained a certain lower expression in N23674BB52and their hybrid seedlings under non-salt stress, but the150mM NaCl treatment could significantly enhance the expression in the tested three seedlings, and this effect could be induced with the processing time extension, and reached the maximum expression after24h treatment. The expression in roots was higher than those in leaves. For the three materials tested, there was different response pattern of GmsSOSl at the mRNA level in the process of24h salt treatment, the most rapid response displayed in BB52populations might be associated with its stronger salt tolerance, the most slow response in N23674cultivar might be related to its weaker salt tolerance; and the response of their hybrid4076strain was between the two parents.