Molecular Markers Analysis Of Suaeda Salsa Populations In Coastal Region

Suaeda salsa,belonging to Suaeda of Chenopodiaceae, is an annual herb, and is a kind of typical halophyte which has prominent salinity resistance. S.salsa can restore environments with high salinity in intertidal zone. They are widely used in industrial,medical,ecological areas.Researches about S.salsa on stresses physiology,molecular and genetic projects,ecological abilities and usability developments have made many advances.Whereas the conservative and using work of S.salsa is lack of the supporting of the theoretical guidance on genetic diversity and genetic variation and structure research. In this study, random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers were used to investigate the genetic structure of eight populations of S.salsa from four provinces.This study obtained the conclusion including:1.improved PCR reaction system and program for RAPD and ISSR:RAPD-PCR reactions were conducted in a total volume of 20μl containing 30-50ngDNA,1.0μmol/L primers,2μl 10×Taq buffer(100mmol/L Tris-HCl PH8.4, 500mmol/L KCl,15mmol/L MgCl2,1mg/ml BSA),0.2mmol/LdNTPs, 1U Taq polymerase Amplification reactions were carried out in a Master Thermal Cycler The profile consisted of an initial predenaturation for 5 min at 94℃, followed by 45 cycles to denature at 94℃,1 min; anneal at 36℃,1 min; extend at 72℃,1.5 min, and a final extention of 10 min at 72℃.ISSR-PCR reactions were conducted in a total volume of 20μl containing about 50ng DNA,0.5μmol/L primers,2μl 10×Taq buffer (100mmol/L Tris-HCl PH8.4,500mmol/L KCl,1 mg/ml BSA),2.0mmol/L MgCl2,0.2mmol/L dNTPs,1U Taq polymerase. Amplification reactions were carried out in a Master Thermal Cycler. The profile consisted of an initial predenaturation for 5 min at 94℃, followed by 35 cycles to denature at 94℃,1 min; anneal at 50-55℃, 45 sec; extend at 72℃,2 min, and a final extention of 5 min at 72℃.2.Used RAPD and ISSR methods to evaluate the genetic diversities of different S.salsa populations:In my study, a moderate level of genetic diversities within each population was revealed by three different indicators (P, H and I) whether using RAPD or ISSR analyses. In RAPD analysis the mean value of them are 63%,0.1933. 0.2915;In ISSR analysis the mean value of them are 59%.0.1718.0.2540.The two methods got similar conclusions,and revealed the moderate or high level of 8 S. salsa populations'genetic diversities.3.Used AMOVA analysis to demonstrate the genetic variation and structure of different S.salsa populations:the mean Fst values for the eight S.salsa populations were, by 0.3985,RAPD and 0.5436 by ISSR respectively, which indicated that most of the total genetic variances (39.85% or 54.36%) were on inter population level. Values of Fst exceeding 0.15 mean high genetic structuring, here the Fst indicated a high level of genetic differentiation and suggested a low level of gene flow among the S.salsa populations. The restricted gene flow (genetic isolation)might result from large geographical distances which limited long distance dispersal of pollens or drifting reproductive fragments. For S.salsa, we believe that long distance dispersals of pollens or seeds of S.salsa is rare among the sample sites, and the large spacing would be large enough to form geographical barrier for gene flow among the four S. salsa populations of different provinces.4.Utilized Mantel test manifested the correlation between the genetic diversities and geographic distance:In RAPD analysis,the relevance of genetic distance and geographic distance is (r=0.617),indicated highly related; In ISSR analysis, the correlation of genetic distance and geographic distance is (r=0.460) manifested moderate related. The result indicated that the genetic diversity of 8 different S.salsa population is correlated with geographical distance, the population far from each other had the big genetic distance,the nearer ones obtained the smaller genetic diversities. This conclusion accorded with the genetic variation and structure study, it manifest that geographic distance cause the low level of gene flowing and high level genetic variation.