Selection Of DNA Markers In Salt-tolerant Alfalfa By Using RAPD

Salt-tolerant alfalfa is adapted to grow in salt soil, and is a good germplasm for animal husbandry in salt soil area. The selection of primers on molecular markers of salt-tolerant alfalfa was conducted with salt-tolerant and sensetive alfalfa varieties by using RAPD in order to decide the position of salt-tolerant gene of alfalfa and provide genetic basis for dtermining sequerence and molecular breeding on alfalfa.To optimalize RAPD reaction system is the prerequisite of improving stability of amplified products. Three alfalfa varieties were used as the materials. They were the Zhong Mu No l,Wei Nan, Da xi yang alfalfa.The step experiences to the concentration of MgCl2,dNTPrandom primer,template DNA,Taq DNA polymerase and buffer on RAPD were conduced.Then a stable reaction system of RAPD was set up.In the optimalization of RAPD system with total volume of 25 u 1 including MgCl2 at 2.7mmol/L,dNTP at 150 w l/L,random primer at 0.192mol/L,TaqDNA polymerase at 2U/25 u l,buffer at 50mmol/L and 120~240ng/l template DNA Therm program is : predenature 94,3min.then 94,15 sec,36,30 sec,72, 1 min,45 circles,and 72, 12min.Above RAPD reaction system was used in selecting 240 lines primer of twelve groups.The salt-tolerant alfalfa and salt-sensitive alfalfa were used in this study.After five turns, we selected out 56 lines of primer in eleven groups (OPA,OPB,OPC,OPD,OPE,OPF,OPJ,OPH,OPG,OPN,OPX) that have produced stable polymorphic DNA fragments.In all of them,the stable degree of OPD-5 primer was the best and it made the DNA groups of Zhong Mu No 1,AZ90NDCST,AZ-SALT-II three salt-tolerant alfalfa varieties produce stable 3000 bp polymorphic DNA fragmentFurthermore, there was the band of DNA in some single plants of cross-breed FI of the Zhong Mu No 1 crossing Daxi Yang and Zhong Mu No 1 crossing Wei Nan .The seven alfalfa varieties showed obvious salt-tolerant or salt-sensitive traits on farm. Thus,we believe that if the 3000 bp polymorphic DNA band is the molecular marker of salt-tolerant trait for alfalfa, the OPD-5 is the best primer of marking salt-tolerant gene of Alfalfa.