Establishment Of EST-SSR, SRAP, ISSR And RAPD Reaction System And Studies On The Application Of Purity Identification In Bottle Gourd

Bottle Gourd(Lagenaria vulgaris Ser.) is an important vegetable with abundant nutritious ingredient and that is of great benefit to human health.The study on the hybrid identification of bottle gourd can benefit manage the seed market,conservation and classification of the bottle gourd germplasm.It is very important to protect intellectual property rights of new varieties and breeders' rights.In this study,the technique system of hybrids purity identification in bottle gourd were established via RAPD,EST-SSR,SRAP and ISSR four markers based on optimization on reaction system.The main results were as follows:1.Using the research results of other cucurbitaceous plants,EST-SSR,SRAP, RAPD and ISSR amplification programs were optimized to set up the reaction system of EST-SSR,SRAP,RAPD and ISSR fitting for the hybrid identification of bottle gourd with the orthogonal design and one-way methods.The optimal reaction systems and amplification programs for every markers as follows:(1) The optimal reaction of EST-SSR,the result showed that the optimum concentration of four important components i.e.TaqDNA polymerase,primer,dNTPs and Mg²⁺ in 10μl EST-SSR reaction system were 0.5U,0.1μmol/L,100μmol/L and 0.75mmol/L,respectively.(2) The optimal reaction of SRAP,the result showed that the optimum concentration of five important components i.e.TaqDNA polymerase,template DNA,primer,dNTPs and Mg²⁺ in 20μl SRAP reaction system were 1.25U,30ng,0.5μmol/L,100μmol/L, 2mmol/L,respectively.The amplification products were separated by 8%polyacrycamide gels at 120V.(3) The optimal reaction of ISSR,the result showed that the optimum concentration of five important components i.e.TaqDNA polymerase,template DNA,primer,dNTPs and Mg²⁺ in 20μl ISSR reaction system were 0.5U,40ng,0.75μmol/L,150μmol/L and 2mmol/L,respectively.The optimum reaction procedure was as follows:pre-denaturation at 94℃for 5rain;denaturation at 92℃for 30s;anneal at 58℃for 45s;extension at 72℃for.5min and in total 35 cycles;extension at 72℃for 7min;preservation at 4℃.The amplification products were separated by electrophoresis in a 1.8%agarose gel.(4) The optimal reaction of RAPD,the result showed that the optimum concentration of five important components i.e.TaqDNA polymerase,template DNA,primer,dNTPs and Mg²⁺ in 20μl RAPD reaction system were 1U,30ng,1.5ng/μl,150μmol/L and 1.75mmol/L,respectively.2.56 pairs of primer in melon,54 pairs of primer in cucumber and 10 pairs of primer in bitter gourd,all EST-SSR primers produced monomorphic bands among 3 hybrid seeds and their parents,but 2 primers pairs produced polymorphic bands among 3 hybrid seeds from our labs and 9 bottle gourd varieties from market.3.Among the 12 primer combinations tested,produced mono-morphic bands among 3 hybrid seeds and their parents.But 4 screened primer combinations produced 46 stable and reproducible bands,of which 20 bands(43.38%) were polymorphic among 3 hybrid seeds from our labs and 9 bottle gourd varieties from market.This 4 primer combinations could distinguished all varieties from market,but 2^# varieties from market can't distinguished between 3 hybrid seeds from our labs.4.A total of 24 primers were used in this study.All primers produced monomorphic bands pattern for all varieties.5.9 primers were screened from 186 the ones that could be beneficial to the purity identification among the parents and their hybrid seeds in bottle gourd.2 primers of partial female type and 2 primers of shortage type showed polymorphic on 06-2×06-6;3 primers of partial male type and 2 primers of shortage type showed polymorphic on 06-1×06-5;1 primers of partial male type and 4 primer of partial female type showed polymorphic on 06-4×06-5.6.In conclusion,RAPD and SRAP markers maybe useful for hybrid identification in bottle gourd.Utilization advantages of RAPD and SRAP markers,improved the reliable results and feasibility of markers using hybrid identification in bottle gourd.