Identification Of Original Relationship Of Varieties In Cucurbita Based On RAPD,SSR And ISSR

RAPD,SSR and ISSR were used to study genetic relationship and diversity of 36 cucurbita accessions.The results were as follows.1.Optimization of methods for genomic DNA extraction on cucurbita was carried out and an improved CTAB method was developed,giving rise to satifactory results in RAPD,SSR and ISSR analyses.2.Optimization of the amplification system of RAPD for cucurbita was performed.The optimal amplification system of RAPD was as follows:The total 20μL contained TaKaRa Taq enzyme 0.2μl,10×GC Bufferâ…¡2μl,2.5 mM Mg²⁺1μl,dNTP1μl,primer 1μl,template 2μl,ddH₂O12.8μl.The temperature profile used for PCR was:94℃for 10min,followed by 40 cycles of 94℃for 40s,annealing temperature for 40s,72℃for 90s,and was terminated with a 10min DNA extension step at 72℃.Amplification products were conserved at 4℃.3.With reference to related species of melon and cucumber,30 pairs of SSR primers for 36 pumpkin varieties SSR amplification were screened,only five pairs of primers detected differences,suggesting that the direct use of the primers for the same family was not appropriate.4.Efforts were made to explore the ISSR primer annealing temperature and the difference between Tm values,finding the best annealing temperature Tm values generally about 2-6℃.But there Were also more serious deviations of more than 10℃, in the primers with a combination of simple nucleotide repeat,for example(CAG) 5.5.Using RAPD,SSR and ISSR molecular markers for analyses of 36 varieties of pumpkin 187 primers amplified polymorphism and better repeatability of 45, co-amplification out of 183 fragments,of which 167 bands were polymorphic, accounting for 91.26%of polymorphism,indicating a rich genetic diversity.6.36 tested materials can be divided into 2 major groups:the main cultivars of domestic and imported varieties and their hybrids,respectively.Genetic distances between domestic varieties were small,indicating the close relationships;genetic distance between the imported varieties were also smaller,showing their close kinship relations; and genetic distances between domestic and foreign carieties were larger,demonstrating their more distant their relationship.7.Based on the recovered stronger specific bands with high brightness from ISSR analusis,cloning and sequencing were performed,and SSR primers were designed using Primer5.0 software.