| SSRs are ideal DNA molecular markers for genetic diversity study among plant germlasm resoures, in this study,40 EST-SSR primers were developed from NCBI EST database.25 markers were operated on genetic diversity and genetic structure study, and constructed the core set of 289 cultivated Wickham clones. the results were as follows:11809 ESTs were assembled into 3932 unigenes,in which 3923 ESTs includes 358 SSRs, about 9.1% unigenes included SSR.93 kinds SSR repeats were identified, 57.5% of SSR-ESTs were dinucleotide repeats,28.8% trinucleotide,12.8% pentanucleotide. the repeats motifs GA/CTã€CT/GAã€AG/TC and TC/AG were the most abundant.Based on the flanking sequences of these 3923 ESTs,158 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed.60 SSRs (38%) were successfully amplified and 40 (66.7%) of them showed polymorphism in 48 germplasms including 31 Wickhanm germplasms,12 wild germplasms,2 H.nitida,2 H. spruceana and 1 H. pauciflora.The results of genetic diversity study showed that there were 103 alleles at the 25 loci investigated. The average number of alleles per locus was 4.12. The average value of polymorphism information content(PIC) and the except heterozygosity (He) were 0.3665 and 0.4106, respectively. The average value of Nei’s genetic diversity index (H) was 0.4098. we can draw a conclusion that the EST-SSR alleles amplified by the 25 primer pairs had moderate level of polymorphism. In addition, According to the results of SPSS software package, the level of genetic diversity among different populations were not significant.This clustering analysis result showed that, in most case, some clones with common parents were not observed to cluster together, only a few descendants which with common ancestors were clustered in the same category and coincided with the pedigree analysis. the average value of genetic identity(GI) between pairewise poputions was 0.9103,and the value of genetic distance(GD) among accessions ranged from 0 to 1.1632, it is clearly indicated that close genetic relationship and small genetic difference among Heava rubber clones.Using the information of 25 EST-SSR markers, the Hardy-Weinberg Equilibrium analyses showed that there are 16 locus in China popution and 12 locus in Malaysia popution were not consistent with linkage disequilibrium. Only a few locus in Indonesia, Sri Lanka, Thailand and Brazil population were not consistent with linkage disequilibrium. Locus in India, X and Vietnam population were consistent with linkage disequilibrium or monomorphism.The analysis of molecular variance (AMOVA) indicated that most of the genetic variations were found among accessions within the population, while interpopulatiot variance component accounted for 2.67% only. Similar to the results of F-statistics, Gst analysis. The average value of gene flow (Nm) among populations was 4.463. Indicating that high and smooth gene flow was the main reason leading to very low genetic differentiation level of six Hevea brasiliensis populations. And the impact of human selection and the plant breeding system were the important reasons. Different living conditions are the reason leading to slight genetic differentiation among populations.All of these 27 coreset were fingerprinied withth esameset of 25 EST-SSR markers, the results showed that the corset captured 100% alleles and rare alleles of the 289 cultivated clones, and there existed no significant differences between the coreset and the 289 cultivated Wickham clones genetic diversity(p>0.05). Phylogentic analysis of all the 27 coreset based on 25 EST-SSRs rather norrow genetic variation among these coreset. We were do our best to contain the coreset as many as qualified characteristics and widely cover the type of ecological distribution.That in stead of 289 cultivated clones, the coreset could be thoroughly used in the inprovement of rubber breeding in China, as well as mining of the favorable alleles contained in these germplasm an other related suceeeding researches on rubber breeding. |
