Development Of EST-SSR Makers In Diospvros L.

Simple Sequence Repeats (SSR) is one of the most powerful molecular markers becauseof its reproducibility，multiallelic nature，codominant inheritance，relative abundance and fullgenome coverage. However，the development of SSR markers is quite time and labor-intensivewhich limits its applications. EST（Expressed Sequence Tag） has been developed withprogress of functional genomics. EST (Sequence tagged sites) derived SSR markers has manyintrinsic advantages with higher transferability among relmed species and can be developedeasily at a lower cost. With these advantages，using ESTs to derive SSR makers has become themost popular way to produce new makers.Diospyros spp. is one of the most important economic forest resources in China. The mostpopular species are D. kaki，D. oleifera，D. lotus，etc. Their fruits and ingredient are widly usedin food，medicine and other relevent industry to achieve sound ecological and economicbenefits. as a result, it is widely planted particularly in mountain areas with its low cost andhigh output. However, systematic analysis and research on genetic relationships amongDiospyros species, cultivars are rare. Development of EST-SSR primers is an urgent approachfor research on genetic diversity and economic evaluation for Diospyros spp. and its cultivars.In this study，9474ESTs of Diospyros L. in the database of NCBI were downloaded.1390contigs and4097singles of ESTs were assembled by removing redundants ESTs andthose with low qualities. In total，540SSRs was obtained in601ESTs，10.96%of ESTs weremined out，and61.06%was dinucleotides. Trinucleotides and tetracleotides were32.61%and6.33%, respectively.158pairs of makers were designed from these sequences，then they werechecked by blast online，and three pairs were removed which cound not be detected fromneither databases. Finally,100pairs with higher score were chosen.6Oriental persimmons（Diospyros kaki）were used to validate these makers. Thirty onepairs showed the amplification，accounting for31%of designed primers，and14pairs showed polymorphism，accounting for45.2%of primers available. Meanwhile，EST-SSR primers fromdinucleotides showed highest polymorphism(64.3%). The primers from the sequences withSSR length longer than20bp（64.3%）showed higher polymorphism.Then the same makers were used to analysis the9genotypes (Diospyros L.).Forty-two pairs showed the amplification, accounting for42%of designed primers，and30pairs showed polymorphism，accounting for71.4%of primers available.Meanwhile，EST-SSR primers from dinucleotides showed highest polymorphism(56.7%).The primers from the sequences with SSR length between14bp and20bp（56.7%）showedhigher polymorphism.After verification at both genus and species levels,35pairs finally showedpolymorphism. Nine of them could be used in two levels. Five of them were specificfor persimmons，and the rest21were specific for Diospyros L.. Polymorphismanalysis showed that the EST-SSR primers from dinucleotides showed highestpolymorphism（60%），next were primers from trinucleotide sequences（37.1%），only twoprimers from tetracleotide sequences were detected（5.7%）. The primers from thesequences with SSR length between14bp and20bp（54.3%）showed higher polymorphism thanthat with SSR length longer than20bp（45.7%）．...