Optimization Of Start Codon Targeted Polymorphism (SCoT) System And Identification Of Korla Fragrant Pear Excellent Clones

Korla fragrant pear native to korla area, and the cultivation of time is very long.With the advantages of good quality, thin skin, flesh crisp, juice abundant, sweet taste,strong fragrance, most resistant to storage. Due to the good quality of korla fragrantpear, it has been exported to domestic and overseas in recent years. It has become oneof the main economic crops in Korla area. With the development of molecular biologytechnology, it has become to a trend of using molecular techniques assisted breedingand identification of new species.In this paper, the excellent clones with the advantage of leaving calyx, high yield,big fruit which selected at the Korla fragrant pear plantation in second division ofXinjiang provience from2006to2009and Korla fragrant pear were used as thematerials. We used the SCoT molecular marker, amplified and clone sequencing,found out the basic group diffirence between tetraploid type, excellent clones withtraditional type materials, and authenticated the excellent clones, then provide thetechnical support for the Korla fragrant pear cultivars‘research. The mainly job werefound the best DNA extraction methods suited for Korla fragrant pear‘s SCoTmolecular marker; established and optimized SCoT-PCR reaction system withorthogonal design method, and used the system selected the primers. Tree of selectedprimers had been used to amplify24materials, cloning sequencs. The sequencingresults were analyzed by appling of biological analysis softwares. The results asfollows:1. DNA secure Plant Kit and improved SDS were suited to DNA extraction,though DNA secure Plant Kit was best for SCoT-Analysis of Korla Fragrant Pear.2. The optimized PCR cocktail of20μL contained10×buffer with Mg2+2.5μL，1.5U Taq polymerase，10μmol·L-1primer，25ng template DNA， and1.6mmol·L-1dNTPs. The optimized reaction procedure was：94℃for1min，followed by35cycles of94℃for1min，52℃annealing temperature for45s，72℃for1min，and was terminated with a8min extension step at72℃, holded at16℃.3. Randomly selected2primers from20primers for amolificating24materials.Analysed the sequencing results by using DNAMAN、Editseq、ORF Finder andNCBI-BLAST-tblastx, the results showed that the sequence had the high homologywith related gene of sand pear, golder delicious, strawberry， flowring peach， winegrape。4. Twenty-four materials were identified with the combination of SCoT primer S8and S12. The results demonstrating that molecular markers of SCoT is able to beused for identification the bud mutation of pears genus excellent clones.