ISSR Analysis Of Pear Resource And Research On Flower Bud Dormancy Release

Pear is a deciduous tree, belongs to Rosaceae, Maloideae,Pyus L. It has a cultivate history for more than3000years. As chinese traditional famous flower, pear flower not only have beautiful shape, simple and elegant smell, but also have deep culture accumulation. All this trait endowed pear flower high ornamental value. However, before burgeon, pear flower bud need a time to pass low temperature dormancy. The southern low-chilling and early-ripe sand pear have character of early-flower and eary-ripe, can occupy the gap of northern market, and have enormous economic benefits. So, it make great sense to study on pear flower resource and flower bud dormancy releasing.Two parts were expanded in this reseach.In one hand, we established an ISSR-PCR reaction system which suit for the pear flower resource of southern subtropics, obtained the relationship between genetic cluster and low-chilling hour on38pear flower resources. On the other hand, we use different periods of flower bud of Huanghua pear(Pyrus pyrifolia var. Huanghua) as experimental material. Huanghua pear is a main cultivar in FuJian province,which have midium-chilling hour. Paraffin method was used to make sure materials are in the correct period and its representation, then established a suppression subtractive Hybridization library. By sequencing, we find some related diversity genes, use NCBI-BLAST to analysis the homologous, use on line sorftware Blast2go, WEGO to make sequence be GO noted and classified. Then find out the possible related genes of dormancy release.It also provide some theory foundation to pear flower bud dormancy release. Concrete results are as follows:1. Pear leaves are rich in tannins, polysaccharied and polyphenol, we optimized the DNA extraction method. Established an ISSR-PCR reaction system which suit for the pear flower resource of southern subtropics. The reaction system is as fellows:The final total volume was20μL, contains2μL10xPCR buffer(Mg2+free),60ng DNA template,0.5U Taq DNA polymerase,1μmol/L of primer concentration,90μmol/L of dNTP concentration,2.25mmol/L of Mg2+concentration.In order to find out the relationship between low-chilling hour and clustering result,clustering analysis of38speices of pear based on ISSR was done.19specific primers were picked out among96ISSR primers,111products amplified and77.5%of them were specific. The genetic similarity was between0.69to0.97, the testing species were divided into western pear and oriental pear. Oriental pear include dou pear, sand pear, zhongbao pear and so on. In most of the sand pear, low-chilling character and its diversity is related to each other according to clustering analysis. So as to provied criteria for the resesrch on low-chilling of pear flower bud in southern subtropics.2. Sampling different dormancy release periods of huanghua flower bud as material. To ensure the buds in corresponging phase, Paraffin method was used to inspect the morphological and anatomical structure. We established a forward suppression subtractive Hybridization library(PZ) by using dormancy released bud as "Tester", under dormancy bud as "Driver" established a reverse suppression subtractive Hybridization library(PF) by using under dormancy bud as "Tester", dormancy released bud as "Driver". In each total inversion product,10μL transformation product of forward suppression subtractive Hybridization library(PZ) can grow378colonies, contains41blue colonies, recombination rate was89%, each microlite transformation efficiency of ligation product can grow28,350colonies; meanwhile,10μL transformation product of reverse suppression subtractive Hybridization library(PF) can grow326colonies, contains31blue colonies, recombination rate was90%, each microlite transformation efficiency of ligation product can grow24,450colonies. The results of colony PCR showed that both library(PZ,PF) have insert fragments size distribute between500bp to2000bp, the avarage size was750bp. We picked150colonies in both forward and reverse suppression subtractive Hybridization library(PZ,PF) randomly.123colonies(82%) was sequencing out successfully in PZ library,127colonies(84%) was sequencing out successfully in PF library. By NCBI-BLAST, some bud dormancy release related genes was found, transcription factor like IAA, MYB1, BZIP, WRKY, dehydrin, protein kinase, structural protein, AP2domain class transcription factor, NADH dehydrogenase, calcium binding protein, most of them are full length sequence gene. By "Blast2go" software noting "GO", all the protein encoded can be classified into three categories by "WEGO", as fellows:Cellular Componen, Molecular Function, Biological Process. This research take the advantage of SMART, single-strand hybridization, DSN, Nest PCR, and other biotechnology to establishe a full length and single strand suppression subtractive Hybridization library about the possible related genes of dormancy release of pear flower bud. It provide some theory foundation to related genes of pear flower bud dormancy release.