Construction And Screening Of SSR-enriched Genomic Library In Persimmon

Persimmon (Diospyros Kaki Thunb) is Chinese native, and, is one of the most popular cultivated fruit crops in China, it ranks first in the world in both acreage and production. The National Persimmon Germplasm Repository located in Northwest University of Agriculture and Forestry. More than 560 accessions were collected and preserved. Among all these materials, only some non-astringent cultivars from Japan were obtained by cross breeding, which pedigree and relative was clear, others' background of inheritance was unknown. In order to investagate, research and utilize these germplasm from profound level, it must be found a powerful tool to evaluate these germplasm. Microsatellites, known as single sequence repeats (SSRs) as markers are highly polymorphic, highly abundant, highly repeating and co-dominant inheritant and are one of the most important molecular markers. But the disadvatage of using SSR markers is that SSR-PCR specific primers is necessary. So far, there is no SSR markers or specific primers in persimmon has been published, and no SSR markers in related species or in same family can be tried and tested. So it is needed to develop SSR primers by our own, however, constructing SSR-enriched small-insert libraries is the base of rapid develping SSR primers.In this study, SSR-enriched small-insert libraries in persimmon have been constructed firstly. A simple and efficient method and system were developed for isolating microsatellite DNA markers from persimmon genome. The methods are: the persimmon genomic DNA was converted into pre-amplified AFLP fragments and hybridized with bio-labeled SSR probes, Then the hybrid mixture was used to incubate with magnetic beads coated with streptavidin for isolation of hybridized complex. After washing to remove the non-SSR fragments, the eluted taget single-strand DNA, which was ligated into pGEM-T Easy Vector system and transform into E.coli competent cells for cloning, was largely enriched for microsatellites. After premarily screening by white/blue phynotype of colonies, white positive colonies were used to construct SSR-enriched libraries. Use Colony PCR technique to screen the library. Positive clones were sequenced and analysised. The main results are:1. Modified CTAB method for persimmon genomic DNA isolation has been set up, genomic DNA isolated by this protocal is complete and pure enough for next step use. The concentration of the DNA is diluted to 100ng/μl. The molecular weight is about 21.23kb.2. Set up the systems of genomic DNA digested by EcoRI/MseI Enzymes and AFLP pre-amplifacation for persimmon genome.3. Establishment of the hybridization system for AFLP fragments with bio-labled SSR probes and isolation of the hybrid complex by magnetic beads coated with streptavidin.4. SSR-enriched small-insert DNA libraries in persimmon have been constructed firstly, and screened by colony PCR techniques. The positive clones cover about 60% after sequencing. It show that constructed libraries is highly SSR-enriched and it is efficient enough for developing SSR marker in persimmon.