Analysis Of Genetic Relationship And Identification Of S-Genotypes And Clone Of S-Rnase Gene In Pyrus

Pear（Pyrus） is a commercially important fruit crop worldwide and most come from China, which resoure is abundant. It’s necessary to classify and analyze the relationship between the different cultivars in heredity. The phylogenetic relationships of 56 Pyrus were analyzed by using SSR and SRAP markers.Self-incompatibility（SI） is a genetic mechanism employed by flowering plants to prevent inbreeding and promoting out-crossing, which involved a complex set of cell-cell interactions between the pistil and the pollen, thence, a model system for studying of intercellular information transmission, cell-cell recognition and gene spatial-temporal expression. Pear exhibits S-RNase-based gametophytic self-incompatiility, as other Rosaceae species do. Therefore, they will not set fruit unless pollinated with cultivars with different S-genotypes. Determination of correct pollen incompatibility groups and assignment of cultivars to the groups are essential for good crop production. Pollen incompatibility groups in pear have been identified by controlled pollination tests and pollen tube growth tests, but these tests is time-consuming and prone to be affected by environmental factors. The allele-specific PCR system was established to identify the S-genotype of 90 pear cultivars in this study.All results were summarized as follows:1. Simple sequence repeat（SSR） markers were used to assess relationship of 56 Pyrus cultivars. Atotal of 38 putative alleles were generated from six primer-pairs. All the SSR markers showed a high level of genetic polymorphism with a mean of 6.3 putative alleles per locus. Four groups were generated from all the accessions by UPGMA clusters analysis at Dice’s similarity coefficient of 0.71. The third group was classified into three subgroup;2. Factors influencing SRAP-PCR analysis were studied using three pear cultivars. A reliable, effective and reproductive PCR reaction system for detecting SRAP was developed and 72 primer-pairs were filtered. Each 20μL PCR reaction mixture consisted of template DNA 30 ng, 2.0 mmol·L^-1 of MgCl₂, 200μmol·L^-1 of dNTPs, 0.2μmol·L^-1 of primer and 1 unit of Taq polymerase;3. Further study on relationship of Pyrus cultivars native mainly to Eastern Asia had been carried out with sequence-related amplified polymorphism markers. The eleven primer could successfully differentiate all the materials and generated 188 fragments of which 184 fragments were polymorphic to 97.9%. Dendrogram analysis clusted 56 Pyrus cultivars into four group at Dice’s similarity coefficient of 0.662. The research supported the close relationship between Japanese pears and Chinese sand pears. All these results showed that SRAP markers were economic, effective, and reliable. Pear has widely genetic diversity;4. Classifying pear S-genotypes are useful for breeding programs and may help selection. Using primers（P1 and P2） based on the conserved regions C1 and C3 of pear S-RNase structure and specific primers（PS2） based on sequence of the pear S₃-RNase, DNA of ninety Chinese-bred pear cultivars were carried on PCR and PAGE. S-genotypes of these cultivars were identified by analysis of DNA sequencing. The results were confirmed by fruit set of some cultivars and microscopic observation of pollen tube. The S-genotypes of ninety cultivars were determined;5. Partial S-RNase genes of pear were amplified and cloned in seven cultivars of unknown S-geontypes. The PCR products were sequenced and blasted in GenBank. S₂₈-RNase, S₃₀-RNase, S₃₁-RNase, S₃₂-RNase, S₃₃-RNase, S₃₅-RNase, S₃₆-RNase and S₃₇-RNase were deposited in NCBI under the accession numbers of AY562394, AY876945, DQ124366, DQ124367, DQ138081, DQ323732, DQ417607, DQ448239.