Optimization Of Pear Self-incompatibility Gene CDNA Microarray Hybridization Assay And Its Application In Identifying S-genotypes Of Pear Cultivars

Pear(Pyrus.spp in subfamily Maloidae of Rosaceae)displays typical gametophytic self-incompatibility(GSI).In order to dispose suitable pollination cultivars in peargrowing and chose reasonable parent in pear breeding,the project of identification of s-genotypes of cultivars&mining and evolutionary analysis of S gene resources in sand pear based on gene chip has been systematically researched.The main results and novel achievement are as follows:(1)S-RNase cDNA microarray were developed.Based on the structure and sequence characteristics of S genes in Oriental pear cultivars,using the specific cDNA probes with PF1-PR1 cDNA sequence which contains hypervariable region of S-RNase,the cDNA microarray was prepared.it comes out that the S-RNase genchip has guaranteed the accuracy and reliability of S-RNase rapid detection using S-RNase genchip detection system.(2)Optimization of S-RNases cDNA microarray hybridization condition.The optimized hybridization condition are the optimized temperature 42? the optimum hybridization time 8?9 h,the optimum purified PCR product 200 ng ?l-1 and the washing conditions which is washing 3 min in solution of 2× SSC + 0.1%SDS?0.1×SSC + 0.1%SDS?0.1×SSC respectively,then washing 2?3 times(at most 10s per times)in distilled water,and finally drying the slide by Slide drying centrifuge.(3)Determination of S-genotypes of 50 pear cultivars with difference germplasm resources using optimized S-RNase genchip detection system.50 pear cultivars have been detected successfully.Simultaneously new S-RNases of cultivars could be inferred according to the hybridization signal.(4)Isolation and identification of 7 new S-alleles from Pyrus Pyrifolia.The 7 new S-alleles PpS53(P.PyrifoliaS53)?PpS54?PpS55?PpS56?PpS57?PpS58?PpS59were identified from pear cultivars by auxiliary enzyme digestion and DNA sequencing and were registered in GenBank(5)Analysis of 7 new S-RNases resources molecular evolution.The relationship of S-RNase sequence polymorphism and molecular evolution was systematically researched.the kind and distribution,the evolution pattern of Pyrus Pyrifolia S-RNases and the evolutionary relationship between Pyrus Pyrifolia S-RNases and other plant S-RNases in Rosaceae were determined.(6)Modification of pear S-RNase identification technology system.Based on the establishment of S-RNases cDNA microarray detection system,the S-RNase identification and the research of S-RNase resources technology system was modified accordingly.Therefore,the research has established scientific foundation for pear high yield cultivation and breeding.