Cloning The New S Genes, Identify The S-genotype And AFLP Analysis Of Some Pear Cultivars

The plant self-incompatibility, a universal biological phenomenon in angiosperm, is an important protective mechanism of preventing close breeding and species retrogression which was formed during the long-term evolution. As a kind of rosaceae plants, Pear is a typical fruit tree of gametophytic self-incompatibility (GSI). Because of its self-incompatibility, it has a low fruiting set of self-pollination and it is necessary to arrange varieties with different S-genotype or to perform artificial pollination to ensure a stable and high yield. In this article, some cultivars'S-genotype was identified and 5 new S genes were isolated and identified. It will provide a theoretical reference for studying the self-incompatibility and for parent's selection when hybridization is carried out. At the same time,30 cultivars such as aiganshuili were studied by fluorescent AFLP. A fingerprinting has been constructed. Relationship and inheritance of the studied cultivars have been analyzed on molecular level. The purpose is to provide basis for pear classification and theoretical references for hybridization parents. Mainly study results are listed as following.1. Identification the S-genotype. After using the method of PCR-RFLP, sequence and genetic relationship analysis etc., some cultivars S-genotype were identified. Such as: baozhuli(S22S33),bingtangli(S19S31),donghuangli(S20S34),huangli(S22S34),jinqiuli(S3S9),kuerleli(S22S34),lvbaoshili(S4S35),daguoshuijingli(S3S9) matihuangli(S17S19),maogongli(S12S13),ningmenghuangli(S31S32),xinyali(S4S17),zaosuli(S22S35),xizilvli(S1S4),huanghuali(S1S2),huangjinli(S3S4),qingxiangli(S4S7),xuefengli(S4S16),xuefangli(S4S16),xueyingli(S3SX),xuefenli(S3SX)。2. Isolation and identification of new S gene.5 new S-RNase genes were isolated from the cultivars such as donghuangli after PCR and sequence analysis. The isolated new S-RNase genes were S31-RNase(DQ072113), S32-RNase(DQ072114), S33-RNase(DQ082897), S34-RNase(DQ224345) and S35-RNase(DQ224344). The fragment of S31-RNase contains 346 nucleotides; the exon and intron of it contain 201 and 145 nucleotides respectively. The fragment of S32-RNase contains 999 nucleotides; the exon and intron of it contain 213 and 786 nucleotides respectively. The fragment of S33-RNase contains 532 nucleotides; the exon and intron of it contain 195 and 337 nucleotides respectively. The fragment of S34-RNase contains 428 nucleotides; the exon and intron of it contain 201 and 227 nucleotides respectively. There are 2 different nucleotides and only one different deduced amino acid between S28-RNase and S34-RNase. There are 9 different nucleotides and no different deduced amino acid between S8-RNase and S34-RNase. We concluded that S8-RNase, S28-RNase and S34-RNase are the same one. The fragment of S35-RNase contains 371 nucleotides; the exon and intron of it contain 204 and 167 nucleotides respectively.3. The full length cDNA of S35-RNase (genebank accession number:DQ224344) were obtained from the cultivars of Zaosu pear after using RACE technique. The analytical result suggested that S35-RNase contains 681 nucleotides, codes 229 amino acids. Furthermore, there are 8 conservative'Cys'residues forming 4 disulfide bonds,2 conservative'His'residues influencing the S-RNase activity, and 1 conservative 'Asn-Xaa-Ser/Thr'motif within RC4, which plays an important role in the folding of core structure. To study the expression characteristic of S35-RNase, the technique of northern blotting was employed and discovered that it is highly expressed in the pistil of flowers at the stage of big ball and only expressed in pistil.4. AFLP fingerprinting analysis of 30 pear cultivars. AFLP fingerprinting technique were used to study the relationship of 30 cultivars such as aiganshuili et al. there were 28 cultivars have more than 1 characteristic bands, which were 93% among all of the cultivars. All of the cultivars were clustered into 4 groups. The cultivars of the 1st group are the Japanese pear or have genetic relationship with Japanese pear. The cultivars of the 2nd group are mainly the cultivars of Chinese pear. Huangguanli and qingxiangli were clustered to the 3rd group; these two cultivars have a common parent of shinseiki. The 4th group has only 1 cultivar of kangdeli. One of its parents is Pyrus communis.