Isolation And Identification Of Self-Incompatible Genes Of Chinese Pyrus Bretschneideri

The plant self-incompatibility, a universal phenomenon during reproductive period, provides the pledge for survival and evolution of species, and relative independency of population by a cross-pollination mechanism that prevents close breeding and species retrogression. Pear, a cosmopolitan fruit tree, belongs to the kind of gametophytic self-incompatibility (GSI). So it is necessary to arrange varieties for pollination or to perform artificial pollination for high and stable yield during the planting of pear. When arranging varieties for pollination, the compatibility of varieties should be considered above all. This compatibility of varieties lies on the S-genotypes of two varieties: compatible for the same S-genotypes, and incompatible for the different. Bretschnider pear (Pyrus bretschnider) is a pear genealogy holding the maximum number of improved variety as a primary planting genealogy of pear in China. At present the researches on incompatible genes and S-genotypes of bretschnider pear still do not appear in papers internal. In this paper, the research on isolation and identification of S-genes from bretschnider pear, and determination of S-genotypes of pear varieties in molecular biology and hybrid compatibility analyses was performed. The findings of this research would provide an important scientific foundation for fertile planting and genetical improvement of pear, and a theoretical elements and practical instruction for relative researches on self-incompatibility of other pear varieties and fruit trees. The key results of this research are following as:Determination of S-alleles and S-genotypes of part varieties of bretschnider pear by PCR-RFLP system. The PCR on genomic DNA of 64 varieties of bretschnider pear were performed using special primers 'FTQQYQ' and 'IIWPNV of pear S1-8-RNase. The result showed that 1 -3 special DNA fragments were obtained from each variety. The result from PCR-RFLP analyses displayed that twelve varieties (Xinxiang, Yali, Dayali, Pingguo, Zhaoxiandayali, Baoshansu, Tianyali, Haitangsu, Jinanxiaohuangli, Sumei, Enli, Jinli) contain alleles of S1-RNase. Specially, variety Jinli should be the homozygote of S1RNase for its two alleles of Si-RNase. Varieties Jinfeng, Xinliyihao, and Donghuang contain an allele of S8-RNase. Varieties Kuerlexiangli, Xiangchun, and Huangli contain an allele of S3-RNase. Varieties Dongguo and Donghuang contain an allele of S7-RNase. None of alleles of S2-RNase, S4-RNase, S5-RNase, S6-RNase, and S9-RNase were found among all 64 varieties.Identification of 7 new genes of S-RNase and determination of S-genotypes of15 varieties by DNA sequencing and bioinformatics analysis. The result from sequence analyses of DNA fragments from PCR on 27 S-genes of 15 varieties showed that the isolated DNA fragments display high homology (similarity of 80-100 %) with those of S1-16-RNase. However, 7 DNA fragments among them display difference with those of S1-16-RNase, and were identified as new S-genes named Sn-RNase, S19-RNase, S20-RNase, S21-RNase, S22-RNase, S26-RNase, S27-RNase (GenBank access numbers: AY249432, AY250987, AY250988, AY250989, AY250990, AY339396, AY339397). The DNA bp numbers of the sum of signal peptide, P1, C2, C2, HV, introns, and P2 of each new S-gene are 439, 542, 458, 433, 442, 441, and 449 respectively. The DNA bp numbers of introns of each new S-gene are 142, 251, 167, 139, 148, 144, and 152 respectively. The lengths of putative amino acids of each new S-gene are 99, 96, 99, 98, 98, 99, and 99 respectively. The lengths of corresponding HV regions are 16, 13, 16, 15, 15, 15, and 15 respectively. The residue lengths of signal peptide, conservative region 1, conservative region 2, and HV region are 27, 11, 10, and 12-16 respectively. The isolated fragments from new S-genes contain signal peptide, conservative region 1, conservative region 2, introns, HV region related to the special recognition function of S-RNase, three cysteines related to the structure of S-RNase, and a histidine related to the activity of RNase. Signal peptide, Cl, an...