S-genotyping Fifteen Pear Cultivars And Cloning Two Full Length CDNAs Encoding S₂₂-RNase And S₄₂-RNase

Pear is a typical kind of gametophytic self-incompatibility plant.Because of the low fruiting during the natural pollination, it is necessary to arrange varieties for pollination to increase the yield. 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. In this paper, the research on isolation and identification of S-genes from fifteen Chinese pear cultivars, and determination of S-genotypes of pear varieties in molecular biology and The findings of this research would provide an important scientific foundation for fertile planting and genetical improvement of pear. The key results of this research are following as:1. Using a pair of primers specific to pear S-RNases, PCR amplification from genomic DNAs of fifteen Chinese pear cultivars were carried out. Then the PCR products were extracted, cloned and sequenced. The amplified fragments were assigned to their respective S-RNases by Blast analysis.The S-genotypes of the fifteen tested self-incompatible cultivars were identified as follows:Lanzhou huachangba(S19S22),Zhangyechangba(S19S22), Guidechangba(S19S22),Longxiang(S16Sx),Qingmian(S1S18),Huangmian(S1S12),Zaomi(S19S29) ,Damianhuang(S1S19), Wuzihuang(S28S16),Daqingpi(S34S19),Jinchuizi(S16S19),Banxinsu(S5S21 ),Shuidonggua(S15Sy),Yunnanbaozhu(S22Sx)and Mandingue(S4S15).2.545-RNase was isolated from Shuidonggua pear. The number of S45-RNase amino acid in signal peptide, HV region, conserved region 1 (C1) and conserved region 2 (C2) was 27, 15,12 and 11.There is an amino acid substitutions in conserved region 1 of S45. S45 shows the highest similar to S26 in the amino acid sequences. But there are five different five amino acids in the hyper variable regions (HV) between S45 and S26.The S-genotype of Shuidonggua pear was identified as S15S45.3. S42-RNase were isolated from Longxiang pear and Yunnan Baozhu pear.S42 shows the highest similar to SaucS27. Furthermore, a 1044bp sequence of S-RNase gene was isolated by PCR from Longxiang pear with a pair of primers designed basing on SaucS27.which suggests the full length genome DNA of S42-RNase.The Sequence was spliced by two exons, the deleted intron was 335bp. The S-genotypes of Longxiang pear and Yunnan Baozhu pear were identified as S16S42 and S22S42.4. The full length cDNA of S22-RNase and S42-RNase were obtained from Yunan Baozhu pear with RT-PCR and RACE technique.The analytical result suggests that S22-RNase has 681bp encoding 227 amino acid and S42-RNase has 678bp encoding 226 amino acid. Furthermore, there are both 8 conservative 'Cys' residues forming 4 disulfide bonds,2 conservative'His'residues influencing the S-RNase activity in S22-RNase and S42-RNase.