Determination Of Genotype Of 15 Main Cultivars And Isolation Of S₂₉-RNase Gene From Chinese Bretschnider Pear (Pyrus Bretschnider)

Self-incompatibility, a common biological phenomenon in angiosperm, is an important protective mechanism of preventing close breeding and species retrogression that formed during long period evolution. Pear, an important fruit tree in China, belongs to the kind of gametophytic self-incompatibility (GS1). At present to arrange varieties for pollination or to perform artificial pollination is mainly adept to overcome SI for high and stable yield during the planting of pear. Brelschnider pear (Pyrus brelschnider) is a primary planting genealogy of pear in China. The researches on self-incompatible genes and S-genotypes of bretschnider pear are still in initial stage, 8 new S genes were isolated and determined and almost 20 cultivars' genotypes were determined in Bretschnider pear (Pyrus bretschnider) until 2003. In this paper, the researches on identification of S-genes and determination of S-genotypes of pear varieties are performed from 15 pear cultivars of bretschnider pear. The main results of this research are following as:The PCR on genomic DNA of 15 varieties of bretschnider pear were performed using special primers 'FTQQYQ'(Pl) or '5 ' -32bp'(P3) and 'IIWPNV'(P2) of pear S1-8-RNase, 1-2 special DNA fragments were obtained from each variety. The genotype of 11 varieties and one S-allelic gene of 4 varieties of Bretschnider pear are determined by PCR-RFLP system, DNA sequencing, and bioinformatics analysis. The genotypes of Dahebai, Yinzhiqing, Enli, Tianshengfu, Jinfeng, Donghuang, Dayali, Mili, Zisu, Yaoxianyinli, and Qoubai are S16S19, S16S19, S7S19, S15S19, S17S19, S12S12,S1S19, S29S12, S16S8, S20S8, S1S1S21S21, S29SX, S19SX, S2lSX, S19SX.A new S-gene, named S29, is isolated and determined from two varieties (Mili and Tianshengfu) by sequencing. The GeneBank accession number is AY601098. The isolated DNA fragments between PI and P2 display high homology (similarity of 79-99 %) with those of S1-16-RNase.and new determined S-genes in bretschnider pear, and the similarity between it's putative amino sequence and S1-28-RNase is up to 62-97%. The DNA bp numbers of the sum of P3, signal peptide, P1, C2, C2, HV, introns, and P2 of the new S-gene is 472. The DNA bp numbers of introns of the new S-gene is 147. The lengths of putative amino acids of the gene fragment is 99, it contains signal peptide, C1 region, C2 region, HV region related to the recognition function of S-RNase, 3 cysteine residues related to the structure of S-RNase and 1 histidine residue related to the activity of RNase. The residue length of signal peptide, C1 region, C2 region, and HV region is 27, 11, 10 and 15 respectively. To summary, the determination of genotypes of these varieties and discovery of new S-RNase gene provide an important scientific foundation for arranging pollination varieties and further study of SI in fruit tree.