Identification Of S Genotypes In Loquat Based On AS-PCR And Study On The Mechanism Of Self Compatibility Of 'Zaohuang' Loquat

Loquat is an evergreen fruit tree and belongs to Eriobotrya in the Rosaceae,which carries the RNase-dependent gametophytic self-incompatibility system(GSI).Self incompatibility(GSI)is a common genetic control mechanism and is widely found in angiosperms,which can induce the style to reject its own pollen,thereby preventing inbreeding.SI is controlled by a single locus(S-locus)allele(S-allele),but the genetic control mechanisms in different plant species are not consistent.Until now,there is little relatively research on the self incompatibility of loquat.In this study,9 S-allele specific primer pairs were designed and S-genotypes of 28 loquat cultivars were identified for the first time and S-genotypes of 11 loquat with known S genes were re-identified.In addition,8 new SFB genes were obtained from 8 different loquat cultivars.And the causes of the self compatibility loquat cultivars were studied.The main results as follow:1.According to the previously reported S-RNase alleles(S2,S7,S6,S8,S9,S10,S11,S12,S13),9 S-RNase alleles specific primer pairs were designed,which located in intron or some areas with high specificity.8 loquat cultivars with known S-genotypes were used for evaluating the specificity of 9 S-RNase allele specific primer pairs.S-genotypes of 39 loquat cultivars were identified efficiently through the 9 S-RNase allele-specific primer pairs.Based on these results,the 9 S allele-specific primer pairs designed in this study will be useful for the 9 S-RNase alleles efficient identifying in loquat.2.'Zaohaung'was determined as a self compatible loquat cultivar that is related to the S-RNase gene and SFB gene based on the results of pollination.Cloning and sequencing analysis of the S2-RNase and S10-RNasee genes in'Zaohaung'and 'Baiyu'revealed that the sequences of the two S-RNase genes in the two species were identical.Using qRT-PCR to analyze the expression of two S-RNase genes in the two varieties,and found that the expression level of S10-RNase in 'Zaohaung'was significantly lower than that of S10-RNase in 'Baiyu'.Thus,we speculated that the mutation of promoter in S10-RNase gene may affect the expression of downstream gene.Then,we cloned the promoter of S10-RNase in'Zaohuang' and 'Baiyu'(2410bp/2462bp).After sequencing and alignment,the lack of 52bp fragment in the upstream position of 1755bp was detected in the promoter of S10-RNase in'Zaohuang'.3.8 new SFB genes of 8 loquat cultivars were identified using the specific primer pair D-SFB1F1/R1 which was designed from the conserved regions of loquat SFB genes obtained earlier.After DNA sequencing and alignment,found that each loquat cultivars obtained a number of different SFB genes,SFB2 appeared in 6 different loquat cultivars,but SFB20 only appeared in 'Ninghaibai'.After protein function analysis,found that the main function of SFB protein is involved in the biosynthesis of amino acids,synthetic cofactors,fatty acid metabolism,in addition SFB13,SFB 16,SFB 17 and SFB20 have lyase function.SFB genes identified in this study and combined with the previous obtained SFB genes in loquat,apple and pear were used to construct phylogenetic tree,found that these SFB genes in loquat was mainly divided into three categories.The homology in class II and? were reach to 98%and 100%,while the classI was 69%.