Cloning And Identification Of Self-incompatiblegenes Of Loquat And The Mechanism Of Self-incompatibility

Loquat(Eriobotrya Lindl.)is evergreen tree which belongs to Maloideae,Rosaceae.It is a typical gametophytic self incompatibility(GSI)species.In this study,20 cultivars of loquat were genotyped.S-RNase and SFB genes were cloned.The possibbe mechanism of self compatible cultivar ’Zaohuang’ was studied.The main results are as follows:1.AS-PCR wes conducted on the genomic DNA of 20 cultivars of loquat.Primers’MaC1F1’ and ’MaC2/3R’were designed based on the Cl and C3 conserved region of Pyrus.S-RNase genes were isolated by PCR amplification.Fourteen varieties were detected two bands on 2%agarose gel electrophoresis.Six varieties were detected only one band,and the bands were further analysed by dCAPs.Finally,through cloning and sequencing,we obtained 7 S-RNase genes(S2,S6,S7,S8,S9,S11,S31)and 2 new S-RNase genes,named Sb and Sk with GenBank Accession Numbers KR149297 and KR149298.The S genotype of 20 loquat cultivars were as follows:S2S31(’Zaohuang’、’Changlvl2’、’Zhaozhong’、’Baiyu’、’Dongshanjibaidan’),S11S31(’Jiefangzhong’),S7S11(’Cuannao’、’Bingtangzhong’),S7Sk(’Qingzhong’、’Jinfengl’),S31Sb(’Xishanjidanbai’),S2S8(’Guanyu’),S2S11(’Tianzhong’),S6S9(’Qianxipeiyu’),S6S7(’Hongdenglong’、’Shanghaizhong’),S6S31(’Meiguozhong’),S2Sx(’Fengyu’、’Ninghaibai’)and S31Sx(’Baihong’)。2.For the identification of SFB genes,PCR was carried out on the genonic DNA of 11 loquats using the primers D-SFB1F1 and D-SFB1R1,which was designed from the conserved regions of loquat SFB genes obtained earlier,we got 11 new SFBs of loquat.Only a 1.1 kb band was detected by agarose gel electrophoresis from each species.The cloning and sequencing results showed that every breed has a number of different SFB genes,and 12 new SFB genes were identified.The GenBank Accession Numbers are as follows:SFB1(KR149288),SFB2(KR149289),SFB3(KR149299),SFB4(KR149300),SFB5(KR 149301),SFB6(KR149302),SFB7(KR149303),SFB8(KR149304),SFB9(KR149305),SFB10(K R149306),SFB11(KR149307),SFB12(KR149308).In addition,S-RNase genes and SFB genes on the identification of loquat were sequenced,and the expressions were verified by RT-PCR.The results showed that both of them have the typical structure of the S gene specific expression,respectively,in the style and pollen.3.In this study,we confirmed SC of‘Zaohuang’through relicated self-pollination tests and fluorescence microscopy.Cross pollination tests showed that SC of‘Zaohuang’was caused by a loss of style function.Sequence analysis and expression analysis of RT-PCR showed S genes of ’Zaohuang’ owned the typical structural characteristics of Rosaceae.However,four amino acids was inserted in the S2-RNase of ’Zaohuang’.’Zaohuang’ was at the same expression level with the same S genotype loquat ’Baiyu’.In addition,several new SFB genes were identified.The above results showed that SC of’Zaohuang’ due to the function of S gene mutation in style,or other factors outside the S-locus,it may also be related to inactivation of S-RNase protein.