Identification S-RNase And SFB Genes Of Japanese Apricot And Analysis Of Self-Compatibility Mechanism Of ’Sichuanbaimei’

The Japanese apricot（Prunus mume Sieb.et Zucc）originated in China.Although it is a typical gametophyte self-incompatible tree species,a few self-compatible varieties have been found in the long-term cultivation process.The study found that at least two genes controlled the self-incompatibility of Prunus mume.In addition to the style S（S-RNase）gene located at the S site,there are also pollen S（SFB/SLF）genes located at the S site.In order to identify the S genotype of Prunus mume germplasm resources,identify the new S-RNase gene and SFB gene,and further identify the self-compatibility variation mechanism of Prunus mume germplasm,in this study,S-RNase gene and SFB/SLF gene were cloned from some Prunus mume germplasm resources resources is located in the National Field Genebank for Prunus mume and Waxberry of Nanjing Agricultural University.The molecular mechanism of self-compatibility variation of’Sichuanbaimei’was analyzed.The results of this study provide a theoretical basis for the efficient allocation of pollination trees and the selection of parents for cross breeding.The main results are as follows:1.According to the second conserved region（C2 region）and the third conserved region（C3region）of Prunus S-RNase gene,Pru-C2 and PCE-R were designed.Using the genomic DNA of 11 Prunus mume cultivars AS templates,the S-RNase gene of style was amplified and cloned by AS-PCR.The obtained sequences were analyzed by BLAST in NCBI,and the results showed that all the 11 varieties obtained two items of products.Sequence analysis found 11 registered Prunus mume S-RNase genes were obtained.These 11 Prunus mume S-RNase genes were S₁-RNase,S₃-RNase,S₅-RNase,S₆-RNase,S₁₃-RNase,S₁₄-RNase,S₁₅-RNase,S₂₀-RNase,S₂₄-RNase,S₃₀-RNase,S₃₃-RNase),In addition,two new S-RNase genes have been found that have not yet been registered,were named S₃₇-RNase and S₃₈-RNase,And log in the NCBI database with the login numbers MT950061 and MT950062 respectively.After comparison and analysis,the style S（S-RNase）genotypes of these 11 Prunus mume varieties were finally clarified,’Ruantiaohongmei’(S₁₃S₁₄),’Xiaoyezhugan’(S₃S₃₃),’Zhonghong’(S₃S₂₄),’Qingjia915’(S₃S₁₅),’Meilinhong’(S₁S₃₇),’Chifenghongmei’(S₁₄S₃₈),’Dalongmei’(S₂₀S₃₀),’Hongguangmei’(S₃S₃₃),’Nannongfengyu’（S₃S₅）,’Nannongfengjiao’（S₃S₆）,’Nannonglongxia’(S₁₅S₃₇),respectively.2.In this study,the pollen SFB/SLF genes of 61 Prunus mume germplasm resources were identified.Genomic DNA was used AS template.According to the conserved sequence of SFB gene of Prunus mume,primers SFB-C1F and PM-VB were designed and amplified by AS-PCR technology and sequenced.The results showed that 61 Prunus mume germplasm resources were amplified into a band of about 1000 bp.Sequencing analysis showed that each sample contained two different SFB genes.namely’Zhaoanbaimei’(SFB₂/SFB₁₄),’Xiaoyezhugan’(SFB₅₁/SFB₅₃),’Zhonghong’(SFB₂/SFB₂₄),’Qingjia 915’(SFB₂/SFB₄₃),’Changxingyeshengmei’(SFB₁₂/SFB₄₁),’AO=meilinhuang’(SFB₂/SFB₄₂),’Meilinhong’(SFB₁/SFB₂₄),’Changnong 17’(SFB₁₄/SFB₄₂),’Yixing No.1’(SFB₁₈/SFB₄₂),’Yixing No.2’(SFB₁₄/SFB₄₁),’Yixing No.3’(SFB₇/SFB₅₄),’Yixing No.4’(SFB₇/SFB₄₂),’Yixing No.5’(SFB₂/SFB₄₂),’Xianjulvmei’(SFB₂/SFB₄₁),’Longyoubaimei’(SFB₂/SFB₁₄),’Lizimei’(SFB₂/SFB₄₁),’Hongding’(SFB₁₈/SFB₄₂),’Xinbaimei’(SFB₂₄/SFB₃₁),’Puningqingzhumei’(SFB₂₄/SFB₄₁),’Guangdonghuangpi’(SFB₄₃/SFB₅₆),’Daheqing’(SFB₂₄/SFB₄₃),’Huanghoumei’(SFB₂₄/SFB₃₁),’Dalizhong’(SFB₁₂/SFB₅₅),’Henghe’(SFB₂/SFB₄₂),’Chifenghongmei’(SFB₇/SFB₄₁),’Ruanjiangroumei’(SFB₂/SFB₁₄),’Ruanjingdaguoqingmei’(SFB₂/SFB₄₇),’Siyuemei‘(SFB₁₄/SFB₁₈),’Yunnanyanmei’(SFB₁₄/SFB₃₁),’Yunnanzhaoshuimei’(SFB₁₄/SFB₅₀),’Zhaoanshuimei’(SFB₂/SFB₁₄),’Dalongmei’(SFB₁₂/SFB₄₉),’Fujianqingmei’(SFB₁₄/SFB₄₄),’Sichuanbaimei’(SFB₃₁/SFB₅₂),’Sichuanhuangmei’(SFB₁₂/SFB₂₄),’Xinxiangxingmei’(SFB₁₄/SFB₂₄),’Kaidi’(SFB₂/SFB₄₅),’Xiaolve’(SFB₁/SFB₁₄),’Hongguangmei’(SFB₂₄/SFB₅₅),’Huangdoumei’(SFB₂/SFB₄₂),’Liuliumei No.1’(SFB₂/SFB₄₆),’Liuliumei No.2’(SFB₂₄/SFB₅₆),’Nannongfengyan’(SFB₁₄/SFB₃₁),’Nannongfengmao’(SFB₁₂/SFB₄₃),’Nannongfengyu(SFB₃₁/SFB₄₃),’Nannongfengjiao’(SFB₂/SFB₄₇),’Nannonglongfeng’(SFB₃₁/SFB₄₃),’Nannonglongxia’(SFB₄₁/SFB₄₃),’Wanhong’(SFB₂/SFB₅₇),’Yunnankumei’(SFB₅₀/SFB₅₉),’Dabaimei’(SFB₄₃/SFB₄₉),’Hangzhoubaimei’(SFB₄₂/SFB₅₈),’Hongnong’(SFB₂/SFB₂₄),’Zhizhimei’(SFB₁₂/SFB₂₄),’Fenbanguomei’（SFB₂/SFB₇）,’Dongshanlimei’(SFB₁₄/SFB₂₄),’Dongqing’(SFB₇/SFB₄₂),’Taihu NO.1’(SFB₂₄/SFB₄₆),’Taihu NO.3’(SFB₂/SFB₂₄),’Huangxiaoda’(SFB₁/SFB₄₃),’Lvmei’(SFB₁₄/SFB₃₁).Of these 11 genes that have been registered in NCBI(Pm SFB₁,Pm SFB₂,Pm SFB₇,Pm SFB₁₂,Pm SFB₁₄,Pm SFB₁₈,Pm SFB₂₄,Pm SFB₃₁,Pm SFB₄₁,Pm SFB₄₂,Pm SFB₄₃),and 16 new SFB genes were identified(SFB₄₄,SFB₄₅,SFB₄₆,SFB₄₇,SFB₄₈,SFB₄₉,SFB₅₀,SFB₅₁,SFB₅₂,SFB₅₃,SFB₅₄,SFB₅₅,SFB₅₆,SFB₅₇,SFB₅₈,SFB₅₉),and the new gene Gen Bank accession numbers are MW186460,MW186461,MW186462,MW186463,MW186464,MW186465,MW186466,MW186467,MW186468,MW186469,MW186470,MW186471,MW186472,MW786959,MW786960,MW786961.The amino acid sequences of S-RNase gene and pollen SFB gene were further analyzed,and their expression sites were verified by RT-PCR.The results showed that they had the typical structural characteristics of Prunus S gene,and S-RNase gene was specifically expressed in style and pollen SFB gene was specifically expressed in pollen.3.Based on field self-affinity pollination and pollen tube growth test in laboratory,’Sichuanbaimei’was clearly identified as self-incompatibility variety.AS-PCR and RT-PCR analysis showed that S-RNase gene and SFB gene were specifically expressed in styles and pollen,respectively.Further amino acid sequence analysis showed that the S-RNase gene and SFB gene of’Sichuanbaimei’had the typical structural characteristics of the S gene of Prunus,and there was no genetic mutation.Sequence analysis of SFB gene revealed a novel F-box gene,although the protein function of this novel gene is still unknown,a detailed analysis of the amino acid sequence of this novel F-box gene revealed that five amino acid residues were inserted into its variable region V1.However,two amino acid residues were inserted in the variable region V2,and three amino acid residues were deleted in the highly variable region HVB.The recognition of pollen and style would be affected by this mutation,resulting in the variation of self-compatibility.In conclusion,the self-compatibility variation of’Sichuanbaimei’has nothing to do with S-RNase gene and SFB gene;the generation of self-compatibility variation in’Sichuanbaimei’was related to other modifiers other than S site,including F-box gene.