控制桃粘/离核PG基因的BAC克隆筛选与序列分析  

Screening and Sequence Analysis of BAC Clone Contained PG Gene Controlling Clingstone/Freestone Characteristic of Peach

作　　者：孟君仁 牛良[1] 邓丽 潘磊[1] 鲁振华[1] 崔国朝[1] 王志强[1] 曾文芳[1] MENG JunRen;NIU Liang;DENG Li;PAN Lei;LU ZhenHua;CUI GuoChao;WANG ZhiQiang;ZENG WenFang(Zhengzhou Fruit Research Institute,Chinese Academy of Agricultural Sciences/National Peach and Grape Improvement Center/Key Laboratory of Fruit Breeding Technology of Ministry of Agriculture,Zhengzhou 450009)

机构地区：[1]中国农业科学院郑州果树研究所/国家桃葡萄改良中心/农业部果树育种技术重点实验室,郑州450009

出　　处：《中国农业科学》2021年第20期4396-4404,共9页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2019YFD1000200);国家自然科学基金(31872085);河南省科技攻关项目(202102110037)。

摘　　要：【目的】控制桃果实粘/离核性状的多聚半乳糖醛酸酶(polygalacturonase,PG)基因存在串联重复和大片段的缺失。本研究对粘核桃所处的F-M基因座序列特征进行分析,为开发相关分子标记提供依据。【方法】本研究利用构建的粘核桃单株‘87-7-1’基因组的细菌人工染色体(bacterial artificial chromosome,BAC)文库,通过PCR筛选出含F-M基因座的阳性克隆,利用单分子纳米孔技术进行全长测序,并对BAC克隆中的插入片段进行基因注释、序列比对和生物信息学分析。【结果】利用已有桃品种的基因组重测序数据设计PCR引物,以BAC文库为模板,扩增F-M基因座上/下游稳定共有的序列,获得了扩增产物上/下游条带均为阳性的目标单克隆46-B-10。全长测序结果表明,BAC克隆的插入片段全长为111612 bp,GC含量为37.03%。利用基因同源共线性方法对Prunus_persica_v2.0桃参考基因组和BAC克隆之间的同源信息进行比对分析,确定了同源区域。而与已知的桃参考基因组(测序品种为‘Lovell’,离核)序列进行比对,发现只有5个基因(Prupe.4G261700、Prupe.4G261800、Prupe.4G261900、Prupe.4G262000、Prupe.4G262500)序列能比对到该单克隆全长的区域,而‘87-7-1’在相应位置缺失了4个基因共34 kb,其中包括控制桃粘/离核的EndoPGF(Prupe.4G262200)。【结论】与桃参考基因组测序品种‘Lovell’相比,粘核桃单株‘87-7-1’的F-M基因座缺失了EndoPGF,只有EndoPGM,本研究明确了粘核桃F-M基因座的结构变异情况,为粘/离核性状分子标记的开发奠定了基础。【Objective】Tandem repeats and large segment deletions of Polygalacturonase(PG)gene was related to the clingstone/freestone characteristic of peach(Prunus persica)fruit.In this study,the sequence character of F-M locus of clingstone peach was analyzed to provide a basis for the development of related molecular markers.【Method】With a Bacterial Artificial Chromosome library of clingstone peach 87-7-1 constructed,the positive BAC clone contained F-M locus was screened from the BAC library by PCR analysis.The screened BAC clone was sequenced by single-molecule nanopore technology.Gene annotation and sequence alignment were performed by bioinformatics.【Result】PCR primers were designed based on re-sequencing data of existing peach varieties,and PCR reactions were performed with all the BAC library clones.Amplification products of the sequences in upstream/downstream of F-M loci were corrected,and then the target clone 46-B-10 was obtained.Full-length sequencing showed that the fragment with the length of 111612 bp and GC content of 37.03%was inserted between upstream and downstream primers.The homologous region of 46-B-10 was determined by sequence alignment with the reference genome Prunus_persica_v2.0.Five genes(Prupe.4G261700,Prupe.4G261800,Prupe.4G261900,Prupe.4G262000,and Prupe.4G262500)in F-M locus were found in the BAC clone 46-B-10.In comparison,87-7-1 with 34 kb sequence including four genes was discarded,and one of them was EndoPGF,which controlled peach freestone and was reported previously.【Conclusion】Compared with the reference genome freestone variety‘Lovell’,there was only EndoPGM(Prupe.4G261900)in F-M locus of clingstone peach individual 87-7-1,while EndoPGF(Prupe.4G262200)was discarded.In this study,the structural variation of F-M locus in clingstone peach was determined,which has laid an important foundation for the development of molecular markers for clingstone/freestone trait in peach.

关 键 词：桃 粘/离核 PG基因 BAC文库 

分 类 号：S662.1[农业科学—果树学]