簇毛麦3V染色体短臂特异分子标记的开发与应用  

Development and Application of Molecular Markers Specific to 3VS Chromosome Arm of Haynaldia villosa

作　　者：陈晓彤 张旭[1] 慕宁 陈丽娟 徐涛[1] 肖进[1] 袁春霞[1] 王宗宽 孙丽[1] 王秀娥[1] 王海燕[1] CHEN Xiaotong;ZHANG Xu;MU Ning;CHEN Lijuan;XU Tao;XIAO Jin;YUAN Chunxia;WANG Zongkuan;SUN Li;WANG Xiue;WANG Haiyan(National Key Laboratory of Crop Genetics&Germplasm Enhancement and Utilization/JCIC-MCP,College of Agriculture,Nanjing Agricultural University,Nanjing,Jiangsu 210095,China)

机构地区：[1]南京农业大学作物遗传与种质创新利用全国重点实验室/细胞遗传所/现代作物生产省部共建协同创新中心,江苏南京210095

出　　处：《麦类作物学报》2023年第12期1504-1513,共10页Journal of Triticeae Crops

基　　金：国家重点研发计划项目(2020YFE0202900);国家自然科学基金项目(32172076);江苏省重点研发计划项目(BE2022346);江苏省现代农业产业技术体系项目(JATS〔2022〕464)。

摘　　要：普通小麦野生近缘物种簇毛麦(Haynaldia villosa L.2 n=14,VV)3V染色体短臂(3VS)携带抗小麦条锈病基因。开发高密度的3VS特异标记是准确鉴定和追踪3VS、推进抗病基因转移和利用的重要基础。为了开发更多的均匀分布于3VS染色体臂不同区段的分子标记,本研究利用簇毛麦和小麦品种中国春参考基因组序列与基因注释信息,通过比较3VS与3AS、3BS、3DS同源基因序列内含子序列差异,选择同一内含子长度差异大于10%的区域开发了104个跨越内含子(intron targeting,IT)的分子标记,在簇毛麦、簇毛麦-硬粒小麦双二倍体、硬粒小麦中引1286、中国春、普通小麦-簇毛麦二体异附加系DA1V-DA7V、小麦-簇毛麦T3VS·3DL易位系中进行扩增,筛选出3VS特异、扩增条带清晰且稳定性好的IT分子标记43个。为了验证上述标记的有效性,选择位于3VS不同区段的12个标记对[DS3V(3D)×中国春ph1b]的F_(3)群体中的148个ph1b纯合的单株进行分析,共筛选得到6种类型涉及3VS的结构变异体。进一步利用GISH/FISH技术对上述材料进行鉴定,结果与分子标记结果相吻合,证明利用外源染色体特异分子标记可有效筛选涉及外源染色体结构变异体。Haynaldia villosa L.(2 n=14,VV)is an important tertiary gene pool of bread wheat and chromosome arm 3VS of H.villosa contained stripe rust resistance gene.Developing high density and evenly distributed molecular markers specific to chromosome 3VS is an important basis for accurate identification and tracing of 3VS in the genetic background of common wheat.DNA sequences used in this work included H.villosa assembly,common wheat cv.Chinese Spring assemblies and annotated genes of Chinese Spring.Intron sizes of the corresponding genes of Chinese Spring 3AS,3BS,3DS and H.villosa 3VS were then calculated and compared against each other.Intron sizes of corresponding genes were then calculated and compared against each other.Genes whose intron sizes in 3VS chromosome differed by at least 10%from those in corresponding allele of short arms of wheat homoeologous group 3 were chosen for marker design.A total of 104 primer pairs were designed in the exons flanking the targeted introns using the online software Primer 3(https://bioinfo.ut.ee/primer3/).PCR was performed with the designed 104 primer sets using genomic DNA from H.villosa,T.durum-H.villosa amphiploid(AABBVV),T.durum,the wheat cv.Chinese Spring,T.aestivum-H.villosa disomic addition lines(DA1V-7V),and a T.aestivum-H.villosa translocation line T3VS·3DL as templates.After PCR amplification,43 IT markers were identified with H.villosa 3VS specific bands.To verify the validity of the above developed markers,we selected 12 markers located in different regions of 3VS to amplify from the individual plants derived from the DS3V×Chinese Spring ph1b F_(3)population.Finally,six types of structural variants involving 3VS chromosome were screened.GISH/FISH was further used to identify the above identified plants,and the results were consistent with the results of IT markers.This result proved that using chromosome-specific markers for identification of alien chromatin in wheat breeding is very effective.

关 键 词：簇毛麦 IT分子标记 结构变异体 同源基因 

分 类 号：S512.9[农业科学—作物学] S330