小麦4个多效抗病基因分子标记的转化和再开发  

Convertion and Redevelopment of Molecular Markers of 4 Pleiotropic Disease Resistance Genes in Wheat(Triticum aestivum)

作　　者：高洁[1,2] 宋国琦 李吉虎[1,2] 李玉莲 张淑娟 张荣志[1,2] 谷田田 李根英[1,2] 李玮 GAO Jie;SONG Guo-Qi;LI Ji-Hu;LI Yu-Lian;ZHANG Shu-Juan;ZHANG Rong-Zhi;GU Tian-Tian;LI Gen-Ying;LI Wei(Crop Research Institute,Shandong Academy of Agricultural Sciences,Jinan 250100,China;Key Laboratory of Wheat Biology and Genetic Improvement in the North Yellow&Huai River Valley,Ministry of Agriculture/National Engineering Laboratory for Wheat&Maize,Jinan 250100,China)

机构地区：[1]山东省农业科学院作物研究所,济南250100 [2]农业部黄淮北部小麦生物学与遗传育种重点实验室/小麦玉米国家工程实验室,济南250100

出　　处：《农业生物技术学报》2021年第5期847-856,共10页Journal of Agricultural Biotechnology

基　　金：山东省重点研发计划(2018GNC110033);山东省农业科学院农业科技创新工程项目(CXGC2019G02);山东省农业良种工程(2019LZGC001;2019LZGC015);国家自然科学基金(31701428)。

摘　　要：我国小麦(Triticum aestivum)分子育种发展已经超过20年,但利用分子标记辅助选择选育的品种仍屈指可数,加快分子育种发展是当务之急。小麦中已发现4个多效抗病基因,在育种过程中表型选择比较困难,可通过分子标记辅助选择。已开发的多效抗病基因标记存在检测效果不佳,类型单一等问题,限制了其使用。本研究针对已报道的多效抗病基因竞争性等位基因特异性PCR(kompetitive allele-specific PCR,KASP)标记基因分型效果较差的问题,将KASP标记所检测基因位点序列与’中国春’小麦参考基因组进行BLAST比对,发现基因组中其他位置存在与目标基因位点高度相似的序列,可能是导致KASP标记检测效果不理想的原因。根据BLAST比对结果重新设计引物,改良抗叶锈基因34(leaf rust resistance gene 34,Lr34)的KASP标记Lr34TCCIND和Lr46的KASP标记Lr46JF2-2获得了分型效果改善的Lr34K2和Lr46K3;再开发了抗秆锈基因2(stem rust resistance 2,Sr2)的KASP标记Sr2K3、Lr34的KASP标记C6K1C2和C6K2C1、Lr46的KASP标记Lr46g22K3,获得了较好的基因分型效果;通过将Lr46JF2-2转化成衍生酶切扩增多态性序列(derived cleaved amplified polymorphic sequences,dCAPS)标记Lr46Rdcaps,将Lr67的KASP标记TM4和TM10转化成dCAPS标记TM4dcaps和TM10dcaps,解除了KASP标记检测的仪器限制,降低了应用门槛。本研究丰富了多效抗病基因可用分子标记的数量和类型,为多效抗病基因的分子标记辅助选择提供便利,对KASP标记开发和改良有重要参考价值。It has been more than 20 years since wheat(Triticum aestivum)molecular breeding was introduced to China,only a few commercial cultivars were released.To promote combination of marker assistant selection and traditional breeding,and accelerate molecular breeding development is urgent.There are 4 well characterized pleiotropic disease resistance genes in wheat.It is difficult to select these genes in breeding program through phenotype,marker assistant selection is an option.Markers developed for pleiotropic disease resistance genes have some shortcoming,such as ambiguous genotyping or single marker type,which limit its use.In this study,Focus on the ambiguous genotyping problem of reported kompetitive allele-specific PCR(KASP)markers of pleiotropic disease resistance genes,the basic local alignment search tool(BLAST)was used to align the KASP marker amplification regions and the’Chinese Spring’reference genome.Except the target regions,several other similar genomic regions were found.Too many similar sequences in the genome may be the reason for genotyping problem of KASP markers.According to the BLAST result,primers were redesigned.The KASP marker genotyping result of improved Lr34 K2 and Lr46 K3 were better than Lr34TCCIND for leaf rust resistance gene 34(Lr34)and Lr46JF2-2 for Lr46,respectively;KASP markers Sr2 K3 for stem rust resistance 2(Sr2),C6 K1 C2 and C6 K2 C1 for Lr34,and Lr46 g22 K3 for Lr46 were redeveloped,the genotyping results were clear;KASP markers Lr46JF2-2 for Lr46,TM4 and TM10 for Lr67 were also converted into derived cleaved amplified polymorphic sequences(dCAPS)markers Lr46 Rdcaps,TM4 dcaps,and TM10 dcaps,respectively.dCAPS markers would release the machinery requirement and lower the application threshold.In conclusion,this study enriches marker type and quantity,and provides convenience for marker assistant selection of pleiotropic disease resistance genes.It also has important reference value for KASP marker development and improvement.

关 键 词：小麦 多效抗病基因 分子标记 衍生酶切扩增多态性序列(dCAPS) 竞争性等位基因特异性PCR(KASP) 

分 类 号：S512.1[农业科学—作物学] Q812[生物学—生物工程]