摘要
【目的】来自小麦-簇毛麦6VS/6AL易位系的抗病基因Pm21对小麦白粉病具有持久和广谱抗性,开发该基因的特异性标记,分析其在全国冬麦区中的应用情况,为Pm21的合理布局及分子标记辅助选择育种提供理论依据和技术支撑。【方法】根据已克隆的与Pm21抗性途径紧密相关的丝氨酸/苏氨酸蛋白激酶基因Stpk-V的序列(GenBank登录号为HQ864471.1),提取其蛋白序列并利用Pfam软件分析其保守结构域起止位点,在其保守结构域外设计开发特异序列标记WS-1;构建Pm21载体品种92R137和感病品种Avcoet S(AvS)的F2群体,以小麦白粉病菌E09对该群体每个单株进行苗期抗白粉病表型鉴定,同时利用WS-1对F2群体进行分子检测,分析检测表型与抗病表型,以验证WS-1标记的准确性;利用WS-1标记对来自中国不同冬麦区的662份小麦品种(系)进行分子标记检测,分析Pm21在不同麦区小麦品种(系)的分布情况,并将检测到含有Pm21的品种(系)在田间进行抗白粉病鉴定;选取WS-1已检测到及没有检测到Pm21的品种(系)各50份,利用曹爱忠等开发的标记NAU/xibao15902进行PCR扩增,进一步证明WS-1的准确性。【结果】(1)开发的Pm21特异标记WS-1为显性标记,含Pm21的小麦材料在8%非变性聚丙烯酰胺凝胶中扩增出一条大小为949 bp的片段,而不含该基因的小麦材料中无该片段。(2)在包含377个株系的F2群体中,286个单株为抗病,91个单株为感病,抗感比符合3﹕1的分离比例,表明在该群体中Pm21表现为显性单基因,WS-1对F2群体的每个株系的检测结果与抗/感表型完全一致。(3)供试的662份小麦材料中,49份携带Pm21,平均分布频率为7.4%,其中,西南冬麦区中检测到33份,占该区参鉴品种(系)数的34.4%,而北部冬麦区、黄淮冬麦区和长江中下游冬麦区,分别检测到4份、9份和3份,各占该区参鉴品种(系)数的5.3%、3.1%和1.5%。【结论】开发的Pm21特异性标记WS-1可以作为该基因的检测标记,也可应用到今后的基因聚合育种中;该基因在不同麦区分布相差很大,其中,西南冬麦区四川、贵州省的小麦品种(系)中Pm21使用频率过高,有促进病原菌定向选择的风险,在当前小麦育种中应给以重视。
Objective Powdery mildew, caused by Blumeria graminis f. sp.tritici （Bgt）, is an important disease that causes substantial yield losses in wheat （Triticum aestivum L.） in China, there is a great significance for using resistance genes to control this disease. Powdery mildew resistance gene Pm21, transferred from Haynaldia villosa by a 6VS/6AL translocation, confers durable and broad-spectrum resistance to the disease. This study is to develop gene specific PCR marker, analyze the distribution of Pm21 in China winter wheat regions, and to provide a theoretical basis and technical support for reasonable arrangement and marker-assisted selection for Pm21.[Method]Based on the sequence of Stpk-V gene which was cloned and related to the resistance pathway （GenBank accession number：HQ864471.1）, the protein sequences were extracted and the conservative start-stop sites were analyzed by Pfam software, in order to amplify this gene specifically, the marker WS-1 was developed excluding conserved domain. For constructing an F2 populations derived from susceptible cultivar Avocet S and the lines 92R137 carrying Pm21, infection types in F2 plants were evaluated by artificial inoculation with isolate E09 during seedling stage, F 2 plants were amplified by WS-1, and the testing results and infection types were analysed to confirm the accuracy of WS-1. A total of 662 wheat cultivars and breeding lines growing in different winter wheat regions in China were detected by WS-1 to analyze the distribution of Pm21, and the materials carrying Pm21 were further tested the resistance to powdery mildew under field conditions. In order to further prove the accuracy of WS-1, the marker NAU/xibao15 902 developed by Cao Aizhong and others was used to amplify the lines with Pm21 and without Pm21 （50 samples from each type）. [Result] As a dominant molecular marker, WS-1 could be amplified a 949 bp fragment in the lines with Pm21 on 8%non-denaturing polyacrylamide gel, but did not in the lines without Pm21. The 377 F2 populations segregated 286 resistant﹕91 susceptible fitting the 3﹕1 ratio, which indicated that Pm21 presents a single dominant gene, and the testing results by WS-1 were consistent with infection types. There were 49 lines showed positive strand in 662 lines, and the average distribution frequency of Pm21 gene was 7.4%;the lines containing Pm21 in Southwestern Winter Wheat Region, Northern China Plain Winter Wheat Region, Yellow and Huai Valley Facultative Wheat Region and Middle＆amp;Lower Yangtze Valley Winter Wheat Region were 33, 4, 9, and 3, and their frequencies were 34.4%, 5.3%, 3.1%and 1.5%, respectively.[Conclusion]WS-1 can be detected wheat cultivars and breeding lines which carry Pm21 accurately, also can be applied to the gene pyramiding breeding in the future. The distribution of Pm21 in winter wheat regions is different, the risk of pathogen directional selection will be promoted because of the high frequency of Pm21 gene in wheat cultivars of Sichuan and Guizhou provinces, and we should pay more attention to it in wheat breeding.
出处
《中国农业科学》
CAS
CSCD
北大核心
2014年第11期2078-2087,共10页
Scientia Agricultura Sinica
基金
国家“973”计划(2013CB127700)
国家小麦产业技术体系(CARS-3-1-11)
国家公益性行业(农业)科研专项(201203014)
高等学校学科创新引智计划项目(B07049)
