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致病疫霉基因组微卫星标记开发
张宏磊1,杨志辉2,胡珍珠1,朱杰华3,张奥川1
1. 河北农业大学植物保护学院
2. 河北农业大学西校区植物保护学院
3. 河北农业大学
Development of Genomic Microsatellite Marker for Phytophthora Infestans
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摘要 致病疫霉(Phytophthora infestans)引起的马铃薯晚疫病是影响马铃薯生产的第一大真菌病害。通过致病疫霉基因组开发微卫星标记,旨为致病疫霉群体遗传结构研究提供更系统、有效的标记。利用软件SciRoKo和Clustal X对致病疫霉基因组中微卫星序列长度≥25 bp的适合标记开发的位点及两端序列进行筛选。然后在这些位点的两端序列上设计专一性PCR扩增引物,选择9株致病疫霉菌对引物的专一性和微卫星的多态性进行检测。最后利用40株不同年份和地理来源的致病疫霉菌株对可行性微卫星标记进行等位基因数的确定及其多态性评估,然后选取部分标记对40株致病疫霉群体进行遗传多样性分析。共获得了33个具多态性的微卫星标记,占开发总数的28.7%,其多态信息含量(PIC)值都在0.164~0.614之间,其中PIC≥0.5的标记有7个,0.25<PIC<0.5的标记有25个,PIC≤0.25的有1个。发现致病疫霉微卫星基因型与其交配型及地理来源有一定相关性,与甲霜灵敏感性不相关。本研究开发出一批具有多态性微卫星标记,为致病疫霉群体遗传学研究机理提供更多多态性高,稳定性强的分子标记。
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张宏磊
杨志辉
胡珍珠
朱杰华
张奥川
关键词 致病疫霉基因组SSR标记    
Abstract:Potato late blight caused by Phytophthora infestans is one of the most destructive diseases. Crop losses and costs of late-blight control constitute a significant financial burden on the potato industry. However, understanding the mechanisms, processes and rates of P. infestans evolution are important factors in predicting the durability and effectiveness of new management practices. Microsatellite marker is regarded as a perfect molecular marker for studying biogenetics, syngenetic succession of population and diversity. Hence, microsatellite marker developed based on the genome system is particularly important for understanding of the genetic diversity of P. infestans. Single-copy microsatellite (more than 25 bp in microsatellite sequence length and more than 20 bp in adjacent interval) sites and sequences at both ends were screened in the genome of P. infestans by using SciRoKo, Clustal X and Primer Pair Specificity Checking from Primer-BLAST. And then the microsatellite specificity and polymorphism were detected by using 9 P. infestans strains of different years and geographical origins. Finally, 115 appropriate sites were obtained for developing microsatellite markers. Among them, 64 microsatellite markers were validated by feasibility test, including 33 polymorphic ones (14 intergenic ones, 13 Flank5', 7 Flank3', 15 exons and 6 introns) and 31 non-polymorphic ones(whose electrophoretograms included 4 situations——cluttering bands, no bands, bands appearing in some strains as well as three or more bands appearing in some strains). The determination of the number of alleles and evaluation of polymorphism were made for polymorphic microsatellite markers developed by using 40 P. infestans strains of different years and geographical origins. It was found that polymorphism–tagged microsatellite polymorphic information content(PIC) values ranged from 0.164 to 0.614. There were 7 markers with PIC≥0.5 (highly polymorphism), most of which belonged to the intergenic region; I-00408, I-10840 and E-04958 had 4 alleles, and I-01408, I-07111, I-06861 and F5-22735 had 3 alleles. There were 25 markers with 0.25<PIC<0.5 (moderately polymorphism) and there was only one with PIC≤0.25 (low polymorphism). The 9 polymorphic microsatellite markers were selected for genetic diversity analyses on 35 (from Hebei, Heilongjiang, Jilin, Yunnan, Sichuan, Chongqing, Ningxia, Inner Mongolia and Fujian) and 5 (from Poland, Switzerland and the U.S.) P. infestans strains of different years. UPGMA cluster analysis found a high genetic diversity of P. infestans in China and a significant interpopulation differentiation in different regions. Moreover, it was also found that P. infestans genome microsatellite genotype was correlated to its mating type and geographical origin to some extent but not correlated to metalaxyl sensitivity. These genomic microsatellites could provide rich and polymorphic markers for gene location, clone and genetic diversity of P.infestans.
Key wordsPhytophthora infestans    Genome    SSR marker
收稿日期: 2013-03-27     
ZTFLH:  S435.32  
通讯作者: 朱杰华   
引用本文:   
张宏磊1,杨志辉2,胡珍珠1,朱杰华3,张奥川1. 致病疫霉基因组微卫星标记开发[J]. , 2013, 21(9): 1110-1118.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2013/V21/I9/1110
 
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