杏树李痘病毒病的快速分子检测和抗PPV连锁标记的筛选  

RT-PCR Detection of Plum pox virus and the Screening of DNA Markers Linked to PPV-resistance in Apricot

作　　者：张俊环 孙浩元 Boris Krska 杨丽 姜凤超 张美玲 王玉柱 ZHANG Junhuan;SUN Haoyuan;Boris Krska;YANG Li;JIANG Fengchao;ZHANG Meiling;WANG Yuzhu(Beijing Academy of Forestry and Pomology Sciences,Beijing 100093,China;Faculty of Horticulture,Mendel University,Brno 69144,Czech Republic)

机构地区：[1]北京市林业果树科学研究院,北京100093 [2]孟德尔大学园艺学院,布尔诺69144

出　　处：《华北农学报》2018年第4期31-38,共8页Acta Agriculturae Boreali-Sinica

基　　金：国家林业局“948”项目(2013-4-50);国家自然科学基金项目(31401836);北京市自然科学基金项目(6162012);北京市农林科学院青年基金项目(QNJJ201702);北京市农林科学院科技创新能力建设专项(KJCX2016)

摘　　要：为快速检测引进杏资源是否携带李痘病毒(PPV),并从国内资源中筛选抗PPV材料,通过RT-PCR和荧光定量PCR 2种方法的比较验证,确定了RT-PCR为快速、准确、经济高效检测杏PPV病毒病的方法,并明确了PCR扩增体系和反应条件的具体参数。扩增体系为20μL,包括10×Buffer 2μL、25 mmol/L Mg^(2+)1. 6μL、10 mmol/L d NTP0. 4μL、0. 5 U/μL Taq酶0. 2μL、上下游引物各0. 4μL、模板c DNA 1μL、dd H_2O 14μL。RT-PCR反应条件:94℃预变性5 min,然后以94℃变性45 s、55℃退火30 s、72℃延伸45 s,进行35个循环,72℃延伸10 min。利用捷克孟德尔大学园艺学院构建的杏PPV抗性分离F1群体,采用SSR分子标记技术和集团分离分析法(BSA)相结合的策略,首先在抗PPV和易感PPV的对等基因间进行多态性标记的筛选,初步筛选得到6个SSR位点(PGS1. 23、PGS1. 24、Pa CITA5、UDAP-414、Pchcms4和Pchgms4)在两亲本间和2个对等基因池间的多态性是一致的,并进一步在F1分离群体间进行复选和验证,最终筛选到1个与PPV抗性性状连锁程度较高的SSR分子标记PGS1. 23,此标记检测杏资源分离群体PPV抗性的符合度达到了78. 3%。进而利用该标记在37份我国杏种质中进行了PPV抗性评价,结果筛选出了5份抗PPV的杏资源,分别是菜籽黄、杨继元、垂枝山杏、大早熟和媳妇杏,为今后杏的抗PPV育种提供较好的亲本材料,也为进一步挖掘抗PPV基因的深度研究奠定了基础。In order to rapidly detect the Plum pox virus （PPV） from the introduced apricot resources, and screening PPV-resistant materials from domestic resources, we employed RT-PCR and quantitative Real-time PCR, and the rapid detection technology system of PPV was established in apricot, by comparing and verifying these two methods. The PCR specific parameters of the PCR amplification system and reaction conditions were determined.RT-PCR amplifications were performed in 20 μL reaction mixture containing 10×PCR Buffer 2 μL, 25 mmol/L Mg^2＋ 1.6 μL, 10 mmol/L dNTP 0.4 μL, 0.5 U/μL Taq DNA polymerase 0.2 μL, both forward and reverse primer 0.4 μL, cDNA 1 μL, and ddH2O 14 μL. Reaction parameters for RT-PCR were as follows：94℃ for 5 min; 35 cycles of 94℃ for 45 s, 55℃ for 30 s, and 72℃ for 45 s; with a final extension step at 72℃ for 10 min. In addition, F1 population crossed from two different PPV-resistant apricot parents had been constructed by Faculty of Horticulture, Mendel University. Based on these F1 individual materials, we used SSR method coupled with BSA method to screen the markers linked to PPV-resistant genes. Firstly, the polymorphic markers were screened between the PPV-resistant gene pool and susceptible one, and six SSR loci （PGS1.23, PGS1.24, PaCITA5, UDAP-414, Pchcms4 and Pchgms4） had been screened out. The polymorphism between two gene pools and between two parents was consistent. Then these six locus were further verified among the F1 progenies. As the final results, one SSR locus PGS1.23 with a high degree of linkage to PPV resistance was identified. There was a high coincidence of 78.3% between expected marker genotype and phenotype for the PGS1.23. Furthermore, this linkaged marker was used to evaluate the PPV resistance for 37 Chinese apricot germplasms, and five cultivars with PPV-resistance including Caizihuang, Yangjiyuan, Chuizhishanxing, Dazaoshu and Xifuxing were screened out. These results would provide better parents materials for PPV-resistant apricot breeding

关 键 词：李痘病毒 杏 分子检测 SSR BSA 

分 类 号：Q78[生物学—分子生物学] S662.03[农业科学—果树学]