分子连锁分析探讨家蚕高抗BmNPV品系的抗性遗传基础

doi:10.3864/j.issn.0578-1752.2017.01.017

摘要/Abstract

摘要： 【目的】家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus,BmNPV）所引发的血液型脓病是一种传染性强的蚕病，极大地影响蚕业生产。本研究旨在定位对BmNPV高抗的家蚕品系中控制其抗性的基因，进而解析其抗性遗传机制，为培育抗性素材和品系提供理论支持。【方法】以BmNPV高抗家蚕品系99R和较感品系Dazao-N为亲本，配制连锁分析（BC₁F）和定位分析（BC₁M）回交群体。首先对两个亲本品系进行浓度梯度添毒，统计感病死亡的家蚕头数，运用SPSS 17.0软件，计算其半致死剂量（LD₅₀），在此基础上确定BC1分离群体的攻毒剂量，并通过单头定量攻毒，选取感病个体作为连锁定位分析材料；利用筛选得到的覆盖家蚕全套常染色体的多态性标记进行分型，并通过T检验计算各标记与抗性的连锁显著性水平（P值），筛选出与抗性相连锁的标记。在这些标记所在的染色体上加密标记，检测各标记在定位分析群体中的基因型，定位抗性基因。【结果】LD₅₀(99R)=2.92×10⁶个多角体/头,LD₅₀(Dazao-N)=9.78×10⁵个多角体/头，基于双亲的半致死剂量，选择介于两者之间且略高于其均值的剂量——2×10⁶个多角体/头作为BC1分离群体的攻毒剂量；先后于2014年秋季和2015年春季处理并检测连锁分析群体，前后两次所进行的连锁分析结果有较大差异，其中第一次找到Chr22上的多态性标记S2205与99R抗性连锁，而第二次的连锁分析显示标记S2205与抗性不连锁，也没有找到其他的连锁关系。通过与前人对BmNPV抗性的连锁定位分析结果进行对比，发现连锁定位分析结果的不可重复性是一个普遍的问题。AY380833是GenBank中已公布的在家蚕高抗品系NB和871C中与其抗性位点紧密连锁的分子标记，本研究调查发现其与99R和871C的抗性位点均不连锁。【结论】分子连锁分析结果证明，家蚕对BmNPV的抗性在不同抗性品系中遗传基础有很大的差异性，同一品系可能具有多个抗性位点；家蚕对BmNPV的抗性是一种复杂性状，在符合“质量-数量性状”结论的同时，其数量性状特征突出。

关键词: 家蚕, BmNPV, 抗性, 分子标记, 连锁分析

Abstract: 【Objective】Bombyx mori nucleopolyhedrovirus (BmNPV) can lead the B. mori nucleopolyhedrosis, which caused a huge loss in the sericulture industry. The objective of this study is to map the controlling genes and understand the genetic basis underlying the BmNPV resistance to serve the theory support for resistant variety breeding. 【Method】Backcross population BC₁F (using for linkage analysis) and BC₁M (using for mapping analysis) were derived from crosses between the highly resistant B. mori strain 99R and the sensitive strain Dazao-N. Concentration gradient of virus was used to treat the parents, and the number of infected individuals was recorded. Then by using SPSS17.0, the LD₅₀ for each parent was calculated. Based on appraisal of resistance performance of two parents, the virus adding concentration for BC₁population was determined and then the virus was fed to larva quantitatively at the start of 4th instar one by one. The infected individuals in the BC₁F were chosen as linkage analysis materials. By using the screened polymorphism markers covering all B. mori autosomes, linkage analysis was conducted and then the genotype data were analyzed by T test to get the linkage significance level of each polymorphism marker to show whether it linked with resistance. Thereafter, polymorphism markers will be enriched on the chromosomes which include the resistance linked polymorphism markers to map resistance locus on them. 【Result】Median lethal dose (LD₅₀) of 99R and Dazao-N is 2.92×10⁶and 9.78×10⁵ polyhedral bodies, respectively, and the dose 2.0×10⁶was chosen as the infection dosage of BC₁ backcross population, which is slightly higher than the average of two parents’ LD₅₀. Two independent linkage analyses were conducted successively in the autumn of 2014 and spring of 2015, which got a discrepant result that the linkage analysis result could not be repeated in the following confirmation; i.e., one marker, S2205, on Chr 22 was identified to link with resistance in the 1st experiment, while no linked marker was identified in the following one. Comparison with the previous reports on the linkage and mapping analysis of BmNPV resistance, it was found that the nonrepeatability was ubiquitous in the related researches in B. mori. Previously, a molecular marker (AY380833) was found to highly link with the resistant trait in NB and 871C, which are both the highly resistance strains to BmNPV. However, the linkage relationship was not detected in the linkage analysis population of 99R and 871C here.【Conclusion】the genetic complexity of resistance by molecular linkage analysis was further proved, including the variation among different resistant strains, as well the plausible multiple resistant loci in one single strain. At the same time, it was proposed that the BmNPV resistance may be a complex trait, as a prerequisite to be one type of qualitative-quantitative traits, the quantitative trait characteristic of BmNPV resistance is very obvious.

Key words: Bombyx mori, BmNPV, resistance, molecular markers, linkage analysis

高瑞，李春林，童晓玲，曹明亚，石美宁，徐安英，鲁成，代方银. 分子连锁分析探讨家蚕高抗BmNPV品系的抗性遗传基础[J]. 中国农业科学, 2017, 50(1): 195-204.

GAO Rui, LI ChunLin, TONG XiaoLing, CAO MingYa, SHI MeiNing, XU AnYing, LU Cheng, DAI FangYin. Insight into Genetic Basis of Bombyx mori Resistant Strains with Resistance to BmNPV by Molecular Linkage Analysis[J]. Scientia Agricultura Sinica, 2017, 50(1): 195-204.

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