基于微卫星和线粒体标记的水稻三化螟地理种群遗传结构分析

The yellow rice borer (Tryporyza incertulas) is one of the most important insect pests in rice production, especially in south part of China. It is also the target pest for chemical control and transgenic Bt rice. Both resistances to pesticides and Bt rice were highly concerned. In this proposal,the population genetic structures of this insct distributed in different rice areas will be analyzed based on microsatellite and mitochondrial markers. The light trap methods will be used to collect insect samples in 17 provinces including 35 counties in main rice areas. The genome of yellow rice borer will be sequenced using the second generation sequencing techniques. The microsatellite sequences in genome will be searched using the software prepared by Per computer language in our laboratory. The genomic microsatellite database will be constructed and characterized. More than 100 pairs of PCR primers will be designed according to the sequences on both sides of microsatellite repeats and a total of 20 high quality markers will be obtained through a series of screening processes. About 30 insect individuals in each sample site will be characterized using these 20 microsatellite markers. The software "Cervus 3.0" will be used to caculate the genetic diversity parameters including mean number of alleles, observed heterozygosity, expected heterozygosity and polymorphism information content in different geographical populations. At the same time, the special PCR primers will be designed to amplify the control region in mitogenome and about 30 insects will be sequenced in each sample site. The genetic diversity parameters including haplotype diversity, nucleotide polgmorphisms will be calculated using software DnaSP Ver.5 and Arlequin 3.1 and the population historical dynamics will also be analyzed. The research results of this proposal will be very important to help us to understand the resistant adaption mechanism and to make control strategy of this pest insect on rice.

三化螟Tryporyza incertulas (walker)是我国长江以南稻区的重要害虫之一，是化学防治和转Bt基因水稻的靶标害虫。本项目利用我国农业病虫测报网络在三化螟重点发生区设点取样，样点覆盖17个省市自治区的35个县市；在对基因组浅测序的基础上，应用实验室开发的微卫星软件比较分析三化螟基因组微卫星的基本特征，建立微卫星序列特征数据库，为微卫星相关研究奠定基础；根据数据库中微卫星序列设计PCR引物100对以上，经多态性和稳定性筛选得到高质量微卫星标记20个以上，建立三化螟标准化微卫星标记系统；采用引物多色荧光标记多位点复合扩增技术和基因扫描分型技术，对三化螟各地理种群的遗传结构变化规律进行深入分析。同时，还采用线粒体高变区测序、单倍型网络分析等技术研究三化螟种群遗传结构及种群间的基因交流、种群扩散和种群历史动态，为深入理解三化螟适应性机制和制定科学防控策略提供理论依据。

1. 三化螟线粒体基因组结构分析：三化螟线粒体基因组全长15,200 bp，共编码13个蛋白编码基因， 22个转运RNA基因及两个核糖体RNA基因，基因组结构表现出典型的鳞翅目特征，以“CR-M-I-Q”结构区别于昆虫纲其它物种。平均AT含量为77.2%，显示出明显的碱基偏倚特征。其蛋白编码基因除COX1外均使用ATT或ATG作为起始密码子，COX1基因为CGA。除COX1、COX2、ND5及ND4外均使用TAA作为终止子。 22个tRNA基因总长1,449 bp，其中 14个编码于重链上，8个编码于轻链上。A-T富集区位于核糖体12S亚基与tRNAMet之间，全长393 bp，AT含量高达92.3%。.2. 基于COI和CYTB基因的三化螟种群遗传结构分析：三化螟COI基因全长为1531bp，编码510个氨基酸。通过DNASP分析，36个三化螟样本共有9个单倍型，17个多态性位点,包括10个单变异位点，7个简约信息位点。单倍型多态性指数为0.763，核苷酸多样性指数为0.00916。通过DNASP分析，45个CYTB基因序列有14个单倍型，18个多态性位点，单倍型多态性指数为0.796，核苷酸多样性指数为0.00576。基于COI基因和CYTB基因构建系统树，结果表明中国三化螟与印尼三化螟聚成一支后与同属的红尾蛀禾螟（Scirpophaga innotata）以及黄尾禾螟（S. nivella）形成姐妹群关系，而来自四川德阳、西昌和海南三亚的三化螟没有发现明显的遗传分化现象。.3. 三化螟基因组微卫星数据库的建立：在三化螟基因组低倍测序的基础上，共得到不同类型的微卫星序列31164条，其中单碱基重复微卫星23954条，占76.86%，四碱基2566条，三碱基1760条，二碱基1608条，六碱基678条，五碱基为598条。在二碱基重复类型中，以AT重复序列740条，占二碱基微卫星的46.02%，其次是AC和AG，分别为704和100。在三碱基重复类型中，ACT数量最多，为499条，占三碱基微卫星序列的28.35%，其次是AGT和ACG，分别为496和194。在四碱基重复类型中，AAAC重复序列1175条，占四碱基微卫星的45.79%，其次是ACAT和ACAG，分别为445和368条。在此基础上设计优化了PCR引物，并进行了多态性筛选。