摘要：小麦近缘物种簇毛麦是普通小麦重要的三级基因资源库，通过对簇毛麦相关NLRs类基因的发掘可以拓展小麦的抗病基因资源。本实验室通过大麦专化NLRs-baits文库富集二倍体簇毛麦品系91C43全基因组NLRs类基因，并结合第三代基因测序技术，获得了簇毛麦全基因组水平的NLRs基因序列数据库。本实验利用生物信息学比对的方法，将已获得的簇毛麦NLRs基因序列的contig与近缘物种大麦，以及普通小麦中国春参考基因组数据进行高通量比对，发掘相应同源序列间因进化过程中的缺失、插入、重复等原因所产生的基因组序列差异。根据这些差异片段设计出簇毛麦序列特异引物715对，在小麦—簇毛麦整臂易位系、中国春、簇毛麦等材料进行PCR扩增，确定对应簇毛麦NLRs类基因在染色体上的分布。根据以定位结果，共开发出簇毛麦各染色体臂特异的Indel分子标记174个，其多态率达到24.3％。这些基于NLRs类基因开发的专化分子标记，既可用于辅助簇毛麦易位系等染色体工程材料的选育和鉴定，同时又为后续功能基因的定位和克隆奠定基础。

关键词：簇毛麦；R基因；分子标记；染色体定位

Chromosomal location and molecular markers development of NLRs type genes in Haynaldia villosa

Abstract：Haynaldia villosa is a wild relativeand an important third level gene resource pool of common wheat, Through  the  development  of  NLRs  gene  in Haynaldia villosa  can expend   the  disease resistance gene resources of wheat r. In previous study of this laboratory, N the full-length NLRs type genes of the diploid Haynaldia villosa cv. 91C43 were enriched by a barley specific NLRs-baits library. In combination with the third-generation PacBiosequencing technology, the full-lengthNLRs genes  database was built on the genomic level. In this study, to explore the corresponding homologous sequences,  the contigs of NLRs gene sequences from Haynaldia villosa  genome  were compared with NLRs from genomic sequence of the close related specie barley and reference of common wheat cv. Chinese Spring by high-throughput bioinformatics method . Differences were found in the comparison of the genome sequences, which caused by  deletions, insertions, and duplications during evolution. Based on these differential fragments, 715 pairs of NLRs gene specific primers were designed and used for PCR amplification among the wheat- Haynaldia villosa whole-arm translocation lines, Chinese spring, andHaynaldia villosa. Based on the results of chromosomal localization, a total of 174 specific Indel molecular markers were developed for each chromosome arm of Haynaldia villosa, and the polymorphism rate reached 24.3%. These special molecular markers developed based on NLRs can be used to assist the selection and identification of chromosomal engineering materials such as the translocation lines. At the same time, it established foundation for the subsequent mapping and cloning of the functional genes.

Key words: Haynaldia villosa;R gene;Molecular marker;Chromosomal localization

目  录

摘要…1

关键词1

Abstract…1

Key words1

1引言…1

2材料与方法…1

2.1实验材料 …2

2.2实验方法 …2

2.2.1簇毛麦NLRs基因的测序2

2.2.2簇毛麦Indel分子标记引物设计2

2.2.3整套小麦—簇毛麦整臂易位系鉴定…3

2.2.4实验材料DNA提取…3

2.2.5簇毛麦NLRs基因分子标记的扩增3

2.2.6扩增产物的检测…3

2.2.7簇毛麦NLRs基因Indel分子标记开发4

3结果与分析 … 4

3.1 所用细胞学材料鉴定4

3.2 Indel分子标记引物设计4

3.3 簇毛麦NLRs基因Indel分子标记的PCR验证…4

3.4 簇毛麦NLRs基因染色体定位6

4讨论 … 6

致谢…7

参考文献7

附录 实验胶图整理8