偃麦草基因组特异重复序列的分离与应用

doi:10.3864/j.issn.0578-1752.2016.19.002

摘要/Abstract

摘要： 【目的】偃麦草（Thinopyrum）是小麦（Triticum aestivum L.）的多年生野生近缘植物，具有许多可用于小麦品种改良的优异基因。利用基因组特异重复序列可以研究物种的进化关系、绘制染色体指纹图谱及检测外源染色质。克隆十倍体长穗偃麦草（Th. ponticum (Host) Liu and Wang）基因组特异重复序列，可用于鉴定和追踪导入到小麦背景中的偃麦草遗传物质。【方法】通过构建十倍体长穗偃麦草小片段质粒文库，并对文库进行高密度点杂交（Dot-blot hybridization）筛选，结合荧光原位杂交（fluorescence in situ hybridization，FISH）技术，获得偃麦草基因组特异的重复序列，分析其在不同基因组及染色体上的分布特点。利用Repeat Masker在小麦族重复序列数据库（Triticeae repeat sequence database，TREP）及NCBI GenBank对特异重复序列进行比对分析，并设计偃麦草基因组特异重复序列的PCR引物。通过FISH分析和特异PCR引物扩增，对小麦-偃麦草衍生后代进行鉴定和选择。【结果】获得7条偃麦草基因组特异的重复序列。FISH分析表明，其在十倍体长穗偃麦草和六倍体中间偃麦草所有染色体两臂上均呈弥散型分布，且在不加小麦封阻DNA的情况下，能明确区分八倍体小偃麦中的偃麦草和小麦染色体。将其应用到小麦-偃麦草代换系和易位系的分子细胞学检测中，特异重复序列同样可以在不加封阻的情况下分辨出偃麦草染色体及染色体片段，而且信号相比基因组原位杂交（genomic in situ hybridization，GISH）更加特异和清晰。基于偃麦草基因组特异重复序列开发了90对引物，通过在中国春、十倍体长穗偃麦草和八倍体小偃麦中的扩增产物比较分析，筛选出36对（40%）偃麦草基因组特异PCR标记；利用这些特异引物对109份小麦-偃麦草衍生材料进行扫描，发现10对扩增效果较好的特异引物，其检测效率为73.3%—95%。【结论】获得偃麦草基因组特异的重复序列，开发了特异PCR扩增引物，可应用于小麦背景下偃麦草遗传物质的高效检测和跟踪。

关键词: 小麦, 偃麦草, 特异重复序列, 荧光原位杂交, PCR

Abstract: 【Objective】As a tertiary gene pool of wheat, Thinopyrum genus has many useful traits, such as tolerance to both abiotic and biotic stresses. DNA repetitive sequence is one major component for genomes of most eukaryotes. Isolation of genome specific repeats is very helpful for understanding of sub-genome component of polyploid species, their evolution and utilization in crop improvement. To identify Th. ponticum chromatin in wheat genetic background more accurately and efficiently, genome specific repetitive sequences were cloned from Th. ponticum.【Method】Screening the plasmid library of Th. ponticum via dot-blot hybridization, combining with fluorescence in situ hybridization (FISH) analysis of the wheat-Th. ponticum partial amphiploids, Th. ponticum genome specific repetitive sequences were isolated and their distribution patterns on chromosomes were characterized by FISH. The Th. ponticum genome specific repeats were analyzed by Repeat Masker, Triticeae Repeat Sequence Database and NCBI. A set of PCR primers was designed for detecting Th. ponticum chromosomal fragments. Wheat-Th. ponticum hybrid derivatives were employed to testify the genome specificity and effectiveness of these repeats in identifying Th. alien fragments in wheat genetic background. 【Result】Seven Th. ponticum genome specific repetitive sequences were obtained. Their FISH signals dispersively covered all chromosomes in Th. ponticum and Th. intermedium. Furthermore, they could discriminate chromosomes of Th. ponticum and Th. intermedium from wheat chromosomes without wheat DNA block by FISH. This was further proved in wheat-Th. ponticum substitution lines and translocation lines. Ten PCR primers were developed for efficient amplification of Th. ponticum segments in wheat. Their detection efficiency ranged from 73.3% to 95% in 109 wheat-Th. ponticum derivatives. 【Conclusion】Th. ponticum genome specific repetitive sequences were isolated, which can be used for detection of wheatgrass fragments in wheat by FISH or PCR amplification.

Key words: T. aestivum, Th. ponticum, genome specific repetitive sequences, FISH, PCR

姚涵，汤才国，赵静，郑琪，李滨，郝晨阳，李振声，张学勇. 偃麦草基因组特异重复序列的分离与应用[J]. 中国农业科学, 2016, 49(19): 3683-3693.

YAO Han, TANG Cai-guo, ZHAO Jing, ZHENG Qi, LI Bin, HAO Chen-yang, LI Zhen-sheng, ZHANG Xue-yong. Isolation of Thinopyrum ponticum Genome Specific Repetitive Sequences and Their Application for Effective Detection of Alien Segments in Wheat[J]. Scientia Agricultura Sinica, 2016, 49(19): 3683-3693.

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