花生自主型DNA转座子派生MITEs的分离及其标记开发利用

Peanut is one of the most important oil and economic crops in the world. Due to its narrow genetic background, high efficient and useful molecular markers were obviously insufficient which restricted the process of peanut molecular breeding. Miniature inverted repeat transposable elements (MITEs), most likely derived from the autonomous DNA transposons, had been proved to be very easily and valuable tool used for the development of the insertion sites specific molecular markers owing to the characteristics of the widely distribution in plant genome, high copy number in plant genome and small size in fragments. So in this project, based on the previous isolated conserved sequences of the peanut autonomous DNA transposons, the full length of the autonomous DNA transposons were isolated using chromosome walking technique. Then the peanut MITE transposons were amplified from the peanut genome through ‘top-down’ strategy with single primer designed according to the TIR sequences of the corresponding full length autonomous DNA transposons. Using the justly isolated MITE sequences and MITE sequence inserted in FAD2B gene as probes, a large number of MITE transposons and their flanking sequences were isolated from the peanut genomic library using magnetic bead-probe capture technique. Finally, the MITE transposons markers were developed and employed to reveal the DNA polymorphism of cultivated peanut and explored the phylogenetic relationships of genus Arachis. It aims both to enrich the type and the number of peanut molecular markers; and to introduce much more efficient and useful molecular markers into peanut for laying the foundation for peanut genetic improvement and molecular breeding.

花生是世界上重要的油料作物和经济作物之一，其遗传基础狭窄，目前分子标记的种类和数量明显不足，进而制约了花生的分子育种进程。微型反向重复转座元件（MITEs）多来源于自主型DNA转座子，广泛分布于植物的基因组中，拷贝数多、片段小，易被用来开发插入位点特异标记。本项目在前期分离到花生自主型DNA转座子保守区序列的基础上，利用染色体步移技术扩增分离出其全长序列，基于“top-down”策略根据其TIR序列设计单引物从基因组中分离出MITE转座子序列，利用该序列及FAD2B基因中插入的MITE序列为探针，采用磁珠探针吸附法从花生基因组文库中吸附分离出一批MITE转座子并获得其侧翼序列，最后开发出花生MITE转座子标记并利用其研究栽培种花生四大类型的DNA多态性和花生属的系统发育。项目的完成将丰富花生分子标记的种类和数量，为花生上增加更多高效实用的分子标记，为花生的遗传改良和分子育种奠定基础。

花生是世界上重要的油料作物和经济作物之一，其遗传基础狭窄，目前分子标记的种类和数量明显不足，进而制约了花生的分子育种进程，而微型反向重复转座元件（MITEs）多来源于自主型DNA转座子，广泛分布于植物的基因组中，拷贝数多、片段小，易被用来开发插入位点特异标记。本项目已通过同源克隆获得多条栽培种花生五大类自主型DNA转座子的保守区序列，并通过同源检索生物信息学分析策略鉴定出大量自主型DNA转座子全长序列，已获得MITE转座子探针序列并采用全新生物信息学分析策略获得大量花生MITE转座子，已设计大量引物对并开发出花生MITE转座子分子标记，对花生属栽培种和野生种进行了分子评价，揭示了花生属丰富的多态性，通过建立的全新生物信息学分析策略获得了来自自主型DNA转座子的大量MITE转座子，明确了自主型DNA转座子和MITE转座子之间的派生起源关系。此外，还特意额外地在花生上率先尝试完成了大量跟本项目直接相关的基础研究，获得了大量研究结果和基础数据，并在研究过程中产生了一系列跟本项目密切相关的创新性思想和新技术，有些创新性思想和新技术已经完全成熟并具体方案化。本项目以第一作者及通讯作者身份发表了3篇研究论文，以第一发明人身份申请发明专利7件（已获授权4件）和实用新型专利9件（已获授权8件），以第一发明人身份登记1项计算机软件著作权，主要参与的三项成果分别获得广西科技进步奖二等奖、广西农业科学院科技进步奖一等奖和二等奖，主要参与制定和颁布广西地方标准8项，主要参与育成6个花生新品种并以第一完成人身份选育出5个优良花生新品系，培养花生分子生物学研究学术骨干4名，储存了一批花生MITEs转座子研究的关键技术。本项目所取得的研究结果将丰富花生分子标记的种类和数量，为花生上增加更多高效实用的分子标记，将为下一步花生MITE转座子分子标记的广泛开发及今后花生的遗传改良和分子育种奠定基础。