马铃薯单倍体诱导基因克隆及功能验证

Haploid induction technology in potato is of great potential in breeding. The bottleneck of this technology lies in the selection of lines with high induction rate (HIR). Reduced ploidy by the haploid induction is also of great significance for the basic research. Studies on the potato haploid induction line itself have lagged behind in recent years, and related haploid induction gene has not been cloned, becoming an important factor limiting this technology and fundamental researches. In this study, relatively excellent haploid induction line through parthenogenesis by ourselves will be used for the following researches: (1) Different tissues during flowering will be used for PacBio sequencing and each tissue will be used for RNA-Seq in haploid induction liens and non-haploid induction lies. The induction genes in potato induction lines will be excavated based on the conserved sequence and tissue-specific expression characteristics in maize and other species, and also the expression characteristics of transcript in each tissue will be analyzed. (2) Sequences of haploid induced genes will be got according to homologous cloning methods. (3) CRISPR/Cas9 will be employed for function validation of haploid induced gene, with the possibility of obtaining HIR lines. The project will provide the theoretical basis and technical support for selecting HIR line.

马铃薯单倍体育种技术是一个具有巨大潜力的育种方法，该技术的瓶颈在于高频诱导系的获得；通过单倍体诱导获得倍性降低的材料对于基础研究同样具有重要意义。近年来，对于马铃薯单倍体诱导系本身的研究较为落后，单倍体诱导相关基因也未被克隆，成为限制该项技术和基础研究的重要因素。本研究将以自行选育的较为优良的马铃薯孤雌生殖单倍体诱导系为材料，从以下研究内容着手：（1）在马铃薯单倍体诱导系和非诱导系的开花期取不同组织进行PacBio测序和分组织RNA-Seq，基于玉米等物种中诱导基因的保守性及组织特异性表达特点，挖掘马铃薯诱导系中的诱导基因，分析各组织转录本表达特征；（2）依据同源克隆的方法对单倍体诱导基因进行克隆；（3）基于CRISPR/Cas9进行单倍体诱导基因功能验证并创制高频诱导系。通过以上研究，本项目将为提高单倍体诱导系的诱导率提供理论依据和技术支持。

马铃薯单倍体育种技术是一个具有巨大潜力的育种方法，同时，通过单倍体诱导系获得倍性减半的材料可以降低基础研究的复杂性，有助于解析马铃薯重要农艺性状，但是高频诱导系的缺乏限制了这两方面的应用。近年来，玉米、水稻、小麦等作物上克隆了单倍体诱导系，而马铃薯单倍体诱导基因还未被克隆，限制了马铃薯单倍体育种和马铃薯单倍体诱导基因的理论基础研究。在前期工作中，本课题自行培育了单倍体诱导系IVF1，对该材料在开花期取不同组织进行Pacbio测序和分组织RNA-Seq，对比玉米等物种中的诱导基因和组织特异性表达，克隆了马铃薯单倍体诱导基因，分析其亚细胞定位，构建基因编辑载体转化后得到1株阳性苗。同时基于群体的表型鉴定筛选得到一个诱导率较IVF1高2-3%的诱导系，且该材料克服了长日照不结薯的缺点。本项目的研究进展为马铃薯单倍体育种的应用和单倍体诱导基因的分子机理奠定了理论基础。