玉米胚性愈伤组织再生能力的遗传基础及分子调控机制解析

Regeneration of embryonic callus is a crucial procedure in maize genetic transformation. Scarcity of genetic resources with high regenerative capacity limits the revelation of maize gene function and the development of transgenic breeding in China. In this study, we plan to identify the significant quantitative trait nucleotides (QTNs) in a natural population of 342 inbred lines by genome-wide association study (GWAS) using 650,000 SNP markers. Meanwhile, the four inbred lines with extreme phenotypes selected from the population will be applied to develop two independent RIL (recombinant inbred line) populations. High density SNP markers derived from restriction-site-associated DNA sequencing (RAD-seq) will be then utilized for genetic typing of the RIL populations and QTL mapping for callus regenerative ability. The candidate genes will be identified by combination of the results from GWAS and QTL mapping, according to gene function annotations. We will further perform candidate gene association analysis based on the natural population and validate the co-segregation between superior allele and high regenerative capability dependent on the RIL populations. Functional validation of candidate genes will be performed using over-expression and CRISPR/Cas9 (or mutants). Finally, expression QTL (eQTL) analysis and chromatin immunoprecipitation-sequencing (ChIP-Seq) as well as the completed RNA-Sequencing (RNA-Seq) would be used to reveal the regulatory network of the functional gene(s). For the present project, the phenotypic investigations of the three populations in three environments have been accomplished, and the association study has also been initiated. These all contribute to the completion of the project.

胚性愈伤组织再生是玉米遗传转化技术的关键环节之一，而我国高再生力玉米种质资源匮乏，阻碍了玉米基因功能研究和转基因育种的发展。本项目采用65万个SNP标记对342份玉米自交系愈伤组织再生能力进行全基因组关联分析，鉴定与目标性状关联的QTN；同时从自然群体中筛选4份极端材料构建2个独立的RIL群体，利用简化基因组测序(RAD)开发高密度SNP标记对群体进行基因分型，开展目标性状的QTL定位；拟合QTN和QTL结果，结合基因功能注释挖掘候选基因；再次用自然群体进行候选基因关联分析，明确基因内显著关联位点；然后用RIL群体验证优异等位位点与优异表型之间的共分离；采用过表达、CRISPR/Cas9 （或突变体）验证基因功能；最后利用eQTL、ChIP-Seq和已有的转录组数据建立功能基因表达调控网络。其中，三个群体在三环境下的表型鉴定已完成，并初步开展了GWAS分析，为任务目标顺利完成奠定了基础。

玉米愈伤组织再生能力是影响玉米遗传转化效率的重要因素，是转基因玉米育种和功能基因研究的关键。然而，玉米愈伤组织再生能力表现出严重的基因型限制，因此，揭示其遗传基础并鉴定控制再生能力的关键基因有助于突破玉米遗传转化的基因型限制，加速转基因玉米育种进程和功能基因组研究。本项目构建了3个高代玉米遗传重组群体，并利用重组群体和自然群体开展了玉米愈伤组织再生相关性状的遗传结构解析，检测到18个控制目标性状的QTL和35个显著关联的SNP。结合遗传定位和共表达网络构建鉴定到2个调控玉米愈伤组织再生能力的核心基因，通过关联分析挖掘了核心基因的优异单倍型。通过遗传学和分子生物学初步验证了核心基因Zm00001d038862和Zm00001d043651的功能，它们分别编码细胞分裂素磷酸核糖水解酶和色氨酸氨基转移酶，是细胞分裂素和生长激素合成的关键酶，从而影响愈伤组织内源细胞分裂素和生长激素的含量，调控玉米愈伤组织的再生能力。Zm00001d038862和Zm00001d043651上游分别受转录因子ERF2和CUC3调控。项目成果为培育高愈伤组织再生率玉米材料提供了基因资源和分子标记资源，推动玉米转基因领域的发展，相关成果在国际知名学术期刊上发表论文共12 篇，SCI累计影响因子67.25，其中IF > 6的共4篇，单篇最高影响因子14.224；获授权发明专利2项；培养博士研究生3名、硕士研究生5名、国家高层次特殊支持计划青年拔尖人才1人，1名讲师晋升副教授。