紫薇显性叶色基因LiGL1的精细定位和克隆

Lagerstroemia indica is a popular woody ornamental plant throughout the world. Leaf coloring is an important direction of landscaping industry transformation and upgrading, but only a few studies of leaf color have been reported in Lagerstroemia indica. The mutant GL1 is the only dominant yellow leaf mutant in Lagerstroemia indica in the world, which is important to illuminate the regulate mechanism of chlorophyll synthesis and photosynthesis. The mutation controlled by a dominant nuclear gene (LiGL1), had been mapped by two SNP markers with 2.3 cM genetic distance. In this study, genome survey of mutant was sequenced by next generation sequencing. The two SNP markers will be mapped to survey sequences. Base on those sequences, new markers will be developed to fine map the gene LiGL1 with a large segregating population..One BAC library of mutant will be constructed. The markers closely linked to LiGL1 will be screened the library. The target BAC clone will be sequenced using high throughput sequencing. Genetic transformation of Lagerstroemia indica will be to identify gene function. This study is mainly to solve the fine mapping and cloning of LiGL1, which has an important theoretical significance for improving leaf color regulate mechanism and photo-protection. The study will lay a foundation for the cultivation of new colorful varieties in Lagerstroemia indica by gene engineering. Meanwhile, the genome sequences will play an important role in the markers development and gene cloning.

紫薇是世界上重要的观赏植物，叶色是其重要观赏性状。植物彩叶化是园林绿化产业转型升级的重要方向，但紫薇叶色研究相对较少。紫薇显性金叶突变体GL1是世界上唯一黄叶突变体，对研究叶片呈色机理具有重要价值。申请人经过持续研究确定其受1对显性核基因（LiGL1）控制，初步定位在2.8 cM的分子标记区间。本研究拟利用“2+3”代测序完成突变体的genome survey，比对到与LiGL1连锁的分子标记后，定向开发新标记结合已构建的大分离群体精细定位LiGL1；构建突变体的BAC文库，连锁标记筛选确定目标BAC，目标BAC经高通量测序后结合qRT-PCR确定候选基因；候选基因转化紫薇进行验证，初步明确其功能。本研究主要解决LiGL1的精细定位和克隆的问题，对完善植物叶色调控机理及光保护具有重要的理论意义，也为基因工程培育紫薇新彩叶品种奠定基础。同时，获得的基因组序列对标记开发、基因克隆等有重要作用。

叶色是观赏植物的重要性状，紫薇是千屈菜科紫薇属中适应性最广、观赏价值最高的园林植物，但是叶色形成机制的研究滞后，限制了彩叶紫薇品种的培育。本项目利用组学、分子生物学、分子遗传学等技术和方法，在世界上率先完成紫薇（2n=48）全基因组测序，图位克隆了叶色基因LiGL1候选基因，初步解析其调控叶色的分子机制，成果包括：（1）利用Illumina+PacBio+Hi-C技术完成紫薇金叶品种‘金幌’全基因组测序，获得了368.39 Mb的染色体水平的参考基因组，重复序列为158.3 Mb，占基因组的43.0%，LTR是最主要的转座子。鉴定了33,357个编码蛋白基因和775个非编码RNA，注释32,541个基因，紫薇经历了三次全基因组复制事件。紫薇的24条染色体间存在部分同源关系，可以明确分为8组，与石榴的8条染色体高度同源。（2）利用BSA-seq数据将LiGL1定位在Chr13的0.77 Mb候选区间内，SSR标记和F1群体将LiGL1定位在6.42kb区间内，仅1个基因Lind13G0831，注释为Rab11A。（3）野生型与‘金幌’的基因序列分析发现在编码区存在3个SNP位点的突变，均为T至C，前2个SNP突变导致T至M和A至V的氨基酸突变，qPCR表明Lind13G0831的表达随着叶色的加深逐渐降低，互补试验发现‘金幌’叶色恢复为绿色而基因编辑研究发现叶色由绿色变为黄色，证明Lind13G0831为LiGL1的候选基因。（4）‘金幌’与野生型转录组分析发现差异基因显著富集在‘蛋白质磷酸化’、‘细胞壁生物合成’、‘核酸结合转录因子活性’等，Lind13G0831与MYB、SPL、CHLH等基因具有共表达趋势表明Lind13G0831可能通过光响应和叶绿素代谢途径信号调控紫薇叶色变化。本项目不仅发现并阐明了一条新的叶色调控通路，而且也为紫薇彩叶品种的精准育种提供了新的基因资源。