菠菜Y染色体雄性特异区域（MSY）的克隆及性别决定候选基因的功能鉴定

The structure and evolution of sex chromosome and the cloning of the sex-determining genes are the hot issues in the research of the current reproduction biology. MSY is the male specific region of Y chromosome, which is the important area for revealing the molecular mechanism of plant sex chromosome evolution and cloning the sex-determining genes. With early stage sex chromosome, the dioecious spinach, is the model materials for the research on plant sex chromosome evolution. In this study, the BAC library of the spinach YY genome was constructed and screened by using the primers designed by sex cosegregating markers, the X chromosome counterparts DNA, and the parts of Male-Specific Regions of Y chromosome (MSY) in spinach. After being verified by Fluorescence In Situ Hybridization (FISH) to the Y or X chromosome, the positive clones were then sequenced, assembled and joined to form the whole length of MSY region in spinach. Based on this, combined with the early stage transcripts of spinach male and female flower, the sex-determining candidate genes will be functionally identified. At the same time, compared with the X chromosome counterpart, the content, distribution and difference of repetitive DNA sequences on the MSY region of spinach will be revealed. The role of repetitive DNA sequences on sex chromosome evolution will be discussed according to the fluorescence signals distribution of the repetitive DNA sequences on the Y chromosome, X chromosome and autosomes. The project will contribute to our understanding the molecular mechanism of early stage sex chromosome evolution and the cloning of sex-determining genes in spinach.

性染色体的结构与演化及性别决定基因的克隆是当前生殖生物学研究的热点问题。MSY是Y染色体雄性特异区域，该区域是揭示植物性染色体的演化及获得性别决定基因的重要区域。菠菜是雌雄异株、处于性染色体演化早期阶段的植物，是研究植物性染色体演化非常好的模式材料。本研究以菠菜为材料，利用菠菜性别连锁分子标记和已克隆X染色体性别决定区、已鉴定的部分MSY区域序列设计引物，筛选菠菜YY株基因组BAC文库，对阳性克隆进行FISH验证及测序、序列拼接，获得菠菜MSY区域全长。在此基础上，结合菠菜雄花、雌花发育早期阶段转录组，确定菠菜性别决定候选基因并进行功能鉴定。同时MSY区域序列与X染色体对应区域序列进行比对分析，揭示重复序列含量、分布、序列差异，探讨重复序列在性染色体演化中的作用。本项目对研究菠菜性染色体早期演化分子机制及获得性别决定基因具有重要意义。

性别决定候选基因的鉴定、重复序列在性染色体演化中作用探讨以及性别决定区的鉴定对于探讨雌雄异株植物性染色体演化分子机制至关重要。菠菜是研究性染色体演化的模式材料，因此本项目以菠菜为材料，主要进行了以下几个方面的工作：对菠菜雌、雄花发育阶段进行二代转录组（RNA-seq）及三代转录组测序，筛选性别决定候选基因；对菠菜栽培种、野生种基因组DNA进行重测序，探讨逆转座子序列和Satellite序列在菠菜雌雄基因组之间、性染色体演化过程中的作用；利用单染色体显微分离与单细胞测序技术结合的方法对菠菜野生种S. turkestanica进行了X/Y染色体的组装，鉴定性别决定区。研究结果表明：初步筛选到性别决定候选基因SpDYT1、SpMYB35、SpAMS、SpMS1、SpAGL18、SpWUS、SpAGL11、SpWOX1、SpYAB4，并且对基因SpDYT1、SpMS1以及SpWUS的功能进行了研究；菠菜属植物基因组中的重复序列比例远远高于甜菜基因组的重复序列比例；Spanicia tetrandra中重复序列所占比例（平均为53.41%）高于S. oleracea（平均为50.5%）和S. turkestanica（平均为49.56%）；菠菜属雌雄株基因组重复序列比率差异不显著；1号染色体上卫星DNA的积累可能在菠菜属植物的性染色体进化中起着重要作用；已获得X染色体二代测序数据32.19 G、三代测序数据14.69G，获得Y染色体二代测序数据46.99G、三代测序数据18.07G；利用Wtdbg2软件对三代测序结果进行组装，目前组装的X染色体长度为14 355 374 bp，Y染色体长度为27 762 727 bp；性别决定区的鉴定正在进行之中。研究结果为进一步揭示菠菜性染色体演化分子机制提供了重要的理论资料。