菠菜性别决定基因Y的克隆及功能分析

As one of the most important vegetable crops in China, the spinach is a typical dioecious species, which is controlled by sex-determining locus Y. The study on the sex-determining gene in the world is still at the stage of identifying the gender-related gene molecular markers and gene mapping. However, there is no report on cloning the Y gene and elucidating the sex determination mechanism. It is very important for us to further cloning the Y gene to be used for spinach breeding in China. Till now, our research group has constructed well the first spinach genome-wide genetic map with high density, based on large scale marker development by specific length amplified fragment (SLAF) sequencing. Using these markers and BSA method, the Y gene has been mapped in interval region with 0.2 cM on LG 4. The 25 Kb male-specific fragment (MSY) has been identified via re-sequencing 2 female lines and 3 male lines. Our research group will continue to verify the MSY information via re-sequencing and assembling contigs of the male and female material Denovo, and to further identify the Y candidate gene, via sequencing RNA-seq, miRNA-seq and comprehensively analyzing the differentially expressed genes between male and female lines at flower organ development stage. Then, the functional verification will be done via constructing the plant expression vector and transferring this candidate gene to Arabidopsis. Finally, the interaction relationship between the Y gene and the flower organ development pathway and the mechanism of Y gene will be elucidated based on transcriptome data and the result of Y2H experiment. The result of this project will provide the theory basis for molecular improvement of spinach female line and inbred lines, and will also play an important role in spinach hybrid breeding.

菠菜为典型雌雄异株植物，是我国重要的蔬菜作物之一，其性别遗传受性别决定基因Y控制。国内外对菠菜性别研究还在性别相关基因分子标记及定位阶段，性别决定基因Y还未克隆，性别决定机制有待阐明。项目组前期构建了菠菜首张高密度遗传图谱，利用BSA法将Y基因定位在4号连锁群0.2cM区间内，结合5份雌雄株重测序初步确定了25Kb的雄性特异片段（MSY）。本项目拟开展菠菜雌雄材料Denovo测序、组装，进一步确定MSY片段；开展菠菜雌雄材料RNA及小RNA测序，分析雌雄花器官发育中的差异表达基因，将MSY片段中差异表达基因确定为Y候选基因；利用候选基因构建植物表达载体，通过拟南芥转基因进行功能验证；结合转录组数据，深入分析Y基因与花器官发育通路基因的相互作用关系，通过酵母双杂等实验进行验证互作关系，对Y基因进行功能分析，为菠菜雌性系及自交系分子改良提供理论依据，对菠菜杂交育种具有重要的科学意义和应用价值。

菠菜为典型的雌雄异株植物，是我国重要的蔬菜作物之一，其性别遗传受性别决定基因Y控制。对菠菜性别的研究不仅有助于菠菜的杂交育种工作，而且对植物性染色体的起源分化研究具有重要的意义。项目组基于5份雌株和5份雄株菠菜的重测序数据的比对，除了在4号染色体鉴定到25Kb的雄性特异片段（MSY）外，在6个scaffold（SpoScf_03028:15001_30000，SpoScf_03123:1_30000，SpoScf_03249:15001_30000，SpoScf_03254:1_15000，SpoScf_04677:1_10294，SpoScf_05066:1_8043）上同样检测到MSY 片段。为了获得完整的MSY片段，项目组利用ONT三代测序技术对一份雌株（XX）和一份雄株（YY）材料进行了40×测序和 denovo组装工作，利用Nextdenovo组装软件结合高密度连锁图将XX基因型和YY基因型组装到978Mb和926Mb，N50分别达到34Mb和41Mb。通过X染色体与Y染色体的序列比对，鉴定到一个约23 Mb的性别决定区域，其中包括2个倒位片段和一个10 Mb的MSY片段，在10 Mb的MSY片段预测到100个候选基因。此外，项目组对高代自交系材料“Sp55”的雌株和雄株进行转录组测序，共鉴定到2278个差异表达基因，其中包括1401个上调基因和877个下调基因。在2278个差异表达基因中，182个基因属于转录因子家族，26个基因属于花器官发育调控基因。KEGG通路分析表明油菜素内酯合成的5个关键基因均为下调基因，显示了油菜素内酯可能参与了调控菠菜的雌雄性别分化。以上100个候选基因的功能验证以及下游基因的调控网络分析仍在进行，为下一步菠菜性别决定基因的确定及调控的机制研究奠定基础。