山桐子(Idesia polycarpa)是我国特有且极具开发潜力的油料树种, 果实含油率高且油品好。在苗期通过分子生物学手段对山桐子进行性别鉴定, 可有效降低种苗培养成本。在前期组装的染色体级别高质量山桐子基因组基础上, 进一步对山桐子雌雄群体进行全基因组重测序, 根据雌雄株系的特异性片段, 确定山桐子属于ZW/ZZ性别决定类型; 并根据3.365×10⁷个SNPs, 结合性别表型进行全基因组关联分析, 找到控制性别的19号染色体, 并将性别决定区段定位到35 Mb附近; 针对显著性位点, 开发了性别共显性CAPS分子标记。该方法可在幼苗期快速、高效鉴定山桐子的性别, 极大地降低了山桐子育苗环节的生产成本, 也可在山桐子种质鉴定及分子标记辅助育种等方面为产业提供技术支撑。

INTRODUCTION Sexual differentiation of plants, as known as dioecy, is the phenomenon of sexually dimorphic traits, which means that the female and male reproductive organs are on different individuals. In agronomic or economic ap- plications, dioecy is not a desired character for various reasons. Therefore, a simple and effective method for distin- guishing female and male individuals is a common goal for the people in many fields. As so far, molecular marker is a gold standard for sex identification genetically. It is necessary to develop a molecular technique to detect sexual systems in the dioecious plants.

RATIONALE In plants, the origin and evolution of sex chromosomes was independent in many species, therefore sex determination region/chromosome was different among them. Idesia polycarpa is a deciduous tree, making it an attrac- tive model system with which to study sex determination mechanism. We characterized the re-sequence data of female and male individuals from natural population. We used these data to identify the sex determination systems by statistics unique sequences in females and males. To detect the sex chromosome, we analysed the single nucleotide polymor- phisms (SNPs) by genome-wide association study (GWAS).

RESULTS The information of genome contains the secret code of various traits, so we mined the re-sequenced data of females and males. We calculated the common and unique reads in females and males, compared the unique reads between them, and found that the unique reads of females were much more than that of males. The result suggested that the sex determination system of I. polycarpa was ZW/ZZ. We then identified 30 million high quality SNPs in the population by mapping to the reference genome, calculated the p value of each SNP, drew a Manhattan plot. The result showed the highest peak on the end of 19 chromosome. We analysed the heterozygote of SNPs in this peak, and the result showed that the SNPs were heterozygotic in females, but homozygotic in males. The result confirmed that the sex determination system in I. polycarpa is ZW/ZZ. Further, a cleave amplified polymorphic sequences (CAPS) mo- lecular marker was developed according to the significant SNPs, and was successfully applied to different female and male individuals.

CONCLUSION In the Salicaceae family, there were two kinds of sex determination system, one is XX/XY (females are homogametic XX, and males are heterogametic XY), another is ZW/ZZ (females are heterogametic ZW, and males are homogametic ZZ). At present, most studies on the sex-determining regions and genes in the Salicaceae family are carried out by methods such as genome, transcriptome, and resequencing analyses. Our results showed that the sex determination system in I. polycarpawas ZW/ZZ, and the 19^th chromosome is most likely associated with sex determination. And a CAPS molecular marker was developed, which is a simple and fast method for efficient identification sex at seedling stage. The research provided a useful way to the planting pattern of I. polycarpa.

Identification of sex determination molecular marker of Idesia polycarpa. Trees of I. polycarpa are indistinguishable as to sex without examination of the flowers. A CAPS molecular marker was developed based on genome wide analysis study on a natural population.