基于BSA混池测序分析开发胡杨性别鉴定的标记

A Novel Method for Gender Identification of Populus euphratica Oliv. Based BSA Mixed Pool Sequencing Analysis

摘要: 胡杨是中国西北地区重要的乔木树种，其在新疆的分布最为广泛，具有较强的耐旱、耐寒、抗盐碱的能力，在遏制沙漠扩展、保护生物多样性、保障工农业生产生态安全等诸多方面具有不可替代的作用。为了能够在胡杨幼苗阶段对雌雄个体进行性别鉴定，本研究提取雌雄胡杨叶片高质量的基因组DNA，分别构建文库以进行Illumina、PacBio和Hi-C测序。基于PacBio测序得到的reads进行初步组装。并采用Illumina测序得到的reads对组装结果进行纠错得到草图版本的基因组。利用Hi-C测序技术将雌雄基因组组装，对雌雄基因组进行注释、评估，获得到染色体水平的高质量雌雄基因组序列。基于BSA混池测序分析，以胡杨雄株高质量基因组为参考，对96个雌株和97个雄株的BSA-seq数据进行关联分析，筛选出雌性混池深度连续为0，同时雄性混池深度连续大于10的区域。对这些区域提取包括上下游150 bp在内的序列作为性别鉴定候选区域，将性别候选区域与胡杨高质量雌株基因组进行Blast同源比对，根据性别候选区最终筛选出胡杨雌雄共有的3对特异性引物和雄性性别连锁的4对特异性引物。结果表明胡杨待测样品在雌雄共有引物对扩增出条带的基础上，若扩增出100~300 bp大小的特有条带则为雄性，若未扩增出目标条带，则为雌性。这为在幼苗期对胡杨性别进行鉴定提供了重要的理论依据，为解决行道树旁胡杨飞絮污染问题和商业化种植与应用提供了可靠的解决方案。

Abstract: Populus euphratica is an important tree species in northwest China, among which Xinjiang is the most widely distributed, with strong drought, cold, and salinity resistance. It plays an irreplacea-ble role in many aspects such as curbing the expansion of deserts, protecting biodiversity, and ensuring the ecological security of industrial and agricultural production. To enable sex identifi-cation of male and female individuals at the seedling stage of P. euphratica, high-quality genomic DNA from male and female P. euphratica leaves was extracted, and libraries were constructed for Illumina, PacBio, and Hi-C sequencing. Preliminary assembly was performed based on the reads obtained by PacBio sequencing, and the reads obtained by Illumina sequencing were used to correct the assembly results to obtain a draft version of the genome. The male and female genomes were assembled by Hi-C sequencing technology, and the male and female genomes were annotated and evaluated to obtain high-quality male and female genome sequences at the chromosome level. Taking the assembled male genome as a reference, the BSA-seq data of 96 female plants and 97 male plants were subjected to association analysis using the BSA mixed pools. The candidate sequences of sex identification are where the depth of the female pool was continuously 0, while the depth of the male pool was continuously greater than 10 was obtained. Sequences including upstream and downstream 150 bp were extracted from these regions as candidate gender markers. Blast the candidate sequences to the assembled female genome. Finally, three pairs of specific primers shared by male and female and four pairs of specific primers linked by male sex were designed according to the screened sex candidate sequences. The results show that, On the basis of amplifying the bands from the male and female common markers, if a 100~300 bp male-specific marker band is amplified, it will be a male; if the target band is not amplified by the primer pair of the male marker, it will be a female. These provide an important theoretical basis for the sex identification mechanism of P. euphratica in the seedling stage, and provide a solution for solving the pollution problem of flying fuzz seeds near street trees and commercial planting and application.