关键词: 广州管圆线虫, 全基因组测序, 性别推断, 群体遗传学

Abstract:

Objective To investigate the genome-wide genetic differentiation between male and female Angiostrongylus cantonensis populations based on whole-genome sequencing, establish a gender inference method for unknown samples, and explore the functional characteristics of potential sex-related genomic regions. Methods A total of 20 adult worms of morphologically identified A. cantonensis were obtained, including 10 males and 10 females. Whole-genome sequencing of adult worms was performed, and single nucleotide polymorphism (SNP) data were subjected to principal component analysis, population structure analysis, nucleotide diversity (π) analysis, and population differentiation index (F_ST) analysis. Candidate X-linked assembly sequences were screened through the difference in sequencing coverage between males and females, and candidate genes in sex-related regions were subjected to functional annotation and gene ontology (GO) enrichment analysis to decipher their potential biological functions. A gender determination threshold was defined by the ratio of the average sequencing depth of X-linked assembly sequences to the average sequencing depth of autosomal regions (X/A). The inference strategy was tested using a training set and a validation set (10 samples each), and the effectiveness was evaluated using machine learning tools. Results Whole-genome analysis revealed no stable gender clustering of male and female worms, with similar overall genetic diversity, consistent changing trends of π values, and similar overall fluctuation range. The average whole-genome F_ST was -0.005 8, and the weighted F_ST was -0.002 7, both approaching to 0. A few windows exhibited significantly increased F_ST values, with multiple extreme high values reaching up to 1.0. Based on coverage analysis screening, 2 631 candidate X-linked assembly sequences with coverage ratios ranging from 0.4 to 0.6 were yielded. The significantly enriched entries in candidate genes involved processes such as chromatin structure regulation, nucleosome assembly, and DNA helicase activity regulation, and the enriched entries shared linker histone H1 family proteins such as H1FOO and H1F0. In the training set, the average X/A ratios of female and male samples were 1.808 ± 0.090 (range, 1.659 to 1.879) and 0.977 ± 0.078 (range, 0.879 to 1.080), with a significant deviation in the distribution intervals, and an X/A coverage ratio of 1.4 was defined as a gender determination indicator. In the validation set, the X/A ratios of female samples were all above the threshold (mean, 1.852 ± 0.047; range, 1.809 to 1.924), while the ratios of male samples were all below the threshold (mean, 1.137 ± 0.073; range, 1.042 to 1.217). Among the 20 samples, the X/A ratio of female samples was higher than that of male samples (t = 18.582, P < 0.01). The classification results using machine learning tools further supported that the X/A coverage ratio had a high stability and accuracy for sex identification. Conclusion Male and female A. cantonensis populations show no remarkable genetic differentiation at a whole-genome level, and the X/A coverage ratio-based approach is stable for sex identification. Chromatin regulation-related genes enriched in candidate sex-related regions suggest their potential involvement in sex-related chromosome structure formation and epigenetic regulation.

Key words: Angiostrongylus cantonensis, Whole-genome sequencing, Gender inference, Population genetics