中国7个不同地理居群野猪的系统发育研究

doi:10.3864/j.issn.0578-1752.2015.S.007

摘要/Abstract

摘要： 【目的】线粒体DNA是真核生物细胞内唯一存在的核外遗传物质，具有相对恒定且不同于核DNA的生物信息量。线粒体DNA序列研究分析为物种的起源和进化研究提供了丰富的理论依据。因此，研究通过分析国内外的野猪线粒体基因组差异，了解中国不同地理居群野猪遗传资源情况，阐明不同亚种之间的亲缘关系。【方法】选取内蒙古、四川、陕西、浙江、江西、湖南、福建7个省或自治区共计26头野猪耳组织/尾组织，通过酚-氯仿法抽提基因组DNA，取3 µg DNA，利用Covaris System(Life Technologies) 打碎至200—800 bp片段，经末端配对，加A-尾巴，以插入片段500 bp建库。使用Hiseq-2500(Illumina)平台测序。为了比较国内野猪与国外野猪在线粒体基因组上的差异，从NCBI上下载6头欧洲野猪、1头日本野猪、1头苏门答腊岛 (Sumatra) 野猪和4份中国野猪的全基因组重测序数据。利用该12头野猪重测序数据与本研究26头国内7个不同地理居群野猪进行线粒体变异检测。通过比较reads对的平均测序质量，挑选出reads平均质量大于Q30的双末端 reads。通过BWA和samtools，检测38头野猪之间的线粒体全基因组SNP。通过次等位基因频率（MAF≥0.05）对群体SNP进行筛选。利用筛选出的可信SNP位点，利用最大似然法（ML）进行系统进化树的构建、主成分分析（principal component analysis; PCA）和核苷酸多态性分析（nucleotide diversity；π）。【结果】对38头野猪的线粒体全基因序列分析发现，群体之间共检测到226个SNP位点（0.014 SNPs/bp），其中D-loop区域共检测到33个SNP (0.028 SNPs/bp)。通过将不同地理位置的野猪进行分类后，共得到9个组，包括Sumatra，欧洲，日本，陕西，四川，湖南，江西，浙江和内蒙古。通过每个类群的线粒体DNA的GC含量进行分析发现，9个亚群内的GC含量趋于相对稳定，其中A含量平均为34.7%， C含量平均为26.2%， G含量为13.3%，T含量为25.8%。在D-loop区域，相对于线粒体全长来说，A含量减少1%，而G含量增加1%。通过这226个SNP进行PCA和进化树分析发现，欧洲类群和亚洲类群存在明显的分化，说明这两个类群是独立进化的；国内野猪的7个不同地理居群可分为蒙古、川陕、华南野猪3个亚种。通过序列多样性分析发现，结果显示国内不同地区野猪之间的遗传距离较小（0.00024-0.0037），π的平均值为0.0022，而欧洲与中国内地之间的差异较大（0.0044-0.0052），平均值为0.0049，Sumatra和日本与中国内地之间的差异平均为0.0025。说明欧洲野猪相对于Sumatra和日本野猪与中国内地野猪有着较大的序列多样性。【结论】中国野猪虽然存在较丰富的单倍型多态性，但是遗传多样性有逐步丧失的风险，提示要加强中国野猪种质资源的科学规划和保护。

关键词: 野猪, 线粒体DNA, 遗传距离, 系统进化

Abstract: 【Objective】 The mitochondria DNA is the unique genetic material outside the nuclear within eukaryotic cells, having a relatively constant and different bioinformation from the nucleus DNA. The analysis of mitochondria DNA sequence could provide theoretical basis for researching the origin and evolution of the species. Therefore, based on the analysis of mitochondria DNA sequence mutation between different wild boars, we could study the genetic resources of wild boar from different geographical populations, and explain genetic relationships between different subspecies in China. 【Method】A total of 26 wild boars belonging to seven regions, including Inner Mongolia, Sichuan, Shanxi, Zhejiang, Jiangxi, Hunan and Fujian, were selected for DNA extraction using ear tissues and/or tail tissues based on Phenol - chloroform method. For each individual, according to Covaris System (Life Technologies), 3μg of DNA was sheared into DNA fragments of 200-800 bp. The DNA fragments were treated based on the Illumina DNA sample preparation protocol. Fragments were ending repaired, A-tailed, ligated to paired-end adaptors and PCR amplified with 500-bp inserts for library construction. Sequencing was performed to generate 125bp paired-end reads on the HiSeq2500 platform (Illumina). To compare the difference between wild boars in China and other countries, we downloaded the resequencing data of 12 wild boars, including six European wild boars, one Japanese wild boar, one Sumatra wild boar and four Chinese wild boars from NCBI website. We used the 12 resequencing data of wild boars and 26 resequencing data sequenced by ourselves to identify the mutation between the 38 wild boars. Based on the average of sequencing quality of paired-end reads, we filtered out the quality of paired-end reads less than the Q30. To identify the SNPs between 38 wild boars in whole mitochondria DNA sequence, the software BWA and samtools were used in this study. And then, we used minor allele frequency (MAF) more than 0.05 to filter SNP in wild boars population. Based on the reliable SNPs, we used maximum likelihood (ML) to construct the phylogenetic tree, principal component analysis (PCA) and nucleotide diversity analysis (π) in 38 wild boars. 【Result】A total of 226 SNP (0.014 SNPs/bp) were identified in 38 wild boars in whole genome sequence of mitochondria DNA, including 33 SNPs (0.028 SNPs/bp) in D-loop region. Based on different geographical populations, the 38 wild boars could be divided into nine groups, including Sumatra, Europe, Japan, Shanxi, Sichuan, Hunan, Jiangxi, Zhejiang and Inner Mongolia. For the analysis of GC content in whole genome sequence of mitochondria DNA in each group, the GC contents were not much changed, 34.7% for A, 26.2% for C, 13.3% for G and 25.8% for T, respectively. For the D-loop region, compared to whole genome sequence of mitochondria DNA , A content reduced 1% and G content raised 1%. The analysis of phylogenetic tree and PCA show that there was significantly divided into two groups between European group and Asian group. It indicates that the two groups were independent evolution. On the other hand, seven different geographical populations in China could be divided into three subspecies (Mongolian, Sichuan-Shaanxi, and South China wild boar). The results of nucleotide diversity analysis showed that the genetic distance were smaller (average π is 0.0022; range from 0.00024 to 0.0037) amongst the wild boars in China. However, there was large nucleotide diversity (average is 0.0049; range from 0.0044 to 0.0052) between European group and Chinese group. Sumatra and Japan groups were average 0.0025 nucleotide diversity compared to Chinese group. It indicates that comparing to Sumatra and Japanese wild boars, the European ones have large nucleotide diversity with Chinese wild boars. 【Conclusion】 Although there were abundant polymorphism haploid type in Chinese wild boars, the risk of losing the genetic diversity were increasing, which suggested that the scientific planning and protection of germplasm resources should be strengthened.

Key words: wild boar, mitochondrial DNA, genetic distance, phylogenetic relationship

白立景，刘博，李林，牟玉莲，李奎. 中国7个不同地理居群野猪的系统发育研究[J]. 中国农业科学, 2015, 48(S): 58-66.

BAI Li-jing, LIU Bo, LI Lin, MU Yu-lian, LI Kui. Phylogenetic Analysis of Seven Different Geographical Populations of Wild Boar in China[J]. Scientia Agricultura Sinica, 2015, 48(S): 58-66.

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