Table_7_Accurate Phylogenetic Relationships Among Mycobacterium bovis Strains Circulating in France Based on Whole Genome Sequencing and Single Nucleotide Polymorphism Analysis.XLSX

In recent years the diversity of the French Mycobacterium bovis population responsible for bovine tuberculosis (bTB) outbreaks since 1970 has been described in detail. To further understand bTB evolution in France, we used single nucleotide polymorphisms (SNPs) based on whole genome sequence versus classical genotyping methods in order to identify accurate phylogenetic relationships between M. bovis strains. Whole genome sequencing was carried out on a selection of 87 strains which reflect the French M. bovis population’s genetic diversity. Sequences were compared to the M. bovis reference genome AF2122/97. Comparison among the 87 genomes revealed 9,170 sites where at least one strain shows a SNP with respect to the reference genome; 1,172 are intergenic and 7,998 in coding sequences, of which 2,880 are synonymous and 5,118 non-synonymous. SNP-based phylogenetic analysis using these 9,170 SNP is congruent with the cluster defined by spoligotyping and multilocus variable number of tandem repeat analysis typing. In addition, some SNPs were identified as specific to genotypic groups. These findings suggest new SNP targets that can be used for the development of high-resolving methods for genotyping as well as for studying M. bovis evolution and transmission patterns. The detection of non-synonymous SNPs on virulence genes enabled us to distinguish different clusters. Our results seem to indicate that genetically differentiated clusters could also display distinctive phenotypic traits.

近年来，1970年以来引发法国牛结核病（bovine tuberculosis, bTB）暴发的牛分枝杆菌（Mycobacterium bovis）种群多样性已得到详尽阐释。为进一步解析法国地区牛结核病的演化规律，本研究采用基于全基因组测序的单核苷酸多态性（single nucleotide polymorphisms, SNPs）分析方法替代传统基因分型手段，以精准鉴定牛分枝杆菌菌株间的系统发育关系。研究选取87株能够代表法国牛分枝杆菌种群遗传多样性的菌株开展全基因组测序，并将所得序列与牛分枝杆菌参考基因组AF2122/97进行比对。对87株菌株的全基因组进行比对后，共发现9170个存在单核苷酸多态性的位点（至少有1株菌株与参考基因组存在该位点的多态性）；其中1172个位点位于基因间区，7998个位点位于编码序列内，而编码区的多态位点中，2880个为同义突变位点，5118个为非同义突变位点。基于这9170个SNP位点的系统发育分析结果，与间隔寡核苷酸分型（spoligotyping）及多位点可变数目串联重复序列分型所界定的菌株簇高度一致。此外，本研究还鉴定出若干基因型群特异性的SNP位点。上述研究结果可为开发高分辨率基因分型方法提供新型SNP靶点，同时也可用于牛分枝杆菌的演化与传播模式研究。通过对毒力基因上的非同义SNP位点进行检测，本研究得以区分不同的菌株簇。本研究结果表明，遗传分化显著的菌株簇或许也会展现出独特的表型特征。