基于全基因组重测序策略对三疣梭子蟹耐副溶血弧菌抗性相关基因进行挖掘
Mining of Genes Associated with V. parahemolytica of the Swimming Crab Resistance Based on the Whole Genome Resequencing Strategy

副溶血弧菌(Vibrio parahaemolyticus)是造成三疣梭子蟹批量死亡的重要病原之一。随着测序技术发展，基于IIlumina测序及生物信息学技术手段筛选耐病基因已成为可能。本研究利用全基因组重测序的方法对三疣梭子蟹易感群体和耐感群体的肌肉组织进行IIlumina测序，过滤获得51.867G的clean reads。将clean reads数据与已有梭子蟹参考基因组进行比对，覆盖比对率高达85%以上，覆盖深度也达到25X，同时还检测到了36,929个单核苷酸多态性位点(single nucleotide polymorohsms, SNP)和145,790个小片段插入缺少位点(insertion/deletion, InDel)。以上位点通过SNP/InDel频率分布，在染色体上作图进行精细定位，获得了257个SNPs和184个InDels。以上位点进一步通过同义突变筛查，获得了55个SNP位点和32个InDel位点。对87个位点设计引物进行验证，利用一代测序技术和ContigExpress软件对变异位点进行SNP分型，结果表明：与参考基因组相比，55个SNP位点和32个InDel位点中，分别有23个SNP标记和10个InDel标记存在碱基变化；继续利用上述引物，分别在易感群体和耐感群体上进行扩增测序，最终筛选出9个SNP标记和2个InDel标记(P < 0.05)，并瞄定到8个基因上，推测其中有5个基因属于抗逆抗病基因可用于改良三疣梭子蟹优良性状。
Vibrio parahaemolyticus is one of the most important pathogens that cause the batch death of the swimming crab. With the development of sequencing technology, it has been possible to screen disease resistance genes based on Illumina sequencing and bioinformatics technology. In this study, high-throughput Illumina sequencing technology was used to perform a whole-genome resequencing of muscle tissues in susceptible and resistant populations of P. trituberculatus, and filtered to obtain 51.867G clean reads. The sequencing results were compared with the genome of swimming crab. The coverage ratio was above 85% and the coverage depth reached 25X. At the same time, 36,929 single nucleotide polymorohsms and 145,790 Insert/missing mutant fragments were detected. Subsequently, the SNPs/InDels frequency distribution was mapped on the chromosome for fine localization, and finally 257 SNPs and 184 InDels were obtained. We functionally annotated the above results and found that these loci are non-synonymous mutations, mainly concentrated in introns and intergenic regions. We screened the sites obtained from the resequencing of P. trituberculatus genomes, selected 55 SNP sites and 32 InDel sites to design primers for verification, and used the first-generation sequencing technology and ContigExpress software to perform SNP genotyping on mutation sites. The results showed that compared with the reference genome, there were base changes in 55 SNP loci and 32 InDel loci, respectively in 23 SNP loci and 10 InDel loci. Continuing to use the above primers to perform amplification and sequencing on susceptible individuals and resistant individuals, respectively, finally, 9 SNP markers and 2 InDel markers were selected (P < 0.05), and 8 genes were targeted. It was speculated that 5 genes belonged to resistance genes and could be used to improve the good traits of Portulus trisulatus.