Abstract:

Objective: In this study, we investigated the genetic variations of Bursaphelenchus xylophilus in Guangdong and Jiangsu Provinces, two major dispersal centers of the pest in China, in order to provide fundamental information for tracking the epidemic source of pine wilt disease and establishing effective measures to control pine wilt disease. Method: The whole genome of all B. xylophilus strains was re-sequenced by Illumina high-throughput sequencing platform with coverage>40×. The possible adapters were removed by Cutadpapt software after the sequencing result was filtered by FastQC pipeline. All sequenced data were aligned to the B. xylophilus genome by BWA software, and then Samtools and Picard were used to sort and remove redundancy from the aligned results. Freebayes was used to perform SNP calling and VCFtools was used to summarize SNP calling results derived from SNP types and genotypes. SNPRelate package was employed to construct PCA and a phylogenetic tree. Pyrosequencing method was also used to validate the accuracy of our sequencing and analysis results. Result: A total of 24 B. xylophilus strains were isolated from the infected wood in Guangdong and Jiangsu, with 12 strains from each Province. A total of 14 559 107 SNPs loci were found in all sequenced strains, including 12 990 503 SNPs loci in Guangdong and 2 109 778 SNPs loci in Jiangsu. Detailed analysis indicated that the SNP counts and homozygotes were significantly higher in Guangdong strains than those in Jiangsu strains. Moreover, the mutation genotypes with highest frequencies found in most of Guangdong strains were A→G、C→T、G→A、T→C, while those found in all of Jiangsu strains, as well as GD09 and GD12, were A→G、C→G、G→C、T→C. Cluster analysis demonstrated a notable genetic differentiation in most of the strains between Guangdong and Jiangsu. All B. xylophilus strains were clearly classified into 2 different groups in accordance with their origins, except for GD09 and GD12 which were classified into Jiangsu group. It was speculated that GD09 and GD12 isolated from Guangdong might be transmitted from Jiangsu through human activities. The subsequent pyro-sequencing results proved the validity of our sequencing and analysis results. Conclusion: There is obvious genetic differentiation of B. xylophilus strains between Jiangsu Province and Guangdong Province. Based on the occurrence timeline of B. xylophilus in China, it is speculated that most of the strains in the two Provinces might have different origins and ancestors. The B. xylophilus strains in Shaoguan and Shantou of Guangdong shared the same transmission source as those in Jiangsu province.

Key words: Bursaphelenchus xylophilus, SNP, genetic diversity, population differentiation