The Comparative Genome Analysis Of Vibrio Alginolyticus Pathogenic Strains For Coral And The Development Of Rapid Detection Method For XSBZ03

Porites andrewsi White Syndrome(PAWS),caused by Vibrio alginolyticus strains(XSBZ03 and XSBZ14),has posed a serious threat to the corals in the South China Sea.The complete genome sequence and comparative genome analysis of XSBZ03 and XSBZ14 were carried out in this paper and a PCR method,based on the IGS sequence analysis,was also established for XSBZ03.The whole genome sequence in this research was carried on the combine of the Illumina Hiseq4000 high throughput sequencing platform and the third generation sequencing instrument Pac Bio RSII system.The corresponding biological information analysis showed that the strain XSBZ03 genome contained 5,126 genes with a genome size of 5,890,205bp and the strain XSBZ14 genome contained 4,705 genes with a genome size of 5,199,476bp.The number of tandem repeat sequences in XSBZ03 and XSBZ14 were 122 and 95.The number of tRNAs were 134 and 130.The number of rRNAs in two strains was 37.The function annotation was accomplished through the four databases:GO,KEGG,COG and SwissProt.Then the prophage genes and the regulation systems such as T6SS,T3SS and two-compnent systems were also obtained.The complete genome sequence and analysis of XSBZ03 and XSBZ14 not only enriched the V.alginolyticus genome database,but also benefited for the exploration of their pathogensis,evolution and environmental adaptability.The comparative genomic analysis was carried on XSBZ03,XSBZ14 and other four V.alginolyticus strains.Each linear synteny analysis of XSBZ03 and XSBZ14 with the reference strain ATCC17749 at nucleic acid level showed that great genetic variations such as reversal and translocation were occurred in XSBZ03 and XSBZ14 when compared to ATCC17749.The Core-Pan gene analysis with reference:ATCC17749 and query:E0666,Vl,XSBZ03,XSBZ14 showed that there were 3846 core genes,690 specific genes for XSBZ03 and 258 specific genes for XSBZ14.Among them,there were 47 specific genes only concurrently belonged to strain XSBZ03 and XSBZ14.The analysis of Ka/Ks for the single copy genes in XSBZ03 and XSBZ14 suggested that there were 95 genes with Ka/Ks>1 in XSBZ03 and 90 genes in XSBZ14.They were all subjected by the positive selection of the environment and most of them were closely related to the pathogenicity of strains.It was noteworthy that among the genes,the transcription factor genes of two strains may be the probably targets for the establishment of a rapid detection method for them.The virulence related genes,such as toxR,tdh and flaA,were summarized by comparation with the ones in other Vibrios and the function annotation through the four iii databases.The comparative genomic analysis found that inversion mutation was occurred in part of the lateral flagellar system of XSBZ14 and there were two T6SS in strain XSBZ14 just as the ones in other Vibrios,while three T6SS systems were found in XSBZ03.This is the first repot about there were three T6SS in V.alginolyticus.The multiple alignments of the homologous IGSs from four strains(XSBZ03,XSBZ14,ATCC17749 and HN08155)were carried out and one specific sequence in IGSAG-697bp of XSBZ03 was found.It could be used to design strain-specific primers for the rapid identification.The results of the comparative genome research revealed the adaptation for the pathogenesis and survival in the unique environment.It would aid in the elucidation of the pathogen evolution,survival strategies and pathogenesis.The strain-specific primers(BZRDF/BZRDR)for XSBZ03 were designed on the specific target obtained by multiple alignments of the homologous IGSs and then the rapid detection for XSBZ03 was establishment.The method was tested among 106 different bacterial strains and only one amplicon,came from the strain XSBZ03,was obtained.The sensitivity of the detection was 1.68x103CFU ml-1 and 1.38ng μL-1.This is the first rapid detection for a coral pathogen of PAWS in the world.