Association between single nucleotide polymorphisms of nLvALF1 and PEN2-1 genes and resistance to Vibrio parahaemolyticus in the Pacific white shrimp Litopenaeus vannamei

Vibrio parahaemolyticus carrying a plasmid encoding the PirAvp and PirBvp toxins is the causative agent of acute hepatopancreatic necrosis disease(AHPND)in penaeid shrimps.In the Pacific white shrimp farming industry,one possible strategy to reduce economic loss due to AHPND is the development of a shrimp line resistant to the disease.In this study,we identified single nucleotide polymorphisms(SNPs)in the Litopenaeus vannamei anti-lipopolysaccharide factor 1(nLvALF1)and penaeidin 2-1(PEN2-1)genes,and we analyzed the associations between these SNPs and resistance/susceptibility to V.parahaemolyticus infection in the Pacific white shrimp.Postlarvae(PL20)shrimp from a local hatchery in Prachuap Khiri Khan Province were challenged with an isolate of VPAHPND and mortality was observed for 14 days.DNA was extracted from susceptible(died within 6 days)and resistant(survived the challenge)shrimp(45 individuals/group)and used for PCR amplifications of nLvALF1(397 bp)and PEN2-1(637 bp)gene fragments.PCR products were sequenced by direct sequencing and SNPs were identified from sequencing chromatograms.Nine and seven SNPs were identified in nLvALF1 and PEN2-1 gene fragments,respectively.Analyses of allele frequencies in susceptible and resistant samples using Chi-square tests revealed that four and six SNPs in nLvALF1 and PEN2-1,respectively,were associated with resistance/susceptibility to V.parahaemolyticus infection(P<0.05).The SNPs in the candidate genes identified here are potential DNA markers for breeding V.parahaemolyticus-resistant Pacific white shrimp in the study population;however,further validation will be required if these SNPs are to be used across populations.