Identificaton of SNP markers in the giant tiger shrimp Penaeus monodon

Abstract

Single nucleotide polymorphism (SNP) by expressed sequence tags (SBE) of genes in functionally important genes of the giant tiger shrimp (Penaeus monodon) was examined. Primers were designed from 101 gene homologues initially isolated by the EST approach and tested against genomic DNA of wild P. monodon. Forty-eight gene homologues were successfully amplified and subjected to SSCP analysis. Forty-four of which were polymorphic. Five genes including ribophorin I, receptor for activated protein kinase C (RACK), defender against cell death 1 (DAD1), thioredoxin peroxidase and calponin 1 were chosen for further studies. SNP of these genes were confirmed by direct sequencing. Simplification of SNP detection was preliminary carried out by PCR-RFLP and PCR allele-specific amplification (PASA). RACE-PCR was carried out and successfully identified the full length of ribophorin I and RACK. In addition, the genomic full length of DAD1, thioredoxin peroxidase andcalponin 1 were also successfully characterized by genome walk analysis. Polymorphism of 5'UTR and 3'UTR of each gene was successfully genotyped by SSCP analysis. Semi-quantitative RT-PCR was carried out. Relative expression levels of ribophorin I, RACK, DAD1, thioredoxin peroxidase and calponin1 in ovaries and testes of juvenile P. monodon were significantly different (P<0.05). However, the expression levels of RACK in male and female P. monodon broodstock were not significantly different. Therefore association analysis between SNP thorough SSCP and gene expression levels on male and female P. monodon should be considered separately. A time course response of these genes following the thermal stress was also examined. Only the expression level of thioredoxin peroxidase in ovaries and RACK in hemocytes of juvenile shrimps was significantly higher than that the normal level at 6 and 12 hours post thermal treatment, respectively (P<0.05). Correlations between SNP through SSCP and the corresponding gene expression in broodstock and juveniles of P. monodon were examined. Due to limited sample sizes, several SSCP genotypes carried by single individuals were therefore excluded from statistical analysis. Non-significant associations between SNP and gene expression levels were found in both broodstock and juvenile P. monodon.