Identification of single nucleotide polymorphism of genes in ovaries and teste[i]s of the giant tiger shrimp Penaeus monodon.

Abstract

Single nucleotide polymorphism (SNP) in functionally important genes of the giant tiger shrimp (Penaeus monodon) was examined by SSCP analysis. A total 108 pairs of primers were designed from ESTs of ovaries and testis of P.monodon and tested against genomic DNA of representative individuals of wild P.monodon (N=15). Fifty-six primer pairs(51.85%) generated the positive amplification product. Thirty-one of which gave larger product sizes than those expected from the coding sequences suggesting the existence of intron (S) in the amplified fragments. The amplification product of 42 genes using genomic DNA of wild P.monodon (N = 15) as the template was analyzed by SSCP. Thirty-seven gene homologues (e.g. splicing factor 3a, subunit 1, phosphoglucose isomerase, solute carrier family 3 member 2, rasputin, RNA helicase, heterogenous nuclear ribonucleoprotein 87F and laminin-beta chain) were polymorphic. Nucleotide sequences of an individual representing each SSCP genotype of X-box binding protein, NADP-dependentleukotriene-12-hydroxy-dehydrogenase (LTB4DH), phosphatidylinositol 4 kinase, RUVB-like protein, phosphatidylserine receptor and solute carrier family 3 member 2 were examined by direct sequencing of the PCR product and showed compatible results with those from SSCP analysis. Therefore, SSCP analysis is an alternative cost-effective and potential for identifying polymorphism of various gene homologues. Nineteen gene homologues initially exhibited polymorphism in wild P. monodon were further tested against the 132-day-old G2 family of P. monodon (N = 10). Seven genes (FIII-7, FII-I4, FIII-8, solute carrier family 3 member 2, DDPG, phosphatidylinositol 4 kinase and phosphatidylserine receptor) did not revealed polymorphic pattern in these specimens. Although nine RAP-PCR fragments (457/OPA01, 428/OPB17, MI-36, MII-51, FI-40, FIV-33, FV-1, FV-27 and FV-42) were polymorphic, they were unknown transcripts and were not further analyzed. Therefore, only homologues of X-box binding protein (N = 76), LTB4DH (N = 60), and RUVB-like protein (N = 61) were subjected to preliminary association analysis. Results indicated no correlation between the body weight of 132-day-old P. monodon and SSCP genotypes of LTB4DH (2 genotypes) and X-box binding protein (3 genotypes)(p is more than 0.05). Nevertheless, a statistical significance between genotypes (SSCP patterns) of RUVB-like protein and the body weight of a 132-day-old shrimp was found in presumably the fast growing shrimps exhibiting the genotype II(28.6875 +- 2.3564) and the genotype I (26.1333+-4.5566; P is less than 0.05). A new sample set of P. monodon juveniles was collected from a commercial farm(17.39+-4.36 g, N = 359).Polymorphism of RUVB-like protein was tested and resulted indicated that the body weight of shrimp carrying the genotype A (19.2768 +- 3.640, N=34) and B (19.2929+-4.5477,N=78) was significantly different from that of shrimp carry the C (16.5277+-3.8466,N= 94) and D (16.3645+-4.3780 , N = 129) genotypes. Three SNP positions (G vector A[subscript81] , A vector T[subscript 196]and G vector T[subscript 248]) were found from cloned nucleotide sequences of RUVB-like protein in representative individuals of these specimens (p is less than 0.05). The full length cDNA of raputin (ORF of 1659 bp with 5’ and 3’UTRs of 132 and 2305 bp) LTB4DH (ORF of 1038 bp with 5’and 3’UTRs of 165 and 1384 bp), RUVB-like protein (ORF of 1395 bp with 5’ and 3’UTRs of 436 and 2413 bp) and X-box binding protein (ORF of 858 bp with 5’and 3’UTRs of 265 and 278 bp) was successfully identified and reported for the first time. A time course effect of 5-HT (single or double injection of 50 ug.g [superscript -1] of the body weight of 5-HT) on expression of LTB4DH, X-box binding protein and RUVB like protein in ovaries of juvenile P. monodon were examined using semiquantitative RT-PCR. Results indicate that 5-HT potentially stimulated the expression levels of all of the investigated gene homologues (p is less than 0.05).