Molecular genetic markers for identification of species, sex, and population of giant tiger shrimp Penaeus monodon

Abstract

DNA-based molecular markers for differentiation of five penaeid shrimps were developed based on PCR-RFLP and SSCP of 16s rDNA[subscript 560]. Differentiation of Peaneus monodon, Litopenaeus vannamei and Fenneropenaeus merguiensis could be unambiguously carried out by PCR-RFLP of 16s rDNA[subscript 560] where P. semisulcatus and M. japonicus shared a BABB mitotype. These shrimps were successfully discriminated by SSCP analysis of 16s rDNA[subscript 560]. Nevertheless, the amplification success for L. and F. merguiensis was not consistent when tested against larger sample sizes. As a result, 16s rDNA[subscript560] of an individual representing the most common mitoype of each species cloned and sequenced. The amplitication success was consistent across all species (N=185) using newly designed primers. PCR-RFLP of 16S rDNA[subscript 312] was as effective as that of 16s rDNA[subscript 560]. Differentiation of all shrimp species were successfully carried out by SSCP analysis. Population genetic studies of P. monodon in Thailand were examined by PCR-RFLP and SSCP analysis of 16s rDNA[subscript 312]. Low genetic diversity and a lack of intraspecific population subdivisions of P. monodon were illustrated (P>0.05). Additionally, 320 AFLP primer combinations were screened against bulked genomic DNA of P. monodon. Twenty two polymorphic AFLP fragments were cloned and sequenced. Fourteen pairs of sequence-specific primers were designed. Four markers (P6M2-370, P6M6-470, E4M6-295 and E7M10-450) were used for population genetic studies of P. monodon. Like results from 16s rDNA[subscript 312], low genetic diversity and a lack of population differentiation was found (p>0.05). Moreover, a COI[subscript 614] gene segment of 100 individuals of P. monodon were unidirectional sequenced. A neighbor-joining tree indicated three phylogenetic lineages of P. monodon. Large nucleotide divergence was observed was observed between inter-lineage haplotypes but limited divergence was found between intra-lineage haptotypes. Distribution frequencies of haplotype clusters indicating the existence of population subdivisions. of P. monodon based on COI polymorphism (p<0.05). Sex determination and differentiation markers of P. monodon were analyzed by RAPD (100 primers) and RAP-PCR (150 primer combinations). Eight candidate genomic sex-specific RAPD bands and twenty-one and fourteen RAP-PCR fragments specifically/differentially expressed in ovaries and testes of P. monodon were successfully cloned and sequenced. Four RAPD-derived markers did not reveal sex-specificity when tested against genomic DNA of P. monodon. Therefore, genomic sex determination markers were not successfully developed in P. monodon. Expression patterns of 25 RAP-PCR derived markers were tested against the first strand cDNA of ovaries and testes of 3-month-old and broodstock-sized P. monodon (N = 5 and N = 7-10 for females and N = 4 and N = 5-7 for males, respectively). Five (FI-4, FI-44, FIII-4, FIII-39 and FIII-58) and two (M457-A01 and MII-51) derived RAP-PCR markers revealed female- and male-specific expression patterns in P. monodon. Surprisingly, MII-5 originally found in testes showed a higher expression level in ovaries than did testes of juvenile shrimps but a temporal female-specific pattern in P. monodon adults.