Identification of molecular genetic markers involed in sex determination in the giant tiger shrimp Penaeus monodon using AFLP analysis

Abstract

AFLP analysis was used to isolate sex-specific markers in P. monodon. A total of 256 primer combinations were tested against 6-10 bulked genomic DNA of P. monodon. Five (FE10M9520, FE10M10725, FE14M16340, FE15M14400 and FE16M8350) and one (ME10M8420) candidate female and male-specific AFLP markers were identified. The former markers were cloned and further characterized. SCAR markers derived from FE10M9520, FE10M10725.1, FE10M10725.2, FE14M16340 did not retain the original sex specificity. SSCP analysis was applied to identify whether fixed SNP was existent in SCAR markers amplified from male and female P. monodon. Polymorphic but not sex-linked pattern were found from FE10M10725.1 and FE14M16340-derived SCAR markers. Additionally, thirty-four primers pairs derived from sex-related AFLP and cDNA fragments of P. monodon, M. rosenbergii and H. asinina from previous studies were tested. Polymorphic SSCP markers that are not linked to sexes of P. monodon(TSP462F+288R, XNP-1, peritrophin, DSI, ZFP, PMO920 and PMT1700 from P. monodon and VCP2 from H. asinina) were found. Four polymorphic markers (PMO920, PMT1700, ZFP and DSI) were selected for population genetic studies of natural P. monodon in Thai waters. Relative high genetic diversity was found at these loci. The number of allele per locus was 6, 5, 12 and 19 respectively. The observed heterozygosity was 0.3043 0.5128, 0.3462 0.4643 and 0.5000 0.8108 for PMO920, PMT1700 and ZFP, respectively. Linkage disequilibrium analysis indicated that genotypes of these loci segregated randomly (P > 0.05). Significant deviation from Hardy-Weinberg expectation was observed in Trang and Trad at PMO920 (P = 0.0002 and P = 0.0005 ; homozygote excess) and Satun and Phangnga (P = 0.0047 and P = 0.0175 ; heterozygote excess). Low genetic distance was found between pairs of geographic samples (P = 0.0077 0.0178). The neighbor-joining tree constructed from the average genetic distance of overall loci allocated Satun, Trang andPhangnga into one cluster and Chumphon and Trad into the other cluster. Population differentiation between Satun Trad and Satun Phangnga was found at ZFP (P < 0.05). In addition, seven polymorphic alleles of DSI revealed significant distribution patterns across overall samples (P < 0.05) implying low but significant genetic heterogeneity of P. monodon in Thailand