Polymorphic microsatellite loci were isolated for Panulirus ornatus using 454 GS-FLX Titanium pyrosequencing. Fifteen markers containing perfect di-, tri-, tetra-, and penta-nucleotide motifs were consistently co-amplified in five multiplexes in a panel of 91 randomly selected samples. Observed number of alleles varied from 2 to 14 per locus. Observed and expected heterozygosity ranged from 0.090 to 0.79 and 0.08 to 0.87, respectively. Ten loci deviated from Hardy-Weinberg equilibrium after sequential Bonferroni correction. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between 11 loci. The microsatellite markers were also amplified successfully in related Panulirus homarus species with adequate level of polymorphism. The successful cross-species primer amplification of the 15 microsatellites indicates the potential of the developed markers to be transferred to other Panulirus species. The 15 novel microsatellite markers reported in this work add to the previously characterized markers by our group, exhibit adequate levels of polymorphism for wide range of future studies investigating population structure, genetic diversity, and evolutionary relationships among Panulirus species.

have contributed equally to the work. AbstractParentage analysis is of vital importance for hatchery production and breeding programmes. Two multiplex PCR protocols including seven tropical spiny lobster (Panulirus homarus) microsatellites (Pho-G06, Pho-G25, Pho-G53, Pho-G62, Pho-G74, Pho-G89 and Pho-G100) were introduced for parentage assignment. All loci were polymorphic with allele sizes from 113 to 337 base pairs (bp), observed alleles (k) from two to seven and polymorphic information content (PIC) of 0.25 to 0.73. Twenty-four stage-3 phyllosoma larvae (39 days posthatching) were collected for paternity exclusion study using selected microsatellites. Exclusion-based parentage analysis unambiguously assigned 83% of fry (20 of 24) to a single female parent. Of ten putative female parents, five have contributed to the 20 allocated offspring, with one being true parent of 11. Four others were assigned to two or more potential female parents, probably due to genotyping error or the presence of null allele. The exclusion power (EP) for the seven loci varied between 13% and 54% with known genotypes of one parent (P1) and 19% and 71% for given the genotypes of both parents (P2). The theoretical combined parentage exclusion (cEP) power for all seven microsatellites was P1 = 95% and P2 = 99%. The power of discrimination for each locus varied between 0.18 and 0.86. This report presents the first study to utilize microsatellite markers for successful parentage assignment of P. homarus. The selected microsatellites provide a practical tool for parentage analysis in hatchery production of juveniles as well as in future commercial breeding programme of tropical spiny lobsters.

ABSTRACT. Fourteen polymorphic microsatellites with perfect di-, tri-, and tetra-nucleotide repeats were identified for Panulirus homarus using Roche 454 whole-genome sequencing method. Microsatellites were efficiently co-amplified in four multiplexes and a singleplex, providing consistent and easily interpretable genotypes. The number of alleles per locus ranged from 2 to 11 with the observed and expected heterozygosity ranging between 0.000-0.532 and 0.031-0.836, respectively. A significant deviation from Hardy-Weinberg equilibrium was observed for majority of the loci, probably due to homozygote excess. Genetic linkage disequilibrium analysis between all the possible pairs of the loci showed significant departure from the null hypothesis in the loci pairs Pho-G11-Pho-G33 and Pho-G33-Pho-G57. High success in primer cross-species amplification of these microsatellite markers indicates their utility for genetic studies of different Panulirus species.