Development of DNA marker multiplex PCR to estimate the genetic diversity of Haliotis tuberculata coccinea broodstock

Haliotis tuberculata coccinea is a promising candidate species for sustainable and integrated aquaculture development in the Canary Islands. Although many studies focused on the technical improvement of culture systems of this species have been conducted in the PCTM of ULPGC, no genetic studies had been developed to investigate the genetic resources of its broodstock population. Therefore, to have insights into the genetic structure and diversity of the existing broodstock, group of microsatellite markers, belonging to other abalone species, were selected and grouped into two multiplex PCRs which were used to develop one single multiplex PCR (MULTHAB) optimized for amplification of 8 microsatellites after the exclusion of monomorphic and non-amplified primer pairs. The usefulness of using this multiplex was assessed. 316 DNA samples were used and included: 16 samples from Fi animals that were part of the breeder group composed of different hatchery generations and 300 DNA samples that belonged to the next F2 generation obtained from the Fi breeder group. A total number of 80 alleles were detected for the 8 selected microsatellite markers in the MULTHAB in all analyzed samples. They showed good allelic polymorphism with moderate allelic number per locus (NA) and expected heterozygosity (He) values, (mean equal to 10 and 0.6299, respectively). High PIC number values (mean equal to 0.5753) for all used loci were observed, suggesting that the selected markers showed a high capability to detect polymorphisms in population of H. tuberculata coccinea. The 8 microsatellite markers were successfully segregated in two identified families produced within this study. The optimized MULTHAB succeed to assign 32 per cent of the F2 analyzed individuals to their possible parent pair or single parent. The mean FST value (0.0103) showed a little genetic differentiation between Fi and F2 animals. There was high deviation from 11W equilibrium for the studied microsatellite loci in ah l the broodstock population. Additionally, the mean FIS was positive (0.0899) and there was linkage disequilibrium in 12 of 28 loci pairwise. This results indicates that the studied population was affected by a somehow loss of genetic diversity due to heterozygosity deficiency, inbreeding and founder problems, which was expected due to the crossing between parents and offspring of different generations for seed production. This study concludes the successful applicability of the developed MULTHAB multiplex for genetic studies in H. tuberculata coccinea broodstock and recommends that adequate genetic maintenance should be implemented for the broodstock population.