Abalone, genus Haliotis, are single shelled nocturnal herbivorous marine gastropods. There are nearly 56 currently described species of haliotids worldwide. About 10% of these are commercially important as wild fisheries or aquaculture species. Significant progress had been made in abalone aquaculture in general but genetic improvement and restocking programmes are still in their infancy. A single tropical species, Haliotis asinina of the Indo-Pacific, is extensively harvested in Southeast Asia. H. asinina, with a shorter generation time than the temperate species and predictable spawning cycle, is well suited for aquaculture and an ideal species to investigate the molecular mechanisms regulating reproduction and growth in abalone.
Molecular genetics and associated technologies have facilitated the analysis and understanding of the underlying genetics of commercially
important traits in plants and animals. The genetic markers, both protein and DNA markers, have been found to be efficient in addressing genetic issues associated with management of fisheries and aquaculture. Among the available genetic markers, microsatellites are increasingly being used to answer genetic questions of fisheries and aquaculture. Microsatellites are short tandem repeats of 2-6 base pairs and are abundant in eukaryotic genome. These markers are highly variable, selectively neutral and co-dominantly inherited. Polymorphic nature of microsatellites is helpful in identification of quantitative trait loci, parentage and pedigrees and genetic diversity between populations. In abalone aquaculture, marker assisted selection for growth has the potential to enhance industry development, by identifying individuals and broodstock with genetic productivity for faster growth. The focus of this study is to develop polymorphic microsatellite DNA markers and demonstrate their
application in H. asinina fisheries and aquaculture.
A partial genomic library of H. asinina, enriched for microsatellite clones, was screened for dinucleotide microsatellite DNA using biotinylated (AC)18 primer. Isolated microsatellite-containing DNA were cloned and sequenced. Out of 27 microsatellite sequences, 7 contained microsatellite motifs and priming sequences for detailed studies on the natural and cultured populations. These microsatellite loci and four other loci that were isolated earlier by Sandie Degnan, were characterised using H. asinina samples from Heron Island Reef. All 11 microsatellite loci were polymorphic with the number of alleles ranging from 2-30 per locus and the observed and expected heterozygosity between 0.25-0.97 and 0.31-0.95 respectively. These primers were also able to amplify homologous loci in other tropical abalone species, H.
ovina and H. varia.
Five of these microsatellite markers were used to investigate the genetic structuring of natural populations of H. asinina in Australia and Southeast Asia. The allelic diversity and average heterozygosities were high at these loci in all the populations. Significant genetic differentiation was observed among all the wild populations analysed. Genetic structuring of H. asinina populations appears to follow isolation-by-distance model. These microsatellite loci were used to test whether the hatchery-reared abalone at SEAFDEC, the Philippines, could be used to restock some of the Philippines populations. The hatchery population did not show significant differentiation with one of the populations but showed a significant differentiation with the other. Based on this observation, suggestions are made regarding the use of hatchery-bred abalone for reseeding the wild populations in
the Philippines. The implications for future restocking programmes are also discussed.
In aquaculture, microsatellite DNA markers are used to genotype parental broodstock, to assess fertilization success and to maintain pedigree information for selective breeding. In this study individual H. asinina larvae were genotyped by analysing a suit of polymorphic microsatellite loci. At least 10 loci can be analysed from a single abalone veliger larvae. Five polymorphic loci were assayed to identify the parents of individual larvae produced in three separate crosses. In all cases, the parents of an individual veliger could be determined from as few as 3 loci. The microsatellite analysis revealed a substantial inequality in the contribution of each broodstock to the new generation of veligers, as only a subset of broodstock males sired most of the offspring. These observations suggest that highly controlled breeding practices may be required to
ensure that the genetic diversity of an abalone population produced for aquaculture.
The individual larval genotyping technique was used to test for sperm competition in abalone crosses. The sperm age was the predominant factor affecting the rate of fertilization success. A difference in fertilization success was evident between two males which spawned at the same time, which might be due to natural variation in viability or genetic differences.
The microsatellite loci isolated and characterised in this study are found to be efficient genetic markers for management of H. asinina fisheries and aquaculture. These markers have the potential to be used in population structure studies and are polymorphic enough to reveal small genetic differences between populations. These markers could be used to make informed decisions on translocation of animals for reseeding and aquaculture purposes. The polymorphism, exhibited at
these microsatellite loci, demonstrates their potential to be used as genetic tags to monitor the success of reseeding programmes and to maintain pedigree information. These microsatellites markers along with development of AFLP markers could be used to develop linkage and genetic maps for H. asinina to identify and characterise quantitative trait loci.