Rouvay Roodt-Wilding

Transcriptome characterization of the South African abalone Haliotis midae using sequencing-by-synthesis

BackgroundWorldwide, the genus Haliotis is represented by 56 extant species and several of these are commercially cultured. Among the six abalone species found in South Africa, Haliotis midae is the only aquacultured species. Despite its economic importance, genomic sequence resources for H. midae, and for abalone in general, are still scarce. Next generation sequencing technologies provide a fast and efficient tool to generate large sequence collections that can be used to characterize the transcriptome and identify expressed genes associated with economically important traits like growth and disease resistance.ResultsMore than 25 million short reads generated by the Illumina Genome Analyzer were de novo assembled in 22,761 contigs with an average size of 260 bp. With a stringent E-value threshold of 10-10, 3,841 contigs (16.8%) had a BLAST homologous match against the Genbank non-redundant (NR) protein database. Most of these sequences were annotated using the gene ontology (GO) and eukaryotic orthologous groups of proteins (KOG) databases and assigned to various functional categories. According to annotation results, many gene families involved in immune response were identified. Thousands of simple sequence repeats (SSR) and single nucleotide polymorphisms (SNP) were detected. Setting stringent parameters to ensure a high probability of amplification, 420 primer pairs in 181 contigs containing SSR loci were designed.ConclusionThis data represents the most comprehensive genomic resource for the South African abalone H. midae to date. The amount of assembled sequences demonstrated the utility of the Illumina sequencing technology in the transcriptome characterization of a non-model species. It allowed the development of several markers and the identification of promising candidate genes for future studies on population and functional genomics in H. midae and in other abalone species.

Historical isolation and hydrodynamically constrained gene flow in declining populations of the South-African abalone, Haliotis midae

Over the past two decades, the South African abalone (Haliotis midae), has been under serious threat mainly due to overexploitation. To assure successful management and conservation of wild stocks, the consideration of species-specific evolutionary and population dynamic aspects is critical. In this study, eight microsatellites and 12 single nucleotide polymorphic loci (SNPs) were applied to determine genetic structure in nine populations sampled throughout the species’ natural distribution range. It spans along three biogeographical regions of the South African coastline: temperate in the West coast, warm temperate in the South coast and subtropical in the East coast. Data analysis applying frequentist and Bayesian-based clustering methods indicated weak genetic differentiation between populations of the West, South and East coast. Spatial Bayesian inference further revealed clinal variation along a longitudinal gradient and a transitional zone in the South coast. Coalescent analysis of long-term migration showed restricted interchange among the sampling locations of the South coast while estimates of effective population size were comparable between coastal regions. Furthermore demographic analysis of microsatellite data suggested population expansion, probably reflecting range expansion that occurred following glacial retreat during the Pleistocene. Overall, population structure analysis suggested contemporary (hydrographical conditions) as well as historical (Pleistocene contraction of habitat) restrictions to gene flow. This study provides the foundation for the establishment of an integrated management policy for preserving the natural diversity and adaptive potential of H. midae.

Differential Growth-Related Gene Expression in Abalone (Haliotis midae)

The slow growth rate of Haliotis midae impedes the optimal commercial production of this most profitable South African aquaculture species. To date, no comprehensive effort has been made to identify genes associated with growth variation in farmed H. midae. The aim of this study was therefore to investigate growth variation in H. midae and to identify and quantify the expression of selected growth-related genes. Towards this aim, molecular methodologies and cell cultures were combined as a time-efficient and economical way of studying abalone transcriptomics and cell biology. Modern Illumina sequencing-by-synthesis technology and subsequent sequence annotation were used to elucidate differential gene expression between two sibling groups of abalone demonstrating significant growth variation. The expression of selected target genes involved in growth was subsequently analysed by quantitative real-time PCR (qPCR). Fast- and slow-growing abalone and in vitro primary haemocyte cultures treated with different growth-stimulating factors were used. The results obtained from transcriptome analysis and qPCR revealed significant differences in gene expression between large and small abalone, and between treated and untreated haemocyte cell cultures. Throughout in vivo and in vitro qPCR experiments, the up-regulation of genes involved in the insulin signalling pathway suggests that insulin may be involved in enhanced growth rate for various H. midae tissues.

Discovery and evaluation of single nucleotide polymorphisms (SNPs) for Haliotis midae: a targeted EST approach

In this study, we describe the first set of SNP markers for the South African abalone, Haliotis midae. A cDNA library was constructed from which ESTs were selected for the screening of SNPs. The observed frequency of SNPs in this species was estimated at one every 185 bp. When characterized in wild-caught abalone, the minor allele frequencies and F(ST) estimates for every SNP indicated that these markers may potentially be useful for population analysis, parentage assignment and linkage mapping in Haliotis midae. No linkage disequilibrium was observed between SNPs originating from different EST sequences. These SNPs, together with additional SNPs currently being developed, will provide a useful complementary set of markers to the currently available genetic markers in abalone.

Preliminary investigation to determine the cytotoxicity of various cryoprotectants on southern African abalone (Haliotis midae) embryos

Cryopreservation could provide stock quantities of embryos for transgenic research. This study aimed to determine the least toxic cryoprotective agent for Haliotis midae embryos. They were exposed for 30min to concentrations varying from 5% to 20% of the following cryopreservatives: methanol (MET), polyethylene glycol (PEG), dimethyl sulfoxide (ME(2)SO) and glycerol (GLY). In contrast to cryopreservation studies done in other molluscs, PEG showed the least toxicity to H. midae embryos in concentrations ranging from 5% to 15%. MET was also less toxic than ME(2)SO and GLY at correlating concentrations. GLY showed the most toxic effects with most embryos dead or abnormal at concentrations above 15%.

Comparison of population genetic estimates amongst wild, F1 and F2 cultured abalone (Haliotis midae)

Haliotis midae is South Africas most important aquaculture species. The reproduction cycle is currently not closed as many farms rely on wild-caught broodstock for seed production. However, there is an increasing interest in genetic improvement in commercial stocks, with a growing number of producers implementing selective breeding strategies. High throughput commercial production and mass spawning make it difficult to maintain breeding records; therefore, mostly mass selection is practised. The high fecundity and unequal parental contributions also often lead to increased levels of inbreeding. This study therefore aimed to assess the genetic effects of such breeding practices on commercial populations of H. midae. Using microsatellite loci, the genetic properties of a wild, an F1 and an F2 population were estimated and compared. Although there was no significant loss of genetic diversity amongst the cultured populations in comparison with the wild progenitor population, there was low-to-moderate genetic differentiation between populations. Relatedness amongst the F2 population was significant, and the rate of inbreeding was high. The effective population size for the F2 (±50) was also comparatively small with respect to the wild (∞) and F1 (±470) populations. These results suggest that farms need to give caution to breeding practices beyond the first (F1) generation and aim to increase effective population sizes and minimise inbreeding to ensure long-term genetic gain and productivity. This study also confirms the usefulness of population genetic analyses for commercial breeding and stock management in the absence of extensive pedigree records.

Karyotype and Genome Size Estimation of Haliotis midae: Estimators to Assist Future Studies on the Evolutionary History of Haliotidae

ABSTRACT The genome size of the abalone Haliotis midae was determined using flow cytometry and calculated relative to the known genome size of the rainbow trout (Oncorhynchus mykiss). The C value for H. midae was estimated to be 1.43 pg, which is smaller in comparison with other abalone species with the same number of chromosomes (2n = 36), namely H. corrugata, 2.14 pg; H. rufescens, 1.82 pg; and H. fulgens, 1.71 pg. In these Californian abalone species, a positive correlation between C value and the number of metacentric/submetacentric chromosomes with a subsequent loss of submetacentric/subtelocentric chromosomes was observed. Karyotypic analysis in H. midae indicates 6M + 10SM +2 ST (metacentric + submetacentric + subtelocentric) chromosome pairs, the lowest number of metacentric and the highest number of subtelocentric chromosomes when compared with the Californian species. This pattern, in the light of recent phylogenetic and cytogenetic evidence, leads us to hypothesize that during the evolution of the genus, there were some preferential karyotypic rearrangements. Research on the genome size of other abalone species in general and specifically South African species, combined with chromosome banding techniques, could provide more insight into the evolution of this genus.

Chromosome number of the South African abalone Haliotis midae

Despite the widespread distribution of the genus Haliotis, chromosome numbers are only known for a small subset of species. In South Africa, no chromosome studies have been conducted on any of the five species occurring in the region. This study is the first report for Haliotis midae, the largest and only economically important species in South Africa, with a somatic chromosome number of 2n = 36. The study also provides the first count for a species in the southernmost radiation of abalone comprising South African, Australian and New Zealand species, and corroborates the hypothesis of the radiation of the genus into the Southern and Northern hemispheres from the Indo-Pacific area.

Transcriptome profiles of wild and cultured South African abalone, Haliotis midae

This report describes the use of pyrosequencing technologies to generate the first comparative analysis of de novo assembled transcriptome data from cultured and wild specimens of the South African abalone. The transcriptome data and database described here provide a significant genomic resource for abalone research. The data set annotated 11,240 genes, which matched genes with known functions in other species. A large number of transmembrane protein domains (4087) that may indicate a high portion of undiscovered gene receptors were identified. Further, we detected an interesting set of transcription factors (516) that are valuable candidates for participating in regulatory events in developmental (such as cell proliferation and differentiation) and reproductive processes.

Microsatellite cross-species amplification and utility in southern African elasmobranchs: A valuable resource for fisheries management and conservation

BackgroundSimilarly to the rest of the world, southern Africa’s diverse chondrichthyan fauna is currently experiencing high fishing pressures from direct and non-direct fisheries to satisfy market demands for shark products such as fins and meat. In this study, the development of microsatellite markers through cross-species amplification of primer sets previously developed for closely related species is reported as an alternative approach to de novo marker development. This included the design of four microsatellite multiplex assays and their cross-species utility in genetic diversity analysis of southern African elasmobranchs. As this study forms part of a larger project on the development of genetic resources for commercially important and endemic southern African species, Mustelus mustelus was used as a candidate species for testing these multiplex assays in down-stream applications.ResultsThirty five microsatellite primer sets previously developed for five elasmobranch species were selected from literature for testing cross-species amplification in 16 elasmobranch species occurring in southern Africa. Cross-species amplification success rates ranged from 28.6%-71.4%. From the successfully amplified microsatellites, 22 loci were selected and evaluated for levels of polymorphism, and four multiplex assays comprising of the 22 microsatellites were successfully constructed, optimised and characterised in a panel of 87 Mustelus mustelus individuals. A total of 125 alleles were observed across all loci, with the number of alleles ranging from 3–12 alleles. Cross-species amplification of the four optimised multiplex assays was further tested on 11 commercially important and endemic southern African elasmobranch species. Percentage of polymorphism ranged from 31.8%-95.5% in these species with polymorphic information content decreasing exponentially with evolutionary distance from the source species.ConclusionsCross-species amplification of the 35 microsatellites proved to be a time- and cost-effective approach to marker development in elasmobranchs and enabled the construction of four novel multiplex assays for characterising genetic diversity in a number of southern African elasmobranch species. This study successfully demonstrated the usefulness of these markers in down-stream applications such as genetic diversity assessment and species identification which could potentially aid in a more integrative, multidisciplinary approach to management and conservation of commercially important cosmopolitan and endemic elasmobranch species occurring in southern Africa.