Browsing by Author "Roodt-Wilding, Rouvay"

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    Microsatellite cross-species amplification and utility in southern African elasmobranchs : a valuable resource for fisheries management and conservation
    (BMC Research Notes, 2014) Maduna, Simo N.; Rossouw, Charne; Roodt-Wilding, Rouvay; Bester-Van der Merwe, Aletta E.
    Background: Similarly 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. Results Thirty 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. Conclusions Cross-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.
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    Molecular characterisation of apple accessions with respect to aminocyclopropane-1-carboxylic acid synthase gene (ACS1) polymorphism
    (Czech Academy of Agricultural Sciences, 2020-02-07) Mhelembe, Khethani; Kriel, Johan; Bester, Cecilia; Louw, Esme; Roodt-Wilding, Rouvay; Tobutt, Kenneth
    The ARC apple gene bank collection was genotyped for the fruit expressed gene ACS1, in which a short-interspersed element (SINE) in the promoter is known, when homozygous, to correlate with the delayed ethylene production. Primers were designed amplifying products less than 500 bp and 224 cultivars of domestic apple were analysed, 169 not previously genotyped. Of these, 82 were aa (homozygous for the high ethylene allele at 202 bp), 73 were ab and 14 bb (homozygous for the low ethylene allele, with the SINE, at 339 bp). The difference between the allele sizes, 137 bp, observed in the current study is consistent with the indel of 138 bp originally described, but differs considerably from the indel of 166 bp reported in literature. In addition, 21 accessions of other Malus species were analysed. Only one, M. ‘Golden Hornet’, had the b allele, which suggests it may have been introgressed from M. pumila.
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    Population genetics of Southern Hemisphere tope shark (Galeorhinus galeus) : Intercontinental divergence and constrained gene flow at different geographical scales
    (Public Library of Science, 2017) Bester-Van Der Merwe, Aletta E.; Bitalo, Daphne; Cuevas, Juan M.; Ovenden, Jennifer; Hernandez, Sebastian; Da Silva, Charlene; McCord, Meaghen; Roodt-Wilding, Rouvay
    The tope shark (Galeorhinus galeus Linnaeus, 1758) is a temperate, coastal hound shark found in the Atlantic and Indo-Pacific oceans. In this study, the population structure of Galeorhinus galeus was determined across the entire Southern Hemisphere, where the species is heavily targeted by commercial fisheries, as well as locally, along the South African coastline. Analysis was conducted on a total of 185 samples using 19 microsatellite markers and a 671 bp fragment of the NADH dehydrogenase subunit 2 (ND2) gene. Across the Southern Hemisphere, three geographically distinct clades were recovered, including one from South America (Argentina, Chile), one from Africa (all the South African collections) and an Australia-New Zealand clade. Nuclear data revealed significant population subdivisions (FST = 0.192 to 0.376, p<0.05) indicating limited gene flow for tope sharks across ocean basins. Marked population connectivity was however evident across the Indian Ocean based on Bayesian clustering analysis. More locally in South Africa, F-statistics and multivariate analysis supported moderate to high gene flow across the Atlantic/Indian Ocean boundary (FST = 0.035 to 0.044, p<0.05), with exception of samples from Struisbaai and Port Elizabeth which differed significantly from the rest. Discriminant and Bayesian clustering analysis indicated admixture in all sampling populations, decreasing from west to east, corroborating possible restriction to gene flow across regional oceanographic barriers. Mitochondrial sequence data recovered seven haplotypes (h = 0.216, π = 0.001) for South Africa, with one major haplotype shared by 87% of the individuals and at least one private haplotype for each sampling location except Port Elizabeth. As with many other coastal shark species with cosmopolitan distribution, this study confirms the lack of both historical dispersal and inter-oceanic gene flow while also implicating contemporary factors such as oceanic currents and thermal fronts to drive local genetic structure of G. galeus on a smaller spatial scale.
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    Transcriptome characterization of the South African abalone Haliotis midae using sequencing-by-synthesis
    (BioMed Central, 2011-03) Franchini, Paolo; Van der Merwe, Mathilde; Roodt-Wilding, Rouvay
    Abstract. Background. Worldwide, 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. Results More 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. Conclusion This 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.
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    Transcriptome-wide single nucleotide polymorphisms (SNPs) for Abalone (Haliotis midae) : validation and application using GoldenGate medium-throughput genotyping assays
    (MDPI, 2013-09) Bester-Van der Merwe, Aletta; Blaauw, Sonja; Du Plessis, Jana; Roodt-Wilding, Rouvay
    Haliotis midae is one of the most valuable commercial abalone species in the world, but is highly vulnerable, due to exploitation, habitat destruction and predation. In order to preserve wild and cultured stocks, genetic management and improvement of the species has become crucial. Fundamental to this is the availability and employment of molecular markers, such as microsatellites and single nucleotide (SNPs). Transcriptome sequences generated through sequencing-by-synthesis technology were utilized for the in vitro and in silico identification of 505 putative SNPs from a total of 316 selected contigs. A subset of 234 SNPs were further validated and characterized in wild and cultured abalone using two Illumina GoldenGate genotyping assays. Combined with VeraCode technology, this genotyping platform yielded a 65%−69% conversion rate (percentage polymorphic markers) with a global genotyping success rate of 76%−85% and provided a viable means for validating SNP markers in a non-model species. The utility of 31 of the validated SNPs in population structure analysis was confirmed, while a large number of SNPs (174) were shown to be informative and are, thus, good candidates for linkage map construction. The non-synonymous SNPs (50) located in coding regions of genes that showed similarities with known proteins will also be useful for genetic applications, such as the marker-assisted selection of genes of relevance to abalone aquaculture.