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Microsatellite marker development and parentage assignment in Haliotis midaeVan den Berg, Nicol-Candice 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: The five leading abalone producers in South Africa have initiated a genetic enhancement program for Haliotis midae in a collaborative effort to improve economically valuable traits. Several independent objective-specific studies were initiated, including the establishment of a Performance Recording Scheme (PRS), utilised in this study, and necessary to monitor the ongoing performance of individuals as the move from mass-selection to marker assisted selection (MAS) is implemented. The primary objective of this study was parentage assignment of F1 offspring mass-selected for size at approximately one year and allocated to either a “faster” or a “slower” growth group. Nine microsatellite markers were used to genotype juveniles and potential parents, with assignment completed using CERVUS 2.0. Average growth results for Abagold and HIK were comparable for both growth groups. Slight environmental effects, although not statistically significant, were evident as growth advantages for juveniles within the faster growth group at two of the five locations and for juveniles within the slower growth group at one of the five rearing locations. Despite measures to standardise environmental influences, variables are difficult to control within the reality of a production environment; and potential genotype x environment interactions may require further investigation and factoring into future breeding programs. The additional costs associated with MAS often make the technology prohibitive to most aquaculture operations, despite the significant genetic gains to be realised from its implementation. Cost-optimising routine processes such as DNA extractions may be one approach to reduce these additional costs. Chelex®100 appears to be a suitable alternative to the CTAB method – being quick and cost-effective to perform. Applying this method in combination with the high throughput of a robotic platform warrants further evaluation. For the microsatellite development, 50% of positive recombinant clones contained inserts. Sequencing of these clones produced 16% perfect repeats and 47% imperfect repeats for which 52 primer sets were designed and tested. In total, 31 polymorphic microsatellite loci of different motifs and composition were developed. Sixty-one percent of sequenced clones were deemed redundant and pre-screening for both uniqueness and the presence of microsatellites would reduce unnecessary sequencing thus improving the efficiency of the FIASCO method and reducing costs. Nine loci were selected for parentage assignments. Null alleles were present for all the selected markers; however, frequencies were below the critical level of 5%. Parentage yielded 91% and 90% successful assignment for Abagold and HIK respectively; however, observations indicate that a measure of relatedness may exist between breeders. Recommendations with regards to future family breeding include, for both Abagold and HIK, retaining selected breeders based on their respective contributions to the F1 progeny while reassessing the potential of remaining breeding stock under more controlled breeding conditions. No obvious trends were observed for growth with most individuals producing both faster and slower growing offspring. Juveniles will be reassessed at two years to determine whether the size advantage or disadvantages were maintained and to ascertain whether growth advantages/disadvantages may be gender specific. / AFRIKAANSE OPSOMMING: Die vyf mees toonaangewende perlemoen produseerders in Suid Afrika het „n genetiese verbeteringsprogram vir Haliotis midae geinisieer in „n gesamentlike poging om ekonomiese belangrike eienskappe te verbeter. Verskeie onafhanklike fokus-spesifieke studies is geinisieer, insluitend die totstandkoming van „n groeiprestasie aantekenstelsel, soos gebruik in hierdie studie, en wat noodsaaklik is om die aaneenlopende prestasie van individue te moniteer soos daar beweeg word van massa seleksie tot merker bemiddelde seleksie. Die primêre fokus van hierdie studie was die ouerskapsbepaling van F1 nageslag wat massa geselekteer is op ouderdom 1 jaar vir grootte en as of “vinniger” of “stadiger” groeiers geklassifiseer is. Nege mikrosatelliet merkers is gebruik om jong perlemoen individue en moontlike ouers te genotipeer, met die ouerskapstoekenning bereken deur CERVUS 2.0. Groei resultate vir Abagold en HIK was vergelykbaar vir beide groei groepe op drie van die lokaliteite. Geringe omgewingseffekte, alhoewel nie statisties betekenisvol nie, was sigbaar as „n groei voordeel vir jong individue op twee van die vyf lokaliteite. Ongeag maatstawe om omgewingsinvloede te standardiseer, is varieerbares moeilik om te beheer in die produksie omgewing en genotipe x omgewings interaksies mag verdere navorsing vereis en behoort in ag geneem te word in toekomstige telingsprogramme. Die onkoste wat met merker bemiddelde seleksie geassosieer word, maak die tegniek soms onaantreklik vir die meeste akwakultuur operasies; nie teen staande die genetiese voordele wat die gebruik daarvan veroorsaak. Die koste-optimiseering van roetine prosesse, soos byvoorbeeld, DNA ekstraksies, is dalk een aanslag om die addisionele koste te verminder. Chelex®100 blyk „n geskikte alternatief tot die CTAB metode te wees – die tegniek is vinnig en koste-effektief om uit te voer. Die gebruik van hierdie metode in kombinasie met die hoë deurvloei van ‟n robotiese sisteem behoort verder ondersoek te word. Vir die mikrosatelliet ontwikkeling het slegs 50% van die positiewe rekombinante klone invoegings bevat. Nukleotiedvolgorde bepaling van hierdie klone het 16% perfekte herhalings en 47% onderbroke herhalings bevat waaruit 52 inleierstelle ontwikkel en getoets is. In totaal is 31 polimorfiese mikrosatelliet loki van verskillende motiewe en samestelling ontwikkel. Een-en-sestig persent van die volgorde bepaalde klone is oortollig geag en vooraf sifting vir beide uniekheid en die teenwoordigheid van mikrosatelliete sal onnodige volgorde bepaling verhoed, die effektiwiteit van die FIASCO tegniek verhoog sowel as addisionele koste verminder. Nege loki is geselekteer vir ouerskapsbepaling. Nul allele was teenwoordig vir al die geselekteerde merkers, maar die frekwensies was egter laer as die 5% kritieke waarde. Ouerskap is 91% en 90% suksesvol bepaal vir Abagold en HIK onderskeidelik. Waarnemings dui egter daarop dat daar verwantskappe mag wees tussen van die broeidiere. Voorstelle in terme van toekomstige familie teling sluit is, vir beide Abagold en HIK, om geselekteerde broei diere te behou gebaseer op hulle onderskeie bydraes tot die F1 nageslag asook die herevaluaring van die potensiaal van die oorblywende broei diere onder meer beheerde teling toestande. Geen voor-die-handliggende tendense is waargeneem vir groei nie met die meeste individue wat beide vinniger en stadiger groeiende nageslag geproduseer het. Jong individue moet geherevalueer word op tweejarige ouderdom om te bepaal of die groei voordeel of nadele behou is en om te bepaal om groei voordele/nadele geslagspesifiek is.
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Mutation analysis of four genes implicated in iron homeostasis in porphyria cutanea tarda (PCT) patientsPanton, Nicola 03 1900 (has links)
Thesis (MSc (Genetics))--University of Stellenbosch, 2008. / The porphyrias are a group of genetic diseases resulting from the accumulation of haem precursors
due to defective enzyme activity in either one of the last seven enzymes in the haem biosynthesis
pathway. One of the common hepatic porphyrias, porphyria cutanea tarda (PCT), arises from the
inhibition of uroporphyrinogen decarboxylase (UROD) activity. It is characterised by excessive
urinary and hepatic excretion of uroporphyrinogens and manifests cutaneously in the form of
dermatitis. Two main forms of PCT have been described, namely familial PCT (fPCT) and sporadic
PCT (sPCT). PCT is a complex disease and a few genetic (including modifier loci) and
environmental precipitating factors have been implicated in the aetiology of PCT. An important
exacerbating factor, iron overload, is observed in the majority of PCT patients.
The aim of this study was to determine whether DNA sequence variation in the 5' untranslated
regulatory region of four genes involved in iron metabolism i.e. CP, CYBRD1, HAMP and
SLC40A1 may in any way be associated with PCT. The study cohort consisted of 74 patients from
three diverse South African populations including 15 Black (eight males and seven females), 30
Caucasian (13 male and 17 females) and 29 Coloured (18 males and 11 females) individuals as well
as 132 population-matched controls. The promoter region of the selected genes were screened for
variation utilising the techniques of polymerase chain reaction (PCR) amplification, heteroduplex
single-stranded conformational polymorphism (HEX-SCCP) analysis, restriction fragment length
polymorphism (RFLP) analysis and bi-directional semi-automated DNA sequencing.
Twenty three previously described and eleven novel variants were identified. The novel variants
comprised CYBRD1: -1540G/A, -1474G/A, -1452T/C, -1346T/C, -1272T/C, -645T/C;
G(T)8G(T)nG(T)nG(T)9; HAMP: -429G/T and SLC40A1: -1461T/C, -1399G/A, -524C/T.
Statistically significant associations were observed at a number of loci. In silico analysis revealed
several putative transcription factor binding sites (TFBSs) spanning the regions of variation. The
disruption of an existing (or creation of a novel) TFBS is thought to occur in the presence of a variant in a number of instances. This may lead to the manipulation of transcription rates, thereby
depicting a possible mechanism for gene dysregulation.
The study presented here was undertaken as a preliminary investigation to determine the
contribution (if any) of variants in the regulatory regions of candidate genes in iron metabolism in
South African PCT patients. Considering the increasing incidence of PCT, in particular the Black
South African population, it is necessary to elucidate the underlying mechanisms of iron overload in
PCT patients. The propitious findings signified in the study, in conjunction with phenotypegenotype
correlations, will assist in clarifying the association between iron overload and PCT. / jfl2010 / Imported from http://etd.sun.ac.za April 2010.
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The molecular and biological characterisation of ORF5 of three South African variants of Grapevine Vitivirus ABlignaut, Marguerite 03 1900 (has links)
Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Grapevine Vitivirus A (GVA), genus Vitivirus, family Flexiviridae is a well
characterised single-stranded RNA virus that has been implicated in the grapevine
diseases, Kober stem grooving and Shiraz disease. The virus infects both its
host, Vitis vinifera and the experimental model plant, Nicotiana spp.. Biological
studies performed on the virus in its herbaceous host, Nicotiana benthami-
ana, revealed that many divergent variants of the virus exists in South Africa
and can induce di erent symptoms in the model plant. Further molecular
analysis divided the variants into three molecular groups based on molecular
heterogeneity and nucleotide identity. The establishment of an infectious
full-length cDNA clone of GVA contributed towards the elucidation of gene
functions for 4 of the 5 open reading frames (ORF's), and indicated ORF5
as the pathogenicity determinant within the genome. Further studies also
showed that ORF5 encodes for a nucleic acid binding protein that exhibits
suppression activity of a plants' natural virus silencing mechanism. Many proteins
that have previously been identi ed as the pathogenicity determinant
within a viral genome have been found to encode for suppression activity.
Although suppression activity has been elucidated within the ORF5 of the
Italian cDNA clone of GVA, IS 151, no such study has yet been performed on
the divergent South African variants of GVA. Three variants, GTR1-1, GTR1-
2 and GTG11-1, which represent each of the molecular groups (Group III, II
and I), were selected for this study. The aim of this study was to visually
elucidate suppression activity of RNA transgene silencing by the ORF5's of
GTR1-1, GTR1-2 and GTG11-1 in a transient expression assays in transgenic
N. benthamiana (line 16c). Pathogenicity studies for these variants were also
performed. The ORF5 of the infectious full-length clone, GVA118, which can
also serve as an expression vector, was deleted and provided with restriction
enzyme sites into which the respective ORF5s and the marker genes, GFP and
GUS could be cloned directionally. Infectivity, symptom development and systemic
movement were compared between the di erent full length clones after
co-in ltration in N. benthamiana. Preliminary results obtained in this study
failed to visually indicate any suppression activity encoded by the ORF5 of
GTR1-1, GTR1-2 and GTG11-1. The deletion of ORF5 within GVA118 was
successful and rendered the infectious full length clone asymptomatic. Directional
cloning of the ORF5 of GTR1-1 into the unique restriction enzymes
provided previously, resulted in much milder symptoms than those observe for
GTR1-2 and GTG11-1. No GFP and GUS accumulation could be detected.
This study has established an infectious full-length cDNA clone, pBINSN-e35SGVA118
ORF5-1-1-pA, that can possibly induce much milder symptoms in
the herbaceous host, N. benthamiana. This construct can be further characterised
as a possible expression vector of foreign proteins in herbaceous hosts
and grapevine.
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The construction of an infectious clone of grapevine virus A (GV A)Du Preez, Jacques 04 1900 (has links)
Thesis (MSc (Genetics))--University of Stellenbosch, 2005. / An infectious clone of a viral RNA genome is one that can be used, either as an in vitro transcript or as
cDNA, to produce an infection in a susceptible plant. Infectious clones serve as a tool to study viral
RNA genomes at a molecular level to gain deeper insight into genome organization, viral gene
function, presence of regulatory sequences and gene expression. In the Western Cape (and elsewhere) a
new crippling grapevine disease, known as Shiraz disease, is emerging of which the aetiology and
pathogenic agents involved are not yet fully understood. Grapevine virus A (GVA), genus Vitivirus,
family Flexiviridae, is thought to be the associated with this disease. The aim of this study was to
construct a full-length infectious cDNA clone of GVA, which will aid in the molecular study of the
viral genome. This clone could ultimately be used to investigate GVA’s involvement in Shiraz disease,
which could lead to the unravelling of the aetiology and control of the disease. A full-length clone of
GVA, named GVA-IC2/T7-2972-3, was constructed in several steps using restriction digestion/ligation
and primer overlap extension PCR. Grapevine virus A cDNA fragments were obtained from GVAinfected
Nicotiana benthamiana and Vitis vinifera plants using three different techniques, of which the
Rapid direct-one-tube RT-PCR was most successful. A 5’ T7 promoter and a 3’ poly-A tail were
incorporated and the full-length clone was cloned into pBluescript II SK (+). Full-length sequencing
of the clone, revealed two significant frameshift mutations. The first mutation was a single base pair
insertion (one G) in a slippery site of 6 G’s at position 1380 – 1385 in open reading frame one (ORF 1)
of the viral genome. This mutation was corrected by PCR-based site-directed mutagenesis, which
resulted in pSK-GVA-mutagen-3 and pSK-GVA-mutagen-4. The second mutation was a single base
pair deletion (one G) at position 6959 in ORF4, which coded for the coat protein (CP). Several
techniques were attempted to correct this mutation, but none were successful. Even though the second
mutation could not be corrected, in vitro transcriptions were performed on three clones followed by
subsequent infections of N. benthamiana plants. The three clones included pSK-GVA-mutagen-3, pSKGVA-
mutagen-4 (both hosting the mutation at position 6959) and GVA-IC2/T7-2972-3 (hosting both
mutations). At 21 days post-inoculation no significant visual symptoms were observed in plants
infected with in vitro RNA or in plants infected with wild type GVA. Rapid direct-one-tube RT-PCR
results revealed the presence of viral RNA in infected leaves and apical leaves of infected plants, and
provided preliminary evidence that the mutated clones were still capable of systemic infection and viral
movement. These results are still inconclusive, and several post-infection studies will have to be
performed to confirm these findings. Koch's postulates will also have to be proved in order to confirm the infectious nature of the clones. The effect of the two mutations in the constructed clones will be
investigated further and post-infection analysis performed to deduce whether the viral progeny are
devoid of the mutations. Three full-length GVA cDNA clones (hosting mutations) seemingly capable
of systemic infection in N. benthamiana plants were constructed in this study and have laid the
foundation for molecular and mutational analysis of the GVA genome. This could lead to the study of
pathogen-host interactions in order to unravel the aetiology of Shiraz disease in the future.
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Iron and multiple sclerosisBloem, Liezl 03 1900 (has links)
Thesis (MSc (Genetics))--University of Stellenbosch, 2007. / Multiple sclerosis (MS) is a disease that causes neurological dysfunction. Studies attempting to elucidate the role of genes in MS development may aid efforts to control the damage caused by the disease that affects two million people worldwide, e.g. improved diagnosis and treatment. Although the association of MS and genes has not been fully characterized the proposed genetic etiology has been supported by the observed association of MS with the Major Histocompatibility Complex (MHC), haplotype HLA-DRB1*1501, DRB5*0101, DQA1*0102, DQB1*0602. Iron, or rather the dysregulation thereof, has also been implicated as a precipitating factor in MS development.
Considering the factors of iron dysregulation and the genes involved in iron regulation, this study aims to identify variation within genes involved in iron metabolism namely the high iron gene (HFE), solute-carrier family 40 (iron regulated transporter) member 1 gene (SLC40A1), hepcidin anti-microbial peptide (HAMP), cytochrome b reductase 1 (CYBRD1) and hemojuvelin (HJV). Screening of 40 patients (33 female, seven male; 33 Caucasian, seven Coloured) for each of the five genes was achieved by the Heteroduplex Single-Stranded Conformation Polymorphism (HEX-SSCP) technique. Semi-automated DNA sequencing allowed for verification and characterization of the variants detected. Results included identification of four novel variants present in only the Caucasian patient group, characterized as IVS4-53G→A (HFE) (one of 33 patients; 3%), IVS2-65delA (CYBRD1) (two of 32 patients; 6.3%), 3’UTR+26delACGTCACGTTTCAAAACTA (CYBRD1) (one of 31 patients; 3.2%) and 219delG (HJV) (two of 33 patients; 6%). In addition, a total of 15 previously described variants were identified (seven intronic and eight exonic) of which three were also prevalent in only the Caucasian patient group. This study aimed to investigate the differences ...
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Genetic association analysis of polymorphisms in four cytochrome P450 genes, the MDR1 gene and treatment-outcome in Xhosa schizophrenia patientsTruter, Erika 03 1900 (has links)
Thesis (MSc (Genetics))--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Rapidly expanding knowledge of the human genome allows new insight into the interaction between drugs and DNA. The heterogeneic nature of schizophrenia is known to cause different patients to display dissimilar drug responses, reflecting distinct genetic profiles. Resulting adverse side effects include tardive dyskinesia (TD), a movement disorder associated with the long-term use of antipsychotic drugs. The identification of a pharmacogenetic basis of TD may have significant clinical implications in the treatment of schizophrenia, allowing individualised prescription of antipsychotic drugs and eventual elimination of undesirable side effects.
The current study focussed on a number of South African Xhosa schizophrenia patients, some of whom have been diagnosed with TD. The investigation sought to establish whether the underlying mechanism causing the disorder to manifest only in some individuals, might be attributed to differences in DNA sequences, i.e. genomic susceptibility. A number of candidate polymorphisms in the CYP and MDR1 genes were evaluated in three separate analyses. (The same approach was followed in each investigation, and only known polymorphisms were selected.) The incidences of the various variants were compared between TD and non-TD patients. In addition, potential predisposing factors, i.e. tobacco and cannabis smoking and anhedonia, were taken into consideration. These were analysed concurrently with DNA data and TD status.
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Mutation screening of pre-eclampsia candidate genes, LEP (ob) and LEPR (obR).Hoek, Kim G.P. 03 1900 (has links)
Thesis (MSc (Genetics))--University of Stellenbosch, 2006. / Pre-eclampsia is a multisystemic disorder with an incidence of ~6-8% in non-Caucasian women in the Western Cape. Trophoblast invasion is vital for adequate anchorage of the placenta to the uterine wall as well as for the optimisation of utero-placental blood flow in uncomplicated pregnancies. This process is facilitated by the fetal trophoblast cells that digest the extracellular matrix of the uterus by secreting various molecules, including the metalloproteinases (MMP), of which MMP-9 has an increased production during the first trimester. Leptin, an autocrine regulator of MMP-9 secretion, functions via the leptin receptor to prevent over-invasion of maternal tissues.
The aim of this study was to investigate the role of the leptin (ob) and leptin receptor (obR) genes in predisposition to pre-eclampsia and involved screening the genes in South African non-Caucasian cohorts and performing statistical analysis to determine whether any variants contributed to the disease profile.
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Identification of molecular markers for the diagnostic identification of the intracellular prokaryote associated with the appearance of withering syndrome in the abalone Haliotis midaeOckert, Candice 03 1900 (has links)
Thesis (MSc (Genetics))--University of Stellenbosch, 2005. / Withering syndrome is a severe disease of abalone, Haliotis species that has been associated with mortality ranging from 99% in black, H. cracherodii Leach and 30% in red abalone, H. rufescens Swainson. The disease was first observed in California, along the west coast of North America and is an economically important disease that has led to the closure of the black abalone fishery throughout the southern California State. The causative agent of withering syndrome is a gram-negative intracellular Rickettsiales-like prokaryote designated Candidatus xenohaliotis californiensis. The geographical range of C. xenohaliotis californiensis is broad, besides red and black abalone it has also been reported in yellow, H. corrugate and blue abalone, H. fulgens all caught in Baja California, Mexico. In 2000 a Rickettsiales-like prokaryote resembling the disease-causing agent was observed in the digestive gland of the South African abalone H. midae. In this study we aimed to determine the relationship of the bacterium observed in the local abalone species, H. midae to the disease-causing agent of withering syndrome.
A specific PCR and in situ hybridization test using primers and probes specific for the C. xenohaliotis californiesis 16S rDNA gene were used to screen H. midae digestive gland tissues showing classical features of the Rickettsiales-like prokaryote. Both analyses indicated that C. xenohaliotis californiensis is not present in the local abalone species. We therefore aimed the identification of the organism parasiting the local abalone species by DNA sequence analysis of the 16S rDNA gene. The 16S rDNA gene was amplified by PCR, cloned and sequenced. Phylogenetic trees, constructed by maximum parsimony analysis revealed a diverse community comprised of α - Proteobacteria, Mollicutes and Spirochaetes. In the class α - Proteobacteria a novel group of sequences showing phylogenetic affinities to the order Rickettsiales was identified as likely candidate for forming the Rickettsiales-like inclusions in the digestive gland of H. midae. Oligonucleotide probes that bind to four variable regions of the novel group were used to confirm their presence in infected H. midae digestive gland tissue by in situ hybridization. Although these probes did not recognize the inclusions formed by the Rickettsiales-like organisms, they revealed the presence of a group of free-living bacteria abundant in the host tissue.
We therefore conclude that (1) C.xenohaliotis californiensis is not present in the South- African abalone, H. midae; (2) the organisms isolated from the digestive gland of H. midae are part of the normal microflora and (3) the group of sequences showing phylogenetic affinities to the order Rickettsiales is not responsible for the Rickettsiales-like inclusions in infected digestive gland tissues but represent a novel group of organisms that are abundant in the host tissue.
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Molecular characterisation of the gene, LGALS13, and its putative involvement in pre-eclampsiaPostma, Alisa 03 1900 (has links)
Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Pre-eclampsia is one of the most common hypertensive disorders of
pregnancy in South Africa. Presently, the only cure for pre-eclampsia is
delivery, which brings with it, additional complications. As an alternative,
clinical management of this disorder relies on timely diagnosis.
The predictive biomarker, Placental Protein 13 (PP13), is currently used
for the early diagnosis of pre-eclampsia, in an ELISA-based diagnostic
kit, developed by Diagnostic Technologies Limited (DTL)1. A decrease in
serum PP13 levels has been reported during the first trimester of
pregnancy in women who later develop pre-eclampsia. The function of
PP13 has not been fully elucidated and it is also not known whether the
reduction in PP13 levels is a cause or an effect of the disease. The use
of PP13 as a predictive biomarker for pre-eclampsia therefore warrants a
comprehensive study of this peptide and the encoding gene, LGALS13.
The aim of this study was firstly to characterise LGALS13 using a range
of in silico tools. PP13 was found to be most homologous to the
predicted protein product of a neighbouring “putative” gene, LOC148003.
A gene conversion event between these two genes most likely underlies
the so-called “hotspot mutation” in LGALS13. Data also demonstrates
that the DelT mutation disrupts functionally and structurally important
features of the gene and peptide sequences. Through the analysis of the
putative promoter region of LGALS13, the presence of a Stimulatory
protein-1 (Sp1) binding sequence element was predicted, which has
implications for regulation of LGALS13.
Secondly, the study aimed to establish a study cohort for the
investigation of the effect that the LGALS13 genotype has on the
expression of its mRNA and protein products. Serum, plasma and whole blood samples were collected and prepared from 316 pregnant women.
Placental tissue samples were obtained from a selected group of these
subjects for RNA extraction. Once the sampling on the two remaining
targeted deliveries has occurred, the collection of samples will be
batched and sent to DTL in Israel, for PP13 measurement.
DNA was extracted from the whole blood samples obtained, and all
study participants were genotyped for seven sequence variants within
the LGALS13 gene using (i) Multiphor Single Stranded Conformational
Polymorphism and Heteroduplex (SSCP/HD) analysis, (ii) restriction
enzyme analysis and (iii) DNA sequencing. The genotype data sets will
be compared with PP13 levels when they become available, and also
with clinical parameters, once the deliveries have all occurred and the
database is complete.
This study demonstrated the power of an in silico approach to direct the
focus of future experimental work. The newly established study cohort
will be used for prospective studies aiming at a better understanding of
the role which LGALS13 and PP13 play in the early prediction of preeclampsia.
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Development of gene-linked molecular markers in South African abalone (Haliotis midae) using an in silico mining approachRhode, Clint 03 1900 (has links)
Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The South African abalone, Haliotis midae, is the only endemic species of commercial value. Aquaculture remains the only avenue for expanding the industry, since the closure of the fishery. The current focus is on implementing a molecular breeding programme; thus the development of molecular markers for linkage mapping and QTL analysis is a priority. Various markers, mainly anonymous, have been developed for H. midae; however emphasis is being placed on the development of gene-linked type I molecular markers. The present study investigates and demonstrates the use of public sequence collections to develop type I markers for a species with limited genomic resources, via three strategies: Surveying anonymous H. midae microsatellite markers’ flanking regions to find homology to gene sequences in public databases, cross-species marker transfer of anonymous markers from H. rubra and H. discus hannai demonstrating putative gene associations and lastly EST marker mining (SNP and microsatellites) from various Haliotids and testing transfer to the target species. Approximately 17% of H. midae anonymous markers showed significant similarity to genes. The current study also reports higher cross-species transferability from both H. rubra and H. discus hannai to H. midae (39% and 20.5%, respectively) than previously demonstrated and 15 EST-microsatellites and 16 EST-SNPs were successfully mined. Furthermore, the non-random distribution of microsatellites and high nucleotide diversity in the H. midae genome was confirmed. This is a low cost and time effective method for marker development and presents a continuous and dynamic resource that could be used for future marker development and characterisation as sequence information in public databases grow exponentially. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse perlemoen, Haliotis midae, is die enigste van vyf inheemse spesies van kommersiële waarde. Na die noodgedwonge sluiting van die vissery, is akwakultuur die mees praktiese oplossing om die perlemoen industrie uit te brei. Die huidige fokus is gerig op die implementering van ‘n molekulêre teel-program en dus is die ontwikkeling van molekulêre merkers vir genetiese kartering en kwantitatiewe kenmerk lokus analise, van uiterste belang. Tipe II merkers is voorheen vir die perlemoen ontwikkel, maar huidige tendense lê klem op die ontwikkeling van geen-gekoppelde tipe I merkers. Die huidige studie ondersoek die gebruik van publieke databasisse vir die ontwikkeling van tipe I molekulêre merkers vir ‘n spesie met beperkte genomiese bronne. Drie strategieë is geïmplementeer: Eerstens is ‘n opname gemaak van die homologie van perlemoen tipe II merker-vleuelende volgordes met geen volgordes in databasisse. Verder is die oordraagbaarheid van tipe II merkers vanaf H. rubra en H. discus hannai wat assosiasie met gene toon ondersoek. Laastens is ‘n Uitgedrukte Volgorde Merk (UVM) (Expressed Sequence Tag, EST) merker-ontginnings metode vanaf verskeie Haliotis spesies en toetsing van oordraagbaarheid na die teiken spesie uitgevoer. Ongeveer 17% van die tipe II H. midae merkers het geniese assosiasie getoon. ‘n Hoër tussen-spesie oordraagbaarheid vanaf beide H. rubra en H. discus hannai na H. midae (39% en 20.5%, onderskeidelik) word gerapporteer in vergelyking met vorige studies en 15 UVM-mikrosatelliete en 16 UVM-enkel nukleotied polimorfismes (single nucleotide polimorphism, SNP) is ontwikkel. Verder bevestig die studie die nie-lukrake verspreiding van mikrosatelliete en hoë nukleotied diversiteit in die perlemoen genoom. Die gebruik van publieke databasise vir die ontwikkeling en karakterisering van tipe I molekulêre merkers is tyd- en koste-besparend en bied ‘n volgehoue en dinamiese bron vir toekomstige gebruik.
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