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Molecular fingerprinting and molecular characterization of the ARC's peach collection in South Africa

Kwalimba, Lawrence (2017-12)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Peaches and nectarines are important deciduous fruits in South Africa, both belonging to the species Prunus persica. The Agricultural Research Council (ARC) at Infruitec-Nietvoorbij in the Western Cape is the primary source of peach cultivars in South Africa. The germplasm from which these cultivars are developed is maintained at Bien Donne Research Farm (Paarl, Western Cape) and includes the reference collection for the Department of Agriculture, Forestry and Fisheries (DAFF). The germplasm collection has only been phenotyped morphologically and could be prone to errors and duplications. This study had two aims; firstly it aimed to utilize molecular marker technology (i.e. microsatellites markers) to fingerprint the germplasm collection to facilitate authentication. Secondly, the study aimed at employing functional markers for two agronomic traits of economic interest i.e. the peach/nectarine trait (hairy fruit epidermis) and white/yellow flesh colour. Nine reported polymorphic microsatellite markers were selected for the fingerprinting of 206 peach accessions, 20 almond accessions and seven hybrid accessions. One marker amplified multiple loci in both peaches and almonds while another marker did not amplify in either the almonds or the hybrids, and these were excluded. Therefore, the ARC peach accessions were successfully fingerprinted with eight microsatellite markers, and the almonds and hybrids with seven. Clustering analysis found fifty-eight accessions, including eighteen accession from the reference collection, were either misidentified or unresolved needing further molecular and morphological analysis. The accessions belonging to the reference collection are maintained by DAFF and were considered authentic prior to this study. The germplasm was characterized for the peach/nectarine trait (hairy fruit epidermis) as controlled by the MYB25 gene. It has been reported that a retrotransposon insertion in the third exon of the MYB25 gene disrupts formation of epidermal hairs in nectarine. The marker indelG was developed and fluorescently labelled and used to detect the presence of the retrotransposon insertion (g allele) or its absence (G allele). Peaches were observed to have at least one G allele while nectarines were homozygous for the g allele. Seventy-five accessions were genotyped as homozygous gg (nectarine), 35 accessions were heterozygous G/g (peach) and 96 were homozygous GG (peach). The heterozygous peaches can be intercrossed to develop new nectarine cultivars from peaches. The G allele, indicative of hairy fruit epidermis, was found in the almonds and some hybrids. Follow up studies for the role of the MYB25 gene in other Prunus species, especially in apricot (hairy), plum (glabrous) and cherry (glabrous), are recommended. The primers used in this study can be multiplexed with other primers and used for characterizing large number of samples at a relatively lower cost. The germplasm collection was also genotyped for the CCD4 gene that control the expression of white or yellow flesh colour. White flesh is the wildtype while yellow flesh results from loss of gene function through any of three mutations: a frameshift mutation at the TC microsatellite region, an A to T substitution (SNP) or a retrotransposon insertion. Three novel primer sets including fluorescently labelled primer pairs were designed to detect these mutations. The primer pair amplifying the TC microsatellite region (CCD4-SSR) in the CCD4 gene identified the wild type allele, a frameshift mutant and a very rare reversion allele in the accessions Overall, 25 accessions had the 122/122 bp genotype associated with white flesh, 138 accessions had the 124/124 bp genotype associated with yellow flesh colour, 42 accessions had the 122/124 bp genotype associated with the white flesh and one accession had the 124/128 bp genotype containing a reversion mutation associated with white flesh. The primer set amplifying the presence of the SNP (CCD-SNP) and its absence (CCD4-NoSNP) detected this SNP in 26 accessions, two of which were shown to be homozygous for the SNP mutation. The primer sets detecting the presence or absence of the retrotransposon (CCD4-Retro and CCD4-NoRetro) were not informative and the accessions could not be genotyped for this mutation. Therefore, the characterization of the flesh colour was incomplete and the deduced flesh colour are mostly tentative: with 33 accessions deduced as white flesh, 172 accessions as yellow flesh and 18 accessions as inconsistent and needing further follow up. Nevertheless, the partial genotypes and deduced phenotypes are useful and informative when designing of crosses in regard to flesh colour. The primers detecting the retrotransposon should be redesigned and used to complete flesh colour genotyping. Overall, the microsatellite fingerprinting gave baseline data useful for future repropagation while molecular characterization for peach/nectarine and flesh colour will aid in the design of crosses with predictable outcomes. This study, therefore, lays a solid foundation for future molecular characterization and utilization in the ARC peach breeding programme.

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