Molecular fingerprinting and molecular characterization of the ARC's peach collection in South Africa
Date
2017-12
Authors
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Publisher
Stellenbosch : Stellenbosch University
Abstract
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.
AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar
AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar
Description
Thesis (MSc)--Stellenbosch University, 2017.
Keywords
Peaches -- Western Cape -- South Africa, Prunus persica -- Molecular fingerprinting, Prunus persica -- Molecular characterization, UCTD