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

[摘要] ENGLISH ABSTRACT: Peaches and nectarines are important deciduous fruits in South Africa, both belonging to thespecies Prunus persica. The Agricultural Research Council (ARC) at Infruitec-Nietvoorbij inthe Western Cape is the primary source of peach cultivars in South Africa. The germplasmfrom 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 phenotypedmorphologically and could be prone to errors and duplications. This study had two aims; firstlyit aimed to utilize molecular marker technology (i.e. microsatellites markers) to fingerprint thegermplasm collection to facilitate authentication. Secondly, the study aimed at employingfunctional 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 206peach accessions, 20 almond accessions and seven hybrid accessions. One marker amplifiedmultiple loci in both peaches and almonds while another marker did not amplify in either thealmonds or the hybrids, and these were excluded. Therefore, the ARC peach accessions weresuccessfully fingerprinted with eight microsatellite markers, and the almonds and hybrids withseven. Clustering analysis found fifty-eight accessions, including eighteen accession from thereference collection, were either misidentified or unresolved needing further molecular andmorphological analysis. The accessions belonging to the reference collection are maintainedby DAFF and were considered authentic prior to this study.The germplasm was characterized for the peach/nectarine trait (hairy fruit epidermis) ascontrolled by the MYB25 gene. It has been reported that a retrotransposon insertion in thethird exon of the MYB25 gene disrupts formation of epidermal hairs in nectarine. The markerindelG was developed and fluorescently labelled and used to detect the presence of theretrotransposon insertion (g allele) or its absence (G allele). Peaches were observed to haveat least one G allele while nectarines were homozygous for the g allele. Seventy-fiveaccessions were genotyped as homozygous gg (nectarine), 35 accessions were heterozygousG/g (peach) and 96 were homozygous GG (peach). The heterozygous peaches can beintercrossed to develop new nectarine cultivars from peaches. The G allele, indicative of hairyfruit epidermis, was found in the almonds and some hybrids. Follow up studies for the role ofthe MYB25 gene in other Prunus species, especially in apricot (hairy), plum (glabrous) andcherry (glabrous), are recommended. The primers used in this study can be multiplexed withother 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 expressionof white or yellow flesh colour. White flesh is the wildtype while yellow flesh results from lossof gene function through any of three mutations: a frameshift mutation at the TC microsatelliteregion, an A to T substitution (SNP) or a retrotransposon insertion. Three novel primer setsincluding fluorescently labelled primer pairs were designed to detect these mutations. Theprimer pair amplifying the TC microsatellite region (CCD4-SSR) in the CCD4 gene identifiedthe wild type allele, a frameshift mutant and a very rare reversion allele in the accessionsOverall, 25 accessions had the 122/122 bp genotype associated with white flesh, 138accessions had the 124/124 bp genotype associated with yellow flesh colour, 42 accessionshad the 122/124 bp genotype associated with the white flesh and one accession had the124/128 bp genotype containing a reversion mutation associated with white flesh. The primerset amplifying the presence of the SNP (CCD-SNP) and its absence (CCD4-NoSNP) detectedthis 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 andCCD4-NoRetro) were not informative and the accessions could not be genotyped for thismutation. Therefore, the characterization of the flesh colour was incomplete and the deducedflesh colour are mostly tentative: with 33 accessions deduced as white flesh, 172 accessionsas 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 ofcrosses in regard to flesh colour. The primers detecting the retrotransposon should beredesigned and used to complete flesh colour genotyping.Overall, the microsatellite fingerprinting gave baseline data useful for future repropagationwhile molecular characterization for peach/nectarine and flesh colour will aid in the design ofcrosses with predictable outcomes. This study, therefore, lays a solid foundation for futuremolecular characterization and utilization in the ARC peach breeding programme.

[发布机构] Stellenbosch University