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Wild-type seeds of Prunoidaeae are bitter and toxic as they accumulate amygdalin, a cyanogenic phytoanticipin thought to have an important role in defense against herbivores and pathogens. The sweet taste of the seeds of cultivated almond (Prunus dulcis Miller D.A. Webb syn. Prunus amygdalus Batsch) originates from a mutation selected during early domestication and maintained in modern cultivars. Previous genetic analyses revealed that the almond seed taste depends on the genotype of the mother plant. Moreover, it is under monogenic control, and the allele associated with sweetness (Sk) is dominant over the allele conferring bitterness (sk). In previous studies, the Sk locus was positioned on almond linkage group (LG) 5. However, the molecular nature of the Sk gene is still elusive. The genome sequence of peach (Prunus persica L.), a species evolutionary close to almond, has been recently released and is publicly available at the Genomic Database of Rosaceae (GDR). In this thesis, we exploited peach genomic resources to identify new molecular markers linked to the Sk locus in almond. Moreover, we provide a fine-map of the Sk genomic region and identified a few candidate genes for kernel taste. The SSR markers BPPCT037, CPDCT028, EPDCU2584 and UDA045 were previously shown to be closely linked to the Sk locus on the almond LG5. BLAST search for peach nucleotide sequences similar to these markers SUMMARY resulted in the identification of best matches all positioned on scaffold 5, suggesting the identification of a synthenic region between the peach and almond genomes. The two markers UDA045 and CPDCT028, previously reported to flank the Sk locus, were found to delimit a region of about 810 Kb in the physical map of peach, likely containg the Sk gene ortholog. Aiming to saturate the Sk region, we designed several primer pairs on peach genes included in the 810 Kb interval flanked by UDA045 and CPDCT028. Sequence analysis of PCR products revealed the presence of six SNPs segregating in a large F1 mapping population of 476 individuals, obtained by crossing the two parental genotypes R1000 and Desmayo Largueta. This information was used to develop CAPS markers linked to the Sk locus. Newly identified CAPS markers and previously reported Sk-linked SSR markers were used to fingerprint the F1 population above mentioned. Marker data were merged with phenotypic scores and used to produce a saturated map of the Sk region. Notably, the marker order on almond LG5 resulted to be fully in accordance with the order of ortholog sequences in peach scaffold 5, indicating collinearity between the two genomes. Having provided strong evidence for genome collinearity between almond and peach and the identification of almond markers flanking the Sk locus, we concluded that the Sk ortholog in peach is likely located in a physical region of about 80 Kb, SUMMARY delimited by the genes referred to as ppa003882m and ppa005388m according to the nomenclature used in the GDR. Primers designed on the seven peach genes included in the interval delimited by the genes ppa003882m and ppa005388m were used on almond cDNA obtained by the kernel tegument tissues of sweet and bitter almonds for a real-time qPCR assay, in order to detect differences in expression levels that would have indicated the implication of these genes in amygdalin accumulation. Two of them were found to be more expressed in sweet genotypes, although difference were not always significant in all the time points of kernel ripening stage under test. Researches performed during the PhD period brought to the identification of a series of molecular markers linked to the Sk locus in almond. This is of great interest for almond breeding, as they can aid the selection of sweet genotypes without the need to wait for a long juvenile phase and carry out phenotypic tests. Specific advantages of the CAPS markers identified in this study regard their close linkage with the Sk locus, which minimizes errors due to recombination, and their co-dominant nature, which allows to distinguish between sweet homozygous and heterozygous individuals. Moreover, CAPS markers can be obtained by means of simple protocols that require relatively inexpensive laboratory equipment. This thesis also provides a fine-scale map of the genomic region around the Sk gene, which lays a foundation for its isolation via positional cloning and SUMMARY corroborates previous reports indicating high level of synteny and collinearity between species of the Prunus genus. According to the map provided in this thesis, and assuming collinearity between peach and almond, the Sk gene should be one of the ortholog of the seven peach genes flanked by the genes ppa003882m and ppa005388m. Five of them (ppa022201m, ppa025417m, ppa027182m, ppa015634m and ppa005343m, according to the GDR nomenclature) encode MYC transcription factors. The remaining two genes, ppa011942m and ppa023406m, respectively encode the MED10 component of the mediator complex, functioning as transcriptional co-activator in eukaryotes, and a putative galactose oxidase. The almond ortholog of the peach MYC transcription factor ppa022201m is a very good candidate for being the Sk gene, as its expression was found to be higher in the tegument tissue of a sweet genotype than in a bitter genotype, as resulting from real-time qPCR. Overall, we believe that this thesis might be of interest for basic research and applied breeding. Currently, we are testing newly identified CAPS polymorphisms on several almond genotypes, in order to test their possible applicability in practical breeding programs, and sequencing the genomic interval presumably containing the Sk locus in sweet and bitter almond genotypes. Moreover, we are carrying out studies addressed to the functional characterization of MYB transcription factor above mentioned which is a candidate for being the Sk gene.
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Dissertation Università di Foggia 2016
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