Table 1.
List of 95 peach/nectarine varieties representing the diversity of cultivated P. persica.
Table 2.
SNP markers.
Table 3.
List of Primers.
Figure 1.
LG 5 CxA map around the G locus.
Linkage map obtained from analysis of the CxA F2 progeny. On the left side distances are indicated in cM; on the right the marker name, the physical position on Peach v1.0 and marker skewedness are reported. The peach/nectarine locus and the indelG marker are shown in bold.
Figure 2.
Alignment of Quetta reads against a 635 kb interval of Peach v1.0 pseudomolecule 5.
Alignment results of reads, obtained by the resequencing of ‘Quetta’, against the peach genome region identified by the mapping interval in LG5 (from 15,853,006 bp to 16,488,104 bp). Top panel: intron-exon structure of ppa023143m. Central panel: plot of ‘Quetta’ paired-end distance (blue) and frequencies of single reads (yellow) at the ppa023143m locus. Bottom panel: blue lines are paired reads, green and red lines correspond to single reads with missing mate on the right and left side, respectively. The orange arrow points to the putative insertion inside exon 3 of ppa023143m.
Figure 3.
Variant discovery in PpeMYB25 (annotation refinement of ppa023143m).
Five nectarine genotypes (‘Madonna di Agosto’, MdA; ‘Quetta’, Q; ‘Stark Red Gold’, SRG; ‘Goldmine’, G; ‘Ambra’, A) were analyzed to confirm the presence of the insertion within exon 3 of PpeMYB25. (A) Long-range amplification products reveal for all the accessions a fragment of about 7 kb (compared to 960 bp expected from the reference genome). (B) Double digestion results of the long-range PCR products show the same pattern for all the genotypes. (C) Position and structure of the Ty-copia retrotransposon deduced by the by the NGS analysis of ‘Quetta’ long-range amplicon. The insertion results in a truncated version of the R2R3-MYB protein.
Figure 4.
Aminoacid alignment of the R2 and R3 MYB repeat sequences.
MYB domains (pfam00249) of peach PpeMYB25, cotton GhMYB25 (ACJ07153.1, [39]) and Antirrhinum AmMYBML1 (CAB433991.1, [54]) were aligned using the Muscle on line tool at EBI (http://www.ebi.ac.uk/Tools/msa/muscle/). Graphic display of the alignment was obtained using BoxShade (http://www.ch.embnet.org/software/BOX_form.html). Black shaded residues are identical, grey shaded residues are similar. Coordinates in the protein sequences are indicated.
Figure 5.
Expression analysis of PpeMYB25 in peach and nectarine flower buds.
(A) The expression patterns of the R2R3-MYB gene were evaluated in ‘Contender’ [C] and ‘Ambra’ [A] buds at seven, five, four and one week before anthesis (WBA). Genomic DNA of the two cultivars was also tested as a control. The same samples were analyzed for expression of RPII as standard (B) and checked for DNA contamination (C).
Figure 6.
A marker assay was developed based on sequence information on the PpeMYB25 gene and the Ty1-copia insertion. Three primers were designed to discriminate peach and nectarine genotypes (A, B). A panel of nectarines including the putative donors of the trait, show a unique fragment of about 200 bp (C). A set of peaches, of diverse pedigree and origins (Table 1) (D), shows homozygous or heterozygous patterns.