DataSheet_1_Resistance to Sharka in Apricot: Comparison of Phase-Reconstructed Resistant and Susceptible Haplotypes of ‘Lito’ Chromosome 1 and Analysis of Candidate Genes.zip

Sharka, a common disease among most stone fruit crops, is caused by the Plum Pox Virus (PPV). Resistant genotypes have been found in apricot (Prunus armeniaca L.), one of which—the cultivar ‘Lito’ heterozygous for the resistance—has been used to map a major quantitative trait locus (QTL) on linkage group 1, following a pseudo-test-cross mating design with 231 individuals. In addition, 19 SNP markers were selected from among the hundreds previously developed, which allowed the region to be limited to 236 kb on chromosome 1. A ‘Lito’ bacterial artificial chromosome (BAC) library was produced, screened with markers of the region, and positive BAC clones were sequenced. Resistant (R) and susceptible (S) haplotypes were assembled independently. To refine the assembly, the whole genome of ‘Lito’ was sequenced to high coverage (98×) using PacBio technology, enabling the development of a detailed assembly of the region that was able to predict and annotate the genes in the QTL region. The selected cultivar ‘Lito’ allowed not only to discriminate structural variants between the two haplotypic regions but also to distinguish specific allele expression, contributing towards mining the PPVres locus. In light of these findings, genes previously indicated (i.e., MATHd genes) to have a possible role in PPV resistance were further analyzed, and new candidates were discussed. Although the results are not conclusive, the accurate and independent assembly of R and S haplotypes of ‘Lito’ is a valuable resource to predict and test alternative transcription and regulation mechanisms underpinning PPV resistance.</p

Variant discovery in <i>PpeMYB25</i> (annotation refinement of <i>ppa023143m</i>).

<p>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 <i>PpeMYB25</i>. (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-<i>copia</i> 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.</p

LG 5 CxA map around the <i>G</i> locus.

<p>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.</p

genotyping data

Genotyping data for the five mapping population

Table_1_Quantitative Trait Loci Mapping and Identification of Candidate Genes Linked to Fruit Acidity in Apricot (Prunus armeniaca L.).XLSX

Apricot breeding programs could be strongly improved by the availability of molecular markers linked to the main fruit quality traits. Fruit acidity is one of the key factors in consumer acceptance, but despite its importance, the molecular bases of this trait are still poorly understood. In order to increase the genetic knowledge on the fruit acidity, an F1 apricot population (‘Lito’ × ‘BO81604311’) has been phenotyped for titratable acidity and juice pH for the three following years. In addition, the contents of the main organic acids of the juice (malate, citrate, and quinate) were also evaluated. A Gaussian distribution was observed for most of the traits in this progeny, confirming their quantitative inheritance. An available simple sequence repeat (SSR)-based molecular map, implemented with new markers in specific genomic regions, was used to perform a quantitative trait loci (QTL) analysis. The molecular map was also anchored to the recently published apricot genome sequence of ‘Stella.’ Several major QTLs linked to fruit acidity-related traits have been identified both in the ‘Lito’ (no. 21) and ‘BO81604311’ (no. 13), distributed in five linkage groups (LG 4, 5, 6, 7, and 8). Some of these QTLs show good stability between years and their linked markers were used to identify candidate genes in specific QTLs genomic regions.</p

Alignment of Quetta reads against a 635 kb interval of Peach v1.0 pseudomolecule 5.

<p>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 <i>ppa023143m</i>. Central panel: plot of ‘Quetta’ paired-end distance (blue) and frequencies of single reads (yellow) at the <i>ppa023143m</i> 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 <i>ppa023143m</i>.</p

Image_1_Quantitative Trait Loci Mapping and Identification of Candidate Genes Linked to Fruit Acidity in Apricot (Prunus armeniaca L.).TIF

Apricot breeding programs could be strongly improved by the availability of molecular markers linked to the main fruit quality traits. Fruit acidity is one of the key factors in consumer acceptance, but despite its importance, the molecular bases of this trait are still poorly understood. In order to increase the genetic knowledge on the fruit acidity, an F1 apricot population (‘Lito’ × ‘BO81604311’) has been phenotyped for titratable acidity and juice pH for the three following years. In addition, the contents of the main organic acids of the juice (malate, citrate, and quinate) were also evaluated. A Gaussian distribution was observed for most of the traits in this progeny, confirming their quantitative inheritance. An available simple sequence repeat (SSR)-based molecular map, implemented with new markers in specific genomic regions, was used to perform a quantitative trait loci (QTL) analysis. The molecular map was also anchored to the recently published apricot genome sequence of ‘Stella.’ Several major QTLs linked to fruit acidity-related traits have been identified both in the ‘Lito’ (no. 21) and ‘BO81604311’ (no. 13), distributed in five linkage groups (LG 4, 5, 6, 7, and 8). Some of these QTLs show good stability between years and their linked markers were used to identify candidate genes in specific QTLs genomic regions.</p

Functional Marker indelG.

<p>A marker assay was developed based on sequence information on the <i>PpeMYB25</i> gene and the Ty1<i>-copia</i> 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090574#pone-0090574-t001" target="_blank">Table 1</a>) (D), shows homozygous or heterozygous patterns.</p

Aminoacid alignment of the R2 and R3 MYB repeat sequences.

<p>MYB domains (pfam00249) of peach PpeMYB25, cotton GhMYB25 (ACJ07153.1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090574#pone.0090574-Li1" target="_blank">[39]</a>) and <i>Antirrhinum</i> AmMYBML1 (CAB433991.1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090574#pone.0090574-Finn1" target="_blank">[54]</a>) were aligned using the Muscle on line tool at EBI (<a href="http://www.ebi.ac.uk/Tools/msa/muscle/" target="_blank">http://www.ebi.ac.uk/Tools/msa/muscle/</a>). Graphic display of the alignment was obtained using BoxShade (<a href="http://www.ch.embnet.org/software/BOX_form.html" target="_blank">http://www.ch.embnet.org/software/BOX_form.html</a>). Black shaded residues are identical, grey shaded residues are similar. Coordinates in the protein sequences are indicated.</p

List of 95 peach/nectarine varieties representing the diversity of cultivated <i>P. persica</i>.

a<p>(B)  =  breeding materials (L)  =  landrace.</p>b<p>-  =  unknown donor of the nectarine allele.</p><p>For each accession the following information is reported: name, genotype at the indelG marker (941 bp reference peach allele, 197 bp nectarine allele carrying the retrotransposon insertion), phenotype, pedigree, country of origin and year of release/discovery when known, putative donor of the nectarine allele in <i>P.persica</i> if pedrigree information was available.</p