Stefano Tartarini

The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

Aligning male and female linkage maps of apple (Malus pumila Mill.) using multi-allelic markers

Abstract Linkage maps for the apple cultivars ‘Prima’ and ‘Fiesta’ were constructed using RFLP, RAPD, isozyme, AFLP, SCAR and microsatellite markers in a ‘Prima’בFiesta’ progeny of 152 individuals. Seventeen linkage groups, putatively corresponding to the seventeen haploid apple chromosomes, were obtained for each parent. These maps were aligned using 67 multi-allelic markers that were heterozygous in both parents. A large number of duplicate RFLP loci was observed and, in several instances, linked RFLP markers in one linkage group showed corresponding linkage in another linkage group. Distorted segregation was observed mainly in two regions of the genome, especially in the male parent alleles. Map positions were provided for resistance genes to scab and rosy leaf curling aphid (Vf and Sd1, respectively) for the fruit acidity gene Ma and for the self-incompatibility locus S. The high marker density and large number of mapped codominant RFLPs and some microsatellite markers make this map an ideal reference map for use in other progenies also and a valuable tool for the mapping of quantitative trait loci.

Quantitative genetic analysis and comparison of physical and sensory descriptors relating to fruit flesh firmness in apple (Malus pumila Mill.)

Abstract Texture is a major component of consumer preference for eating-quality in apple. A quantitative genetic analysis of traits associated with fruit-flesh firmness was carried out. This was based on segregation in an unselected mapping population replicated at six sites and harvested over 2 years. Different methods of assessment were compared, and a principal components analysis carried out. Instrumental measures used were Magness-Taylor penetrometer readings, stiffness by acoustic resonance, and a range of sensory descriptors assessed by a trained panel. There were good correlations between some measures, although stiffness was poorly correlated. Whilst genotype by environment effects were large, significant effects were attributable to the genotype, and these were used to detect QTLs. Significant QTLs were detected on seven linkage groups, with large effects on linkage groups L01, L10 and L16. Whilst there was a poor correlation between acoustic stiffness and other measures, the significant and suggestive QTL detected for stiffness on linkage group L10 did represent a subset of significant QTLs detected for the penetrometer measure. The use of sensory assessment proved valuable in detecting QTLs representing different attributes of fruit texture. The possibility of interaction between significant QTLs for fruit texture and other strongly selected traits such as scab resistance and fruit acidity is addressed.

Construction of a 550 kb BAC contig spanning the genomic region containing the apple scab resistance gene Vf

Abstract A positional cloning project was started in apple with the aim of isolating the Vf resistance gene of Malus floribunda 821. Vf confers resistance against apple scab, the most important disease in apple orchards. A chromosome walk starting from two molecular markers (M18-CAPS and AM19-SCAR) flanking Vf was performed, using a bacterial artificial chromosome (BAC) library containing inserts of the cultivar Florina, which is heterozygous for Vf. Thirteen BAC clones spanning the region between the two markers were identified in nine chromosome walking steps. The size of the resulting contig is approximately 550 kb. In order to map the Vf region in more detail, we analyzed over 2000 plants from different populations segregating for Vf with markers produced from BAC end sequences. In this way, we were able to restrict the possible location of the Vf gene to a minimum of five clones spanning an interval of approximately 350 kb.

RFLP and RAPD markers linked to the rosy leaf curling aphid resistance gene (Sd1) in apple. T

Abstract Sd1 is a dominant gene for resistance to biotypes 1 and 2 of the rosy leaf curling aphid, Dysaphis devecta Wlk., which can cause economic damage to apple trees. This report describes the identification of three RFLP and four RAPD markers linked to Sd1 in a cross between the D. devecta susceptible variety ‘Prima’ (sd1sd1) and the resistant variety ‘Fiesta’ (Sd1sd1). Potted trees were artificially infested in the glasshouse, and the ratio of resistant:susceptible plants supported the hypothesis that the resistance was under the control of a single dominant gene. The position of the gene was mapped to a single locus on a ‘Fiesta’ chromosome, within 2 cM of three tightly linked RFLP markers (MC064a, 2B12a and MC029b); the four RAPD markers were located further away (between 13 and 46 cM). This is the first report of molecular markers for an aphid resistance gene in tree fruit crops. The potential application of these markers in a marker-assisted resistance breeding programme is discussed.

RAPD markers linked to the Vf gene for scab resistance in apple.

Scab (Venturia inaequalis) is one of the most harmful diseases of apple, significantly affecting world apple production. The identification and early selection of resistant genotypes by molecular markers would greatly improve breeding strategies. Bulked segregant analysis was chosen for the identification of RAPD markers linked to the Vf scab resistant gene. Five different RAPD markers, derived from the wild species Malus floribunda. 821, were identified, and their genetic distance from Vf gene was estimated. The markers OPAM192200 and OPAL07580 were found to be very closely linked to the Vf gene. This result was indirectly confirmed by the analysis of resistant genotypes collected from various breeding programmes. Except for cv ‘Murray’, which carries the Vm gene, all these resistant genotypes showed the markers OPAM192200 and OPAL07580.

S-Allele characterization in self-incompatible pear (Pyrus communis L.)

Abstract. Gametophytic self-incompatibility, a natural mechanism occurring in pear and other fruit-tree species, is usually controlled by the S-locus with allelic variants (S1, S2, Sn). Recently, biochemical and molecular tools have determined the S-genotype of cultivars in various species. The present study determined the S-locus composition of ten European pear cultivars via S-PCR molecular assay, thereby obviating time-consuming fieldwork whose results are often ambiguous because of environmental effects. To verify the S-PCR assay, two putative S-allele DNA fragments of Japanese pear were isolated; their sequences proved to be identical to those reported in the databank. Six S-allele fragments of European pear were then sequenced. While field data confirmed the molecular results, fully and half-compatible field crosses were not distinguishable.

Fine mapping and identification of a candidate gene for a major locus controlling maturity date in peach

BackgroundMaturity date (MD) is a crucial factor for marketing of fresh fruit, especially those with limited shelf-life such as peach (Prunus persica L. Batsch): selection of several cultivars with differing MD would be advantageous to cover and extend the marketing season. Aims of this work were the fine mapping and identification of candidate genes for the major maturity date locus previously identified on peach linkage group 4. To improve genetic resolution of the target locus two F2 populations derived from the crosses Contender x Ambra (CxA, 306 individuals) and PI91459 (NJ Weeping) x Bounty (WxBy, 103 individuals) were genotyped with the Sequenom and 9K Illumina Peach Chip SNP platforms, respectively.ResultsRecombinant individuals from the WxBy F2 population allowed the localisation of maturity date locus to a 220 kb region of the peach genome. Among the 25 annotated genes within this interval, functional classification identified ppa007577m and ppa008301m as the most likely candidates, both encoding transcription factors of the NAC (NAM/ATAF1, 2/CUC2) family. Re-sequencing of the four parents and comparison with the reference genome sequence uncovered a deletion of 232 bp in the upstream region of ppa007577m that is homozygous in NJ Weeping and heterozygous in Ambra, Bounty and the WxBy F1 parent. However, this variation did not segregate in the CxA F2 population being the CxA F1 parent homozygous for the reference allele. The second gene was thus examined as a candidate for maturity date. Re-sequencing of ppa008301m, showed an in-frame insertion of 9 bp in the last exon that co-segregated with the maturity date locus in both CxA and WxBy F2 populations.ConclusionsUsing two different segregating populations, the map position of the maturity date locus was refined from 3.56 Mb to 220 kb. A sequence variant in the NAC gene ppa008301m was shown to co-segregate with the maturity date locus, suggesting this gene as a candidate controlling ripening time in peach. If confirmed on other genetic materials, this variant may be used for marker-assisted breeding of new cultivars with differing maturity date.

A unique mutation in a MYB gene cosegregates with the nectarine phenotype in peach.

Nectarines play a key role in peach industry; the fuzzless skin has implications for consumer acceptance. The peach/nectarine (G/g) trait was described as monogenic and previously mapped on chromosome 5. Here, the position of the G locus was delimited within a 1.1 cM interval (635 kb) based on linkage analysis of an F2 progeny from the cross ‘Contender’ (C, peach) x ‘Ambra’ (A, nectarine). Careful inspection of the genes annotated in the corresponding genomic sequence (Peach v1.0), coupled with variant discovery, led to the identification of MYB gene PpeMYB25 as a candidate for trichome formation on fruit skin. Analysis of genomic re-sequencing data from five peach/nectarine accessions pointed to the insertion of a LTR retroelement in exon 3 of the PpeMYB25 gene as the cause of the recessive glabrous phenotype. A functional marker (indelG) developed on the LTR insertion cosegregated with the trait in the CxA F2 progeny and was validated on a broad panel of genotypes, including all known putative donors of the nectarine trait. This marker was shown to efficiently discriminate between peach and nectarine plants, indicating that a unique mutational event gave rise to the nectarine trait and providing a useful diagnostic tool for early seedling selection in peach breeding programs.

Identifying a Carotenoid Cleavage Dioxygenase (ccd4) Gene Controlling Yellow/White Fruit Flesh Color of Peach

Peach flesh color is a monogenic trait with the white phenotype being dominant over the yellow; its expression has been reported to be determined by a carotenoid degradative enzyme. In the present study, a carotenoid cleavage dioxygenase (ccd4) gene was analyzed to test whether it can be responsible for the flesh color determinism. The analysis was conducted on chimeric mutants with white and yellow sectors of the fruit mesocarp; it was then extended to a pool of cultivars and a segregating F1 population. A ccd4 functional allele is consistently associated with the ancestral white flesh color; on the other hand, the yellow phenotype originated from at least three independent mutations disrupting ccd4 function, thus preventing carotenoid degradation. In addition, retro-mutations recovering ccd4 function and re-establishing the ancestral white flesh color were detected. Our results show that ccd4 is the gene controlling flesh color in peach; its expression results in the degradation of carotenoids in white-fleshed genotypes, while the yellow color arises as a consequence of its inactivation.