Genetic mapping of the evergrowing gene in peach [Prunus persica (L.) Batsch

In temperate locations, terminal apices on evergrowing (also called evergreen) peach trees keep growing in winter until killed by low temperatures, while the lateral buds go into dormancy. A recessive allele of a single gene (evergrowing or evg) controls this trait in peach. The amplified fragment length polymorphism (AFLP) technique and bulked segregant analysis were applied to construct a local genetic linkage map for the evg gene from the cross Empress op op dwarf x Evergrowing (P.I. 442380). This map, comprising nine AFLP markers and the evg locus, covers a total genetic distance of 79.3 cM. Four dominant AFLP markers (EAT/MCAC, ETT/MCCA2, EAT/MCTA, and ETT/MACC) were linked to the evg locus at distances of 1, 5.3, 6.7, and 11.7 cM, respectively. EAT/MCAC and EAT/MCTA were converted into polymorphic sequence-tagged sites. Microsatellite markers in the evg region were developed from peach bacterial artificial chromosome (BAC) clones that hybridized to the AFLP marker fragments. Using three microsatellite anchor markers (pchgms12, pchgms17, and pchgms19), the local genetic linkage map was integrated into one minor linkage group of a previously constructed peach rootstock genetic linkage map. Three AFLP markers from the rootstock genetic linkage map were found linked to the evg locus.     

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)_n- and (CA)_n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L. Received: 18 June 1999 / Accepted: 6 December 1999     

A first insight into peach [Prunus persica (L.) Batsch] SNP variability

Three factors may have reduced the diversity at both individual gene and whole genome levels in cultivated peach: its self-compatible mating system, the narrow genetic basis of most commercial cultivars, and the recent strong selection towards agronomically interesting traits. Previous diversity analyses with markers such as simple sequence repeats (SSRs) have revealed low levels of genetic variability. Here, we sequenced 23 genome-wide distributed DNA fragments in 47 occidental peach varieties, also observing reduced variability levels. On average, there was one single nucleotide polymorphism (SNP) every 598 bp and one indel every 4,189 bp. As expected, variability was higher in non-coding than in coding regions (one SNP every 390 non-coding bp versus one in 1,850 bp in coding DNA). In general, SNPs were observed at relatively high frequency, mean minor allele frequency?=?0.225, meaning that a large proportion of the SNPs discovered by sequencing similar germplasm will be useful for other purposes, such as association mapping. The average heterozygosity of the varieties was 0.28, with a low correlation between SSR and SNP heterozygosity. The whole sequence of two candidate genes, a pectate lyase 1 candidate for fruit firmness (CGPAA2668) and a sucrose synthase 1 candidate for sugar content (CGPPB6189), in the 47 varieties revealed that they both may have suffered a process of balancing selection.     

In vitro callus induction from adult tissues of peach (Prunus persica L. Batsch)

We describe an efficient protocol for callus induction from adult tissues of Prunus persica (L.) Batsch. Three different commercial peach genotypes, Early May®, Zise May®, and UFO-3®, plus three other genotypes from hybrid crosses performed in February 2006, PS108, PS208, and PS708, were used in the study. Thirteen explant treatments were tested using nine different plant parts. Murashige and Skoog and woody plant medium salts were assayed with several concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KN), and thidiazuron, and two different photoperiods were tested, a 16-h photoperiod or continuous darkness. In terms of the quantitative response, two parameters were assessed: the number of d to callus induction and relative callus growth recorded after 30 d. Woody plant medium supplemented with 2,4-D and KN significantly increased the rates of callus induction in the majority of treatments. And no significant differences among the P. persica genotypes were found. The explants derived from stem and calyx produced up to 85 and 96% callus induction, respectively. The protocol described here could be used for efficient callus induction in a range of Prunus spp.     

Mapping QTLs controlling fruit quality in peach (Prunus persica (L.) Batsch)

 The organoleptic quality of fleshy fruits is in a large part defined by their composition of soluble sugars and organic acids. An F₂ population issuing from a cross between two peach varieties, ‘Ferjalou Jalousia’, a non-acid peach, and ‘Fantasia’, an acid nectarine, was analysed over 2 successive years for agronomic characters and for molecular-marker (isoenzymes, RFLPs, RAPDs, IMAs and AFLPs) segregations. Blooming and maturity dates, as well as productivity, were noted for each tree. Four fruits per tree were analysed at maturity for fresh weight, colour, pH, titratable acidity, soluble-solids content (SSC), acid (malic, citric and quinic acids) and sugar (sucrose, glucose, fructose, sorbitol) contents. QTLs were detected for all fruit components analysed, except for fruit colour. The QTLs for nearly all components were present on two linkage groups. For productivity, fresh weight, pH, quinic acid, sucrose and sorbitol content, all the detected QTLs displayed the same effect as the parental phenotypes. By contrast, for maturity date, titratable acidity, malic and citric acids and fructose, some QTLs displayed the same effect as the parental phenotypes while others displayed the opposite effect. The fraction of the total variation in each trait throughout the population explained by the QTLs was very high and reached more than 90% for some characters. For most of the characters analysed, epistasis was observed between QTLs. Received: 10 October 1997 / Accepted: 18 August 1998     

Distinct nuclear organization, DNA methylation pattern and cytokinin distribution mark juvenile, juvenile-like and adult vegetative apical meristems in peach (Prunus persica (L.) Batsch)

Chromatin organization, nuclear DNA methylation and endogenous zeatin localization were investigated in shoot apical meristems (SAM) during juvenile and adult phases of peach (Prunus persica (L.) Batsch). The aim was to examine the extent to which these parameters could discriminate the juvenile and adult SAMs. Seedlings (juvenile, cannot flower), basal shoots (called juvenile-like, because they exhibit juvenile macroscopic traits) and apical shoots (competent to form flowers) of adult plants were chosen. Nuclear chromatin exhibited chromocentres that were peripherally distributed in SAMs of juvenile and juvenile-like shoots, but were diffusely spread in those of adult shoots. These patterns coincided with a peripheral labelling of DNA methylation in juvenile and juvenile-like meristem nuclei versus a diffuse labelling pattern in adult meristem nuclei. During vegetative growth (from March to June), the level of nuclear DNA methylation was higher in adult meristems than in juvenile and juvenile-like ones. The immunolocalization of zeatin in juvenile SAM was in the subapical region, but adult meristems exhibited a widespread localization or a signal confined within the boundaries of the central zone. The extent to which the acquisition of a strongly zonated pattern of these parameters as markers of floral competence in adult SAMs is discussed.     

Purification and characterization of a beta-galactosidase from peach (Prunus persica)

A beta-galactosidase (EC 3.2.1.23) from peach (Prunus persica cv Mibackdo) was purified and characterized. The purified peach beta-galactosidase was 42 kDa in molecular mass and showed high enzyme activity against a the beta-galactosidase substrate, rho-nitrophenyl-beta-D-galactopyranoside. The Km and Vmax values of the enzyme activity of the peach beta-galactosidase were 5.16 and 0.19 mM for rho-nitrophenyl-beta-D-galactopyranoside mM/h, respectively. The optimum pH of the enzyme activity was pH 3.0, but it was relatively stable from pH 3.0-10.0. The temperature optimum was 50 degrees C. The enzyme activities were not improved in the buffers that contained Ca2+, Cu2+, Zn2+, and Mg2+, which indicates that the purified peach beta-galactosidase did not require these cations as co-factors. However, the enzyme was completely inhibited by Hg2+. The purified protein was cross-reacted with an antibody against the persimmon fruit beta-galactosidase. A further comparison of the N-terminal amino acid sequence of the purified protein showed high homologies to those of beta-galactosidase in apple (87%), persimmon (80%), and tomato (87%). Therefore, enzymatic, immunological, and molecular evidences in this study indicate that the purified 42-kDa protein is a peach beta-galactosidase.     

Mapping quantitative trait loci associated with blush in peach [Prunus persica (L.) Batsch]

Blush is an important trait for marketing peaches. The red skin pigmentation develops through the flavonoid and anthocyanin pathways, and both genetic and environmental stimuli, and their interaction, control the regulation of these pathways. The molecular basis of blush development in peach is yet to be understood. An F₂ blush population (ZC²) derived from a cross between two peach cultivars with contrasting phenotypes for blush, “Zin Dai” (～30 % red) and “Crimson Lady” (～100 % red), was used for linkage map construction and quantitative trait loci (QTLs) mapping. The segregating population was phenotyped for blush for 4 years using a visual rating scale and quantified using a colorimeter (L*, a*, and b*) 1 year. The ZC² population was genotyped with the IPSC 9 K peach single-nucleotide polymorphism (SNP) array v1, and a high-density ZC² genetic linkage map was constructed. The map covers genetic a distance of ～452.51 cM with an average marker spacing of 2.38 cM/marker. Four QTLs were detected: one major QTL on LG3 (Blush.Pp.ZC-3.1) and three minor QTLs on LG 4 and 7 (Blush.Pp.ZC-4.1; Blush.Pp.ZC-4.2; Blush.Pp.ZC-7.1), indicating the presence of major and minor genes involved in blush development. Candidate genes involved in skin and flesh coloration of peach (PprMYB10), cherry (PavMYB10), and apple (MdMYB1/MdMYBA/MdMYB10) are located within the interval of the major QTL on LG3, suggesting the same genetic control for color development in the Rosaceae family. Marker-assisted selection (MAS) for blush is discussed.     

Genetic linkage map of peach [Prunus persica (L.) Batsch] using morphological and molecular markers

A genetic linkage map of peach [Prunus persica (L.) Batch] was constructed in order to identify molecular markers linked to economically important agronomic traits that would be particularly useful for long-lived perennial species. An intraspecific F₂ population was generated from self-pollinating a single F₁ plant from a cross between a flat non-acid peach, ‘Ferjalou Jalousia^®’ and an acid round nectarine ‘Fantasia’. Mendelian segregations were observed for 270 markers including four agronomic characters (peach/nectarine, flat/round fruit, acid/non-acid fruit, and pollen sterility) and 1 isoenzyme, 50 RFLP, 92 RAPD, 8 inter-microsatellite amplification (IMA), and 115 amplified fragment length polymorphism (AFLP) markers. Two hundred and forty-nine markers were mapped to 11 linkage groups covering 712 centiMorgans (cM). The average density between pairs of markers is 4.5?cM. For the four agronomic characters studied, molecular markers were identified. This map will be used for the detection of QTL controlling fruit quality in peach and, particularly, the acid and sugar content.     

Molecular characterisation of sweet cherry (Prunus avium L.) genotypes using peach [Prunus persica (L.) Batsch] SSR sequences

A total of 76 sweet cherry genotypes were screened with 34 microsatellite primer pairs previously developed in peach. Amplification of SSR loci was obtained for 24 of the microsatellite primer pairs, and 14 of them produced polymorphic amplification patterns. On the basis of polymorphism and quality of amplification, a set of nine primer pairs and the resulting 27 informative alleles were used to identify 72 genotype profiles. Of these, 68 correspond to unique cultivar genotypes, and the remaining four correspond to three cultivars that could not be differentiated from the two original genotypes of which they are mutants, and two very closely related cultivars. The mean number of alleles per locus was 3.7 while the mean heterozygosity over the nine polymorphic loci averaged 0.49. The results demonstrate the usefulness of cross-species transferability of microsatellite sequences allowing the discrimination of different genotypes of a fruit tree species with sequences developed in other species of the same genus. UPGMA cluster analysis of the similarity data divided the ancient genotypes studied into two fairly well-defined groups that reflect their geographic origin, one with genotypes originating in southern Europe and the other with the genotypes from northern Europe and North America.     

Microsatellite markers in peach [Prunus persica (L.) Batsch] derived from an enriched genomic and cDNA libraries

Twenty‐four and 12 microsatellite loci were developed in peach [Prunus persica (L) Batsch cv. Akatsuki] by using an enriched genomic and fruit cDNA libraries, respectively. The microsatellite loci obtained from an enriched library produced 1–9 alleles per locus, 24 in total, of which 22 showed polymorphisms. The average values of observed and expected heterozygosities among the 24 loci were 0.15 and 0.68, respectively. The microsatellite loci derived from cDNA showed 1–7 alleles per locus. Eight sequences showed significant homology to the registered genes in a database.     

The potential of Prunus davidiana for introgression into peach [Prunus persica (L.) Batsch] assessed by comparative mapping

The potential for introgression of Prunus davidiana, a wild species related to peach, was evaluated with respect to problems of non-Mendelian segregation or suppressed recombination which often hamper breeding processes based on interspecific crosses. Three connected (F1, F2 and BC2) populations, derived from a cross between P. davidiana clone P1908 and the peach cultivar Summergrand were used. The intraspecific map of P. davidiana already established using the F1 progeny was complemented, and two interspecific maps, for the F2 and BC2 progenies, were built with a set of markers selected from the Prunus reference map. With the molecular data collected for the F2 map construction, regions with distorted marker segregation were detected on the genome; one third of all loci deviated significantly from the expected Mendelian ratios. However, some of these distorted segregations were probably not due to the interspecific cross. On linkage group 6, a skewed area under gametic selection was most likely influenced by the self-incompatibility gene of P. davidiana. Using anchor loci, a good colinearity between the three maps built and the Prunus reference map was demonstrated. Comparative mapping also revealed that homologous recombination occurred normally between P. davidiana and the Prunus persica genome. This confirmed the closeness of the two species. Higher recombination rates were generally observed between P. davidiana and P. persica than between Prunus amygdalus and P. persica. The consequences for plant breeding strategy are discussed. The three maps of the F1, F2 and BC2 progenies provide useful tools for QTL detection and marker-assisted selection, as well as for assessing the efficiency of the peach breeding scheme applied to introgress P. davidiana genes into peach cultivated varieties.     

Candidate genes and QTLs for sugar and organic acid content in peach [ Prunus persica (L.) Batsch

The identification of genes involved in variation of peach fruit quality would assist breeders in creating new cultivars with improved fruit quality. Major genes and quantitative trait loci (QTLs) for physical and chemical components of fruit quality have already been detected, based on the peach [Prunus persica (L.) Batsch] cv. Ferjalou Jalousia^® (low-acid peach) 2 cv. Fantasia (normally-acid nectarine) F₂ intraspecific cross. Our aim was to associate these QTLs to structural genes using a candidate gene/QTL approach. Eighteen cDNAs encoding key proteins in soluble sugar and organic acid metabolic pathways as well as in cell expansion were isolated from peach fruit. A single-strand conformation polymorphism strategy based on specific cDNA-based primers was used to map the corresponding genes. Since no polymorphism could be detected in the Ferjalou Jalousia^® 2 Fantasia population, gene mapping was performed on the almond [Prunus amygdalus (P. dulcis)] cv. Texas 2 peach cv. Earlygold F₂ interspecific cross from which a saturated map was available. Twelve candidate genes were assigned to four linkage groups of the peach genome. In a second step, the previous QTL detection was enhanced by integrating anchor loci between the Ferjalou Jalousia^® 2 Fantasia and Texas 2 Earlygold maps and data from a third year of trait assessment on the Ferjalou Jalousia^® 2 Fantasia population. Comparative mapping allowed us to detect a candidate gene/QTL co-location. It involved a cDNA encoding a vacuolar H⁺-pyrophosphatase (PRUpe;Vp2) that energises solute accumulation, and QTLs for sucrose and soluble solid content. This preliminary result may be the first step in the future development of marker-assisted selection for peach fruit sucrose and soluble solid content.     

Contribution of vegetative storage proteins to seasonal nitrogen variations in the young shoots of peach trees (Prunus persica L. Batsch)

Qualitative and quantitative variations in the level of two low molecular weight vegetative storage proteins (VSP 19 kDa and 16.5 kDa) in peach shoots were compared with annual variations in total nitrogen and total soluble proteins. Protein patterns were obtained by SDS-PAGE and silver staining on each of the 12 kinetic samples collected between October 1995 and November 1996. VSP 16.5 kDa and 19 kDa exhibited typical annual VSP variations in both parenchyma and phloem. In wood, VSP 16.5 kDa was only present in November. All N compounds tested were stored in the autumn and their levels fell in the spring. Parenchyma was the principal stem storage tissue for all N compounds tested, even if proteins were more often highly concentrated in phloem and even if wood was the major shoot constituent. In winter, the two VSP accounted for 13% of bark proteins and 11% of wood proteins. Their storage yield, given by the winter/summer (W/S) ratio was higher (18.5) than that of total proteins (4). Between August to March, i.e. during the storage phase, N fractions obtained from VSP (N3) and total soluble proteins minus VSP (N2) accounted, respectively, for only 3% and 21% of total N accumulation in the bark, the remainder being due to the fraction not extracted (N1). A marked drop in all N compound levels characterized the mobilization phase (March to April), particularly for N3 (-84% between March and April) which were mobilized slightly before other N compounds. Although N3 exhibited the best mobilization yield, it represented only 5% of the total N mobilized. So, in spite of a similarity between VSP and N annual variation patterns, there was no tight correlation between their contents in bark. N2 supplied a high proportion of the N used for spring regrowth (40%), but the larger share (55%) came from N1 which was probably made up of free amino acids. Very tight positive correlations have been observed between these two N fractions and the N status. The lower bark total N content measured in August (6.4 mg N g(-1 )DW) during the assimilation phase (April to August) was equal to the unavailable N fraction, and the bark N mobilization potential (between March and August) was estimated at 6.35 mg N g(-1) DW. VSP did not quantitatively represent the main stored N pool. But, because of their high W/S ratio and their early remobilization, they seemed to play an important role in spring regrowth initiation.