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Background Using cultivars based on a single, partly heterozygous, genotype is one of the bases of agriculture, frequently used in seed-propagated species such as vegetables and field crops (F1 hybrid varieties), and in most fruit trees and ornamentals (clonal varieties) that reproduce vegetatively. Producing two inbred lines that fully or partly reconstruct a given high-value heterozygous individual is now possible with the Resynthesis method, which uses molecular markers to select pairs of complementary genotypes in the F2 and subsequent selfing generations. The objective of this paper was to develop a software to facilitate the selection of such complementary pairs from a large number of segregating genotyped individuals. Results The ResynPy tool we developed screens the genotyping data of a segregating population, originating from selfing a top-performing partly heterozygous individual, and detects pairs of individuals that could produce progeny containing individuals nearly identical to the initial elite genotype. Our software selects candidate pairs of individuals in a small timeframe window with a low memory footprint. ResynPy tool could be part of a breeding toolkit to accelerate breeding programs in any plant species of economic importance capable of producing self-pollinated progeny. Conclusions ResynPy is a software developed in Python language and is freely available on GitHub (HYPERLINK “ https://github.com/kostasgalexiou/ResynPy/tree/main ”). ResynPy automates the process of individual selection implemented by the Resynthesis method, in a highly efficient manner through parallelization, allowing to plant breeders for detection of pairs of complementary individuals in a selfing population that could be suitable parents to a plant variety with economically important agronomic traits. With ResynPy , breeders are provided with a fast and easy-to-use tool that will aid them to advance their plant breeding programs.

In peach, the flat phenotype is caused by a partially dominant allele in heterozygosis ( Ss ), fruits from homozygous trees ( SS ) abort a few weeks after fruit setting. Previous research has identified a SSR marker (UDP98–412) highly associated with the trait, found suitable for marker assisted selection (MAS). Here we report a ∼10 Kb deletion affecting the gene PRUPE.6G281100 , 400 Kb upstream of UDP98-412, co-segregating with the trait. This gene is a leucine-rich repeat receptor-like kinase (LRR-RLK) orthologous to the Brassinosteroid insensitive 1-associated receptor kinase 1 ( BAK1 ) group. PCR markers suitable for MAS confirmed its strong association with the trait in a collection of 246 cultivars. They were used to evaluate the DNA from a round fruit derived from a somatic mutation of the flat variety ‘UFO-4’, revealing that the mutation affected the flat associated allele ( S ). Protein BLAST alignment identified significant hits with genes involved in different biological processes. Best protein hit occurred with AtRLP12 , which may functionally complement CLAVATA2 , a key regulator that controls the stem cell population size. RT-PCR analysis revealed the absence of transcription of the partially deleted allele. The data support PRUPE.6G281100 as a candidate gene for flat shape in peach.

Powdery mildew is one of the major diseases of peach ( Prunus persica) , caused by the ascomycete Podosphaera pannosa . Currently, it is controlled through calendar-based fungicide treatments starting at petal fall, but an alternative is to develop resistant peach varieties. Previous studies mapped a resistance gene ( Vr3 ) in interspecific populations between almond (‘Texas’) and peach (‘Earlygold’). To obtain molecular markers highly linked to Vr3 and to reduce the number of candidate genes, we fine-mapped Vr3 to a genomic region of 270 kb with 27 annotated genes. To find evidence supporting one of these positional candidate genes as being responsible of Vr3 , we analyzed the polymorphisms of the resequences of both parents and used near-isogenic lines (NILs) for expression analysis of the positional candidate genes in symptomatic or asymptomatic leaves. Genes differentially expressed between resistant and susceptible individuals were annotated as a Disease Resistance Protein RGA2 ( Prupe2G111700 ) or an Eceriferum 1 protein involved in epicuticular wax biosynthesis ( Prupe2G112800 ). Only Prupe2G111700 contained a variant predicted to have a disruptive effect on the encoded protein, and was overexpressed in both heterozygous and homozygous individuals containing the Vr3 almond allele, compared with susceptible individuals. This information was also useful to identify and validate molecular markers tightly linked and flanking Vr3 . In addition, the NILs used in this work will facilitate the introgression of this gene into peach elite materials, alone or pyramided with other known resistance genes such as peach powdery mildew resistance gene Vr2 .

Blooming in temperate fruit species is triggered by chilling and heat requirements (CR and HR), with a wide range of requirements within the same species. CR for flower bud dormancy release has become a limiting factor for geographical adaptation of fruit trees in warmer regions. The present study investigated the genetic basis of CR and HR to break dormancy and flowering time (FT) in an almond x peach F2 progeny. FT, HR and CR were evaluated over two consecutive years (2015/2016 and 2016/2017). Seven out of the eight identified quantitative trait loci (QTLs) were found in both periods of analysis. They affected eight traits, and included a consistent QTL for breaking dormancy, CR and HR. Two of them, affecting FT and HR for FT (GDHF), colocalized in G1, and the remaining QTLs, affecting chilling and heat requirements, both influenced by dormancy breaking (DB), were located in G6. These results indicate that factors not related to DB affect flowering time in this population. Implications of the results in peach breeding are discussed.

Background Maturity 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 F 2 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. Results Recombinant individuals from the WxBy F 2 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 ( N AM/ A TAF1, 2/ C UC2) 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 F 1 parent. However, this variation did not segregate in the CxA F 2 population being the CxA F 1 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 F 2 populations. Conclusions Using 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 qPCR-based method was developed to detect and quantify Podosphaera pannosa, the main causal agent of peach powdery mildew. A primer pair was designed to target part of the ITS region of the fungal ribosomal DNA, which proved to be highly specific and sensitive. A minimum of 2.81 pg µL− 1 of P. pannosa DNA and 6 conidia mL− 1 in artificially-prepared conidia suspensions were found to be the limit of detection. Moreover, a quantification of conidia placed on plastic tapes commonly used in volumetric air samplers was performed. Regression equations on conidia quantification obtained either from aqueous conidia suspensions or conidia placed on plastic tapes were similar. The protocol was further validated in field conditions by estimating the number of P. pannosa conidia obtained with an air sampler, by both microscopic and molecular quantification. Both techniques detected the peaks of conidia production during a 4-month sampling period, and a significant correlation (r = 0.772) was observed between both quantification methods. Additionally, the molecular method was applied to detect latent fungal inoculum in different plant parts of peach trees. The pathogen was detected mainly on the bark of affected twigs, and to a lesser extent, in foliar buds. The method developed here can be applied in the study of P. pannosa epidemiology and can help in improving the management of this pathogen through its early detection and quantification.

A mapping F2 population from the cross ‘Piel de Sapo’ × PI124112 was selectively genotyped to study the genetic control of morphological fruit traits by QTL (Quantitative Trait Loci) analysis. Ten QTL were identified, five for FL (Fruit Length), two for FD (Fruit Diameter) and three for FS (Fruit Shape). At least one robust QTL per character was found, flqs8.1 (LOD = 16.85, R2 = 34%), fdqs12.1 (LOD = 3.47, R2 = 11%) and fsqs8.1 (LOD = 14.85, R2 = 41%). flqs2.1 and fsqs2.1 cosegregate with gene a (andromonoecious), responsible for flower sex determination and with pleiotropic effects on FS. They display a positive additive effect (a) value, so the PI124112 allele causes an increase in FL and FS, producing more elongated fruits. Conversely, the negative a value for flqs8.1 and fsqs8.1 indicates a decrease in FL and FS, what results in rounder fruits, even if PI124112 produces very elongated melons. This is explained by a significant epistatic interaction between fsqs2.1 and fsqs8.1, where the effects of the alleles at locus a are attenuated by the additive PI124112 allele at fsqs8.1. Roundest fruits are produced by homozygous for PI124112 at fsqs8.1 that do not carry any dominant A allele at locus a (PiPiaa). A significant interaction between fsqs8.1 and fsqs12.1 was also detected, with the alleles at fsqs12.1 producing more elongated fruits. fsqs8.1 seems to be allelic to QTL discovered in other populations where the exotic alleles produce elongated fruits. This model has been validated in assays with backcross lines along 3 years and ultimately obtaining a fsqs8.1-NIL (Near Isogenic Line) in ‘Piel de Sapo’ background which yields round melons.

We propose a method for marker-based selection of cultivars of clonally-reproducing plant species which keeps the basic genetic architecture of a top-performing cultivar (usually a partly heterozygous genotype), with the addition of some agronomically relevant differences (such as production time, product appearance or quality), providing added value to the product or cultivation process. The method is based on selecting a) two complementary nearly-inbred lines from successive selfing generations (ideally only F and F ) of large size, that may generate individuals with most of their genome identical to the original cultivar but being homozygous for either of the two component haplotypes in the rest, and b) individuals with such characteristics already occurring in the F . Option a) allows for introgressing genes from other individuals in one or both of these nearly-inbred lines. Peach, a woody-perennial, clonally-reproduced species, was chosen as a model for a proof of concept of the Resynthesis process due to its biological characteristics: self-compatibility, compact and genetically well-known genome, low recombination rates and relatively short intergeneration time (3-4 years). From 416 F seedlings from cultivar Sweet Dream (SD), we obtained seven individuals with 76-94% identity with SD, and selected five pairs of complementary lines with average homozygosity of the two parents ≥0.70 such that crossing would produce some individuals highly similar to SD. The application of this scheme to other species with more complex genomes or biological features, including its generalization to F hybrids, is discussed.

Two intraspecific peach breeding populations have been used to conduct a quantitative trait locus (QTL) analysis of fruit quality traits: an F₁ from the cross Bolero (B) x OroA (O) and an F₂ from the cross Contender (C) x Ambra (A). A total of 344 Prunus simple sequence repeats (SSRs) were analyzed in B, O, C, A parents and CxA F₁ hybrid. Eight SSR were mapped for the first time in peach. A multiplex-ready polymerase chain reaction (PCR) protocol has allowed considerable time and cost saving during genotyping steps. Two maps (B map and O map) were produced for BxO population following the pseudo-test cross strategy and one for CxA. No marker could be mapped on G6 for the B map, on G4 and G8 for the O map and on G5 for the CxA map. Both populations were phenotyped over 2 years for maturity date (MD), fruit weight, external fruit skin overcolor, juice total soluble solids (SSC, Brix degree), juice titrable acidity and juice pH. Data for blooming time and flower type were scored only for BxO in 2007. All traits had a normal distribution, except for MD which was bimodal in BxO and trimodal in CxA, where it was scored as a co-dominant trait. Up to two QTLs per trait were detected in each population, and most of them were located in the same region forming clusters of QTLs, especially on G4. This is likely due to a major pleiotropic effect of MD masking the identification of other QTLs for different traits.