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The vast majority of traditional almond varieties are self-incompatible, and the level of variability of the species is very high, resulting in a high-heterozygosity genome. Therefore, information on the different haplotypes is particularly relevant to understand the genetic basis of trait variability in this species. However, although reference genomes for several almond varieties exist, none of them is phased and has genome information at the haplotype level. Here, we present a phased assembly of genome of the almond cv. Texas. This new assembly has 13% more assembled sequence than the previous version of the Texas genome and has an increased contiguity, in particular in repetitive regions such as the centromeres. Our analysis shows that the 'Texas' genome has a high degree of heterozygosity, both at SNPs, short indels, and structural variants level. Many of the SVs are the result of heterozygous transposable element insertions, and in many cases, they also contain genic sequences. In addition to the direct consequences of this genic variability on the presence/absence of genes, our results show that variants located close to genes are often associated with allele-specific gene expression, which highlights the importance of heterozygous SVs in almond.
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Peach is a model for Prunus genetics and genomics, however, identifying and validating genes associated to peach breeding traits is a complex task. A gene coexpression network (GCN) capable of capturing stable gene-gene relationships would help researchers overcome the intrinsic limitations of peach genetics and genomics approaches and outline future research opportunities. In this study, we created four GCNs from 604 Illumina RNA-Seq libraries. We evaluated the performance of every GCN in predicting functional annotations using an algorithm based on the 'guilty-by-association' principle. The GCN with the best performance was COO300, encompassing 21 956 genes. To validate its performance predicting gene function, we performed two case studies. In case study 1, we used two genes involved in fruit flesh softening: the endopolygalacturonases PpPG21 and PpPG22. Genes coexpressing with both genes were extracted and referred to as melting flesh (MF) network. Finally, we performed an enrichment analysis of MF network and compared the results with the current knowledge regarding peach fruit softening. The MF network mostly included genes involved in cell wall expansion and remodeling, and with expressions triggered by ripening-related phytohormones, such as ethylene, auxin, and methyl jasmonate. In case study 2, we explored potential targets of the anthocyanin regulator PpMYB10.1 by comparing its gene-centered coexpression network with that of its grapevine orthologues, identifying a common regulatory network. These results validated COO300 as a powerful tool for peach and Prunus research. This network, renamed as PeachGCN v1.0, and the scripts required to perform a function prediction analysis are available at https://github.com/felipecobos/PeachGCN.
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SUMMARY: Pedigree-based analyses' prime role is to unravel relationships between individuals in breeding programs and germplasms. This is critical information for decoding the genetics underlying main inherited traits of relevance, and unlocking the genotypic variability of a species to carry out genomic selections and predictions. Despite the great interest, current lineage visualizations become quite limiting in terms of public display, exploration, and tracing of traits up to ancestral donors. PERSEUS is a user-friendly, intuitive, and interactive web-based tool for pedigree visualizations represented as directed graph networks distributed using a force-repulsion method. The visualizations do not only showcase individual relationships among accessions, but also facilitate a seamless search and download of phenotypic traits along the pedigrees. PERSEUS is a promising tool for breeders and scientists, advantageous for evolutionary, genealogy, and diversity analyses among related accessions and species. AVAILABILITY AND IMPLEMENTATION: PERSEUS is freely accessible at https://bioinformatics.cragenomica.es/perseus and GitHub code is available at https://github.com/aranzana-lab/PERSEUS.
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Genómica , Programas Informáticos , Humanos , Linaje , Genoma , InternetRESUMEN
Domestication drastically changed crop genomes, fixing alleles of interest and creating different genetic populations. Genome-wide association studies (GWASs) are a powerful tool to detect these alleles of interest (and so QTLs). In this study, we explored the genetic structure as well as additive and non-additive genotype-phenotype associations in a collection of 243 almond accessions. Our genetic structure analysis strongly supported the subdivision of the accessions into five ancestral groups, all formed by accessions with a common origin. One of these groups was formed exclusively by Spanish accessions, while the rest were mainly formed by accessions from China, Italy, France, and the USA. These results agree with archaeological and historical evidence that separate modern almond dissemination into four phases: Asiatic, Mediterranean, Californian, and southern hemisphere. In total, we found 13 independent QTLs for nut weight, crack-out percentage, double kernels percentage, and blooming time. Of the 13 QTLs found, only one had an additive effect. Through candidate gene analysis, we proposed Prudul26A013473 as a candidate gene responsible for the main QTL found in crack-out percentage, Prudul26A012082 and Prudul26A017782 as candidate genes for the QTLs found in double kernels percentage, and Prudul26A000954 as a candidate gene for the QTL found in blooming time. Our study enhances our knowledge of almond dissemination history and will have a great impact on almond breeding.
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A high-density single nucleotide polymorphism (SNP) array is essential to enable faster progress in plant breeding for new cultivar development. In this regard, we have developed an Axiom 60K almond SNP array by resequencing 81 almond accessions. For the validation of the array, a set of 210 accessions were genotyped and 82.8% of the SNPs were classified in the best recommended SNPs. The rate of missing data was between 0.4% and 2.7% for the almond accessions and less than 15.5% for the few peach and wild accessions, suggesting that this array can be used for peach and interspecific peach × almond genetic studies. The values of the two SNPs linked to the RMja (nematode resistance) and SK (bitterness) genes were consistent. We also genotyped 49 hybrids from an almond F2 progeny and could build a genetic map with a set of 1159 SNPs. Error rates, less than 1%, were evaluated by comparing replicates and by detection of departures from Mendelian inheritance in the F2 progeny. This almond array is commercially available and should be a cost-effective genotyping tool useful in the search for new genes and quantitative traits loci (QTL) involved in the control of agronomic traits.
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Fruit color is an important trait in peach from the point of view of consumer preference, nutritional content, and diversification of fruit typologies. Several genes and phenotypes have been described for peach flesh and skin color, and although peach color knowledge has increased in the last few years, some fruit color patterns observed in peach breeding programs have not been carefully described. In this work, we first describe some peach mesocarp color patterns that have not yet been described in a collection of commercial peach cultivars, and we also study the genetic inheritance of the red dots present in the flesh (RDF) and red color around the stone (CAS) in several intra- and interspecific segregating populations for both traits. For RDF, we identified a QTL at the beginning of G5 in two intraspecific populations, and for CAS we identified a major QTL in G4 in both an intraspecific and an interspecific population between almond and peach. Finally, we discuss the interaction between these QTLs and some other genes previously identified in peach, such as dominant blood flesh (DBF), color around the stone (Cs), subacid (D) and the maturity date (MD), and the implications for peach breeding. The results obtained here will help peach germplasm curators and breeders to better characterize their plant materials and to develop an integrated system of molecular markers to select these traits.
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Peach [Prunus persica L. Batsch] is one of the major temperate fruit tree species, the commercial materials of which have a low level of genetic variability. Almond [P. dulcis (Mill) DA Webb], a close relative of peach cultivated for its kernels, has a much higher level of diversity. The species are inter-compatible and often produce fertile hybrids, almond being a possible source of new genes for peach that could provide biotic and abiotic stress tolerance traits. In this paper we describe the development of a collection of peach-almond introgression lines (ILs) having a single fragment of almond (cv. Texas) in the peach background (cv. Earlygold). Lines with few introgressions were selected with markers from successive generations from a "Texas" × "Earlygold" F1 hybrid, initially using a set of SSRs and later with the 18 k peach SNP chip, allowing for the final extraction of 67 lines, 39 with almond heterozygous introgressions covering 99% of the genome, and 28 with homozygous introgressions covering 83% of the genome. As a proof of concept, four major genes and four quantitative characters were examined in the selected ILs giving results generally consistent with previous information on the genetics of these characters. This collection is the first of its kind produced in a woody perennial species and promises to be a valuable tool for genetic analyses, including dissection of quantitative traits, positional cloning, epistasis and as prebreeding material to introgress almond genes of interest into the peach commercial gene pool.
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BACKGROUND: Peach (Prunus persica) is an economically important stone fruit crop in Rosaceae and widely cultivated in temperate and subtropical regions, emerging as an excellent material to study the interaction between plant and environment. During its genus, there are four wild species of peach, all living in harsh environments. For example, one of the wild species, P. mira, originates from the Qinghai-Tibet Plateau (QTP) and exhibits strong cold/ultraviolet ray environmental adaptations. Although remarkable progresses in the gene discovery of fruit quality-related traits in peach using previous assembled genome were obtained, genomic basis of the response of these wild species to different geographical environments remains unclear. RESULTS: To uncover key genes regulating adaptability in different species and analyze the role of genetic variations in resistance formation, we performed de novo genome assembling of four wild relatives of peach (P. persica), P. mira, P. davidiana, P. kansuensis, and P. ferganensis and resequenced 175 peach varieties. The phylogenetic tree showed that the divergence time of P. mira and other wild relatives of peach was 11.5 million years ago, which was consistent with the drastic crustal movement of QTP. Abundant genetic variations were identified in four wild species when compared to P. persica, and the results showed that plant-pathogen interaction pathways were enriched in genes containing small insertions and deletions and copy number variations in all four wild relatives of peach. Then, the data were used to identify new genes and variations regulating resistance. For example, presence/absence variations which result from a hybridization event that occurred between P. mira and P. dulcis enhanced the resistance of their putative hybrid, P. davidiana. Using bulked segregant analysis, we located the nematode resistance locus of P. kansuensis in chromosome 2. Within the mapping region, a deletion in the promoter of one NBS-LRR gene was found to involve the resistance by regulating gene expression. Furthermore, combined with RNA-seq and selective sweeps analysis, we proposed that a deletion in the promoter of one CBF gene was essential for high-altitude adaptation of P. mira through increasing its resistance to low temperature. CONCLUSIONS: In general, the reference genomes assembled in the study facilitate our understanding of resistance mechanism of perennial fruit crops, and provide valuable resources for future breeding and improvement.
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Prunus persica , Cromosomas , Variaciones en el Número de Copia de ADN , Evolución Molecular , Genoma de Planta , Filogenia , Fitomejoramiento , Prunus persica/genéticaRESUMEN
BACKGROUND: Pru p 1 is a major allergen in peach and nectarine, and the different content in varieties may affect the degree of allergic reactions. This study aimed to quantify Pru p 1 levels in representative peach varieties and select hypoallergenic Pru p 1 varieties. METHODS: To obtain monoclonal and polyclonal antibodies, mice and rabbits, respectively, were immunized with recombinant Pru p 1.01 and Pru p 1.02. The Pru p 1 levels in fruits from 83 representative peach varieties was quantified by sandwich enzyme-linked immunosorbent assay (sELISA). nPru p 1 was obtained through specific monoclonal antibody affinity purification and confirmed by Western blot and mass spectrometry. The variable Pru p 1 content of selected varieties was evaluated by Western blot and the expression level of encoding Pru p 1 genes by quantitative polymerase chain reaction. RESULTS: A sELISA method with monoclonal and polyclonal antibodies was built for quantifying Pru p 1 levels in peach. Pru p 1 was mainly concentrated in the peel (0.20-73.44 µg/g, fresh weight), being very low in the pulp (0.05-9.62 µg/g) and not detected in wild peach. For the 78 peach and nectarine varieties, Pru p 1 content varied widely from 0.12 to 6.45 µg/g in whole fruit. We verified that natural Pru p 1 is composed of 1.01 and 1.02 isoallergens, and the Pru p 1 expression level and Pru p 1 band intensity in the immunoblots were in agreement with protein quantity determined by ELISA for some tested varieties. In some cases, the reduced levels of Pru p 1 did not coincide with low Pru p 3 in the same variety in whole fruit, while some ancient wild peach and nectarines contained low levels of both allergens, and late-ripening yellow flesh varieties were usually highly allergenic. CONCLUSION: Pru p 1 content is generally low in peach compared to Pru p 3. Several hypoallergenic Pru p 1 and Pru p 3 varieties, "Zi Xue Tao," "Wu Yue Xian," and "May Fire," were identified, which could be useful in trials for peach allergy patients.
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The environment has constantly shaped plant genomes, but the genetic bases underlying how plants adapt to environmental influences remain largely unknown. We constructed a high-density genomic variation map of 263 geographically representative peach landraces and wild relatives. A combination of whole-genome selection scans and genome-wide environmental association studies (GWEAS) was performed to reveal the genomic bases of peach adaptation to diverse climates. A total of 2092 selective sweeps that underlie local adaptation to both mild and extreme climates were identified, including 339 sweeps conferring genomic pattern of adaptation to high altitudes. Using genome-wide environmental association studies (GWEAS), a total of 2755 genomic loci strongly associated with 51 specific environmental variables were detected. The molecular mechanism underlying adaptive evolution of high drought, strong UVB, cold hardiness, sugar content, flesh color, and bloom date were revealed. Finally, based on 30 yr of observation, a candidate gene associated with bloom date advance, representing peach responses to global warming, was identified. Collectively, our study provides insights into molecular bases of how environments have shaped peach genomes by natural selection and adds candidate genes for future studies on evolutionary genetics, adaptation to climate changes, and breeding.
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Adaptación Fisiológica/genética , Cambio Climático , Genoma de Planta/genética , Genómica , Prunus persica/genéticaRESUMEN
Loss of genetic variability is an increasing challenge in tree breeding programs due to the repeated use of a reduced number of founder genotypes. However, in almond, little is known about the genetic variability in current breeding stocks, although several cases of inbreeding depression have been reported. To gain insights into the genetic structure in modern breeding programs worldwide, marker-verified pedigree data of 220 almond cultivars and breeding selections were analyzed. Inbreeding coefficients, pairwise relatedness, and genetic contribution were calculated for these genotypes. The results reveal two mainstream breeding lines based on three cultivars: "Tuono", "Cristomorto", and "Nonpareil". Descendants from "Tuono" or "Cristomorto" number 76 (sharing 34 descendants), while "Nonpareil" has 71 descendants. The mean inbreeding coefficient of the analyzed genotypes was 0.041, with 14 genotypes presenting a high inbreeding coefficient, over 0.250. Breeding programs from France, the USA, and Spain showed inbreeding coefficients of 0.075, 0.070, and 0.037, respectively. According to their genetic contribution, modern cultivars from Israel, France, the USA, Spain, and Australia trace back to a maximum of six main founding genotypes. Among the group of 65 genotypes carrying the Sf allele for self-compatibility, the mean relatedness coefficient was 0.125, with "Tuono" as the main founding genotype (24.7% of total genetic contribution). The results broaden our understanding about the tendencies followed in almond breeding over the last 50 years and will have a large impact into breeding decision-making process worldwide. Increasing current genetic variability is required in almond breeding programs to assure genetic gain and continuing breeding progress.
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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.
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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 F2 and F3) 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 F2. 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 F2 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 F1 hybrids, is discussed.
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Plants have evolved a range of adaptive mechanisms that adjust their development and physiology to variable external conditions, particularly in perennial species subjected to long-term interplay with the environment. Exploiting the allelic diversity within available germplasm and leveraging the knowledge of the mechanisms regulating genotype interaction with the environment are crucial to address climatic challenges and assist the breeding of novel cultivars with improved resilience. The development of multisite collections is of utmost importance for the conservation and utilization of genetic materials and will greatly facilitate the dissection of genotype-by-environment interaction. Such resources are still lacking for perennial trees, especially with the intrinsic difficulties of successful propagation, material exchange, and living collection maintenance. This work describes the concept, design, and realization of the first multisite peach (Prunus persica) reference collection (PeachRefPop) located across different European countries and sharing the same experimental design. Other than an invaluable tool for scientific studies in perennial species, PeachRefPop provides a milestone in an international collaborative project for the conservation and exploitation of European peach germplasm resources and, ultimately, as a true heritage for future generations.
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Prunus persica , Banco de Semillas , Europa (Continente)Asunto(s)
Hipersensibilidad a los Alimentos , Prunus persica , Alérgenos , Antígenos de Plantas , Frutas , Humanos , Proteínas de PlantasRESUMEN
We sequenced the genome of the highly heterozygous almond Prunus dulcis cv. Texas combining short- and long-read sequencing. We obtained a genome assembly totaling 227.6 Mb of the estimated almond genome size of 238 Mb, of which 91% is anchored to eight pseudomolecules corresponding to its haploid chromosome complement, and annotated 27 969 protein-coding genes and 6747 non-coding transcripts. By phylogenomic comparison with the genomes of 16 additional close and distant species we estimated that almond and peach (Prunus persica) diverged around 5.88 million years ago. These two genomes are highly syntenic and show a high degree of sequence conservation (20 nucleotide substitutions per kb). However, they also exhibit a high number of presence/absence variants, many attributable to the movement of transposable elements (TEs). Transposable elements have generated an important number of presence/absence variants between almond and peach, and we show that the recent history of TE movement seems markedly different between them. Transposable elements may also be at the origin of important phenotypic differences between both species, and in particular for the sweet kernel phenotype, a key agronomic and domestication character for almond. Here we show that in sweet almond cultivars, highly methylated TE insertions surround a gene involved in the biosynthesis of amygdalin, whose reduced expression has been correlated with the sweet almond phenotype. Altogether, our results suggest a key role of TEs in the recent history and diversification of almond and its close relative peach.
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Secuencia de Bases , Elementos Transponibles de ADN/genética , Genoma de Planta , Prunus dulcis/genética , Prunus persica/genética , Mapeo Cromosómico , Metilación de ADN , Domesticación , Evolución Molecular , Genes de Plantas/genética , Filogenia , Semillas , Especificidad de la EspecieRESUMEN
Significant differences in softening rate have been reported between melting flesh in peach and nectarine varieties. This trait seems to be controlled by several genes. We aimed to identify candidate genes involved in fruit softening rate by integrating quantitative trait loci (QTL) and expression QTL (eQTL) analyses, comparing siblings with contrasting softening rates. We used a segregating population derived from nectarine cv. 'Venus' selfing, which was phenotyped for softening rate during three seasons. Six siblings with high (HSR) and six with low softening rate (LSR) were sequenced using RNA-Seq. A group of 5,041 differentially expressed genes was identified. Also, we found a QTL with a LOD (logarithm of odds) score of 9.7 on LG4 in all analyzed seasons. Furthermore, we detected 1,062 eQTLs, of which 133 were found co-localizing with the identified QTL. Gene Ontology (GO) analysis showed 'Response to auxin' as one the main over-represented categories. Our findings suggest over-expression of auxin biosynthetic related genes in the HSR group, which implies a higher expression and/or accumulation of auxin, thereby triggering fast softening. Conversely, the LSR phenotype might be explained by an altered auxin-homeostasis associated with low auxin levels. This work will contribute to unraveling the genetic mechanisms responsible for the softening rate in peaches and nectarines and lead to the development of molecular markers.
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Prior to the availability of whole-genome sequences, our understanding of the structural and functional aspects of Prunus tree genomes was limited mostly to molecular genetic mapping of important traits and development of EST resources. With public release of the peach genome and others that followed, significant advances in our knowledge of Prunus genomes and the genetic underpinnings of important traits ensued. In this review, we highlight key achievements in Prunus genetics and breeding driven by the availability of these whole-genome sequences. Within the structural and evolutionary contexts, we summarize: (1) the current status of Prunus whole-genome sequences; (2) preliminary and ongoing work on the sequence structure and diversity of the genomes; (3) the analyses of Prunus genome evolution driven by natural and man-made selection; and (4) provide insight into haploblocking genomes as a means to define genome-scale patterns of evolution that can be leveraged for trait selection in pedigree-based Prunus tree breeding programs worldwide. Functionally, we summarize recent and ongoing work that leverages whole-genome sequences to identify and characterize genes controlling 22 agronomically important Prunus traits. These include phenology, fruit quality, allergens, disease resistance, tree architecture, and self-incompatibility. Translationally, we explore the application of sequence-based marker-assisted breeding technologies and other sequence-guided biotechnological approaches for Prunus crop improvement. Finally, we present the current status of publically available Prunus genomics and genetics data housed mainly in the Genome Database for Rosaceae (GDR) and its updated functionalities for future bioinformatics-based Prunus genetics and genomics inquiry.
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BACKGROUND: Monilinia spp. are responsible for brown rot, one of the most significant stone fruit diseases. Planting resistant cultivars seems a promising alternative, although most commercial cultivars are susceptible to brown rot. The aim of this study was to explore resistance to Monilinia fructicola over two seasons in a backcross one interspecific population between almond 'Texas' and peach 'Earlygold' (named T1E). RESULTS: 'Texas' almond was resistant to brown rot inoculation, whereas peach was highly susceptible. Phenotypic data from the T1E population indicated wide differences in response to M. fructicola. Additionally, several non-wounded individuals exhibited resistance to brown rot. Quantitative trait loci (QTLs) were identified in several linkage groups, but only two proximal QTLs in G4 were detected over both seasons and accounted for 11.3-16.2% of the phenotypic variation. CONCLUSION: Analysis of the progeny allowed the identification of resistant genotypes that could serve as a source of resistance in peach breeding programs. The finding of loci associated with brown rot resistance would shed light on implementing a strategy based on marker-assisted selection (MAS) for introgression of this trait into elite peach materials. New peach cultivars resistant to brown rot may contribute to the implementation of more sustainable crop protection strategies. © 2019 Society of Chemical Industry.