Exploring large-scale gene coexpression networks in peach (Prunus persica L.): a new tool for predicting gene function.

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.

Almond population genomics and non-additive GWAS reveal new insights into almond dissemination history and candidate genes for nut traits and blooming time.

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.

Pedigree analysis of 220 almond genotypes reveals two world mainstream breeding lines based on only three different cultivars.

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.

Cultivar Susceptibility and Environmental Parameters Affecting Symptom Expression of Red Leaf Blotch of Almond in Spain.

Red leaf blotch (RLB) of almond, caused by Polystigma amygdalinum, is an important foliar disease of this nut tree in the Mediterranean basin and Middle East regions. In recent years, the incidence of this disease has increased in Spain, corresponding to increases in the area of newly planted orchards and the use of susceptible cultivars. In 2009, an experimental orchard including 21 almond cultivars was planted at Les Borges Blanques, Lleida, in northeastern Spain. No fungicide treatments were applied during the 10-year experimental period (2009 to 2018) in order to allow natural disease development. Cultivar susceptibility to RLB was assessed each year, from 2011 to 2018, through visual observations of symptoms in naturally infected trees. The experimental results led us to classify the cultivars into five susceptibility groups. The most susceptible were Tarraco, Guara, Tuono, Marinada, Desmayo Largueta, and Soleta, whereas Mardía was the most tolerant. The annual incidence of disease was positively correlated with accumulated rainfall in spring, and especially in April, while it was negatively correlated with high spring and summer temperatures, especially in May. These findings could be used to improve disease management strategies by identifying the most susceptible cultivars and improving the timing of fungicide application.

Chemical Markers to Distinguish the Homo- and Heterozygous Bitter Genotype in Sweet Almond Kernels.

Bitterness in almonds is controlled by a single gene (Sk dominant for sweet kernel, sk recessive for bitter kernel) and the proportions of the offspring genotypes (SkSk, Sksk, sksk) depend on the progenitors' genotype. Currently, the latter is deduced after crossing by recording the phenotype of their descendants through kernel tasting. Chemical markers to early identify parental genotypes related to bitter traits can significantly enhance the efficiency of almond breeding programs. On this basis, volatile metabolites related to almond bitterness were investigated by Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry coupled to univariate and multivariate statistics on 244 homo- and heterozygous samples from 42 different cultivars. This study evidenced the association between sweet almonds' genotype and some volatile metabolites, in particular benzaldehyde, and provided for the first time chemical markers to discriminate between homo- and heterozygous sweet almond genotypes. Furthermore, a multivariate approach based on independent variables was developed to increase the reliability of almond classification. The Partial Least Square-Discriminant Analysis classification model built with selected volatile metabolites that showed discrimination capacity allowed a 98.0% correct classification. The metabolites identified, in particular benzaldehyde, become suitable markers for the early genotype identification in almonds, while a DNA molecular marker is not yet available.

High-density mapping suggests cytoplasmic male sterility with two restorer genes in almond × peach progenies.

Peach (Prunus persica) and almond (Prunus dulcis) are two sexually compatible species that produce fertile offspring. Almond, a highly polymorphic species, is a potential source of new genes for peach that has a strongly eroded gene pool. Here we describe the genetics of a male sterile phenotype that segregated in two almond ('Texas') × peach ('Earlygold') progenies: an F2 (T×E) and a backcross one (T1E) to the 'Earlygold' parent. High-density maps were developed using a 9k peach SNP chip and 135 simple-sequence repeats. Three highly syntenic and collinear maps were obtained: one for the F2 (T×E) and two for the backcross, T1E (for the hybrid) and E (for 'Earlygold'). A major reduction of recombination was observed in the interspecific maps (T×E and T1E) compared to the intraspecific parent (E). The E map also had extensive monomorphic genomic regions suggesting the presence of large DNA fragments identical by descent. Our data for the male sterility character were consistent with the existence of cytoplasmic male sterility, where individuals having the almond cytoplasm required the almond allele in at least one of two independent restorer genes, Rf1 and Rf2, to be fertile. The restorer genes were located in a 3.4 Mbp fragment of linkage group 2 (Rf1) and 1.4 Mbp of linkage group 6 (Rf2). Both fragments contained several genes coding for pentatricopeptide proteins, demonstrated to be responsible for restoring fertility in other species. The implications of these results for using almond as a source of novel variability in peach are discussed.

Self-incompatibility genotypes in almond re-evaluated by PCR, stylar ribonucleases, sequencing analysis and controlled pollinations.

As part of the almond breeding programme at IRTA, we investigated the S genotypes of several cultivars using a combination of RNase zymograms, testcrosses, pollen-tube growth analysis and molecular identification by PCR analysis. For some of the cultivars examined, discrepancies appeared between their S alleles as reported in the literature and those found in this investigation, leading to a re-evaluation of their S genotypes. Analysis of the stylar ribonucleases (RNases), which are known to correlate with S alleles, of cvs. Achaak, Ardechoise, Desmayo Largueta, Ferrastar, Gabaix, Garbi, Glorieta, Languedoc, Primorskiy and Texas revealed inconsistencies with respect to the S5 and S10 alleles. However, PCR with the conserved primer pair AS1II/AmyC5R failed to detect any of these inconsistencies. When the S alleles from Desmayo Largueta, Gabaix, Primorskiy and Texas were sequenced, Texas and Primorskiy were found to carry the reported S5 allele, while Desmayo Largueta and Gabaix carried a new allele, which has been tentatively denoted as S25 This new S allele, previously reported to be S10, was also identified in Achaak, Ardechoise and Ferrastar. The proposed new S genotypes are Achaak (S2S25), Ardechoise (S1S25), Desmayo Largueta (S1S25), Ferrastar (S2S25) and Gabaix (S10S25). The S alleles of Garbi, Glorieta, Languedoc, Texas and Primorskiy remain as reported in the literature. Testcrosses in the field and laboratory confirmed the new S genotypes. One cultivar (Gabaix) could be assigned to the existing cross-incompatibility group O of unique genotypes, and two new groups were established (XVI and XVII) consisting of two cultivars each. The clarification of these S alleles will be useful in almond breeding programmes and for planning new commercial orchards in the future.