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.

Development and Evaluation of an AxiomTM 60K SNP Array for Almond (Prunus dulcis).

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.

The Rm1 and Rm2 Resistance Genes to Green Peach Aphid (Myzus persicae) Encode the Same TNL Proteins in Peach (Prunus persica L.).

The green peach aphid (GPA), Myzus persicae, is an important pest of the peach crop. Three major dominant resistance genes have already been detected, Rm1 in the Weeping Flower Peach (WFP) clone, Rm2 in the Rubira clone, and Rm3 in the Fen Shouxing clone. In this study, after NGS resequencing of WFP and Rubira, we found that their genomic sequences in the Rm1 and Rm2 region were similar but very different from that of the susceptible reference peach Lovell. We constructed a BAC library for the GPA-resistant WFP and screened four BAC clones to sequence the target region. The new sequence was 61.7 Kb longer than Lovell and was annotated with four different TIR_NBS_LRR genes. Among them, the TNL1 gene was very overexpressed in WFP leaves 24 h after GPA infestation. This gene was also present and expressed in the Rubira clone and had the same sequence as the candidate Rm3 gene, supporting the hypothesis that the three genes share the same origin. In addition, we identified a second TNL, TNL2, located at 35.4 Kb from TNL1 and slightly overexpressed after GPA infestation. Kasp and size molecular markers were designed for use in marker-assisted selection and were validated in a peach segregating population.

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.

Transposons played a major role in the diversification between the closely related almond and peach genomes: results from the almond genome sequence.

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.

New Data Completing the Spectrum of the Ma, RMia, and RMja Genes for Resistance to Root-Knot Nematodes (Meloidogyne spp.) in Prunus.

Root-knot nematodes (RKN) (Meloidogyne spp.) are worldwide pests that affect a considerable number of plants, among which stone fruit (Prunus spp.) are severely attacked. Prevalent RKN species are Meloidogyne arenaria, M. incognita, and M. javanica in stone fruit but the emergent M. ethiopica and M. enterolobii are also reported to challenge perennial crops. In Prunus spp., the complete-spectrum resistance (R) gene Ma from plum and the more restricted-spectrum R genes RMia from peach and RMja from almond completely inhibit nematode multiplication and gall formation of the RKN species that they control. This study aimed to update the resistance spectra of these three major genes by evaluating their activity toward one isolate of the yet-untested RKN species mentioned above. To state whether a given gene controls a particular species, the principle of our experiment was to genotype with appropriate markers a number of individuals segregating for this gene and then to phenotype these individuals. A perfect matching of the genotype and the phenotype of individuals indicates that the gene of interest is active against and, thus, controls the corresponding isolate of this RKN species. Segregating materials used were an Ma F1 plum progeny, an RMia F2 peach progeny, and an RMja F2 almond progeny. In addition to previous data, our results establish a clear spectrum for each of the three genes toward isolates from both the three prevalent species and the two emerging species. Ultimately, our results reveal that (i) Ma controls all of them, (ii) RMja controls all species except M. incognita and M. floridensis, and (iii) RMia controls M. arenaria, M. incognita, and M. ethiopica but not M. javanica or M. enterolobii. Our data should have wide implications for RKN resistance management and breeding and for deciphering the molecular mechanisms of the spectrum of RKN R genes.

Ma Orthologous Genes in Prunus spp. Shed Light on a Noteworthy NBS-LRR Cluster Conferring Differential Resistance to Root-Knot Nematodes.

Root-knot nematodes (RKNs) are considerable polyphagous pests that severely challenge plants worldwide and especially perennials. The specific genetic resistance of plants mainly relies on the NBS-LRR genes that are pivotal factors for pathogens control. In Prunus spp., the Ma plum and RMja almond genes possess different spectra for resistance to RKNs. While previous works based on the Ma gene allowed to clone it and to decipher its peculiar TIR-NBS-LRR (TNL) structure, we only knew that the RMja gene mapped on the same chromosome as Ma. We carried out a high-resolution mapping using an almond segregating F2 progeny of 1448 seedlings from resistant (R) and susceptible (S) parental accessions, to locate precisely RMja on the peach genome, the reference sequence for Prunus species. We showed that the RMja gene maps in the Ma resistance cluster and that the Ma ortholog is the best candidate for RMja. This co-localization is a crucial step that opens the way to unravel the molecular determinants involved in the resistance to RKNs. Then we sequenced both almond parental NGS genomes and aligned them onto the RKN susceptible reference peach genome. We produced a BAC library of the R parental accession and, from two overlapping BAC clones, we obtained a 336-kb sequence encompassing the RMja candidate region. Thus, we could benefit from three Ma orthologous regions to investigate their sequence polymorphism, respectively, within plum (complete R spectrum), almond (incomplete R spectrum) and peach (null R spectrum). We showed that the Ma TNL cluster has evolved orthologs with a unique conserved structure comprised of five repeated post-LRR (PL) domains, which contain most polymorphism. In addition to support the Ma and RMja orthologous relationship, our results suggest that the polymorphism contained in the PL sequences might underlie differential resistance interactions with RKNs and an original immune mechanism in woody perennials. Besides, our study illustrates how PL exon duplications and losses shape TNL structure and give rise to atypical PL domain repeats of yet unknown role.

Resistance to root-knot nematodes Meloidogyne spp. in woody plants.

I. 42 II. 43 III. 44 IV. 47 V. 49 VI. 50 VII. 50 VIII. 50 IX. 52 52 References 52 SUMMARY: Root-knot nematodes (RKNs) Meloidogyne spp. cause major damage to cultivated woody plants. Among them, Prunus, grapevine and coffee are the crops most infested by worldwide polyphagous species and species with a more limited distribution and/or narrower host range. The identification and characterization of natural sources of resistance are important steps to develop RKN control strategies. In woody crops, resistant rootstocks genetically different from the scion of agronomical interest may be engineered. We describe herein the interactions between RKNs and different woody crops, and highlight the plant species in which resistance and corresponding resistance (R) genes have been discovered. Even though grapevine and, to a lesser extent, coffee have a history of rootstock selection for RKN resistance, few cases of resistance have been documented. By contrast, in Prunus, R genes with different spectra have been mapped in plums, peach and almond and can be pyramided for durable resistance in interspecific rootstocks. We particularly discuss here the Ma Toll/interleukin-1 receptor-like-nucleotide binding-leucine-rich repeat gene from Myrobalan plum, one of the longest plant R genes cloned to date, due to its unique biological and structural properties. RKN R genes in Prunus will enable us to carry out molecular studies aimed at improving our knowledge of plant immunity in woody plants.

Protein and mineral nutrient contents in kernels from 72 sweet almond cultivars and accessions grown in France, Greece and Italy.

Almond protein and potassium (K), phosphorus (P), calcium (Ca) and magnesium (Mg) contents were determined in 72 cultivars and accessions grown in France, Greece and Italy, as part of the networking of European SAFENUT AGRI GEN RES project, which aimed to explore and valorize the almond genetic resources in Europe. Great variation was found in the nutrient content and the amount of nutrient supplied when consuming the recommended daily amount of one serving of almond, among the different genotypes assayed. The variation among the different genotypes was greatest for Ca, followed by the protein content; the latter also exhibited the lowest variation considering the harvest year. Results from a principal component analysis showed that P and Mg were the most discriminant elements for categorizing samples. Cluster analysis showed groups of samples with interesting characteristics for breeding. There was no clear distinction among the different origins of samples. Correlation analyses between weather conditions and the nutrients assayed showed that the mean temperature recorded in the period between March and September was positively correlated with Ca and P only in France, a place where the greatest climatic difference between years was observed.