Phylogenomic analyses and comparative genomics of Pseudomonas syringae associated with almond (Prunus dulcis) in California.

We sequenced and comprehensively analysed the genomic architecture of 98 fluorescent pseudomonads isolated from different symptomatic and asymptomatic tissues of almond and a few other Prunus spp. Phylogenomic analyses, genome mining, field pathogenicity tests, and in vitro ice nucleation and antibiotic sensitivity tests were integrated to improve knowledge of the biology and management of bacterial blast and bacterial canker of almond. We identified Pseudomonas syringae pv. syringae, P. cerasi, and P. viridiflava as almond canker pathogens. P. syringae pv. syringae caused both canker and foliar (blast) symptoms. In contrast, P. cerasi and P. viridiflava only caused cankers, and P. viridiflava appeared to be a weak pathogen of almond. Isolates belonging to P. syringae pv. syringae were the most frequently isolated among the pathogenic species/pathovars, composing 75% of all pathogenic isolates. P. cerasi and P. viridiflava isolates composed 8.3 and 16.7% of the pathogenic isolates, respectively. Laboratory leaf infiltration bioassays produced results distinct from experiments in the field with both P. cerasi and P. syringae pv. syringae, causing significant necrosis and browning of detached leaves, whereas P. viridiflava conferred moderate effects. Genome mining revealed the absence of key epiphytic fitness-related genes in P. cerasi and P. viridiflava genomic sequences, which could explain the contrasting field and laboratory bioassay results. P. syringae pv. syringae and P. cerasi isolates harboured the ice nucleation protein, which correlated with the ice nucleation phenotype. Results of sensitivity tests to copper and kasugamycin showed a strong linkage to putative resistance genes. Isolates harbouring the ctpV gene showed resistance to copper up to 600 µg/ml. In contrast, isolates without the ctpV gene could not grow on nutrient agar amended with 200 µg/ml copper, suggesting ctpV can be used to phenotype copper resistance. All isolates were sensitive to kasugamycin at the label-recommended rate of 100µg/ml.

Plant height variation and genetic diversity between Prunus ledebouriana (Schlecht.) YY Yao and Prunus tenella Batsch based on using SSR markers in East Kazakhstan.

Background: Genetic differences between isolated endemic populations of plant species and those with widely known twin species are relevant for conserving the biological diversity of our planet's flora. Prunus ledebouriana (Schlecht.) YY Yao is an endangered and endemic species of shrub almond from central Asia. Few studies have explored this species, which is closely related and morphologically similar to the well-known Prunus tenella Batsch. In this article, we present a comparative analysis of studies of three P. ledebouriana populations and one close population of P. tenella in Eastern Kazakhstan in order to determine the particular geographic mutual replacement of the two species. Methods: The populations were collected from different ecological niches, including one steppe population near Ust-Kamenogorsk (P. tenella) and three populations (P. ledebouriana) in the mountainous area. Estimation of plant height using a t-test suggested a statistically significant difference between the populations and the two species (P < 0.0001). DNA simple sequence repeat (SSR) markers were applied to study the two species' genetic diversity and population structure. Results: A total of 19 polymorphic SSR loci were analyzed, and the results showed that the population collected in mountainous areas had a lower variation level than steppe populations. The highest level of Nei's genetic diversity index was demonstrated in the 4-UK population (0.622) of P. tenella. The lowest was recorded in population 3-KA (0.461) of P. ledebouriana, collected at the highest altitude of the four populations (2,086 meters above sea level). The total genetic variation of P. ledebouriana was distributed 73% within populations and 27% between populations. STRUCTURE results showed that two morphologically similar species diverged starting at step K = 3, with limited population mixing. The results confirmed the morphological and genetic differences between P. tenella and P. ledebouriana and described the level of genetic variation for P. ledebouriana. The study's results proved that the steppe zone and mountain altitude factor between P. tenella and isolated mountain samples of P. ledebouriana.

Genome-wide analysis of the ABC gene family in almond and functional predictions during flower development, freezing stress, and salt stress.

ABC (ATP-binding cassette) transporter proteins are one of the most extensive protein families known to date and are ubiquitously found in animals, plants, and microorganisms. ABCs have a variety of functions, such as plant tissue development regulation, hormone transport, and biotic and abiotic stress resistance. However, the gene characterization and function of the ABC gene family in almond (Prunus dulcis) have not been thoroughly studied. In this study, we identified 117 PdABC genes using the whole genome of 'Wanfeng' almond obtained by sequencing and explored their protein characterization. The PdABC family members were classified into eight subfamilies. The members of the same subfamily had conserved motifs but poorly conserved numbers of exons and introns and were unevenly distributed among the eight subfamilies and on the eight chromosomes. Expression patterns showed that PdABC family members were significantly differentially expressed during almond development, dormant freezing stress, and salt stress. We found that PdABC59 and PdABC77 had extremely high expression levels in pollen. PdABC63 and PdABC64 had high expression levels during almond petal development and multiple stages of flower development. PdABC98 was highly expressed in annual dormant branches after six temperature-freezing stress treatments. PdABC29, PdABC69, and PdABC98 were highly expressed under different concentrations of salt stress. This study preliminarily investigated the expression characteristics of ABC genes in different tissues of almond during flower development, freezing stress and salt stress, and the results will provide a reference for further in-depth research and breeding of almond in the future.

Survey on Latent Infection of Canker-Causing Pathogens in Budwood and Young Trees from Almond and Prune Nurseries in California.

Almond band canker and prune Cytospora canker have become more severe in the last decade, especially in young orchards, in California. To test our hypothesis that young trees from nurseries could carry the canker-causing pathogens at latency phase to new orchards through transplanting, a multiyear survey on latent infection of canker-causing pathogens of budwood and young trees of almond and prune nurseries in California was conducted. A total of more than 1,730 samples including shoots of rootstocks and scions and grafting union cuttings were collected from 11 nurseries. A real-time quantitative PCR assay was applied to quantify the latent infection levels by six canker-causing pathogen taxa: Botryosphaeria dothidea and species of Cytospora, Diplodia, Lasiodiplodia, Neofusicoccum, and Phomopsis. For almond, the average incidences of latent infection caused by Lasiodiplodia spp. (43.6%) and Neofusicoccum spp. (24.2%) were significantly greater than those by the other four pathogen taxa. The molecular severity (MS) of latent infection caused by Neofusicoccum spp. (3.6) was significantly greater than those caused by other pathogen taxa, except for Lasiodiplodia spp. (2.6). For prune, the average incidence of latent infection caused by Cytospora spp. (13.5%) was significantly higher than those caused by B. dothidea (1.5%) and Diplodia spp. (1.3%) but not significantly higher than those caused by Lasiodiplodia spp. (6.9%), Neofusicoccum spp. (6.3%), and Phomopsis spp. (7.7%), respectively. Moreover, the average MS values caused by Cytospora spp. (3.8) and Neofusicoccum spp. (3.2) were the highest followed by those caused by B. dothidea (1.4), Lasiodiplodia spp. (2.2), and Phomopsis spp. (2.3). Different almond varieties showed various levels of susceptibilities to different canker-causing pathogens. This study concluded that Lasiodiplodia and Neofusicoccum are the predominant pathogen species in almond, and Cytospora is the most important canker-causing pathogen species in prune in nurseries. These findings confirmed the observations of predominancy of canker-causing pathogens in almond and prune orchards in California.

Hypermethylation and small RNA expression are associated with increased age in almond (Prunus dulcis [Mill.] D.A. Webb) accessions.

The focus of this study is to profile changes in DNA methylation and small RNA expression occurring with increased age in almond breeding germplasm to identify possible biomarkers of age that can be used to assess the potential of individuals to develop aging-related disorders. To profile DNA methylation in almond germplasm, 70 methylomes were generated from almond individuals representing three age cohorts (11, 7, and 2 years old) using an enzymatic methyl-seq approach followed by analysis to call differentially methylated regions (DMRs) within these cohorts. Small RNA (sRNA) expression was profiled in three breeding selections, each from two age cohorts (1 and 6 years old), using sRNA-Seq followed by differential expression analysis. Weighted chromosome-level methylation analysis reveals hypermethylation in 11-year-old almond breeding selections when compared to 2-year-old selections in the CG and CHH contexts. Seventeen consensus DMRs were identified in all age contrasts. sRNA expression differed significantly between the two age cohorts tested, with significantly decreased expression in sRNAs in the 6-year-old selections compared to the 1-year-old. Almond shows a pattern of hypermethylation and decreased sRNA expression with increased age. Identified DMRs and differentially expressed sRNAs could function as putative biomarkers of age following validation in additional age groups.

Genetic diversity of Salmonella enterica isolated over 13 years from raw California almonds and from an almond orchard.

A comparative genomic analysis was conducted for 171 Salmonella isolates recovered from raw inshell almonds and raw almond kernels between 2001 and 2013 and for 30 Salmonella Enteritidis phage type (PT) 30 isolates recovered between 2001 and 2006 from a 2001 salmonellosis outbreak-associated almond orchard. Whole genome sequencing was used to measure the genetic distance among isolates by single nucleotide polymorphism (SNP) analyses and to predict the presence of plasmid DNA and of antimicrobial resistance (AMR) and virulence genes. Isolates were classified by serovars with Parsnp, a fast core-genome multi aligner, before being analyzed with the CFSAN SNP Pipeline (U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition). Genetically similar (≤18 SNPs) Salmonella isolates were identified among several serovars isolated years apart. Almond isolates of Salmonella Montevideo (2001 to 2013) and Salmonella Newport (2003 to 2010) differed by ≤9 SNPs. Salmonella Enteritidis PT 30 isolated between 2001 and 2013 from survey, orchard, outbreak, and clinical samples differed by ≤18 SNPs. One to seven plasmids were found in 106 (62%) of the Salmonella isolates. Of the 27 plasmid families that were identified, IncFII and IncFIB plasmids were the most predominant. AMR genes were identified in 16 (9%) of the survey isolates and were plasmid encoded in 11 of 16 cases; 12 isolates (7%) had putative resistance to at least one antibiotic in three or more drug classes. A total of 303 virulence genes were detected among the assembled genomes; a plasmid that harbored a combination of pef, rck, and spv virulence genes was identified in 23% of the isolates. These data provide evidence of long-term survival (years) of Salmonella in agricultural environments.

Whole genome sequencing and analysis of multiple isolates of Ceratocystis destructans, the causal agent of Ceratocystis canker of almond in California.

Ceratocystis canker caused by Ceratocystis destructans is a severe disease of almond, reducing the longevity and productivity of infected trees. Once the disease has established in an individual tree, there is no cure, and management efforts are often limited to removing the infected area of cankers. In this study, we present the genome assemblies of five C. destructans isolates isolated from symptomatic almond trees. The genomes were assembled into a genome size of 27.2 ± 0.9 Mbp with an average of 6924 ± 135 protein-coding genes and an average GC content of 48.8 ± 0.02%. We concentrated our efforts on identifying putative virulence factors of canker pathogens. Analysis of the secreted carbohydrate-active enzymes showed that the genomes harbored 83.4 ± 1.8 secreted CAZymes. The secreted CAZymes covered all the known categories of CAZymes. AntiSMASH revealed that the genomes had at least 7 biosynthetic gene clusters, with one of the non-ribosomal peptide synthases encoding dimethylcoprogen, a conserved virulence determinant of plant pathogenic ascomycetes. From the predicted proteome, we also annotated cytochrome P450 monooxygenases, and transporters, these are well-established virulence determinants of canker pathogens. Moreover, we managed to identify 57.4 ± 2.1 putative effector proteins. Gene Ontology (GO) annotation was applied to compare gene content with two closely related species C. fimbriata, and C. albifundus. This study provides the first genome assemblies for C. destructans, expanding genomic resources for an important almond canker pathogen. The acquired knowledge provides a foundation for further advanced studies, such as molecular interactions with the host, which is critical for breeding for resistance.

Detection of Hazelnut and Almond Adulteration in Olive Oil: An Approach by qPCR.

Virgin olive oil (VOO), characterized by its unique aroma, flavor, and health benefits, is subject to adulteration with the addition of oils obtained from other edible species. The consumption of adulterated olive oil with nut species, such as hazelnut or almond, leads to health and safety issues for consumers, due to their high allergenic potential. To detect almond and hazelnut in olive oil, several amplification systems have been analyzed by qPCR assay with a SYBR Green post-PCR melting curve analysis. The systems selected were Cora1F2/R2 and Madl, targeting the genes coding the allergenic protein Cor a 1 (hazelnut) and Pru av 1 (almond), respectively. These primers revealed adequate specificity for each of the targeted species. In addition, the result obtained demonstrated that this methodology can be used to detect olive oil adulteration with up to 5% of hazelnut or almond oil by a single qPCR assay, and with a level as low as 2.5% by a nested-qPCR assay. Thus, the present research has shown that the SYBR-based qPCR assay can be a rapid, precise, and accurate method to detect adulteration in olive oil.

Genome-Wide Identification and Analysis of the MADS-Box Gene Family in Almond Reveal Its Expression Features in Different Flowering Periods.

The MADS-box gene family is an important family of transcription factors involved in multiple processes, such as plant growth and development, stress, and in particular, flowering time and floral organ development. Almonds are the best-selling nuts in the international fruit trade, accounting for more than 50% of the world's dried fruit trade, and one of the main economic fruit trees in Kashgar, Xinjiang. In addition, almonds contain a variety of nutrients, such as protein and dietary fiber, which can supplement nutrients for people. They also have the functions of nourishing the yin and kidneys, improving eyesight, and strengthening the brain, and they can be applied to various diseases. However, there is no report on the MADS-box gene family in almond (Prunus dulcis). In this study, a total of 67 PdMADS genes distributed across 8 chromosomes were identified from the genome of almond 'Wanfeng'. The PdMADS members were divided into five subgroups-Mα, Mß, Mγ, Mδ, and MIKC-and the members in each subgroup had conserved motif types and exon and intron numbers. The number of exons of PdMADS members ranged from 1 to 20, and the number of introns ranged from 0 to 19. The number of exons and introns of different subfamily members varied greatly. The results of gene duplication analysis showed that the PdMADS members had 16 pairs of segmental duplications and 9 pairs of tandem duplications, so we further explored the relationship between the MADS-box gene members in almond and those in Arabidopsis thaliana, Oryza sativa, Malus domestica, and Prunus persica based on colinear genes and evolutionary selection pressure. The results of the cis-acting elements showed that the PdMADS members were extensively involved in a variety of processes, such as almond growth and development, hormone regulation, and stress response. In addition, the expression patterns of PdMADS members across six floral transcriptome samples from two almond cultivars, 'Wanfeng' and 'Nonpareil', had significant expression differences. Subsequently, the fluorescence quantitative expression levels of the 15 PdMADS genes were highly similar to the transcriptome expression patterns, and the gene expression levels increased in the samples at different flowering stages, indicating that the two almond cultivars expressed different PdMADS genes during the flowering process. It is worth noting that the difference in flowering time between 'Wanfeng' and 'Nonpareil' may be caused by the different expression activities of PdMADS47 and PdMADS16 during the dormancy period, resulting in different processes of vernalization. We identified a total of 13,515 target genes in the genome based on the MIKC DNA-binding sites. The GO and KEGG enrichment results showed that these target genes play important roles in protein function and multiple pathways. In summary, we conducted bioinformatics and expression pattern studies on the PdMADS gene family and investigated six flowering samples from two almond cultivars, the early-flowering 'Wanfeng' and late-flowering 'Nonpareil', for quantitative expression level identification. These findings lay a foundation for future in-depth studies on the mechanism of PdMADS gene regulation during flowering in different almond cultivars.

Characterization of the WRKY gene family reveals its contribution to the adaptability of almond (Prunus dulcis).

Background: WRKY (WRKY DNA-binding domain) transcription factors an important gene family that widely regulates plant resistance to biological and abiotic stresses, such as drought, salt and ion stresses. However, research on the WRKY family in almond has not yet been reported. Almond is an economically important fruit tree in Xinjiang that have strong resistance to various stresses. Results: A total of 62 PdWRKY genes were identified (including six pairs of homologous genes), and the phylogenetic tree was divided into three groups according to the WRKY domain and zinc finger motifs. The members of each group had a significant number of conserved motifs and exons/introns distributed unevenly across eight chromosomes, as well as 24 pairs of fragment duplicates and nine pairs of tandem duplicates. Moreover, the synteny and Ka/Ks analyses of the WRKY genes among almond and distinct species provided more detailed evidence for PdWRKY genes evolution. The examination of different tissue expression patterns showed that PdWRKY genes have tissue-specific expression characteristics. The qRT-PCR results showed that PdWRKY genes participate in the resistance of almond to the effects of low-temperature, drought and salt stress and that the expression levels of these genes change over time, exhibiting spatiotemporal expression characteristics. It is worth noting that many genes play a significant role in low-temperature stress resistance. In addition, based on the conserved WRKY motif, 321 candidate target genes were identified as having functions in multiple pathways. Conclusions: We conducted systematic bioinformatics analysis and abiotic stress research on the WRKY gene family in almond, laying the foundation for future PdWRKY genes research and improvements to almond production and breeding.

Development of PCR-Based Assays for Rapid and Reliable Detection and Identification of Canker-Causing Pathogens from Symptomatic Almond Trees.

Trunk and scaffold canker diseases (TSCDs) of almond cause significant yield and tree losses and reduce the lifespan of orchards. In California, several pathogens cause TSCDs, including Botryosphaeriaceae, Ceratocystis destructans, Eutypa lata, Collophorina hispanica, Pallidophorina paarla, Cytospora, Diaporthe, and Phytophthora spp. Field diagnosis of TSCDs is challenging because symptom delineation among the diseases is not clear. Accurate diagnosis of the causal species requires detailed examination of symptoms and subsequent isolation on medium and identification using morphological criteria and subsequent confirmation using molecular tools. The process is time-consuming and difficult, particularly as morphological characteristics are variable and overlap among species. To facilitate diagnosis of TSCD, we developed PCR assays using 23 species-specific primers designed by exploiting sequence differences in the translation elongation factor, ß-tubulin, or internal transcribed spacer gene. Using genomic DNA from pure cultures of each fungal and oomycete species, each primer pair successfully amplified a single DNA fragment from the target pathogen but not from selected nontarget pathogens or common endophytes. Although 10-fold serial dilution of fungal DNA extracted from either pure cultures or infected wood samples detected as little as 0.1 pg of DNA sample, consistent detection required 10 ng of pathogen DNA from mycelial samples or from wood chips or drill shavings from artificially or naturally infected almond wood samples with visible symptoms. The new PCR assay represents an improved tool for diagnostic laboratories and will be critical to implement effective disease surveillance and control measures.

Whole-genome sequence and methylome profiling of the almond [Prunus dulcis (Mill.) D.A. Webb] cultivar 'Nonpareil'.

Almond [Prunus dulcis (Mill.) D.A. Webb] is an economically important, specialty nut crop grown almost exclusively in the United States. Breeding and improvement efforts worldwide have led to the development of key, productive cultivars, including 'Nonpareil,' which is the most widely grown almond cultivar. Thus far, genomic resources for this species have been limited, and a whole-genome assembly for 'Nonpareil' is not currently available despite its economic importance and use in almond breeding worldwide. We generated a 571X coverage genome sequence using Illumina, PacBio, and optical mapping technologies. Gene prediction revealed 49,321 putative genes using MinION Oxford nanopore and Illumina RNA sequencing, and genome annotation found that 68% of predicted models are associated with at least one biological function. Furthermore, epigenetic signatures of almond, namely DNA cytosine methylation, have been implicated in a variety of phenotypes including self-compatibility, bud dormancy, and development of noninfectious bud failure. In addition to the genome sequence and annotation, this report also provides the complete methylome of several almond tissues, including leaf, flower, endocarp, mesocarp, exocarp, and seed coat. Comparisons between methylation profiles in these tissues revealed differences in genome-wide weighted % methylation and chromosome-level methylation enrichment.

sRNA Analysis Evidenced the Involvement of Different Plant Viruses in the Activation of RNA Silencing-Related Genes and the Defensive Response Against Plum pox virus of 'GF305' Peach Grafted with 'Garrigues' Almond.

Plum pox virus (PPV) causes sharka disease in Prunus trees. Peach (P. persica) trees are severely affected by PPV, and no definitive source of genetic resistance has been identified. However, previous results showed that PPV-resistant 'Garrigues' almond (P. dulcis) was able to transfer its resistance to 'GF305' peach through grafting, reducing symptoms and viral load in PPV-infected plants. A recent study tried to identify genes responsible for this effect by studying messenger RNA expression through RNA sequencing in peach and almond plants, before and after grafting and before and after PPV infection. In this work, we used the same peach and almond samples but focused the high-throughput analyses on small RNA (sRNA) expression. We studied massive sequencing data and found an interesting pattern of sRNA overexpression linked to antiviral defense genes that suggested activation of these genes followed by downregulation to basal levels. We also discovered that 'Garrigues' almond plants were infected by different plant viruses that were transferred to peach plants. The large amounts of viral sRNA found in grafted peaches indicated a strong RNA silencing antiviral response and led us to postulate that these plant viruses could be collaborating in the observed "Garrigues effect."

Susceptibility of Almond (Prunus dulcis) Cultivars to Twig Canker and Shoot Blight Caused by Diaporthe amygdali.

Twenty-five almond cultivars were assessed for susceptibility to Diaporthe amygdali, causal agent of twig canker and shoot blight disease. In laboratory experiments, growing twigs were inoculated with four D. amygdali isolates. Moreover, growing shoots of almond cultivars grafted onto INRA 'GF-677' rootstock were used in 4-year field inoculations with one D. amygdali isolate. In both types of experiments, inoculum consisted of agar plugs with mycelium, which were inserted underneath the bark, and the lesion lengths caused by the fungus were measured. Necrotic lesions were observed in the inoculated almond cultivars in both laboratory and field tests, confirming the susceptibility of all evaluated cultivars to all inoculated isolates of D. amygdali. Cultivars were grouped as susceptible or very susceptible according to a cluster analysis. The relationship between some agronomic traits and cultivar susceptibility was also investigated. Blooming and ripening times were found to be relevant variables explaining cultivar performance related to D. amygdali susceptibility. Late and very late blooming and early and medium ripening cultivars were highly susceptible to D. amygdali. Our results may provide valuable information that could assist in ongoing breeding programs of this crop and in the selection of cultivars for new almond plantations.

Identification and verification of seed development related miRNAs in kernel almond by small RNA sequencing and qPCR.

Many studies have investigated the role of miRNAs on the yield of various plants, but so far, no report is available on the identification and role of miRNAs in fruit and seed development of almonds. In this study, preliminary analysis by high-throughput sequencing of short RNAs of kernels from the crosses between almond cultivars 'Sefid' × 'Mamaee' (with small and large kernels, respectively) and 'Sefid' × 'P. orientalis' (with small kernels) showed that the expressions of several miRNAs such as Pdu-miR395a-3p, Pdu-miR8123-5p, Pdu-miR482f, Pdu-miR6285, and Pdu-miR396a were significantly different. These miRNAs targeted genes encoding different proteins such as NYFB-3, SPX1, PGSIP3 (GUX2), GH3.9, and BEN1. The result of RT-qPCR revealed that the expression of these genes showed significant differences between the crosses and developmental stages of the seeds, suggesting that these genes might be involved in controlling kernel size because the presence of these miRNAs had a negative effect on their target genes. Pollen source can influence kernel size by affecting hormonal signaling and metabolic pathways through related miRNAs, a phenomenon known as xenia.

Polymorphisms and gene expression in the almond IGT family are not correlated to variability in growth habit in major commercial almond cultivars.

Almond breeding programs aimed at selecting cultivars adapted to intensive orchards have recently focused on the optimization of tree architecture. This multifactorial trait is defined by numerous components controlled by processes such as hormonal responses, gravitropism and light perception. Gravitropism sensing is crucial to control the branch angle and therefore, the tree habit. A gene family, denominated IGT family after a shared conserved domain, has been described as involved in the regulation of branch angle in several species, including rice and Arabidopsis, and even in fruit trees like peach. Here we identified six members of this family in almond: LAZY1, LAZY2, TAC1, DRO1, DRO2, IGT-like. After analyzing their protein sequences in forty-one almond cultivars and wild species, little variability was found, pointing a high degree of conservation in this family. To our knowledge, this is the first effort to analyze the diversity of IGT family proteins in members of the same tree species. Gene expression was analyzed in fourteen cultivars of agronomical interest comprising diverse tree habit phenotypes. Only LAZY1, LAZY2 and TAC1 were expressed in almond shoot tips during the growing season. No relation could be established between the expression profile of these genes and the variability observed in the tree habit. However, some insight has been gained in how LAZY1 and LAZY2 are regulated, identifying the IPA1 almond homologues and other transcription factors involved in hormonal responses as regulators of their expression. Besides, we have found various polymorphisms that could not be discarded as involved in a potential polygenic origin of regulation of architectural phenotypes. Therefore, we have established that neither the expression nor the genetic polymorphism of IGT family genes are correlated to diversity of tree habit in currently commercialized almond cultivars, with other gene families contributing to the variability of these traits.

Production of Haploid and Doubled Haploid Lines in Nut Crops: Persian Walnut, Almond, and Hazelnut.

This chapter deals with induction of haploidy via parthenogenesis in Persian walnut and via microspore embryogenesis in almond and hazelnut. Haploid induction through in situ parthenogenesis using pollination with irradiated pollen to stimulate the embryogenic development of the egg cell, followed by in vitro culture of the immature haploid embryos. Microspore embryogenesis allows the induction of immature pollen grains (microspores), to move away from the normal gametophytic developmental route in the direction of the sporophytic one, yielding homozygous organisms (embryos in this case). Unlike other fruit crops (such as Citrus), regeneration of entire plants has not yet been obtained in our studied nut crops; however, it gives the methodology should be used to continue the roadmap.

Gene Expression Analysis of Induced Plum pox virus (Sharka) Resistance in Peach (Prunus persica) by Almond (P. dulcis) Grafting.

No natural sources of resistance to Plum pox virus (PPV, sharka disease) have been identified in peach. However, previous studies have demonstrated that grafting a "Garrigues" almond scion onto "GF305" peach rootstock seedlings heavily infected with PPV can progressively reduce disease symptoms and virus accumulation. Furthermore, grafting a "Garrigues" scion onto the "GF305" rootstock has been shown to completely prevent virus infection. This study aims to analyse the rewiring of gene expression associated with this resistance to PPV transmitted by grafting through the phloem using RNA-Seq and RT-qPCR analysis. A total of 18 candidate genes were differentially expressed after grafting "Garrigues" almond onto healthy "GF305" peach. Among the up-regulated genes, a HEN1 homolog stands out, which, together with the differential expression of RDR- and DCL2-homologs, suggests that the RNA silencing machinery is activated by PPV infection and can contribute to the resistance induced by "Garrigues" almond. Glucan endo-1,3-beta D-glucosidase could be also relevant for the "Garrigues"-induced response, since its expression is much higher in "Garrigues" than in "GF305". We also discuss the potential relevance of the following in PPV infection and "Garrigues"-induced resistance: several pathogenesis-related proteins; no apical meristem proteins; the transcription initiation factor, TFIIB; the speckle-type POZ protein; in addition to a number of proteins involved in phytohormone signalling.

Developmental transcriptome profiling uncovered carbon signaling genes associated with almond fruit drop.

Almond is one of the most featured nut crops owing to its high nutritional value. However, due to three different waves of flower and fruitlet drop, fruit drop is a major concern for growers. In this study, we carried out a time-course transcriptome analysis to investigate gene expression differences between normal and abnormal fruitlet development. By de novo assembly analysis, we identified 33,577 unigenes and provided their functional annotations. In total, we identified 7,469 differentially expressed genes and observed the most apparent difference between normal and abnormal fruits at 12 and 17 days after flowering. Their biological functions were enriched in carbon metabolism, carbon fixation in photosynthetic organisms and plant hormone signal transduction. RT-qPCR validated the expression pattern of 14 representative genes, including glycosyltransferase like family 2, MYB39, IAA13, gibberellin-regulated protein 11-like and POD44, which confirmed the reliability of our transcriptome data. This study provides an insight into the association between abnormal fruit development and carbohydrate signaling from the early developmental stages and could be served as useful information for understanding the regulatory mechanisms related to almond fruit drop.

Linking diverse salinity responses of 14 almond rootstocks with physiological, biochemical, and genetic determinants.

Fourteen commercial almond rootstocks were tested under five types of irrigation waters to understand the genetic, physiological, and biochemical bases of salt-tolerance mechanisms. Treatments included control (T1) and four saline water treatments dominant in sodium-sulfate (T2), sodium-chloride (T3), sodium-chloride/sulfate (T4), and calcium/magnesium-chloride/sulfate (T5). T3 caused the highest reduction in survival rate and trunk diameter, followed by T4 and T2, indicating that Na and, to a lesser extent, Cl were the most toxic ions to almond rootstocks. Peach hybrid (Empyrean 1) and peach-almond hybrids (Cornerstone, Bright's Hybrid 5, and BB 106) were the most tolerant to salinity. Rootstock's performance under salinity correlated highly with its leaf Na and Cl concentrations, indicating that Na+ and Cl- exclusion is crucial for salinity tolerance in Prunus. Photosynthetic rate correlated with trunk diameter and proline leaf ratio (T3/T1) significantly correlated with the exclusion of Na+ and Cl-, which directly affected the survival rate. Expression analyses of 23 genes involved in salinity stress revealed that the expression differences among genotypes were closely associated with their performance under salinity. Our genetic, molecular, and biochemical analyses allowed us to characterize rootstocks based on component traits of the salt-tolerance mechanisms, which may facilitate the development of highly salt-tolerant rootstocks.