Genome-wide association analysis of Monilinia fructicola lesion in a collection of Spanish peach landraces.

Brown rot, caused by the Monilinia spp., is the disease that causes the greatest losses in stone fruit worldwide. Currently, M. fructicola has become the dominant species in the main peach production area in Spain. The fruit cuticle is the first barrier of protection against external aggressions and may have a key role in the susceptibility to brown rot. However, information on the role of skin fruit on the resistance to brown rot in peach is scarce. Previous genetic analyses in peach have demonstrated that brown rot resistance is a complex and quantitative trait in which different fruit parts and resistance mechanisms are involved. To search for genomic areas involved in the control of the cultivar susceptibility to brown rot and to elucidate the role of fruit skin against this infection, we have studied, for two consecutive seasons (2019 and 2020), the fruit susceptibility to M. fructicola, together with fruit cuticle thickness (CT) and density (CD), in a collection of 80 Spanish and 5 foreign peach cultivars from the National Peach Collection at CITA (Zaragoza, Spain). Brown rot incidence, lesion diameter, and severity index were calculated after 5 days of inoculation on non-wounded fruit. The peach collection has also been genotyped using the new peach SNP chip (9 + 9K). Genotypic and phenotypic data have been used to perform a genome-wide association analysis (GWAS). Phenotyping has shown a wide variability on the brown rot susceptibility within the Spanish germplasm as well as on CD and CT. The GWAS results have identified several significant SNPs associated with disease severity index (DSI), CD, and CT, five of which were considered as reliable SNP-trait associations. A wide protein network analysis, using 127 genes within the regions of the reliable SNPs and previously identified candidate genes (169) associated with Monilinia spp. resistance, highlighted several genes involved in classical hypersensitive response (HR), genes related to wax layers as ceramidases and lignin precursors catalyzers, and a possible role of autophagy during brown rot infection. This work adds relevant information on the complexity resistance mechanisms to brown rot infection in peach fruits and the genetics behind them.

Orchard Management and Incorporation of Biochemical and Molecular Strategies for Improving Drought Tolerance in Fruit Tree Crops.

Water scarcity is one of the greatest concerns for agronomy worldwide. In recent years, many water resources have been depleted due to multiple factors, especially mismanagement. Water resource shortages lead to cropland expansion, which likely influences climate change and affects global agriculture, especially horticultural crops. Fruit yield is the final aim in commercial orchards; however, drought can slow tree growth and/or decrease fruit yield and quality. It is therefore necessary to find approaches to solve this problem. The main objective of this review is to discuss the most recent horticultural, biochemical, and molecular strategies adopted to improve the response of temperate fruit crops to water stress. We also address the viability of cultivating fruit trees in dry areas and provide precise protection methods for planting fruit trees in arid lands. We review the main factors involved in planting fruit trees in dry areas, including plant material selection, regulated deficit irrigation (DI) strategies, rainwater harvesting (RWH), and anti-water stress materials. We also provide a detailed analysis of the molecular strategies developed to combat drought, such as Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) through gene overexpression or gene silencing. Finally, we look at the molecular mechanisms associated with the contribution of the microbiome to improving plant responses to drought.

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.

Identification of early and late flowering time candidate genes in endodormant and ecodormant almond flower buds.

Flower bud dormancy in temperate fruit tree species, such as almond [Prunus dulcis (Mill.) D.A. Webb], is a survival mechanism that ensures that flowering will occur under suitable weather conditions for successful flower development, pollination and fruit set. Dormancy is divided into three sequential phases: paradormancy, endodormancy and ecodormancy. During the winter, buds need cultivar-specific chilling requirements (CRs) to overcome endodormancy and heat requirements to activate the machinery to flower in the ecodormancy phase. One of the main factors that enables the transition from endodormancy to ecodormancy is transcriptome reprogramming. In this work, we therefore monitored three almond cultivars with different CRs and flowering times by RNA sequencing during the endodormancy release of flower buds and validated the data by quantitative real-time PCR in two consecutive seasons. We were thus able to identify early and late flowering time candidate genes in endodormant and ecodormant almond flower buds associated with metabolic switches, transmembrane transport, cell wall remodeling, phytohormone signaling and pollen development. These candidate genes were indeed involved in the overcoming of the endodormancy in almond. This information may be used for the development of dormancy molecular markers, increasing the efficiency of temperate fruit tree breeding programs in a climate-change context.

A Cost Reduced Variant of Epi-Genotyping by Sequencing for Studying DNA Methylation in Non-model Organisms.

Reference-free reduced representation bisulfite sequencing uses enzymatic digestion for reducing genome complexity and allows detection of markers to study DNA methylation of a high number of individuals in natural populations of non-model organisms. Current methods like epiGBS enquire the use of a higher number of methylated DNA oligos with a significant cost (especially for small labs and first pilot studies). In this paper, we present a modification of this epiGBS protocol that requires the use of only one hemimethylated P2 (common) adapter, which is combined with unmethylated barcoded adapters. The unmethylated cytosines of one chain of the barcoded adapter are replaced by methylated cytosines using nick translation with methylated cytosines in dNTP solution. The basic version of our technique uses only one restriction enzyme, and as a result, genomic fragments are integrated into two orientations with respect to the adapter sequences. Comparing the sequences of two chain orientations makes it possible to reconstruct the original sequence before bisulfite treatment with the help of standard software and newly developed software written in C and described here. We provide a proof of concept via data obtained from almond (Prunus dulcis). Example data and a detailed description of the complete software pipeline starting from the raw reads up until the final differentially methylated cytosines are given in Supplementary Material making this technique accessible to non-expert computer users. The adapter design showed in this paper should allow the use of a two restriction enzyme approach with minor changes in software parameters.

DNA Methylation Analysis of Dormancy Release in Almond (Prunus dulcis) Flower Buds Using Epi-Genotyping by Sequencing.

DNA methylation and histone post-translational modifications have been described as epigenetic regulation mechanisms involved in developmental transitions in plants, including seasonal changes in fruit trees. In species like almond (Prunus dulcis (Mill.) D.A: Webb), prolonged exposure to cold temperatures is required for dormancy release and flowering. Aiming to identify genomic regions with differential methylation states in response to chill accumulation, we carried out Illumina reduced-representation genome sequencing on bisulfite-treated DNA from floral buds. To do this, we analyzed almond genotypes with different chilling requirements and flowering times both before and after dormancy release for two consecutive years. The study was performed using epi-Genotyping by Sequencing (epi-GBS). A total of 7317 fragments were sequenced and the samples compared. Out of these fragments, 677 were identified as differentially methylated between the almond genotypes. Mapping these fragments using the Prunus persica (L.) Batsch v.2 genome as reference provided information about coding regions linked to early and late flowering methylation markers. Additionally, the methylation state of ten gene-coding sequences was found to be linked to the dormancy release process.

In silico search, characterization and validation of new EST-SSR markers in the genus Prunus.

BACKGROUND: Simple sequence repeats (SSRs) are defined as sequence repeat units between 1 and 6 bp that occur in both coding and non-coding regions abundant in eukaryotic genomes, which may affect the expression of genes. In this study, expressed sequence tags (ESTs) of eight Prunus species were analyzed for in silico mining of EST-SSRs, protein annotation, and open reading frames (ORFs), and the identification of codon repetitions. RESULTS: A total of 316 SSRs were identified using MISA software. Dinucleotide SSR motifs (26.31 %) were found to be the most abundant type of repeats, followed by tri- (14.58 %), tetra- (0.53 %), and penta- (0.27 %) nucleotide motifs. An attempt was made to design primer pairs for 316 identified SSRs but these were successful for only 175 SSR sequences. The positions of SSRs with respect to ORFs were detected, and annotation of sequences containing SSRs was performed to assign function to each sequence. SSRs were also characterized (in terms of position in the reference genome and associated gene) using the two available Prunus reference genomes (mei and peach). Finally, 38 SSR markers were validated across peach, almond, plum, and apricot genotypes. This validation showed a higher transferability level of EST-SSR developed in P. mume (mei) in comparison with the rest of species analyzed. CONCLUSIONS: Findings will aid analysis of functionally important molecular markers and facilitate the analysis of genetic diversity.