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.

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.

Uncovering early response to drought by proteomic, physiological and biochemical changes in the almond × peach rootstock 'Garnem'.

Drought affects growth and metabolism in plants. To investigate the changes in root protein function involved in the early response to drought stress, a proteomic analysis in combination to a physiological and biochemical analysis was performed in plants of 'Garnem', an almond × peach hybrid rootstock, subjected to short-term drought stress. Abscisic acid (ABA) accumulation levels increased during the drought exposure, which induced stomatal closure, and thus, minimised water losses. These effects were reflected in stomatal conductance and leaf water potential levels. However, 'Garnem' was able to balance water content and maintain an osmotic adjustment in cell membranes, suggesting a dehydration avoidance strategy. The proteomic analysis revealed significant abundance changes in 29 and 24 spots after 2 and 24 h of drought stress respectively. Out of these, 15 proteins were identified by LC-ESI-MS/MS. The abundance changes of these proteins suggest the influence in drought-responsive mechanisms present in 'Garnem', allowing its adaptation to drought conditions. Overall, our study improves existing knowledge on the root proteomic changes in the early response to drought. This will lead to a better understanding of dehydration avoidance and tolerance strategies, and finally, help in new drought-tolerance breeding approaches.

Preformed and induced mechanisms underlies the differential responses of Prunus rootstock to hypoxia.

Analysis of the transcriptomic changes produced in response to hypoxia in root tissues from two rootstock Prunus genotypes differing in their sensitivity to waterlogging: resistant Myrobalan 'P.2175' (P. cerasifera Erhr.), and sensitive 'Felinem' hybrid [P. amygdalus Batsch × P. persica (L.) Batsch] revealed alterations in both metabolism and regulatory processes. Early hypoxia response in both genotypes is characterized by a molecular program aimed to adapt the cell metabolism to the new conditions. Upon hypoxia conditions, tolerant Myrobalan represses first secondary metabolism gene expression as a strategy to prevent the waste of resources/energy, and by the up-regulation of protein degradation genes probably leading to structural adaptations to long-term response to hypoxia. In response to the same conditions, sensitive 'Felinem' up-regulates a core of signal transduction and transcription factor genes. A combination of PLS-DA and qRT-PCR approaches revealed a set of transcription factors and signalling molecules as differentially regulated in the sensitive and tolerant genotypes including the peach orthologs for oxygen sensors. Apart from providing insights into the molecular processes underlying the differential response to waterlogging of two Prunus rootstocks, our approach reveals a set of candidate genes to be used expression biomarkers for biotech or breeding approaches to waterlogging tolerance.

Application of Genomic Technologies to the Breeding of Trees.

The recent introduction of next generation sequencing (NGS) technologies represents a major revolution in providing new tools for identifying the genes and/or genomic intervals controlling important traits for selection in breeding programs. In perennial fruit trees with long generation times and large sizes of adult plants, the impact of these techniques is even more important. High-throughput DNA sequencing technologies have provided complete annotated sequences in many important tree species. Most of the high-throughput genotyping platforms described are being used for studies of genetic diversity and population structure. Dissection of complex traits became possible through the availability of genome sequences along with phenotypic variation data, which allow to elucidate the causative genetic differences that give rise to observed phenotypic variation. Association mapping facilitates the association between genetic markers and phenotype in unstructured and complex populations, identifying molecular markers for assisted selection and breeding. Also, genomic data provide in silico identification and characterization of genes and gene families related to important traits, enabling new tools for molecular marker assisted selection in tree breeding. Deep sequencing of transcriptomes is also a powerful tool for the analysis of precise expression levels of each gene in a sample. It consists in quantifying short cDNA reads, obtained by NGS technologies, in order to compare the entire transcriptomes between genotypes and environmental conditions. The miRNAs are non-coding short RNAs involved in the regulation of different physiological processes, which can be identified by high-throughput sequencing of RNA libraries obtained by reverse transcription of purified short RNAs, and by in silico comparison with known miRNAs from other species. All together, NGS techniques and their applications have increased the resources for plant breeding in tree species, closing the former gap of genetic tools between trees and annual species.

Physiological and biochemical parameters controlling waterlogging stress tolerance in Prunus before and after drainage.

Waterlogging is associated with poor soil drainage. As a consequence oxygen levels decrease in the root environment inducing root asphyxia and affecting plant growth. Some plants can survive under these conditions triggering complex anatomical and biochemical adaptations, mostly in the roots. Long- and short-term responses to waterlogging stress were compared in two trials using a set of two myrobalans (Prunus cerasifera Erhr), 'P.2175' and 'P.2980', as tolerant rootstocks and two almond × peach [Prunus amygdalus Batsch ×Prunus persica (L.) Batsch] interspecific hybrids, 'Garnem' and 'Felinem', as sensitive ones in two consecutive years. Stomatal conductance and chlorophyll content were measured in the long-term trials to assess survival performance, while the enzyme activities of superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (POD, EC 1.11.1.7), and catalase (CAT, EC 1.11.1.6) were measured in the short-term trials to study early antioxidant response. The incidence of the stress in the root environment was different as a result of the different plant development at the moment of the treatment, as a consequence of different environmental conditions both before and during the treatment between the 2 years. The activity of the different enzymes was higher in the sensitive genotype 'Felinem' than in the tolerant 'P.2175'. This result shows an activation of the antioxidant system and has been observed to depend of the different nature of the roots between the 2 years. As the antioxidant enzymes seem to work more efficiently when roots are more aerated, we cannot conclude that they are responsible for the higher tolerance observed in the myrobalan plums.

Over-expression of a tomato N-acetyl-L-glutamate synthase gene (SlNAGS1) in Arabidopsis thaliana results in high ornithine levels and increased tolerance in salt and drought stresses.

A single copy of the N-acetyl-L-glutamate synthase gene (SlNAGS1) has been isolated from tomato. The deduced amino acid sequence consists of 604 amino acids and shows a high level of similarity to the predicted Arabidopsis NAGS1 and NAGS2 proteins. Furthermore, the N-terminus ArgB domain and the C-terminus ArgA domain found in SlNAGS1 are similar to the structural arrangements that have been reported for other predicted NAGS proteins. SlNAGS1 was expressed at high levels in all aerial organs, and at basic levels in seeds, whereas it was not detected at all in roots. SlNAGS1 transcript accumulation was noticed transiently in tomato fruit at the red-fruit stage. In addition, an increase of SlNAGS1 transcripts was detected in mature green tomato fruit within the first hour of exposure to low oxygen concentrations. Transgenic Arabidopsis plants have been generated expressing the SlNAGS1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Three homozygous transgenic lines expressing the transgene (lines 1-7, 3-8, and 6-5) were evaluated further. All three transgenic lines showed a significant accumulation of ornithine in the leaves with line 3-8 exhibiting the highest concentration. The same lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to 250 mM NaCl. Similarly, mature plants of all three transgenic lines displayed a higher tolerance to salt and drought stress compared to WT plants. Under most experimental conditions, transgenic line 3-8 performed best, while the responses obtained from lines 1-7 and 6-5 depended on the applied stimulus. To our knowledge, this is the first plant NAGS gene to be isolated, characterized, and genetically modified.