Physiological Changes and Transcriptomic Analysis throughout On-Tree Fruit Ripening Process in Persimmon (Diospyros kaki L.).

The involvement of effectors and transcriptional regulators in persimmon fruit maturation has been mostly approached by the literature under postharvest conditions. In order to elucidate the participation of these genes in the on-tree fruit maturation development, we have collected samples from seven persimmon germplasm accessions at different developmental stages until physiological maturation. This study has focused on the expression analysis of 13 genes involved in ethylene biosynthesis and response pathways, as well as the evolution of important agronomical traits such as skin colour, weight, and firmness. Results revealed different gene expression patterns, with genes up- and down-regulated during fruit development progression. A principal component analysis was performed to correlate gene expression with agronomical traits. The decreasing expression of the ethylene biosynthetic genes DkACO1, DkACO2, and DkACS2, in concordance with other sensing (DkERS1) and transduction genes (DkERF18), provides a molecular mechanism for the previously described high production of ethylene in immature detached fruits. On the other side, DkERF8 and DkERF16 are postulated to induce fruit softening and skin colour change during natural persimmon fruit ripening via DkXTH9 and DkPSY activation, respectively. This study provides valuable information for a better understanding of the ethylene signalling pathway and its regulation during on-tree fruit ripening in persimmon.

Single-Bud Expression Analysis of Bud Dormancy Factors in Peach.

Transcriptomic and gene expression analysis have greatly facilitated the identification and characterization of transcriptional regulatory factors and effectors involved in dormancy progression and other physiological processes orchestrated during bud development in peach and other temperate fruit species. Gene expression measurements are most usually based on average values from several or many individual buds. We have performed single-bud gene analysis in flower buds of peach across dormancy release using amplicons from the master regulatory DORMANCY-ASSOCIATED MADS-BOX (DAM) factors, several jasmonic acid biosynthetic genes, other genes related to flowering development, cell growth resumption, and abiotic stress tolerance. This analysis provides a close view on gene-specific, single-bud variability throughout the developmental shift from dormant to dormancy-released stages, contributing to the characterization of putative co-expression modules and other regulatory aspects in this particular tissue.

Characterization of the Spanish Pomegranate Germplasm Collection Maintained at the Agricultural Experiment Station of Elche to Identify Promising Breeding Materials.

Pomegranates were one of the first domesticated fruit crops, and their long history resulted in the development of local cultivars all over the world. Spain is one of the main producers and exporters of this crop in the Mediterranean Basin, but in order to maintain the competitiveness of this crop, new varieties should be developed. For this purpose, the pomegranate germplasm collection hold at the Agricultural Experiment Station of Elche, a public institution dependent on the Valencian regional government, is an interesting tool. However, the detailed characterization of any germplasm collection is a fundamental requirement to be able to make the most of these resources, allowing to identify putative promising accessions and to optimize the design of the future crosses. In this work, the genetic diversity of 94 accessions of this collection was analyzed using 19 microsatellite markers. As a result, 85 different genotypes were identified. These genetic profiles could be useful for varietal identification. Despite this genetic diversity, no clear substructure was observed, except for the ornamental accessions, that could be related to the vegetative propagation of the species. Additionally, the morphological characterization of this collection has made it possible to identify some materials that may be of interest as a source of traits for breeding. Results presented here pave the way for further genetic analyses, allowing the selection of parents to obtain segregating populations, as well as their descendants by the use of molecular assisted selection.

Regulatory circuits involving bud dormancy factor PpeDAM6.

DORMANCY-ASSOCIATED MADS-BOX (DAM) genes have recently emerged as key potential regulators of the dormancy cycle and climate adaptation in perennial species. Particularly, PpeDAM6 has been proposed to act as a major repressor of bud dormancy release and bud break in peach (Prunus persica). PpeDAM6 expression is downregulated concomitantly with the perception of a given genotype-dependent accumulation of winter chilling time, and the coincident enrichment in H3K27me3 chromatin modification at a specific genomic region. We have identified three peach BASIC PENTACYSTEINE PROTEINs (PpeBPCs) interacting with two GA-repeat motifs present in this H3K27me3-enriched region. Moreover, PpeBPC1 represses PpeDAM6 promoter activity by transient expression experiments. On the other hand, the heterologous overexpression of PpeDAM6 in European plum (Prunus domestica) alters plant vegetative growth, resulting in dwarf plants tending toward shoot meristem collapse. These alterations in vegetative growth of transgenic lines associate with impaired hormone homeostasis due to the modulation of genes involved in jasmonic acid, cytokinin, abscisic acid, and gibberellin pathways, and the downregulation of shoot meristem factors, specifically in transgenic leaf and apical tissues. The expression of many of these genes is also modified in flower buds of peach concomitantly with PpeDAM6 downregulation, which suggests a role of hormone homeostasis mechanisms in PpeDAM6-dependent maintenance of floral bud dormancy and growth repression.

Insights of Phenolic Pathway in Fruits: Transcriptional and Metabolic Profiling in Apricot (Prunus armeniaca).

There is an increasing interest in polyphenols, plant secondary metabolites, in terms of fruit quality and diet, mainly due to their antioxidant effect. However, the identification of key gene enzymes and their roles in the phenylpropanoid pathway in temperate fruits species remains uncertain. Apricot (Prunus armeniaca) is a Mediterranean fruit with high diversity and fruit quality properties, being an excellent source of polyphenol compounds. For a better understanding of the phenolic pathway in these fruits, we selected a set of accessions with genetic-based differences in phenolic compounds accumulation. HPLC analysis of the main phenolic compounds and transcriptional analysis of the genes involved in key steps of the polyphenol network were carried out. Phenylalanine ammonia-lyase (PAL), dihydroflavonol-4-reductase (DFR) and flavonol synthase (FLS) were the key enzymes selected. Orthologous of the genes involved in transcription of these enzymes were identified in apricot: ParPAL1, ParPAL2, ParDFR, ParFLS1 and ParFLS2. Transcriptional data of the genes involved in those critical points and their relationships with the polyphenol compounds were analyzed. Higher expression of ParDFR and ParPAL2 has been associated with red-blushed accessions. Differences in expression between paralogues could be related to the presence of a BOXCOREDCPAL cis-acting element related to the genes involved in anthocyanin synthesis ParFLS2, ParDFR and ParPAL2.

Self-(In)compatibility Systems: Target Traits for Crop-Production, Plant Breeding, and Biotechnology.

Self-incompatibility (SI) mechanisms prevent self-fertilization in flowering plants based on specific discrimination between self- and non-self pollen. Since this trait promotes outcrossing and avoids inbreeding it is a widespread mechanism of controlling sexual plant reproduction. Growers and breeders have effectively exploited SI as a tool for manipulating domesticated crops for thousands of years. However, only within the past thirty years have studies begun to elucidate the underlying molecular features of SI. The specific S-determinants and some modifier factors controlling SI have been identified in the sporophytic system exhibited by Brassica species and in the two very distinct gametophytic systems present in Papaveraceae on one side and in Solanaceae, Rosaceae, and Plantaginaceae on the other. Molecular level studies have enabled SI to SC transitions (and vice versa) to be intentionally manipulated using marker assisted breeding and targeted approaches based on transgene integration, silencing, and more recently CRISPR knock-out of SI-related factors. These scientific advances have, in turn, provided a solid basis to implement new crop production and plant breeding practices. Applications of self-(in)compatibility include widely differing objectives such as crop yield and quality improvement, marker-assisted breeding through SI genotyping, and development of hybrids for overcoming intra- and interspecific reproductive barriers. Here, we review scientific progress as well as patented applications of SI, and also highlight future prospects including further elucidation of SI systems, deepening our understanding of SI-environment relationships, and new perspectives on plant self/non-self recognition.

Resistance to Plum Pox Virus (PPV) in apricot (Prunus armeniaca L.) is associated with down-regulation of two MATHd genes.

BACKGROUND: Plum pox virus (PPV), causing Sharka disease, is one of the main limiting factors for Prunus production worldwide. In apricot (Prunus armeniaca L.) the major PPV resistance locus (PPVres), comprising ~ 196 kb, has been mapped to the upper part of linkage group 1. Within the PPVres, 68 genomic variants linked in coupling to PPV resistance were identified within 23 predicted transcripts according to peach genome annotation. Taking into account the predicted functions inferred from sequence homology, some members of a cluster of meprin and TRAF-C homology domain (MATHd)-containing genes were pointed as PPV resistance candidate genes. RESULTS: Here, we have characterized the global apricot transcriptome response to PPV-D infection identifying six PPVres locus genes (ParP-1 to ParP-6) differentially expressed in resistant/susceptible cultivars. Two of them (ParP-3 and ParP-4), that encode MATHd proteins, appear clearly down-regulated in resistant cultivars, as confirmed by qRT-PCR. Concurrently, variant calling was performed using whole-genome sequencing data of 24 apricot cultivars (10 PPV-resistant and 14 PPV-susceptible) and 2 wild relatives (PPV-susceptible). ParP-3 and ParP-4, named as Prunus armeniaca PPVres MATHd-containing genes (ParPMC), are the only 2 genes having allelic variants linked in coupling to PPV resistance. ParPMC1 has 1 nsSNP, while ParPMC2 has 15 variants, including a 5-bp deletion within the second exon that produces a frameshift mutation. ParPMC1 and ParPMC2 are adjacent and highly homologous (87.5% identity) suggesting they are paralogs originated from a tandem duplication. Cultivars carrying the ParPMC2 resistant (mutated) allele show lack of expression in both ParPMC2 and especially ParPMC1. CONCLUSIONS: Accordingly, we hypothesize that ParPMC2 is a pseudogene that mediates down-regulation of its functional paralog ParPMC1 by silencing. As a whole, results strongly support ParPMC1 and/or ParPMC2 as host susceptibility genes required for PPV infection which silencing may confer PPV resistance trait. This finding may facilitate resistance breeding by marker-assisted selection and pave the way for gene edition approaches in Prunus.

A disulfide bond A-like oxidoreductase is a strong candidate gene for self-incompatibility in apricot (Prunus armeniaca) pollen.

S-RNase based gametophytic self-incompatibility (SI) is a widespread prezygotic reproductive barrier in flowering plants. In the Solanaceae, Plantaginaceae and Rosaceae gametophytic SI is controlled by the pistil-specific S-RNases and the pollen S-locus F-box proteins but non-S-specific factors, namely modifiers, are also required. In apricot, Prunus armeniaca (Rosaceae), we previously mapped two pollen-part mutations that confer self-compatibility in cultivars Canino and Katy at the distal end of chromosome 3 (M-locus) unlinked to the S-locus. Here, we used high-resolution mapping to identify the M-locus with an ~134 kb segment containing ParM-1-16 genes. Gene expression analysis identified four genes preferentially expressed in anthers as modifier gene candidates, ParM-6, -7, -9 and -14. Variant calling of WGS Illumina data from Canino, Katy, and 10 self-incompatible cultivars detected a 358 bp miniature inverted-repeat transposable element (MITE) insertion in ParM-7 shared only by self-compatible apricots, supporting ParM-7 as strong candidate gene required for SI. ParM-7 encodes a disulfide bond A-like oxidoreductase protein, which we named ParMDO. The MITE insertion truncates the ParMDO ORF and produces a loss of SI function, suggesting that pollen rejection in Prunus is dependent on redox regulation. Based on phylogentic analyses we also suggest that ParMDO may have originated from a tandem duplication followed by subfunctionalization and pollen-specific expression.

Self-(in)compatibility in apricot germplasm is controlled by two major loci, S and M.

BACKGROUND: Apricot (Prunus armeniaca L.) exhibits a gametophytic self-incompatibility (GSI) system and it is mostly considered as a self-incompatible species though numerous self-compatible exceptions occur. These are mainly linked to the mutated S C-haplotype carrying an insertion in the S-locus F-box gene that leads to a truncated protein. However, two S-locus unlinked pollen-part mutations (PPMs) termed m and m' have also been reported to confer self-compatibility (SC) in the apricot cultivars 'Canino' and 'Katy', respectively. This work was aimed to explore whether other additional mutations might explain SC in apricot as well. RESULTS: A set of 67 cultivars/accessions with different geographic origins were analyzed by PCR-screening of the S- and M-loci genotypes, contrasting results with the available phenotype data. Up to 20 S-alleles, including 3 new ones, were detected and sequence analysis revealed interesting synonymies and homonymies in particular with S-alleles found in Chinese cultivars. Haplotype analysis performed by genotyping and determining linkage-phases of 7 SSR markers, showed that the m and m' PPMs are linked to the same m 0-haplotype. Results indicate that m 0-haplotype is tightly associated with SC in apricot germplasm being quite frequent in Europe and North-America. However, its prevalence is lower than that for S C in terms of frequency and geographic distribution. Structures of 34 additional M-haplotypes were inferred and analyzed to depict phylogenetic relationships and M 1-2 was found to be the closest haplotype to m 0. Genotyping results showed that four cultivars classified as self-compatible do not have neither the S C- nor the m 0-haplotype. CONCLUSIONS: According to apricot germplasm S-genotyping, a loss of genetic diversity affecting the S-locus has been produced probably due to crop dissemination. Genotyping and phenotyping data support that self-(in)compatibility in apricot relies mainly on the S- but also on the M-locus. Regarding this latter, we have shown that the m 0-haplotype associated with SC is shared by 'Canino', 'Katy' and many other cultivars. Its origin is still unknown but phylogenetic analysis supports that m 0 arose later in time than S C from a widely distributed M-haplotype. Lastly, other mutants putatively carrying new mutations conferring SC have also been identified deserving future research.

Genomic analysis reveals MATH gene(s) as candidate(s) for Plum pox virus (PPV) resistance in apricot (Prunus armeniaca†L.).

Sharka disease, caused by Plum pox virus (PPV), is the most important viral disease affecting Prunus species. A major PPV resistance locus (PPVres) has been mapped to the upper part of apricot (Prunus armeniaca) linkage group 1. In this study, a physical map of the PPVres locus in the PPV-resistant cultivar 'Goldrich' was constructed. Bacterial artificial chromosome (BAC) clones belonging to the resistant haplotype contig were sequenced using 454/GS-FLX Titanium technology. Concurrently, the whole genome of seven apricot varieties (three PPV-resistant and four PPV-susceptible) and two PPV-susceptible apricot relatives (P. sibirica var. davidiana and P. mume) were obtained using the Illumina-HiSeq2000 platform. Single nucleotide polymorphisms (SNPs) within the mapped interval, recorded from alignments against the peach genome, allowed us to narrow down the PPVres locus to a region of ∼196 kb. Searches for polymorphisms linked in coupling with the resistance led to the identification of 68 variants within 23 predicted transcripts according to peach genome annotation. Candidate resistance genes were ranked combining data from variant calling and predicted functions inferred from sequence homology. Together, the results suggest that members of a cluster of meprin and TRAF-C homology domain (MATHd)-containing proteins are the most likely candidate genes for PPV resistance in apricot. Interestingly, MATHd proteins are hypothesized to control long-distance movement (LDM) of potyviruses in Arabidopsis, and restriction for LDM is also a major component of PPV resistance in apricot. Although the PPV resistance gene(s) remains to be unambiguously identified, these results pave the way to the determination of the underlying mechanism and to the development of more accurate breeding strategies.

An S-locus independent pollen factor confers self-compatibility in 'Katy' apricot.

Loss of pollen-S function in Prunus self-compatible cultivars has been mostly associated with deletions or insertions in the S-haplotype-specific F-box (SFB) genes. However, self-compatible pollen-part mutants defective for non-S-locus factors have also been found, for instance, in the apricot (Prunus armeniaca) cv. 'Canino'. In the present study, we report the genetic and molecular analysis of another self-compatible apricot cv. termed 'Katy'. S-genotype of 'Katy' was determined as S(1)S(2) and S-RNase PCR-typing of selfing and outcrossing populations from 'Katy' showed that pollen gametes bearing either the S(1)- or the S(2)-haplotype were able to overcome self-incompatibility (SI) barriers. Sequence analyses showed no SNP or indel affecting the SFB(1) and SFB(2) alleles from 'Katy' and, moreover, no evidence of pollen-S duplication was found. As a whole, the obtained results are compatible with the hypothesis that the loss-of-function of a S-locus unlinked factor gametophytically expressed in pollen (M'-locus) leads to SI breakdown in 'Katy'. A mapping strategy based on segregation distortion loci mapped the M'-locus within an interval of 9.4 cM at the distal end of chr.3 corresponding to ∼1.29 Mb in the peach (Prunus persica) genome. Interestingly, pollen-part mutations (PPMs) causing self-compatibility (SC) in the apricot cvs. 'Canino' and 'Katy' are located within an overlapping region of ∼273 Kb in chr.3. No evidence is yet available to discern if they affect the same gene or not, but molecular markers seem to indicate that both cultivars are genetically unrelated suggesting that every PPM may have arisen independently. Further research will be necessary to reveal the precise nature of 'Katy' PPM, but fine-mapping already enables SC marker-assisted selection and paves the way for future positional cloning of the underlying gene.

Physical mapping of a pollen modifier locus controlling self-incompatibility in apricot and synteny analysis within the Rosaceae.

S-locus products (S-RNase and F-box proteins) are essential for the gametophytic self-incompatibility (GSI) specific recognition in Prunus. However, accumulated genetic evidence suggests that other S-locus unlinked factors are also required for GSI. For instance, GSI breakdown was associated with a pollen-part mutation unlinked to the S-locus in the apricot (Prunus armeniaca L.) cv. 'Canino'. Fine-mapping of this mutated modifier gene (M-locus) and the synteny analysis of the M-locus within the Rosaceae are here reported. A segregation distortion loci mapping strategy, based on a selectively genotyped population, was used to map the M-locus. In addition, a bacterial artificial chromosome (BAC) contig was constructed for this region using overlapping oligonucleotides probes, and BAC-end sequences (BES) were blasted against Rosaceae genomes to perform micro-synteny analysis. The M-locus was mapped to the distal part of chr.3 flanked by two SSR markers within an interval of 1.8 cM corresponding to ~364 Kb in the peach (Prunus persica L. Batsch) genome. In the integrated genetic-physical map of this region, BES were mapped against the peach scaffold_3 and BACs were anchored to the apricot map. Micro-syntenic blocks were detected in apple (Malus × domestica Borkh.) LG17/9 and strawberry (Fragaria vesca L.) FG6 chromosomes. The M-locus fine-scale mapping provides a solid basis for self-compatibility marker-assisted selection and for positional cloning of the underlying gene, a necessary goal to elucidate the pollen rejection mechanism in Prunus. In a wider context, the syntenic regions identified in peach, apple and strawberry might be useful to interpret GSI evolution in Rosaceae.

Narrowing down the apricot Plum pox virus resistance locus and comparative analysis with the peach genome syntenic region.

Sharka disease, caused by the Plum pox virus (PPV), is one of the main limiting factors for stone fruit crops worldwide. Only a few resistance sources have been found in apricot (Prunus armeniaca L.), and most studies have located a major PPV resistance locus (PPVres) on linkage group 1 (LG1). However, the mapping accuracy was not sufficiently reliable and PPVres was predicted within a low confidence interval. In this study, we have constructed two high-density simple sequence repeat (SSR) improved maps with 0.70 and 0.68 markers/cm, corresponding to LG1 of 'Lito' and 'Goldrich' PPV-resistant cultivars, respectively. Using these maps, and excluding genotype-phenotype incongruent individuals, a new binary trait locus (BTL) analysis for PPV resistance was performed, narrowing down the PPVres support intervals to 7.3 and 5.9 cm in 'Lito' and 'Goldrich', respectively. Subsequently, 71 overlapping oligonucleotides (overgo) probes were hybridized against an apricot bacterial artificial chromosome (BAC) library, identifying 870 single BACs from which 340 were anchored onto a map region of approximately 30-40 cm encompassing PPVres. Partial BAC contigs assigned to the two allelic haplotypes (resistant/susceptible) of the PPVres locus were built by high-information content fingerprinting (HICF). In addition, a total of 300 BAC-derived sequences were obtained, and 257 showed significant homology with the peach genome scaffold_1 corresponding to LG1. According to the peach syntenic genome sequence, PPVres was predicted within a region of 2.16 Mb in which a few candidate resistance genes were identified.

AFLP and DNA sequence variation in an Andean domesticate, pepino (Solanum muricatum, Solanaceae): implications for evolution and domestication.

The pepino (Solanum muricatum) is a vegetatively propagated, domesticated native of the Andes, where it grows with wild relatives. We used AFLPs and a 1-kb sequence of the 3-methylcrotonyl-CoA carboxylase gene to study variation of 27 accessions of S. muricatum and 35 collections of 10 species of wild relatives (Solanum section Basarthrum). A total of 298 AFLP fragments and 29 DNA sequence haplotypes were detected. Cluster and principal coordinate analyses and other genetic parameters estimated from both types of markers, show that S. muricatum is closely related to the species from one of the series (Caripensia) of section Basarthrum and that >90% of the variation of the cultigen is also represented in that series. Pepino is highly diverse, either because it is not monophyletic or it has been subjected to regular introgression with wild species, or both. Although a continuous distribution of the genetic variation occurred within the cultivated species, three genetic clusters were recognized. Cluster 1 is mostly centered in Ecuador, cluster 2 in Ecuador and Peru, and cluster 3 in Colombia and Ecuador. Cluster 3 also includes all modern cultivars studied. These results and other evidence suggest that northern Ecuador/southern Colombia is the main center of pepino diversity and the center of origin. The high genetic variation of this cultigen indicates that domestication does not always produce a genetic bottleneck.