Comparative Stem Transcriptome Analysis Reveals Pathways Associated with Drought Tolerance in Maritime Pine Grafts.

The maritime pine (Pinus pinaster Ait.) is a highly valuable Mediterranean conifer. However, recurrent drought events threaten its propagation and conservation. P. pinaster populations exhibit remarkable differences in drought tolerance. To explore these differences, we analyzed stem transcriptional profiles of grafts combining genotypes with contrasting drought responses under well-watered and water-stress regimes. Our analysis underscored that P. pinaster drought tolerance is mainly associated with constitutively expressed genes, which vary based on genotype provenance. However, we identified key genes encoding proteins involved in water stress response, abscisic acid signaling, and growth control including a PHD chromatin regulator, a histone deubiquitinase, the ABI5-binding protein 3, and transcription factors from Myb-related, DOF NAC and LHY families. Additionally, we identified that drought-tolerant rootstock could enhance the drought tolerance of sensitive scions by regulating the accumulation of transcripts involved in carbon mobilization, osmolyte biosynthesis, flavonoid and terpenoid metabolism, and reactive oxygen species scavenging. These included genes encoding galactinol synthase, CBL-interacting serine/threonine protein kinase 5, BEL1-like homeodomain protein, dihydroflavonol 4-reductase, and 1-deoxy-D-xylulose-5-phosphate. Our results revealed several hub genes that could help us to understand the molecular and physiological response to drought of conifers. Based on all the above, grafting with selected drought-tolerant rootstocks is a promising method for propagating elite recalcitrant conifer species, such as P. pinaster.

The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box Gene VviAGL11.

Seedlessness is greatly prized by consumers of fresh grapes. While stenospermocarpic seed abortion determined by the SEED DEVELOPMENT INHIBITOR (SDI) locus is the usual source of seedlessness in commercial grapevine (Vitis vinifera) cultivars, the underlying sdi mutation remains unknown. Here, we undertook an integrative approach to identify the causal mutation. Quantitative genetics and fine-mapping in two 'Crimson Seedless'-derived F1 mapping populations confirmed the major effect of the SDI locus and delimited the sdi mutation to a 323-kb region on chromosome 18. RNA-sequencing comparing seed traces of seedless and seeds of seeded F1 individuals identified processes triggered during sdi-determined seed abortion, including the activation of salicylic acid-dependent autoimmunity. The RNA-sequencing data set was investigated for candidate genes, and while no evidence for causal cis-acting regulatory mutations was detected, deleterious nucleotide changes in coding sequences of the seedless haplotype were predicted in two genes within the sdi fine-mapping interval. Targeted resequencing of the two genes in a collection of 124 grapevine cultivars showed that only the point variation causing the arginine-197-to-leucine substitution in the seed morphogenesis regulator gene AGAMOUS-LIKE11 (VviAGL11) was fully linked with stenospermocarpy. The concurrent postzygotic variation identified for this missense polymorphism and seedlessness phenotype in seeded somatic variants of the original stenospermocarpic cultivar supports a causal effect. We postulate that seed abortion caused by this amino acid substitution in VviAGL11 is the major cause of seedlessness in cultivated grapevine. This information can be exploited to boost seedless grape breeding.

Genetic control of functional traits related to photosynthesis and water use efficiency in Pinus pinaster Ait. drought response: integration of genome annotation, allele association and QTL detection for candidate gene identification.

BACKGROUND: Understanding molecular mechanisms that control photosynthesis and water use efficiency in response to drought is crucial for plant species from dry areas. This study aimed to identify QTL for these traits in a Mediterranean conifer and tested their stability under drought. RESULTS: High density linkage maps for Pinus pinaster were used in the detection of QTL for photosynthesis and water use efficiency at three water irrigation regimes. A total of 28 significant and 27 suggestive QTL were found. QTL detected for photochemical traits accounted for the higher percentage of phenotypic variance. Functional annotation of genes within the QTL suggested 58 candidate genes for the analyzed traits. Allele association analysis in selected candidate genes showed three SNPs located in a MYB transcription factor that were significantly associated with efficiency of energy capture by open PSII reaction centers and specific leaf area. CONCLUSIONS: The integration of QTL mapping of functional traits, genome annotation and allele association yielded several candidate genes involved with molecular control of photosynthesis and water use efficiency in response to drought in a conifer species. The results obtained highlight the importance of maintaining the integrity of the photochemical machinery in P. pinaster drought response.

Maritime Pine Rootstock Genotype Modulates Gene Expression Associated with Stress Tolerance in Grafted Stems.

Climate change-induced hazards, such as drought, threaten forest resilience, particularly in vulnerable regions such as the Mediterranean Basin. Maritime pine (Pinus pinaster Aiton), a model species in Western Europe, plays a crucial role in the Mediterranean forest due to its genetic diversity and ecological plasticity. This study characterizes transcriptional profiles of scion and rootstock stems of four P. pinaster graft combinations grown under well-watered conditions. Our grafting scheme combined drought-sensitive and drought-tolerant genotypes for scions (GAL1056: drought-sensitive scion; and Oria6: drought-tolerant scion) and rootstocks (R1S: drought-sensitive rootstock; and R18T: drought-tolerant rootstock). Transcriptomic analysis revealed expression patterns shaped by genotype provenance and graft combination. The accumulation of differentially expressed genes (DEGs) encoding proteins, involved in defense mechanisms and pathogen recognition, was higher in drought-sensitive scion stems and also increased when grafted onto drought-sensitive rootstocks. DEGs involved in drought tolerance mechanisms were identified in drought-tolerant genotypes as well as in drought-sensitive scions grafted onto drought-tolerant rootstocks, suggesting their establishment prior to drought. These mechanisms were associated with ABA metabolism and signaling. They were also involved in the activation of the ROS-scavenging pathways, which included the regulation of flavonoid and terpenoid metabolisms. Our results reveal DEGs potentially associated with the conifer response to drought and point out differences in drought tolerance strategies. These findings suggest genetic trade-offs between pine growth and defense, which could be relevant in selecting more drought-tolerant Pinus pinaster trees.

Annotated genetic linkage maps of Pinus pinaster Ait. from a Central Spain population using microsatellite and gene based markers.

BACKGROUND: Pinus pinaster Ait. is a major resin producing species in Spain. Genetic linkage mapping can facilitate marker-assisted selection (MAS) through the identification of Quantitative Trait Loci and selection of allelic variants of interest in breeding populations. In this study, we report annotated genetic linkage maps for two individuals (C14 and C15) belonging to a breeding program aiming to increase resin production. We use different types of DNA markers, including last-generation molecular markers. RESULTS: We obtained 13 and 14 linkage groups for C14 and C15 maps, respectively. A total of 211 and 215 markers were positioned on each map and estimated genome length was between 1,870 and 2,166 cM respectively, which represents near 65% of genome coverage. Comparative mapping with previously developed genetic linkage maps for P. pinaster based on about 60 common markers enabled aligning linkage groups to this reference map. The comparison of our annotated linkage maps and linkage maps reporting QTL information revealed 11 annotated SNPs in candidate genes that co-localized with previously reported QTLs for wood properties and water use efficiency. CONCLUSIONS: This study provides genetic linkage maps from a Spanish population that shows high levels of genetic divergence with French populations from which segregating progenies have been previously mapped. These genetic maps will be of interest to construct a reliable consensus linkage map for the species. The importance of developing functional genetic linkage maps is highlighted, especially when working with breeding populations for its future application in MAS for traits of interest.

Molecular study of drought response in the Mediterranean conifer Pinus pinaster Ait.: Differential transcriptomic profiling reveals constitutive water deficit-independent drought tolerance mechanisms.

Adaptation of long-living forest trees to respond to environmental changes is essential to secure their performance under adverse conditions. Water deficit is one of the most significant stress factors determining tree growth and survival. Maritime pine (Pinus pinaster Ait.), the main source of softwood in southwestern Europe, is subjected to recurrent drought periods which, according to climate change predictions for the years to come, will progressively increase in the Mediterranean region. The mechanisms regulating pine adaptive responses to environment are still largely unknown. The aim of this work was to go a step further in understanding the molecular mechanisms underlying maritime pine response to water stress and drought tolerance at the whole plant level. A global transcriptomic profiling of roots, stems, and needles was conducted to analyze the performance of siblings showing contrasted responses to water deficit from an ad hoc designed full-sib family. Although P. pinaster is considered a recalcitrant species for vegetative propagation in adult phase, the analysis was conducted using vegetatively propagated trees exposed to two treatments: well-watered and moderate water stress. The comparative analyses led us to identify organ-specific genes, constitutively expressed as well as differentially expressed when comparing control versus water stress conditions, in drought-sensitive and drought-tolerant genotypes. Different response strategies can point out, with tolerant individuals being pre-adapted for coping with drought by constitutively expressing stress-related genes that are detected only in latter stages on sensitive individuals subjected to drought.

High throughput SNP discovery and genotyping in grapevine (Vitis vinifera L.) by combining a re-sequencing approach and SNPlex technology.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) are the most abundant type of DNA sequence polymorphisms. Their higher availability and stability when compared to simple sequence repeats (SSRs) provide enhanced possibilities for genetic and breeding applications such as cultivar identification, construction of genetic maps, the assessment of genetic diversity, the detection of genotype/phenotype associations, or marker-assisted breeding. In addition, the efficiency of these activities can be improved thanks to the ease with which SNP genotyping can be automated. Expressed sequence tags (EST) sequencing projects in grapevine are allowing for the in silico detection of multiple putative sequence polymorphisms within and among a reduced number of cultivars. In parallel, the sequence of the grapevine cultivar Pinot Noir is also providing thousands of polymorphisms present in this highly heterozygous genome. Still the general application of those SNPs requires further validation since their use could be restricted to those specific genotypes. RESULTS: In order to develop a large SNP set of wide application in grapevine we followed a systematic re-sequencing approach in a group of 11 grape genotypes corresponding to ancient unrelated cultivars as well as wild plants. Using this approach, we have sequenced 230 gene fragments, what represents the analysis of over 1 Mb of grape DNA sequence. This analysis has allowed the discovery of 1573 SNPs with an average of one SNP every 64 bp (one SNP every 47 bp in non-coding regions and every 69 bp in coding regions). Nucleotide diversity in grape (pi = 0.0051) was found to be similar to values observed in highly polymorphic plant species such as maize. The average number of haplotypes per gene sequence was estimated as six, with three haplotypes representing over 83% of the analyzed sequences. Short-range linkage disequilibrium (LD) studies within the analyzed sequences indicate the existence of a rapid decay of LD within the selected grapevine genotypes. To validate the use of the detected polymorphisms in genetic mapping, cultivar identification and genetic diversity studies we have used the SNPlextrade mark genotyping technology in a sample of grapevine genotypes and segregating progenies. CONCLUSION: These results provide accurate values for nucleotide diversity in coding sequences and a first estimate of short-range LD in grapevine. Using SNPlextrade mark genotyping we have shown the application of a set of discovered SNPs as molecular markers for cultivar identification, linkage mapping and genetic diversity studies. Thus, the combination a highly efficient re-sequencing approach and the SNPlextrade mark high throughput genotyping technology provide a powerful tool for grapevine genetic analysis.

Analysis of DNA Cytosine Methylation Patterns Using Methylation-Sensitive Amplification Polymorphism (MSAP).

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is the methylation-sensitive amplified polymorphism technique (MSAP) which is a modification of amplified fragment length polymorphism (AFLP). It has been used to study methylation of anonymous CCGG sequences in different fungi, plants, and animal species. The main variation of this technique resides on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent-cutter restriction enzyme. For each sample, MSAP analysis is performed using both EcoRI/HpaII- and EcoRI/MspI-digested samples. A comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) methylation-insensitive polymorphisms that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples and (2) methylation-sensitive polymorphisms which are associated with the amplified fragments that differ in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses the modifications that can be applied to adjust the technology to different species of interest.

Single-Copy Genes as Molecular Markers for Phylogenomic Studies in Seed Plants.

Phylogenetic relationships among seed plant taxa, especially within the gymnosperms, remain contested. In contrast to angiosperms, for which several genomic, transcriptomic and phylogenetic resources are available, there are few, if any, molecular markers that allow broad comparisons among gymnosperm species. With few gymnosperm genomes available, recently obtained transcriptomes in gymnosperms are a great addition to identifying single-copy gene families as molecular markers for phylogenomic analysis in seed plants. Taking advantage of an increasing number of available genomes and transcriptomes, we identified single-copy genes in a broad collection of seed plants and used these to infer phylogenetic relationships between major seed plant taxa. This study aims at extending the current phylogenetic toolkit for seed plants, assessing its ability for resolving seed plant phylogeny, and discussing potential factors affecting phylogenetic reconstruction. In total, we identified 3,072 single-copy genes in 31 gymnosperms and 2,156 single-copy genes in 34 angiosperms. All studied seed plants shared 1,469 single-copy genes, which are generally involved in functions like DNA metabolism, cell cycle, and photosynthesis. A selected set of 106 single-copy genes provided good resolution for the seed plant phylogeny except for gnetophytes. Although some of our analyses support a sister relationship between gnetophytes and other gymnosperms, phylogenetic trees from concatenated alignments without 3rd codon positions and amino acid alignments under the CAT + GTR model, support gnetophytes as a sister group to Pinaceae. Our phylogenomic analyses demonstrate that, in general, single-copy genes can uncover both recent and deep divergences of seed plant phylogeny.

Nucleotide polymorphisms in a pine ortholog of the Arabidopsis degrading enzyme cellulase KORRIGAN are associated with early growth performance in Pinus pinaster.

We have carried out a candidate-gene-based association genetic study in Pinus pinaster Aiton and evaluated the predictive performance for genetic merit gain of the most significantly associated genes and single nucleotide polymorphisms (SNPs). We used a second generation 384-SNP array enriched with candidate genes for growth and wood properties to genotype mother trees collected in 20 natural populations covering most of the European distribution of the species. Phenotypic data for total height, polycyclism, root-collar diameter and biomass were obtained from a replicated provenance-progeny trial located in two sites with contrasting environments (Atlantic vs Mediterranean climate). General linear models identified strong associations between growth traits (total height and polycyclism) and four SNPs from the korrigan candidate gene, after multiple testing corrections using false discovery rate. The combined genomic breeding value predictions assessed for the four associated korrigan SNPs by ridge regression-best linear unbiased prediction (RR-BLUP) and cross-validation accounted for up to 8 and 15% of the phenotypic variance for height and polycyclic growth, respectively, and did not improve adding SNPs from other growth-related candidate genes. For root-collar diameter and total biomass, they accounted for 1.6 and 1.1% of the phenotypic variance, respectively, but increased to 15 and 4.1% when other SNPs from lp3.1, lp3.3 and cad were included in RR-BLUP models. These results point towards a desirable integration of candidate-gene studies as a means to pre-select relevant markers, and aid genomic selection in maritime pine breeding programs.

Evidence of intense chromosomal shuffling during conifer evolution.

While recent advances have been gained on genome evolution in angiosperm lineages, virtually nothing is known about karyotype evolution in the other group of seed plants, the gymnosperms. Here we used high density gene-based linkage mapping to compare the karyotype structure of two families of conifers (the most abundant group of gymnosperms) separated around 290 million years ago: Pinaceae and Cupressaceae. We propose for the first time a model based on the fusion of 20 ancestral chromosomal blocks that may have shaped the modern karyotpes of Pinaceae (with n=12) and Cupressaceae (with n=11). The considerable difference in modern genome organization between these two lineages contrasts strongly with the remarkable level of synteny already reported within the Pinaceae. It also suggests a convergent evolutionary mechanism of chromosomal block shuffling that has shaped the genomes of the spermatophytes.

Isoschizomers and amplified fragment length polymorphism for the detection of specific cytosine methylation changes.

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is a modification of the Amplified Fragment Length Polymorphism (AFLP) technique that has been used to study methylation of anonymous CCGG sequences in different fungi, plant and animal species. The main variation of this technique is based on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent cutter restriction enzyme. For each sample, AFLP analysis is performed using both EcoRI/HpaII and EcoRI/MspI digested samples. Comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) "Methylation-insensitive polymorphisms" that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples; and (2) "Methylation-sensitive polymorphisms" that are associated with amplified fragments differing in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses modifications that can be applied to adjust the technology to different species of interest.