Comparative transcriptional analysis of Persea americana MYB, WRKY and AP2/ERF transcription factors following Phytophthora cinnamomi infection.

Plant cells undergo extensive transcriptional reprogramming following pathogen infection, with these reprogramming patterns becoming more complex when pathogens, such as hemibiotrophs, exhibit different lifestyles. These transcriptional changes are often orchestrated by MYB, WRKY and AP2/ERF transcription factors (TFs), which modulate both growth and defence-related gene expression. Transcriptional analysis of defence-related genes in avocado (Persea americana) infected with Phytophthora cinnamomi indicated differential immune response activation when comparing a partially resistant and susceptible rootstock. This study identified 226 MYB, 82 WRKY, and 174 AP2/ERF TF-encoding genes in avocado, using a genome-wide approach. Phylogenetic analysis revealed substantial sequence conservation within TF groups underscoring their functional significance. RNA-sequencing analysis in a partially resistant and susceptible avocado rootstock infected with P. cinnamomi was indicative of an immune response switch occurring in either rootstock after 24 and 6 h post-inoculation, respectively. Different clusters of co-expressed TF genes were observed at these times, suggesting the activation of necrotroph-related immune responses at varying intervals between the two rootstocks. This study aids our understanding of avocado immune response activation following P. cinnamomi infection, and the role of the TFs therein, elucidating the transcriptional reprogramming disparities between partially resistant and susceptible rootstocks.

HISTONE DEACETYLASE19 Controls Ovule Number Determination and Transmitting Tract Differentiation.

The gynoecium is critical for the reproduction of flowering plants as it contains the ovules and the tissues that foster pollen germination, growth, and guidance. These tissues, known as the reproductive tract (ReT), comprise the stigma, style, and transmitting tract (TT). The ReT and ovules originate from the carpel margin meristem (CMM) within the pistil. SHOOT MERISTEMLESS (STM) is a key transcription factor for meristem formation and maintenance. In all above-ground meristems, including the CMM, local STM downregulation is required for organ formation. However, how this downregulation is achieved in the CMM is unknown. Here, we have studied the role of HISTONE DEACETYLASE 19 (HDA19) in Arabidopsis (Arabidopsis thaliana) during ovule and ReT differentiation based on the observation that the hda19-3 mutant displays a reduced ovule number and fails to differentiate the TT properly. Fluorescence-activated cell sorting coupled with RNA-sequencing revealed that in the CMM of hda19-3 mutants, genes promoting organ development are downregulated while meristematic markers, including STM, are upregulated. HDA19 was essential to downregulate STM in the CMM, thereby allowing ovule formation and TT differentiation. STM is ectopically expressed in hda19-3 at intermediate stages of pistil development, and its downregulation by RNA interference alleviated the hda19-3 phenotype. Chromatin immunoprecipitation assays indicated that STM is a direct target of HDA19 during pistil development and that the transcription factor SEEDSTICK is also required to regulate STM via histone acetylation. Thus, we identified factors required for the downregulation of STM in the CMM, which is necessary for organogenesis and tissue differentiation.

A multi-omic Nicotiana benthamiana resource for fundamental research and biotechnology.

Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today.

Genomic Approaches for the Study of Flower Development in Floriculture Crops.

The advances in genomics and bioinformatics have made possible the study in non-model plants of phenotypes associated to flower development. Floriculture crops are an interesting source of traits associated to flower development such as the transition between zygomorphic and actinomorphic flowers or the production of flowers with double and triple corollas. In this chapter, we summarize the material and methods for the use of floriculture crops to study flower development using genomic tools, from the sequencing and assembly of a reference genome to QTL and RNA-Seq analysis to search candidate genes associated to specific traits.

A snapshot of the transcriptome of Medicago truncatula (Fabales: Fabaceae) shoots and roots in response to an arbuscular mycorrhizal fungus and the pea aphid (Acyrthosiphon pisum) (Hemiptera: Aphididae).

Plants simultaneously interact with belowground symbionts such as arbuscular mycorrhizal (AM) fungi and aboveground antagonists such as aphids. Generally, plants gain access to valuable resources including nutrients and water through the AM symbiosis and are more resistant to pests. Nevertheless, aphids' performance improves on mycorrhizal plants, and it remains unclear whether a more nutritious food source and/or attenuated defenses are the contributing factors. This study examined the shoot and root transcriptome of barrel medic (Medicago truncatula Gaertn.) plants highly colonized by the AM fungus Rhizophagus irregularis (Blaszk., Wubet, Renker, and Buscot) C. Walker and A. Schüßler (Glomerales: Glomeraceae) and exposed to 7 days of mixed age pea aphid (Acyrthosiphon pisum (Harris)) herbivory. The RNA-seq samples chosen for this study showed that aphids were heavier when fed mycorrhizal plants compared to nonmycorrhizal plants. We hypothesized that (i) insect-related plant defense pathways will be downregulated in shoots of mycorrhizal plants with aphids compared to nonmycorrhizal plants with aphids; (ii) pathways involved in nutrient acquisition, carbohydrate-related and amino acid transport will be upregulated in shoots of mycorrhizal plants with aphids compared to nonmycorrhizal plants with aphids; and (iii) roots of mycorrhizal plants with aphids will exhibit mycorrhiza-induced resistance. The transcriptome data revealed that the gene repertoire related to defenses, nutrient transport, and carbohydrates differs between nonmycorrhizal and mycorrhizal plants with aphids, which could explain the weight gain in aphids. We also identified novel candidate genes that are differentially expressed in nonmycorrhizal plants with aphids, thus setting the stage for future functional studies.

Polyploid Nicotiana section Suaveolentes originated by hybridization of two ancestral Nicotiana clades.

Introduction: Nicotiana section Suaveolentes is an almost all-Australian clade of allopolyploid tobacco species that emerged through hybridization between diploid relatives of the genus. In this study, we aimed to assess the phylogenetic relationship of the Suaveolentes section with several Nicotiana diploid species based on both plastidial and nuclear genes. Methods: The Nicotiana plastome-based phylogenetic analysis representing 47 newly re-built plastid genomes suggested that an ancestor of N. section Noctiflorae is the most likely maternal donor of the Suaveolentes clade. Nevertheless, we found clear evidence of plastid recombination with an ancestor from the Sylvestres clade. We analyzed 411 maximum likelihood-based phylogenetic trees from a set of conserved nuclear diploid single copy gene families following an approach that assessed the genomic origin of each homeolog. Results: We found that Nicotiana section Suaveolentes is monophyletic with contributions from the sections Alatae, Sylvestres, Petunioides and Noctiflorae. The dating of the divergence between these sections indicates that the Suaveolentes hybridization predates the split between Alatae/Sylvestres, and Noctiflorae/Petunioides. Discussion: We propose that Nicotiana section Suaveolentes arose from the hybridization of two ancestral species from which the Noctiflorae/Petunioides and Alatae/Sylvestres sections are derived, with Noctiflorae the maternal parent. This study is a good example in which the use of genome wide data provided additional evidence about the origin of a complex polyploid clade.

Pangenomic analysis identifies structural variation associated with heat tolerance in pearl millet.

Pearl millet is an important cereal crop worldwide and shows superior heat tolerance. Here, we developed a graph-based pan-genome by assembling ten chromosomal genomes with one existing assembly adapted to different climates worldwide and captured 424,085 genomic structural variations (SVs). Comparative genomics and transcriptomics analyses revealed the expansion of the RWP-RK transcription factor family and the involvement of endoplasmic reticulum (ER)-related genes in heat tolerance. The overexpression of one RWP-RK gene led to enhanced plant heat tolerance and transactivated ER-related genes quickly, supporting the important roles of RWP-RK transcription factors and ER system in heat tolerance. Furthermore, we found that some SVs affected the gene expression associated with heat tolerance and SVs surrounding ER-related genes shaped adaptation to heat tolerance during domestication in the population. Our study provides a comprehensive genomic resource revealing insights into heat tolerance and laying a foundation for generating more robust crops under the changing climate.

How genomics can help biodiversity conservation.

The availability of public genomic resources can greatly assist biodiversity assessment, conservation, and restoration efforts by providing evidence for scientifically informed management decisions. Here we survey the main approaches and applications in biodiversity and conservation genomics, considering practical factors, such as cost, time, prerequisite skills, and current shortcomings of applications. Most approaches perform best in combination with reference genomes from the target species or closely related species. We review case studies to illustrate how reference genomes can facilitate biodiversity research and conservation across the tree of life. We conclude that the time is ripe to view reference genomes as fundamental resources and to integrate their use as a best practice in conservation genomics.

RNA Polymerases IV and V Are Involved in Olive Fruit Development.

Transcription is carried out in most eukaryotes by three multimeric complexes (RNA polymerases I, II and III). However, plants contain two additional RNA polymerases (IV and V), which have evolved from RNA polymerase II. RNA polymerases II, IV and V contain both common and specific subunits that may specialise some of their functions. In this study, we conducted a search for the genes that putatively code for the specific subunits of RNA polymerases IV and V, as well as those corresponding to RNA polymerase II in olive trees. Based on the homology with the genes of Arabidopsis thaliana, we identified 13 genes that putatively code for the specific subunits of polymerases IV and V, and 16 genes that code for the corresponding specific subunits of polymerase II in olives. The transcriptomic analysis by RNA-Seq revealed that the expression of the RNA polymerases IV and V genes was induced during the initial stages of fruit development. Given that RNA polymerases IV and V are involved in the transcription of long non-coding RNAs, we investigated their expression and observed relevant changes in the expression of this type of RNAs. Particularly, the expression of the intergenic and intronic long non-coding RNAs tended to increase in the early steps of fruit development, suggesting their potential role in this process. The positive correlation between the expression of RNA polymerases IV and V subunits and the expression of non-coding RNAs supports the hypothesis that RNA polymerases IV and V may play a role in fruit development through the synthesis of this type of RNAs.

EasyGDB: a low-maintenance and highly customizable system to develop genomics portals.

SUMMARY: EasyGDB is an easy-to-implement low-maintenance tool developed to create genomic data management web platforms. It can be used for any species, group of species, or multiple genome or annotation versions. EasyGDB provides a framework to develop a web portal that includes the general information about species, projects and members, and bioinformatics tools such as file downloads, BLAST, genome browser, annotation search, gene expression visualization, annotation and sequence download, and gene ids and orthologs lookup. The code of EasyGDB facilitates data maintenance and update for non-experienced bioinformaticians, using BLAST databases to store and retrieve sequence data in gene annotation pages and bioinformatics tools, and JSON files to customize metadata. EasyGDB is a highly customizable tool. Any section and tool can be enabled or disabled like a switch through a single configuration file. This tool aims to simplify the development of genomics portals in non-model species, providing a modern web style with embedded interactive bioinformatics tools to cover all the common needs derived from genomics projects. AVAILABILITY AND IMPLEMENTATION: The code and manual to use EasyGDB can be found at https://github.com/noefp/easy_gdb.

Exploring transposable element-based markers to identify allelic variations underlying agronomic traits in rice.

Transposable elements (TEs) are a major force in the production of new alleles during domestication; nevertheless, their use in association studies has been limited because of their complexity. We have developed a TE genotyping pipeline (TEmarker) and applied it to whole-genome genome-wide association study (GWAS) data from 176 Oryza sativa subsp. japonica accessions to identify genetic elements associated with specific agronomic traits. TE markers recovered a large proportion (69%) of single-nucleotide polymorphism (SNP)-based GWAS peaks, and these TE peaks retained ca. 25% of the SNPs. The use of TEs in GWASs may reduce false positives associated with linkage disequilibrium (LD) among SNP markers. A genome scan revealed positive selection on TEs associated with agronomic traits. We found several cases of insertion and deletion variants that potentially resulted from the direct action of TEs, including an allele of LOC_Os11g08410 associated with plant height and panicle length traits. Together, these findings reveal the utility of TE markers for connecting genotype to phenotype and suggest a potential role for TEs in influencing phenotypic variations in rice that impact agronomic traits.

Partially Resistant Avocado Rootstock Dusa® Shows Prolonged Upregulation of Nucleotide Binding-Leucine Rich Repeat Genes in Response to Phytophthora cinnamomi Infection.

Avocado is an important agricultural food crop in many countries worldwide. Phytophthora cinnamomi, a hemibiotrophic oomycete, remains one of the most devastating pathogens within the avocado industry, as it is near impossible to eradicate from areas where the pathogen is present. A key aspect to Phytophthora root rot disease management is the use of avocado rootstocks partially resistant to P. cinnamomi, which demonstrates an increased immune response following infection. In plant species, Nucleotide binding-Leucine rich repeat (NLR) proteins form an integral part of pathogen recognition and Effector triggered immune responses (ETI). To date, a comprehensive set of Persea americana NLR genes have yet to be identified, though their discovery is crucial to understanding the molecular mechanisms underlying P. americana-P. cinnamomi interactions. In this study, a total of 161 PaNLR genes were identified in the P. americana West-Indian pure accession genome. These putative resistance genes were characterized using bioinformatic approaches and grouped into 13 distinct PaNLR gene clusters, with phylogenetic analysis revealing high sequence similarity within these clusters. Additionally, PaNLR expression levels were analyzed in both a partially resistant (Dusa®) and a susceptible (R0.12) avocado rootstock infected with P. cinnamomi using an RNA-sequencing approach. The results showed that the partially resistant rootstock has increased expression levels of 84 PaNLRs observed up to 24 h post-inoculation, while the susceptible rootstock only showed increased PaNLR expression during the first 6 h post-inoculation. Results of this study may indicate that the partially resistant avocado rootstock has a stronger, more prolonged ETI response which enables it to suppress P. cinnamomi growth and combat disease caused by this pathogen. Furthermore, the identification of PaNLRs may be used to develop resistant rootstock selection tools, which can be employed in the avocado industry to accelerate rootstock screening programs.

Modifying Anthocyanins Biosynthesis in Tomato Hairy Roots: A Test Bed for Plant Resistance to Ionizing Radiation and Antioxidant Properties in Space.

Gene expression manipulation of specific metabolic pathways can be used to obtain bioaccumulation of valuable molecules and desired quality traits in plants. A single-gene approach to impact different traits would be greatly desirable in agrospace applications, where several aspects of plant physiology can be affected, influencing growth. In this work, MicroTom hairy root cultures expressing a MYB-like transcription factor that regulates the biosynthesis of anthocyanins in Petunia hybrida (PhAN4), were considered as a testbed for bio-fortified tomato whole plants aimed at agrospace applications. Ectopic expression of PhAN4 promoted biosynthesis of anthocyanins, allowing to profile 5 major derivatives of delphinidin and petunidin together with pelargonidin and malvidin-based anthocyanins, unusual in tomato. Consistent with PhAN4 features, transcriptomic profiling indicated upregulation of genes correlated to anthocyanin biosynthesis. Interestingly, a transcriptome reprogramming oriented to positive regulation of cell response to biotic, abiotic, and redox stimuli was evidenced. PhAN4 hairy root cultures showed the significant capability to counteract reactive oxygen species (ROS) accumulation and protein misfolding upon high-dose gamma irradiation, which is among the most potent pro-oxidant stress that can be encountered in space. These results may have significance in the engineering of whole tomato plants that can benefit space agriculture.

Neutral and outlier single nucleotide polymorphisms disentangle the evolutionary history of a coastal Solanaceae species.

Speciation begins with the isolation of some individuals or subpopulations due to drivers promoting a diverging genetic distribution. Such isolation may occur, followed by different processes and pressures. Isolation-by-distance (IBD), isolation-by-adaptation (IBA), and isolation-by-colonization (IBC) have been recognized as the main divergence patterns. Still, it is not easy to distinguish which one is the main pattern as each one may act at different points in time or even simultaneously. Using an extensive genome coverage from a Petunia species complex with coastal and inland distribution and multiple analytical approaches on population genomics and phylogeography, we showed a complex interplay between neutral and selective forces acting on the divergence process. We found 18,887 SNPs potentially neutral and 924 potentially under selection (outlier) loci. All analyses pointed that each subspecies displays its own genetic component and evolutionary history. We suggested plausible ecological drivers for such divergence in a southernmost South Atlantic coastal plain in Brazil and Uruguay and identified a connection between adaptation and environment heterogeneity.

The era of reference genomes in conservation genomics.

Progress in genome sequencing now enables the large-scale generation of reference genomes. Various international initiatives aim to generate reference genomes representing global biodiversity. These genomes provide unique insights into genomic diversity and architecture, thereby enabling comprehensive analyses of population and functional genomics, and are expected to revolutionize conservation genomics.

Identification of Genetic Markers and Genes Putatively Involved in Determining Olive Fruit Weight.

The fruit size of a cultivated olive tree is consistently larger than its corresponding wild relatives because fruit size is one of the main traits associated with olive tree domestication. Additionally, large fruit size is one of the main objectives of modern olive breeding programs. However, as the long juvenile period is one main hindrance in classic breeding approaches, obtaining genetic markers associated with this trait is a highly desirable tool. For this reason, GWAS analysis of both genetic markers and the genes associated with fruit size determination, measured as fruit weight, was herein carried out in 50 genotypes, of which 40 corresponded to cultivated and 10 to wild olive trees. As a result, 113 genetic markers were identified, which showed a very high statistically significant correlation with fruit weight variability, p < 10−10. These genetic markers corresponded to 39 clusters of genes in linkage disequilibrium. The analysis of a segregating progeny of the cross of "Frantoio" and "Picual" cultivars allowed us to confirm 10 of the 18 analyzed clusters. The annotation of the genes in each cluster and the expression pattern of the samples taken throughout fruit development by RNAseq enabled us to suggest that some studied genes are involved in olive fruit weight determination.

Elucidation of the Origin of the Monumental Olive Tree of Vouves in Crete, Greece.

The olive tree of Vouves in Crete, is considered the oldest producing olive tree in the world with an estimated age exceeding 4000 years. In the present study, we sequenced two samples (from the bottom and the top of the tree) to elucidate the genetic relation of this ancient tree with other olive cvs as well as to gain some insights about its origin. Our results showed that both samples have different genetic origins, proving that this ancient tree has been grafted at least one time. On the basis of whole genome sequences the sample from the top of the Vouves tree showed relation of the same order than half-siblings to one accession corresponding to the present-day Greek cv 'Mastoidis'. Nevertheless, in the framework of a microsatellite analysis it was found to cluster with the 'Mastoidis' samples. The Vouves rootstock (bottom sample) showed a clear grouping with the oleaster samples in a similar way to that of 'Megaritiki' Greek cv although it does not show any signal of introgression from them. The genomic analyses did not show a strong relation of this sample with the present-day Greek cvs analyzed in this study so it cannot be proved that it has been used as a source for cultivated olive tree populations represented by available genome sequences. Nevertheless, on the basis of microsatellite analyses, the Vouves rootstock showed affinity with two present-day Greek cvs, one "ancient" rootstock from continental Greece as well as monumental trees from Cyprus. The analysis of the impact of the variants in the gene space revealed an enrichment of genes associated to pathways related with carbohydrate and amino acid metabolism. This is in agreement with what has been found before in the sweep regions related with the process of domestication. The absence of oleaster gene flow, its old age and its variant profile, similar to other cultivated populations, makes it an excellent reference point for domestication studies.

Comparative Analysis of Genotyping by Sequencing and Whole-Genome Sequencing Methods in Diversity Studies of Olea europaea L.

Olive, Olea europaea L., is a tree of great economic and cultural importance in the Mediterranean basin. Thousands of cultivars have been described, of which around 1200 are conserved in the different olive germplasm banks. The genetic characterisation of these cultivars can be performed in different ways. Whole-genome sequencing (WGS) provides more information than the reduced representation methods such as genotype by sequencing (GBS), but at a much higher cost. This may change as the cost of sequencing continues to drop, but, currently, genotyping hundreds of cultivars using WGS is not a realistic goal for most research groups. Our aim is to systematically compare both methodologies applied to olive genotyping and summarise any possible recommendations for the geneticists and molecular breeders of the olive scientific community. In this work, we used a selection of 24 cultivars from an olive core collection from the World Olive Germplasm Collection of the Andalusian Institute of Agricultural and Fisheries Research and Training (WOGBC), which represent the most of the cultivars present in cultivated fields over the world. Our results show that both methodologies deliver similar results in the context of phylogenetic analysis and popular population genetic analysis methods such as clustering. Furthermore, WGS and GBS datasets from different experiments can be merged in a single dataset to perform these analytical methodologies with proper filtering. We also tested the influence of the different olive reference genomes in this type of analysis, finding that they have almost no effect when estimating genetic relationships. This work represents the first comparative study between both sequencing techniques in olive. Our results demonstrate that the use of GBS is a perfectly viable option for replacing WGS and reducing research costs when the goal of the experiment is to characterise the genetic relationship between different accessions. Besides this, we show that it is possible to combine variants from GBS and WGS datasets, allowing the reuse of publicly available data.