Target enrichment sequencing coupled with GWAS identifies MdPRX10 as a candidate gene in the control of budbreak in apple.

The timing of floral budbreak in apple has a significant effect on fruit production and quality. Budbreak occurs as a result of a complex molecular mechanism that relies on accurate integration of external environmental cues, principally temperature. In the pursuit of understanding this mechanism, especially with respect to aiding adaptation to climate change, a QTL at the top of linkage group (LG) 9 has been identified by many studies on budbreak, but the genes underlying it remain elusive. Here, together with a dessert apple core collection of 239 cultivars, we used a targeted capture sequencing approach to increase SNP resolution in apple orthologues of known or suspected A. thaliana flowering time-related genes, as well as approximately 200 genes within the LG9 QTL interval. This increased the 275 223 SNP Axiom® Apple 480 K array dataset by an additional 40 857 markers. Robust GWAS analyses identified MdPRX10, a peroxidase superfamily gene, as a strong candidate that demonstrated a dormancy-related expression pattern and down-regulation in response to chilling. In-silico analyses also predicted the residue change resulting from the SNP allele associated with late budbreak could alter protein conformation and likely function. Late budbreak cultivars homozygous for this SNP allele also showed significantly up-regulated expression of C-REPEAT BINDING FACTOR (CBF) genes, which are involved in cold tolerance and perception, compared to reference cultivars, such as Gala. Taken together, these results indicate a role for MdPRX10 in budbreak, potentially via redox-mediated signaling and CBF gene regulation. Moving forward, this provides a focus for developing our understanding of the effects of temperature on flowering time and how redox processes may influence integration of external cues in dormancy pathways.

Genomic evidence of genuine wild versus admixed olive populations evolving in the same natural environments in western Mediterranean Basin.

Crop-to-wild gene flow is a mechanism process widely documented, both in plants and animals. This can have positive or negative impacts on the evolution of admixed populations in natural environments, yet the phenomenon is still misunderstood in long-lived woody species, contrary to short-lived crops. Wild olive Olea europaea L. occurs in the same eco-geographical range as domesticated olive, i.e. the Mediterranean Basin (MB). Moreover, it is an allogamous and anemophilous species whose seeds are disseminated by birds, i.e. factors that drive gene flow between crops and their wild relatives. Here we investigated the genetic structure of western MB wild olive populations in natural environments assuming a homogenous gene pool with limited impact of cultivated alleles, as previously suggested. We used a target sequencing method based on annotated genes from the Farga reference genome to analyze 27 western MB olive tree populations sampled in natural environments in France, Spain and Morocco. We also target sequenced cultivated olive tree accessions from the Worldwide Olive Germplasm Bank of Marrakech and Porquerolles and from an eastern MB wild olive tree population. We combined PCA, sNMF, pairwise FST and TreeMix and clearly identified genuine wild olive trees throughout their natural distribution range along a north-south gradient including, for the first time, in southern France. However, contrary to our assumption, we highlighted more admixed than genuine wild olive trees. Our results raise questions regarding the admixed population evolution pattern in this environment, which might be facilitated by crop-to-wild gene flow.

Dual domestications and origin of traits in grapevine evolution.

We elucidate grapevine evolution and domestication histories with 3525 cultivated and wild accessions worldwide. In the Pleistocene, harsh climate drove the separation of wild grape ecotypes caused by continuous habitat fragmentation. Then, domestication occurred concurrently about 11,000 years ago in Western Asia and the Caucasus to yield table and wine grapevines. The Western Asia domesticates dispersed into Europe with early farmers, introgressed with ancient wild western ecotypes, and subsequently diversified along human migration trails into muscat and unique western wine grape ancestries by the late Neolithic. Analyses of domestication traits also reveal new insights into selection for berry palatability, hermaphroditism, muscat flavor, and berry skin color. These data demonstrate the role of the grapevines in the early inception of agriculture across Eurasia.

A chromosome-level, haplotype-phased Vanilla planifolia genome highlights the challenge of partial endoreplication for accurate whole-genome assembly.

Vanilla planifolia, the species cultivated to produce one of the world's most popular flavors, is highly prone to partial genome endoreplication, which leads to highly unbalanced DNA content in cells. We report here the first molecular evidence of partial endoreplication at the chromosome scale by the assembly and annotation of an accurate haplotype-phased genome of V. planifolia. Cytogenetic data demonstrated that the diploid genome size is 4.09 Gb, with 16 chromosome pairs, although aneuploid cells are frequently observed. Using PacBio HiFi and optical mapping, we assembled and phased a diploid genome of 3.4 Gb with a scaffold N50 of 1.2 Mb and 59 128 predicted protein-coding genes. The atypical k-mer frequencies and the uneven sequencing depth observed agreed with our expectation of unbalanced genome representation. Sixty-seven percent of the genes were scattered over only 30% of the genome, putatively linking gene-rich regions and the endoreplication phenomenon. By contrast, low-coverage regions (non-endoreplicated) were rich in repeated elements but also contained 33% of the annotated genes. Furthermore, this assembly showed distinct haplotype-specific sequencing depth variation patterns, suggesting complex molecular regulation of endoreplication along the chromosomes. This high-quality, anchored assembly represents 83% of the estimated V. planifolia genome. It provides a significant step toward the elucidation of this complex genome. To support post-genomics efforts, we developed the Vanilla Genome Hub, a user-friendly integrated web portal that enables centralized access to high-throughput genomic and other omics data and interoperable use of bioinformatics tools.

The Identification of Small RNAs Differentially Expressed in Apple Buds Reveals a Potential Role of the Mir159-MYB Regulatory Module during Dormancy.

Winter dormancy is an adaptative mechanism that temperate and boreal trees have developed to protect their meristems against low temperatures. In apple trees (Malus domestica), cold temperatures induce bud dormancy at the end of summer/beginning of the fall. Apple buds stay dormant during winter until they are exposed to a period of cold, after which they can resume growth (budbreak) and initiate flowering in response to warmer temperatures in spring. It is well-known that small RNAs modulate temperature responses in many plant species, but however, how small RNAs are involved in genetic networks of temperature-mediated dormancy control in fruit tree species remains unclear. Here, we have made use of a recently developed ARGONAUTE (AGO)-purification technique to isolate small RNAs from apple buds. A small RNA-seq experiment resulted in the identification of 17 micro RNAs (miRNAs) that change their pattern of expression in apple buds during dormancy. Furthermore, the functional analysis of their predicted target genes suggests a main role of the 17 miRNAs in phenylpropanoid biosynthesis, gene regulation, plant development and growth, and response to stimulus. Finally, we studied the conservation of the Arabidopsis thaliana regulatory miR159-MYB module in apple in the context of the plant hormone abscisic acid homeostasis.

Genome ancestry mosaics reveal multiple and cryptic contributors to cultivated banana.

Hybridizations between closely related species commonly occur in the domestication process of many crops. Banana cultivars are derived from such hybridizations between species and subspecies of the Musa genus that have diverged in various tropical Southeast Asian regions and archipelagos. Among the diploid and triploid hybrids generated, those with seedless parthenocarpic fruits were selected by humans and thereafter dispersed through vegetative propagation. Musa acuminata subspecies contribute to most of these cultivars. We analyzed sequence data from 14 M. acuminata wild accessions and 10 M. acuminata-based cultivars, including diploids and one triploid, to characterize the ancestral origins along their chromosomes. We used multivariate analysis and single nucleotide polymorphism clustering and identified five ancestral groups as contributors to these cultivars. Four of these corresponded to known M. acuminata subspecies. A fifth group, found only in cultivars, was defined based on the 'Pisang Madu' cultivar and represented two uncharacterized genetic pools. Diverse ancestral contributions along cultivar chromosomes were found, resulting in mosaics with at least three and up to five ancestries. The commercially important triploid Cavendish banana cultivar had contributions from at least one of the uncharacterized genetic pools and three known M. acuminata subspecies. Our results highlighted that cultivated banana origins are more complex than expected - involving multiple hybridization steps - and also that major wild banana ancestors have yet to be identified. This study revealed the extent to which admixture has framed the evolution and domestication of a crop plant.

Genome-wide association mapping of QTLs implied in potato virus Y population sizes in pepper: evidence for widespread resistance QTL pyramiding.

In this study, we looked for genetic factors in the pepper (Capsicum annuum) germplasm that control the number of potato virus Y (PVY) particles entering the plant (i.e. effective population size at inoculation) and the PVY accumulation at the systemic level (i.e. census population size). Using genotyping-by-sequencing (GBS) in a core collection of 256 pepper accessions, we obtained 10 307 single nucleotide polymorphisms (SNPs) covering the whole genome. Genome-wide association studies (GWAS) detected seven SNPs significantly associated with the virus population size at inoculation and/or systemic level on chromosomes 4, 6, 9 and 12. Two SNPs on chromosome 4 associated with both PVY population sizes map closely to the major resistance gene pvr2 encoding the eukaryotic initiation factor 4E. No obvious candidates for resistance were identified in the confidence intervals for the other chromosomes. SNPs detected on chromosomes 6 and 12 colocalized with resistance quantitative trait loci (QTLs) previously identified with a biparental population. These results show the efficiency of GBS and GWAS in C. annuum, indicate highly consistent results between GWAS and classical QTL mapping, and suggest that resistance QTLs identified with a biparental population are representative of a much larger collection of pepper accessions. Moreover, the resistance alleles at these different loci were more frequently combined than expected by chance in the core collection, indicating widespread pyramiding of resistance QTLs and widespread combination of resistance QTLs and major effect genes. Such pyramiding may increase resistance efficiency and/or durability.

Evolutionary transcriptomics reveals the origins of olives and the genomic changes associated with their domestication.

The olive (Olea europaea L. subsp. europaea) is one of the oldest and most socio-economically important cultivated perennial crop in the Mediterranean region. Yet, its origins are still under debate and the genetic bases of the phenotypic changes associated with its domestication are unknown. We generated RNA-sequencing data for 68 wild and cultivated olive trees to study the genetic diversity and structure both at the transcription and sequence levels. To localize putative genes or expression pathways targeted by artificial selection during domestication, we employed a two-step approach in which we identified differentially expressed genes and screened the transcriptome for signatures of selection. Our analyses support a major domestication event in the eastern part of the Mediterranean basin followed by dispersion towards the West and subsequent admixture with western wild olives. While we found large changes in gene expression when comparing cultivated and wild olives, we found no major signature of selection on coding variants and weak signals primarily affected transcription factors. Our results indicated that the domestication of olives resulted in only moderate genomic consequences and that the domestication syndrome is mainly related to changes in gene expression, consistent with its evolutionary history and life history traits.

Pervasive hybridizations in the history of wheat relatives.

Cultivated wheats are derived from an intricate history of three genomes, A, B, and D, present in both diploid and polyploid species. It was recently proposed that the D genome originated from an ancient hybridization between the A and B lineages. However, this result has been questioned, and a robust phylogeny of wheat relatives is still lacking. Using transcriptome data from all diploid species and a new methodological approach, our comprehensive phylogenomic analysis revealed that more than half of the species descend from an ancient hybridization event but with a more complex scenario involving a different parent than previously thought-Aegilops mutica, an overlooked wild species-instead of the B genome. We also detected other extensive gene flow events that could explain long-standing controversies in the classification of wheat relatives.

A novel high-density grapevine (Vitis vinifera L.) integrated linkage map using GBS in a half-diallel population.

KEY MESSAGE: A half-diallel population involving five elite grapevine cultivars was generated and genotyped by GBS, and highly-informative segregation data was used to construct a high-density genetic map for Vitis vinifera L. Grapevine is one of the most relevant fruit crops in the world. Deeper genetic knowledge could assist modern grapevine breeding programs to develop new wine grape varieties able to face climate change effects. To assist in the rapid identification of markers for crop yield components, grape quality traits and adaptation potential, we generated a large Vitis vinifera L. population (N = 624) by crossing five red wine cultivars in a half-diallel scheme, which was subsequently sequenced by an efficient GBS procedure. A high number of fully informative genetic variants was detected using a novel mapping approach capable of reconstructing local haplotypes from adjacent biallelic SNPs, which were subsequently used to construct the densest consensus genetic map available for the cultivated grapevine to date. This 1378.3-cM map integrates 10 bi-parental consensus maps and orders 4437 markers in 3353 unique positions on 19 chromosomes. Markers are well distributed all along the grapevine reference genome, covering up to 98.8% of its genomic sequence. Additionally, a good agreement was observed between genetic and physical orders, adding confidence in the quality of this map. Collectively, our results pave the way for future genetic studies (such as fine QTL mapping) aimed to understand the complex relationship between genotypic and phenotypic variation in the cultivated grapevine. In addition, the method used (which efficiently delivers a high number of fully informative markers) could be of interest to other outbred organisms, notably perennial fruit crops.

Transcriptome data from three endemic Myrtaceae species from New Caledonia displaying contrasting responses to myrtle rust (Austropuccinia psidii).

The myrtle rust disease, caused by the fungus Austropuccinia psidii, infects a wide range of host species within the Myrtaceae family worldwide. Since its first report in 2013 in New Caledonia, it was found on various types of native environments where Myrtaceae are the dominant or codominant species, as well as in several commercial nurseries. It is now considered as a significant threat to ecosystems biodiversity and Myrtaceae-related economy. The use of predictive molecular markers for resistance against myrtle rust is currently the most cost-effective and ecological approach to control the disease. Such an approach for neo Caledonian endemic Myrtaceae species was not possible because of the lack of genomic resources. The recent advancement in new generation sequencing technologies accompanied with relevant bioinformatics tools now provide new research opportunity for work in non-model organism at the transcriptomic level. The present study focuses on transcriptome analysis on three Myrtaceae species endemic to New Caledonia (Arillastrum gummiferum, Syzygium longifolium and Tristaniopsis glauca) that display contrasting responses to the pathogen (non-infected vs infected). Differential gene expression (DGE) and variant calling analysis were conducted on each species. We combined a dual approach by using 1) the annotated reference genome of a related Myrtaceae species (Eucalyptus grandis) and 2) a de novo transcriptomes of each species.

Evolutionary forces affecting synonymous variations in plant genomes.

Base composition is highly variable among and within plant genomes, especially at third codon positions, ranging from GC-poor and homogeneous species to GC-rich and highly heterogeneous ones (particularly Monocots). Consequently, synonymous codon usage is biased in most species, even when base composition is relatively homogeneous. The causes of these variations are still under debate, with three main forces being possibly involved: mutational bias, selection and GC-biased gene conversion (gBGC). So far, both selection and gBGC have been detected in some species but how their relative strength varies among and within species remains unclear. Population genetics approaches allow to jointly estimating the intensity of selection, gBGC and mutational bias. We extended a recently developed method and applied it to a large population genomic dataset based on transcriptome sequencing of 11 angiosperm species spread across the phylogeny. We found that at synonymous positions, base composition is far from mutation-drift equilibrium in most genomes and that gBGC is a widespread and stronger process than selection. gBGC could strongly contribute to base composition variation among plant species, implying that it should be taken into account in plant genome analyses, especially for GC-rich ones.

Domestication rewired gene expression and nucleotide diversity patterns in tomato.

Plant domestication has led to considerable phenotypic modifications from wild species to modern varieties. However, although changes in key traits have been well documented, less is known about the underlying molecular mechanisms, such as the reduction of molecular diversity or global gene co-expression patterns. In this study, we used a combination of gene expression and population genetics in wild and crop tomato to decipher the footprints of domestication. We found a set of 1729 differentially expressed genes (DEG) between the two genetic groups, belonging to 17 clusters of co-expressed DEG, suggesting that domestication affected not only individual genes but also regulatory networks. Five co-expression clusters were enriched in functional terms involving carbohydrate metabolism or epigenetic regulation of gene expression. We detected differences in nucleotide diversity between the crop and wild groups specific to DEG. Our study provides an extensive profiling of the rewiring of gene co-expression induced by the domestication syndrome in one of the main crop species.

A large set of 26 new reference transcriptomes dedicated to comparative population genomics in crops and wild relatives.

We produced a unique large data set of reference transcriptomes to obtain new knowledge about the evolution of plant genomes and crop domestication. For this purpose, we validated a RNA-Seq data assembly protocol to perform comparative population genomics. For the validation, we assessed and compared the quality of de novo Illumina short-read assemblies using data from two crops for which an annotated reference genome was available, namely grapevine and sorghum. We used the same protocol for the release of 26 new transcriptomes of crop plants and wild relatives, including still understudied crops such as yam, pearl millet and fonio. The species list has a wide taxonomic representation with the inclusion of 15 monocots and 11 eudicots. All contigs were annotated using BLAST, prot4EST and Blast2GO. A strong originality of the data set is that each crop is associated with close relative species, which will permit whole-genome comparative evolutionary studies between crops and their wild-related species. This large resource will thus serve research communities working on both crops and model organisms. All the data are available at http://arcad-bioinformatics.southgreen.fr/.

Temperature desynchronizes sugar and organic acid metabolism in ripening grapevine fruits and remodels their transcriptome.

BACKGROUND: Fruit composition at harvest is strongly dependent on the temperature during the grapevine developmental cycle. This raises serious concerns regarding the sustainability of viticulture and the socio-economic repercussions of global warming for many regions where the most heat-tolerant varieties are already cultivated. Despite recent progress, the direct and indirect effects of temperature on fruit development are far from being understood. Experimental limitations such as fluctuating environmental conditions, intra-cluster heterogeneity and the annual reproductive cycle introduce unquantifiable biases for gene expression and physiological studies with grapevine. In the present study, DRCF grapevine mutants (microvine) were grown under several temperature regimes in duly-controlled environmental conditions. A singly berry selection increased the accuracy of fruit phenotyping and subsequent gene expression analyses. The physiological and transcriptomic responses of five key stages sampled simultaneously at day and nighttime were studied by RNA-seq analysis. RESULTS: A total of 674 millions reads were sequenced from all experiments. Analysis of differential expression yielded in a total of 10 788 transcripts modulated by temperature. An acceleration of green berry development under higher temperature was correlated with the induction of several candidate genes linked to cell expansion. High temperatures impaired tannin synthesis and degree of galloylation at the transcriptomic levels. The timing of malate breakdown was delayed to mid-ripening in transgressively cool conditions, revealing unsuspected plasticity of berry primary metabolism. Specific ATPases and malate transporters displayed development and temperature-dependent expression patterns, besides less marked but significant regulation of other genes in the malate pathway. CONCLUSION: The present study represents, to our knowledge the first abiotic stress study performed on a fleshy fruits model using RNA-seq for transcriptomic analysis. It confirms that a careful stage selection and a rigorous control of environmental conditions are needed to address the long-term plasticity of berry development with respect to temperature. Original results revealed temperature-dependent regulation of key metabolic processes in the elaboration of berry composition. Malate breakdown no longer appears as an integral part of the veraison program, but as possibly triggered by an imbalance in cytoplasmic sugar, when efficient vacuolar storage is set on with ripening, in usual temperature conditions. Furthermore, variations in heat shock responsive genes that will be very valuable for further research on temperature adaptation of plants have been evidenced.

Gigwa-Genotype investigator for genome-wide analyses.

BACKGROUND: Exploring the structure of genomes and analyzing their evolution is essential to understanding the ecological adaptation of organisms. However, with the large amounts of data being produced by next-generation sequencing, computational challenges arise in terms of storage, search, sharing, analysis and visualization. This is particularly true with regards to studies of genomic variation, which are currently lacking scalable and user-friendly data exploration solutions. DESCRIPTION: Here we present Gigwa, a web-based tool that provides an easy and intuitive way to explore large amounts of genotyping data by filtering it not only on the basis of variant features, including functional annotations, but also on genotype patterns. The data storage relies on MongoDB, which offers good scalability properties. Gigwa can handle multiple databases and may be deployed in either single- or multi-user mode. In addition, it provides a wide range of popular export formats. CONCLUSIONS: The Gigwa application is suitable for managing large amounts of genomic variation data. Its user-friendly web interface makes such processing widely accessible. It can either be simply deployed on a workstation or be used to provide a shared data portal for a given community of researchers.

SNiPlay3: a web-based application for exploration and large scale analyses of genomic variations.

SNiPlay is a web-based tool for detection, management and analysis of genetic variants including both single nucleotide polymorphisms (SNPs) and InDels. Version 3 now extends functionalities in order to easily manage and exploit SNPs derived from next generation sequencing technologies, such as GBS (genotyping by sequencing), WGRS (whole gre-sequencing) and RNA-Seq technologies. Based on the standard VCF (variant call format) format, the application offers an intuitive interface for filtering and comparing polymorphisms using user-defined sets of individuals and then establishing a reliable genotyping data matrix for further analyses. Namely, in addition to the various scaled-up analyses allowed by the application (genomic annotation of SNP, diversity analysis, haplotype reconstruction and network, linkage disequilibrium), SNiPlay3 proposes new modules for GWAS (genome-wide association studies), population stratification, distance tree analysis and visualization of SNP density. Additionally, we developed a suite of Galaxy wrappers for each step of the SNiPlay3 process, so that the complete pipeline can also be deployed on a Galaxy instance using the Galaxy ToolShed procedure and then be computed as a Galaxy workflow. SNiPlay is accessible at http://sniplay.southgreen.fr.

Transcriptome population genomics reveals severe bottleneck and domestication cost in the African rice (Oryza glaberrima).

The African cultivated rice (Oryza glaberrima) was domesticated in West Africa 3000 years ago. Although less cultivated than the Asian rice (O. sativa), O. glaberrima landraces often display interesting adaptation to rustic environment (e.g. drought). Here, using RNA-seq technology, we were able to compare more than 12,000 transcripts between 9 O. glaberrima, 10 wild O. barthii and one O. meridionalis individuals. With a synonymous nucleotide diversity πs = 0.0006 per site, O. glaberrima appears as the least genetically diverse crop grass ever documented. Using approximate Bayesian computation, we estimated that O. glaberrima experienced a severe bottleneck during domestication. This demographic scenario almost fully accounts for the pattern of genetic diversity across O. glaberrima genome as we detected very few outliers regions where positive selection may have further impacted genetic diversity. Moreover, the large excess of derived nonsynonymous substitution that we detected suggests that the O. glaberrima population suffered from the 'cost of domestication'. In addition, we used this genome-scale data set to demonstrate that (i) O. barthii genetic diversity is positively correlated with recombination rate and negatively with gene density, (ii) expression level is negatively correlated with evolutionary constraint, and (iii) one region on chromosome 5 (position 4-6 Mb) exhibits a clear signature of introgression with a yet unidentified Oryza species. This work represents the first genome-wide survey of the African rice genetic diversity and paves the way for further comparison between the African and the Asian rice, notably regarding the genetics underlying domestication traits.

PHIV-RootCell: a supervised image analysis tool for rice root anatomical parameter quantification.

We developed the PHIV-RootCell software to quantify anatomical traits of rice roots transverse section images. Combined with an efficient root sample processing method for image acquisition, this program permits supervised measurements of areas (those of whole root section, stele, cortex, and central metaxylem vessels), number of cell layers and number of cells per cell layer. The PHIV-RootCell toolset runs under ImageJ, an independent operating system that has a license-free status. To demonstrate the usefulness of PHIV-RootCell, we conducted a genetic diversity study and an analysis of salt stress responses of root anatomical parameters in rice (Oryza sativa L.). Using 16 cultivars, we showed that we could discriminate between some of the varieties even at the 6 day-olds stage, and that tropical japonica varieties had larger root sections due to an increase in cell number. We observed, as described previously, that root sections become enlarged under salt stress. However, our results show an increase in cell number in ground tissues (endodermis and cortex) but a decrease in external (peripheral) tissues (sclerenchyma, exodermis, and epidermis). Thus, the PHIV-RootCell program is a user-friendly tool that will be helpful for future genetic and physiological studies that investigate root anatomical trait variations.