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.

Genomicus in 2022: comparative tools for thousands of genomes and reconstructed ancestors.

Genomicus is a database and web-server dedicated to comparative genomics in eukaryotes. Its main functionality is to graphically represent the conservation of genomic blocks between multiple genomes, locally around a specific gene of interest or genome-wide through karyotype comparisons. Since 2010 and its first release, Genomicus has synchronized with 60 Ensembl releases and seen the addition of functions that have expanded the type of analyses that users can perform. Today, five public instances of Genomicus are supporting a total number of 1029 extant genomes and 621 ancestral reconstructions from all eukaryotes kingdoms available in Ensembl and Ensembl Genomes databases complemented with four additional instances specific to taxonomic groups of interest. New visualization and query tools are described in this manuscript. Genomicus is freely available at http://www.genomicus.bio.ens.psl.eu/genomicus.

RapGreen, an interactive software and web package to explore and analyze phylogenetic trees.

RapGreen is a modular software package targeted at scientists handling large datasets for phylogenetic analysis. Its primary function is the graphical visualization and exploration of large trees. In addition, RapGreen offers a tree pattern search function to seek evolutionary scenarios among large collections of phylogenetic trees. Other functionalities include tree reconciliation with a given species tree: the detection of duplication or loss events during evolution and tree rooting. Last but not least, RapGreen features the ability to integrate heterogeneous data while visualizing and otherwise analyzing phylogenetic trees.

Badnaviruses and banana genomes: a long association sheds light on Musa phylogeny and origin.

Badnaviruses are double-stranded DNA pararetroviruses of the family Caulimoviridae. Badnaviral sequences found in banana are distributed over three main clades of the genus Badnavirus and exhibit wide genetic diversity. Interestingly, the nuclear genome of many plants, including banana, is invaded by numerous badnaviral sequences although badnaviruses do not require an integration step to replicate, unlike animal retroviruses. Here, we confirm that banana streak viruses (BSVs) are restricted to clades 1 and 3. We also show that only BSVs from clade 3 encompassing East African viral species are not integrated into Musa genomes, unlike BSVs from clade 1. Finally, we demonstrate that sequences from clade 2 are definitively integrated into Musa genomes with no evidence of episomal counterparts; all are phylogenetically distant from BSVs known to date. Using different molecular approaches, we dissected the coevolution between badnaviral sequences of clade 2 and banana by comparing badnavirus integration patterns across a banana sampling representing major Musa speciation events. Our data suggest that primary viral integrations occurred millions of years ago in banana genomes under different possible scenarios. Endogenous badnaviral sequences can be used as powerful markers to better characterize the Musa phylogeny, narrowing down the likely geographical origin of the Musa ancestor.

Post-transcriptional regulation of several biological processes involved in latex production in Hevea brasiliensis.

BACKGROUND: Small RNAs modulate plant gene expression at both the transcriptional and post-transcriptional level, mostly through the induction of either targeted DNA methylation or transcript cleavage, respectively. Small RNA networks are involved in specific plant developmental processes, in signaling pathways triggered by various abiotic stresses and in interactions between the plant and viral and non-viral pathogens. They are also involved in silencing maintenance of transposable elements and endogenous viral elements. Alteration in small RNA production in response to various environmental stresses can affect all the above-mentioned processes. In rubber trees, changes observed in small RNA populations in response to trees affected by tapping panel dryness, in comparison to healthy ones, suggest a shift from a transcriptional to a post-transcriptional regulatory pathway. This is the first attempt to characterise small RNAs involved in post-transcriptional silencing and their target transcripts in Hevea. METHODS: Genes producing microRNAs (MIR genes) and loci producing trans-activated small interfering RNA (ta-siRNA) were identified in the clone PB 260 re-sequenced genome. Degradome libraries were constructed with a pool of total RNA from six different Hevea tissues in stressed and non-stressed plants. The analysis of cleaved RNA data, associated with genomics and transcriptomics data, led to the identification of transcripts that are affected by 20-22 nt small RNA-mediated post-transcriptional regulation. A detailed analysis was carried out on gene families related to latex production and in response to growth regulators. RESULTS: Compared to other tissues, latex cells had a higher proportion of transcript cleavage activity mediated by miRNAs and ta-siRNAs. Post-transcriptional regulation was also observed at each step of the natural rubber biosynthesis pathway. Among the genes involved in the miRNA biogenesis pathway, our analyses showed that all of them are expressed in latex. Using phylogenetic analyses, we show that both the Argonaute and Dicer-like gene families recently underwent expansion. Overall, our study underlines the fact that important biological pathways, including hormonal signalling and rubber biosynthesis, are subject to post-transcriptional silencing in laticifers.

Transcriptional Regulation of Sorghum Stem Composition: Key Players Identified Through Co-expression Gene Network and Comparative Genomics Analyses.

Most sorghum biomass accumulates in stem secondary cell walls (SCW). As sorghum stems are used as raw materials for various purposes such as feed, energy and fiber reinforced polymers, identifying the genes responsible for SCW establishment is highly important. Taking advantage of studies performed in model species, most of the structural genes contributing at the molecular level to the SCW biosynthesis in sorghum have been proposed while their regulatory factors have mostly not been determined. Validation of the role of several MYB and NAC transcription factors in SCW regulation in Arabidopsis and a few other species has been provided. In this study, we contributed to the recent efforts made in grasses to uncover the mechanisms underlying SCW establishment. We reported updated phylogenies of NAC and MYB in 9 different species and exploited findings from other species to highlight candidate regulators of SCW in sorghum. We acquired expression data during sorghum internode development and used co-expression analyses to determine groups of co-expressed genes that are likely to be involved in SCW establishment. We were able to identify two groups of co-expressed genes presenting multiple evidences of involvement in SCW building. Gene enrichment analysis of MYB and NAC genes provided evidence that while NAC SECONDARY WALL THICKENING PROMOTING FACTOR NST genes and SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN gene functions appear to be conserved in sorghum, NAC master regulators of SCW in sorghum may not be as tissue compartmentalized as in Arabidopsis. We showed that for every homolog of the key SCW MYB in Arabidopsis, a similar role is expected for sorghum. In addition, we unveiled sorghum MYB and NAC that have not been identified to date as being involved in cell wall regulation. Although specific validation of the MYB and NAC genes uncovered in this study is needed, we provide a network of sorghum genes involved in SCW both at the structural and regulatory levels.

Comprehensive classification of the plant non-specific lipid transfer protein superfamily towards its sequence-structure-function analysis.

BACKGROUND: Non-specific Lipid Transfer Proteins (nsLTPs) are widely distributed in the plant kingdom and constitute a superfamily of related proteins. Several hundreds of different nsLTP sequences-and counting-have been characterized so far, but their biological functions remain unclear. It has been clear for years that they present a certain interest for agronomic and nutritional issues. Deciphering their functions means collecting and analyzing a variety of data from gene sequence to protein structure, from cellular localization to the physiological role. As a huge and growing number of new protein sequences are available nowadays, extracting meaningful knowledge from sequence-structure-function relationships calls for the development of new tools and approaches. As nsLTPs show high evolutionary divergence, but a conserved common right handed superhelix structural fold, and as they are involved in a large number of key roles in plant development and defense, they are a stimulating case study for validating such an approach. METHODS: In this study, we comprehensively investigated 797 nsLTP protein sequences, including a phylogenetic analysis on canonical protein sequences, three-dimensional structure modeling and functional annotation using several well-established bioinformatics programs. Additionally, two integrative methodologies using original tools were developed. The first was a new method for the detection of (i) conserved amino acid residues involved in structure stabilization and (ii) residues potentially involved in ligand interaction. The second was a structure-function classification based on the evolutionary trace display method using a new tree visualization interface. We also present a new tool for visualizing phylogenetic trees. RESULTS: Following this new protocol, an updated classification of the nsLTP superfamily was established and a new functional hypothesis for key residues is suggested. Lastly, this work allows a better representation of the diversity of plant nsLTPs in terms of sequence, structure and function.

Corrigendum: New Insights on Leucine-Rich Repeats Receptor-Like Kinase Orthologous Relationships in Angiosperms.

[This corrects the article on p. 381 in vol. 8, PMID: 28424707.].

New Insights on Leucine-Rich Repeats Receptor-Like Kinase Orthologous Relationships in Angiosperms.

Leucine-Rich Repeats Receptor-Like Kinase (LRR-RLK) genes represent a large and complex gene family in plants, mainly involved in development and stress responses. These receptors are composed of an LRR-containing extracellular domain (ECD), a transmembrane domain (TM) and an intracellular kinase domain (KD). To provide new perspectives on functional analyses of these genes in model and non-model plant species, we performed a phylogenetic analysis on 8,360 LRR-RLK receptors in 31 angiosperm genomes (8 monocots and 23 dicots). We identified 101 orthologous groups (OGs) of genes being conserved among almost all monocot and dicot species analyzed. We observed that more than 10% of these OGs are absent in the Brassicaceae species studied. We show that the ECD structural features are not always conserved among orthologs, suggesting that functions may have diverged in some OG sets. Moreover, we looked at targets of positive selection footprints in 12 pairs of OGs and noticed that depending on the subgroups, positive selection occurred more frequently either in the ECDs or in the KDs.

Evolutionary Dynamics of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) Subfamily in Angiosperms.

Gene duplications are an important factor in plant evolution, and lineage-specific expanded (LSE) genes are of particular interest. Receptor-like kinases expanded massively in land plants, and leucine-rich repeat receptor-like kinases (LRR-RLK) constitute the largest receptor-like kinases family. Based on the phylogeny of 7,554 LRR-RLK genes from 31 fully sequenced flowering plant genomes, the complex evolutionary dynamics of this family was characterized in depth. We studied the involvement of selection during the expansion of this family among angiosperms. LRR-RLK subgroups harbor extremely contrasting rates of duplication, retention, or loss, and LSE copies are predominantly found in subgroups involved in environmental interactions. Expansion rates also differ significantly depending on the time when rounds of expansion or loss occurred on the angiosperm phylogenetic tree. Finally, using a dN/dS-based test in a phylogenetic framework, we searched for selection footprints on LSE and single-copy LRR-RLK genes. Selective constraint appeared to be globally relaxed at LSE genes, and codons under positive selection were detected in 50% of them. Moreover, the leucine-rich repeat domains, and specifically four amino acids in them, were found to be the main targets of positive selection. Here, we provide an extensive overview of the expansion and evolution of this very large gene family.

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.

In silico analysis of usher encoding genes in Klebsiella pneumoniae and characterization of their role in adhesion and colonization.

Chaperone/usher (CU) assembly pathway is used by a wide range of Enterobacteriaceae to assemble adhesive surface structures called pili or fimbriae that play a role in bacteria-host cell interactions. In silico analysis revealed that the genome of Klebsiella pneumoniae LM21 harbors eight chromosomal CU loci belonging to γκп and Ï­ clusters. Of these, only two correspond to previously described operons, namely type 1 and type 3-encoding operons. Isogenic usher deletion mutants of K. pneumoniae LM21 were constructed for each locus and their role in adhesion to animal (Intestine 407) and plant (Arabidopsis thaliana) cells, biofilm formation and murine intestinal colonization was investigated. Type 3 pili usher deleted mutant was impaired in all assays, whereas type 1 pili usher deleted mutant only showed attenuation in adhesion to plant cells and in intestinal colonization. The LM21ΔkpjC mutant was impaired in its capacity to adhere to Arabidopsis cells and to colonize the murine intestine, either alone or in co-inoculation experiments. Deletion of LM21kpgC induced a significant decrease in biofilm formation, in adhesion to animal cells and in colonization of the mice intestine. The LM21∆kpaC and LM21∆kpeC mutants were only attenuated in biofilm formation and the adhesion abilities to Arabidopsis cells, respectively. No clear in vitro or in vivo effect was observed for LM21∆kpbC and LM21∆kpdC mutants. The multiplicity of CU loci in K. pneumoniae genome and their specific adhesion pattern probably reflect the ability of the bacteria to adhere to different substrates in its diverse ecological niches.

The genome sequence of the orchid Phalaenopsis equestris.

Orchidaceae, renowned for its spectacular flowers and other reproductive and ecological adaptations, is one of the most diverse plant families. Here we present the genome sequence of the tropical epiphytic orchid Phalaenopsis equestris, a frequently used parent species for orchid breeding. P. equestris is the first plant with crassulacean acid metabolism (CAM) for which the genome has been sequenced. Our assembled genome contains 29,431 predicted protein-coding genes. We find that contigs likely to be underassembled, owing to heterozygosity, are enriched for genes that might be involved in self-incompatibility pathways. We find evidence for an orchid-specific paleopolyploidy event that preceded the radiation of most orchid clades, and our results suggest that gene duplication might have contributed to the evolution of CAM photosynthesis in P. equestris. Finally, we find expanded and diversified families of MADS-box C/D-class, B-class AP3 and AGL6-class genes, which might contribute to the highly specialized morphology of orchid flowers.

Impact of recurrent gene duplication on adaptation of plant genomes.

BACKGROUND: Recurrent gene duplication and retention played an important role in angiosperm genome evolution. It has been hypothesized that these processes contribute significantly to plant adaptation but so far this hypothesis has not been tested at the genome scale. RESULTS: We studied available sequenced angiosperm genomes to assess the frequency of positive selection footprints in lineage specific expanded (LSE) gene families compared to single-copy genes using a dN/dS-based test in a phylogenetic framework. We found 5.38% of alignments in LSE genes with codons under positive selection. In contrast, we found no evidence for codons under positive selection in the single-copy reference set. An analysis at the branch level shows that purifying selection acted more strongly on single-copy genes than on LSE gene clusters. Moreover we detect significantly more branches indicating evolution under positive selection and/or relaxed constraint in LSE genes than in single-copy genes. CONCLUSIONS: In this - to our knowledge -first genome-scale study we provide strong empirical support for the hypothesis that LSE genes fuel adaptation in angiosperms. Our conservative approach for detecting selection footprints as well as our results can be of interest for further studies on (plant) gene family evolution.

The banana genome hub.

Banana is one of the world's favorite fruits and one of the most important crops for developing countries. The banana reference genome sequence (Musa acuminata) was recently released. Given the taxonomic position of Musa, the completed genomic sequence has particular comparative value to provide fresh insights about the evolution of the monocotyledons. The study of the banana genome has been enhanced by a number of tools and resources that allows harnessing its sequence. First, we set up essential tools such as a Community Annotation System, phylogenomics resources and metabolic pathways. Then, to support post-genomic efforts, we improved banana existing systems (e.g. web front end, query builder), we integrated available Musa data into generic systems (e.g. markers and genetic maps, synteny blocks), we have made interoperable with the banana hub, other existing systems containing Musa data (e.g. transcriptomics, rice reference genome, workflow manager) and finally, we generated new results from sequence analyses (e.g. SNP and polymorphism analysis). Several uses cases illustrate how the Banana Genome Hub can be used to study gene families. Overall, with this collaborative effort, we discuss the importance of the interoperability toward data integration between existing information systems. Database URL: http://banana-genome.cirad.fr/

Identification of the Hevea brasiliensis AP2/ERF superfamily by RNA sequencing.

BACKGROUND: Rubber tree (Hevea brasiliensis) laticifers are the source of natural rubber. Rubber production depends on endogenous and exogenous ethylene (ethephon). AP2/ERF transcription factors, and especially Ethylene-Response Factors, play a crucial role in plant development and response to biotic and abiotic stresses. This study set out to sequence transcript expressed in various tissues using next-generation sequencing and to identify AP2/ERF superfamily in the rubber tree. RESULTS: The 454 sequencing technique was used to produce five tissue-type transcript libraries (leaf, bark, latex, embryogenic tissues and root). Reads from all libraries were pooled and reassembled to improve mRNA lengths and produce a global library. One hundred and seventy-three AP2/ERF contigs were identified by in silico analysis based on the amino acid sequence of the conserved AP2 domain from the global library. The 142 contigs with the full AP2 domain were classified into three main families (20 AP2 members, 115 ERF members divided into 11 groups, and 4 RAV members) and 3 soloist members. Fifty-nine AP2/ERF transcripts were found in latex. Alongside the microRNA172 already described in plants, eleven additional microRNAs were predicted to inhibit Hevea AP2/ERF transcripts. CONCLUSIONS: Hevea has a similar number of AP2/ERF genes to that of other dicot species. We adapted the alignment and classification methods to data from next-generation sequencing techniques to provide reliable information. We observed several specific features for the ERF family. Three HbSoloist members form a group in Hevea. Several AP2/ERF genes highly expressed in latex suggest they have a specific function in Hevea. The analysis of AP2/ERF transcripts in Hevea presented here provides the basis for studying the molecular regulation of latex production in response to abiotic stresses and latex cell differentiation.

Some ethylene biosynthesis and AP2/ERF genes reveal a specific pattern of expression during somatic embryogenesis in Hevea brasiliensis.

BACKGROUND: Ethylene production and signalling play an important role in somatic embryogenesis, especially for species that are recalcitrant in in vitro culture. The AP2/ERF superfamily has been identified and classified in Hevea brasiliensis. This superfamily includes the ERFs involved in response to ethylene. The relative transcript abundance of ethylene biosynthesis genes and of AP2/ERF genes was analysed during somatic embryogenesis for callus lines with different regeneration potential, in order to identify genes regulated during that process. RESULTS: The analysis of relative transcript abundance was carried out by real-time RT-PCR for 142 genes. The transcripts of ERFs from group I, VII and VIII were abundant at all stages of the somatic embryogenesis process. Forty genetic expression markers for callus regeneration capacity were identified. Fourteen markers were found for proliferating calli and 35 markers for calli at the end of the embryogenesis induction phase. Sixteen markers discriminated between normal and abnormal embryos and, lastly, there were 36 markers of conversion into plantlets. A phylogenetic analysis comparing the sequences of the AP2 domains of Hevea and Arabidopsis genes enabled us to predict the function of 13 expression marker genes. CONCLUSIONS: This first characterization of the AP2/ERF superfamily in Hevea revealed dramatic regulation of the expression of AP2/ERF genes during the somatic embryogenesis process. The gene expression markers of proliferating callus capacity to regenerate plants by somatic embryogenesis should make it possible to predict callus lines suitable to be used for multiplication. Further functional characterization of these markers opens up prospects for discovering specific AP2/ERF functions in the Hevea species for which somatic embryogenesis is difficult.

Survey of branch support methods demonstrates accuracy, power, and robustness of fast likelihood-based approximation schemes.

Phylogenetic inference and evaluating support for inferred relationships is at the core of many studies testing evolutionary hypotheses. Despite the popularity of nonparametric bootstrap frequencies and Bayesian posterior probabilities, the interpretation of these measures of tree branch support remains a source of discussion. Furthermore, both methods are computationally expensive and become prohibitive for large data sets. Recent fast approximate likelihood-based measures of branch supports (approximate likelihood ratio test [aLRT] and Shimodaira-Hasegawa [SH]-aLRT) provide a compelling alternative to these slower conventional methods, offering not only speed advantages but also excellent levels of accuracy and power. Here we propose an additional method: a Bayesian-like transformation of aLRT (aBayes). Considering both probabilistic and frequentist frameworks, we compare the performance of the three fast likelihood-based methods with the standard bootstrap (SBS), the Bayesian approach, and the recently introduced rapid bootstrap. Our simulations and real data analyses show that with moderate model violations, all tests are sufficiently accurate, but aLRT and aBayes offer the highest statistical power and are very fast. With severe model violations aLRT, aBayes and Bayesian posteriors can produce elevated false-positive rates. With data sets for which such violation can be detected, we recommend using SH-aLRT, the nonparametric version of aLRT based on a procedure similar to the Shimodaira-Hasegawa tree selection. In general, the SBS seems to be excessively conservative and is much slower than our approximate likelihood-based methods.