Coconut genome assembly enables evolutionary analysis of palms and highlights signaling pathways involved in salt tolerance.

Coconut (Cocos nucifera) is the emblematic palm of tropical coastal areas all around the globe. It provides vital resources to millions of farmers. In an effort to better understand its evolutionary history and to develop genomic tools for its improvement, a sequence draft was recently released. Here, we present a dense linkage map (8402 SNPs) aiming to assemble the large genome of coconut (2.42 Gbp, 2n = 32) into 16 pseudomolecules. As a result, 47% of the sequences (representing 77% of the genes) were assigned to 16 linkage groups and ordered. We observed segregation distortion in chromosome Cn15, which is a signature of strong selection among pollen grains, favouring the maternal allele. Comparing our results with the genome of the oil palm Elaeis guineensis allowed us to identify major events in the evolutionary history of palms. We find that coconut underwent a massive transposable element invasion in the last million years, which could be related to the fluctuations of sea level during the glaciations at Pleistocene that would have triggered a population bottleneck. Finally, to better understand the facultative halophyte trait of coconut, we conducted an RNA-seq experiment on leaves to identify key players of signaling pathways involved in salt stress response. Altogether, our findings represent a valuable resource for the coconut breeding community.

Improving transcriptome de novo assembly by using a reference genome of a related species: Translational genomics from oil palm to coconut.

The palms are a family of tropical origin and one of the main constituents of the ecosystems of these regions around the world. The two main species of palm represent different challenges: coconut (Cocos nucifera L.) is a source of multiple goods and services in tropical communities, while oil palm (Elaeis guineensis Jacq) is the main protagonist of the oil market. In this study, we present a workflow that exploits the comparative genomics between a target species (coconut) and a reference species (oil palm) to improve the transcriptomic data, providing a proteome useful to answer functional or evolutionary questions. This workflow reduces redundancy and fragmentation, two inherent problems of transcriptomic data, while preserving the functional representation of the target species. Our approach was validated in Arabidopsis thaliana using Arabidopsis lyrata and Capsella rubella as references species. This analysis showed the high sensitivity and specificity of our strategy, relatively independent of the reference proteome. The workflow increased the length of proteins products in A. thaliana by 13%, allowing, often, to recover 100% of the protein sequence length. In addition redundancy was reduced by a factor greater than 3. In coconut, the approach generated 29,366 proteins, 1,246 of these proteins deriving from new contigs obtained with the BRANCH software. The coconut proteome presented a functional profile similar to that observed in rice and an important number of metabolic pathways related to secondary metabolism. The new sequences found with BRANCH software were enriched in functions related to biotic stress. Our strategy can be used as a complementary step to de novo transcriptome assembly to get a representative proteome of a target species. The results of the current analysis are available on the website PalmComparomics (http://palm-comparomics.southgreen.fr/).

Identification of an ERECTA gene and its drought adaptation associations with wild and cultivated common bean.

In this research, we cloned and accessed nucleotide diversity in the common bean ERECTA gene which has been implicated in drought tolerance and stomatal patterning. The homologous gene segment was isolated with degenerate primer and was found to be located on Chromosome 1. The gene had at least one paralog on Chromosome 9 and duplicate copies in soybean for each homolog. ERECTA-like genes were also discovered but the function of these was of less interest due to low similarity with the ERECTA gene from Arabidopsis. The diversity of the 5' end of the large Chr. 1 PvERECTA gene was evaluated in a collection of 145 wild and cultivated common beans that were also characterized by geographic source and drought tolerance, respectively. Our wild population sampled a range of wet to dry habitats, while our cultivated samples were representative of landrace diversity and the patterns of nucleotide variation differed between groups. The 5' region exhibited lower levels of diversity in the cultivated collection, which was indicative of population bottlenecks associated with the domestication process, compared to the wild collection where diversity was associated with ecological differences. We discuss associations of nucleotide diversity at PvERECTA with drought tolerance prediction for the genotypes.

Allele diversity for abiotic stress responsive candidate genes in chickpea reference set using gene based SNP markers.

Chickpea is an important food legume crop for the semi-arid regions, however, its productivity is adversely affected by various biotic and abiotic stresses. Identification of candidate genes associated with abiotic stress response will help breeding efforts aiming to enhance its productivity. With this objective, 10 abiotic stress responsive candidate genes were selected on the basis of prior knowledge of this complex trait. These 10 genes were subjected to allele specific sequencing across a chickpea reference set comprising 300 genotypes including 211 genotypes of chickpea mini core collection. A total of 1.3 Mbp sequence data were generated. Multiple sequence alignment (MSA) revealed 79 SNPs and 41 indels in nine genes while the CAP2 gene was found to be conserved across all the genotypes. Among 10 candidate genes, the maximum number of SNPs (34) was observed in abscisic acid stress and ripening (ASR) gene including 22 transitions, 11 transversions and one tri-allelic SNP. Nucleotide diversity varied from 0.0004 to 0.0029 while polymorphism information content (PIC) values ranged from 0.01 (AKIN gene) to 0.43 (CAP2 promoter). Haplotype analysis revealed that alleles were represented by more than two haplotype blocks, except alleles of the CAP2 and sucrose synthase (SuSy) gene, where only one haplotype was identified. These genes can be used for association analysis and if validated, may be useful for enhancing abiotic stress, including drought tolerance, through molecular breeding.

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/

Nucleotide diversity patterns at the drought-related DREB2 encoding genes in wild and cultivated common bean (Phaseolus vulgaris L.).

Common beans are an important food legume faced with a series of abiotic stresses the most severe of which is drought. The crop is interesting as a model for the analysis of gene phylogenies due to its domestication process, race structure, and origins in a group of wild common beans found along the South American Andes and the region of Mesoamerica. Meanwhile, the DREB2 transcription factors have been implicated in controlling non-ABA dependent responses to drought stress. With this in mind our objective was to study in depth the genetic diversity for two DREB2 genes as possible candidates for association with drought tolerance through a gene phylogenetic analysis. In this genetic diversity assessment, we analyzed nucleotide diversity at the two candidate genes Dreb2A and Dreb2B, in partial core collections of 104 wild and 297 cultivated common beans with a total of 401 common bean genotypes from world-wide germplasm analyzed. Our wild population sample covered a range of semi-mesic to very dry habitats, while our cultivated samples presented a wide spectrum of low to high drought tolerance. Both genes showed very different patterns of nucleotide variation. Dreb2B exhibited very low nucleotide diversity relative to neutral reference loci previously surveyed in these populations. This suggests that strong purifying selection has been acting on this gene. In contrast, Dreb2A exhibited higher levels of nucleotide diversity, which is indicative of adaptive selection and population expansion. These patterns were more distinct in wild compared to cultivated common beans. These approximations suggested the importance of Dreb2 genes in the context of drought tolerance, and constitute the first steps towards an association study between genetic polymorphism of this gene family and variation in drought tolerance traits. We discuss the utility of allele mining in the DREB gene family for the discovery of new drought tolerance traits from wild common bean.

Molecular ecology and selection in the drought-related Asr gene polymorphisms in wild and cultivated common bean (Phaseolus vulgaris L.).

BACKGROUND: The abscisic acid (ABA) pathway plays an important role in the plants' reaction to drought stress and ABA-stress response (Asr) genes are important in controlling this process. In this sense, we accessed nucleotide diversity at two candidate genes for drought tolerance (Asr1 and Asr2), involved in an ABA signaling pathway, in the reference collection of cultivated common bean (Phaseolus vulgaris L.) and a core collection of wild common bean accessions. RESULTS: Our wild population samples covered a range of mesic (semi-arid) to very dry (desert) habitats, while our cultivated samples presented a wide spectrum of drought tolerance. Both genes showed very different patterns of nucleotide variation. Asr1 exhibited very low nucleotide diversity relative to the neutral reference loci that were previously surveyed in these populations. This suggests that strong purifying selection has been acting on this gene. In contrast, Asr2 exhibited higher levels of nucleotide diversity, which is indicative of adaptive selection. These patterns were more notable in wild beans than in cultivated common beans indicting that natural selection has played a role over long time periods compared to farmer selection since domestication. CONCLUSIONS: Together these results suggested the importance of Asr1 in the context of drought tolerance, and constitute the first steps towards an association study between genetic polymorphism of this gene family and variation in drought tolerance traits. Furthermore, one of our major successes was to find that wild common bean is a reservoir of genetic variation and selection signatures at Asr genes, which may be useful for breeding drought tolerance in cultivated common bean.

Structure, allelic diversity and selection of Asr genes, candidate for drought tolerance, in Oryza sativa L. and wild relatives.

Asr (ABA, stress, ripening) genes represent a small gene family potentially involved in drought tolerance in several plant species. To analyze their interest for rice breeding for water-limited environments, this gene family was characterized further. Genomic organization of the gene family reveals six members located on four different chromosomes and with the same exon-intron structure. The maintenance of six members of the Asr gene family, which are the result of combination between tandem duplication and whole genome duplication, and their differential regulation under water stress, involves probably some sub-functionalization. The polymorphism of four members was studied in a worldwide collection of 204 accessions of Oryza sativa L. and 14 accessions of wild relatives (O. rufipogon and O. nivara). The nucleotide diversity of the Asr genes was globally low, but contrasted for the different genes, leading to different shapes of haplotype networks. Statistical tests for neutrality were used and compared to their distribution in a set of 111 reference genes spread across the genome, derived from another published study. Asr3 diversity exhibited a pattern concordant with a balancing selection at the species level and with a directional selection in the tropical japonica sub-group. This study provides a thorough description of the organization of the Asr family, and the nucleotide and haplotype diversity of four Asr in Oryza sativa species. Asr3 stood out as the best potential candidate. The polymorphism detected here represents a first step towards an association study between genetic polymorphisms of this gene family and variation in drought tolerance traits.

Leaf-level water use efficiency determined by carbon isotope discrimination in rice seedlings: genetic variation associated with population structure and QTL mapping.

Increasing the water use efficiency (WUE) of our major crop species is an important target of agricultural research. Rice is a major water consumer in agriculture and it is also an attractive genetic model. We evaluated leaf-level WUE in young rice seedlings using carbon isotope discrimination (Delta(13)C) as an indicator of the trait. A survey of Delta(13)C was undertaken in 116 diverse germplasm accessions representing O. sativa, O. glaberrima and four wild Oryza species. O. sativa cultivars were classified into sub-populations based on SSR markers, and significant differences in Delta(13)C were observed among the five genetically defined groups. While individual accessions explained a greater proportion of the variation than did sub-population, indica rice varieties had the lowest Delta(13)C values overall, indicating superior WUE, while temperate japonica had the highest Delta(13)C. O sativa accessions had a similar or greater range of Delta(13)C values than wild Oryza species, while domesticated O. glaberrima had a narrower range. Correlation analysis identified leaf morphological and physiological traits that were significantly associated with Delta(13)C, including longer leaves, more drooping leaves, higher tillering ability, and lower leaf nitrogen content. These trait associations were investigated by quantitative trait locus (QTL) mapping using backcross inbred lines derived from a cross between Nipponbare (temperate japonica) and Kasalath (aus). Seven QTL for Delta(13)C were identified using composite interval analysis, located in five chromosomal regions. The QTL with the largest additive effect came from Kasalath and co-localized with QTL for leaf length, tiller number and nitrogen content.

Model-assisted physiological analysis of Phyllo, a rice architectural mutant.

Studies of phenotype of knockout mutants can provide new insights into physiological, phenological and architectural feedbacks in the plant system. Phyllo, a mutant of Nippon Bare rice (Oryza sativa L.) producing small leaves in rapid succession, was isolated during multiplication of a T-DNA insertion library. Phyllo phenotype was compared with the wild type (WT) during vegetative development in hydroponics culture using a wide range of physiological and biometric measurements. These were integrated with the help of the functional-structural model EcoMeristem, explicitly designed to study interactions between morphogenesis and carbon assimilation. Although the phenotype of the mutant was caused by a single recessive gene, it differed in many ways from the WT, suggesting a pleiotropic effect of this mutation. Phyllochron was 25 (1-4 leaf stage) to 38% (>>4 leaf stage) shorter but showed normal transition from juvenile to adult phase after leaf 4. Leaf size also increased steadily with leaf position as in WT. The mutant had reduced leaf blade length : width and blade : sheath length ratios, particularly during the transition from heterotrophic to autotrophic growth. During the same period, root : shoot dry weight ratio was significantly diminished. Specific leaf area (SLA) was strongly increased in the mutant but showed normal descending patterns with leaf position. Probably related to high SLA, the mutant had much lower light-saturated leaf photosynthetic rates and lower radiation use efficiency (RUE) than the WT. Leaf extension rates were strongly reduced in absolute terms but were high in relative terms (normalised by final leaf length). The application of the EcoMeristem model to these data indicated that the mutant was severely deficient in assimilate, resulting from low RUE and high organ initiation rate causing high assimilate demand. This was particularly pronounced during the heterotrophic-autotrophic transition, probably causing shorter leaf blades relative to sheaths, as well as a temporary reduction of assimilate partitioning to roots. The model accurately simulated the mutant's high leaf mortality and absence of tillering. The simulated assimilate shortage was supported by observed reductions in starch storage in sheaths. Soluble sugar concentrations differed between mutant and WT in roots but not in shoots. Specifically, the hexose : sucrose ratio was 50% lower in the roots of the mutant, possibly indicating low invertase activity. Furthermore, two OsCIN genes coding for cell wall invertases were not expressed in roots, and others were expressed weakly. This was interpreted as natural silencing via sugar signalling. In summary, the authors attributed the majority of observed allometric and metabolic modifications in the mutant to an extreme assimilate shortage caused by hastened shoot organogenesis and inefficient leaf morphology.

Identification of drought-inducible genes and differentially expressed sequence tags in barley.

Drought limits cereal yields in several regions of the world and plant water status plays an important role in tolerance to drought. To investigate and understand the genetic and physiological basis of drought tolerance in barley, differentially expressed sequence tags (dESTs) and candidate genes for the drought response were mapped in a population of 167 F8 recombinant inbred lines derived from a cross between "Tadmor" (drought tolerant) and "Er/Apm" (adapted only to specific dry environments). One hundred sequenced probes from two cDNA libraries previously constructed from drought-stressed barley (Hordeum vulgare L., var. Tokak) plants and 12 candidate genes were surveyed for polymorphism, and 33 loci were added to a previously published map. Composite interval mapping was used to identify quantitative trait loci (QTL) associated with drought tolerance including leaf relative water content, leaf osmotic potential, osmotic potential at full turgor, water-soluble carbohydrate concentration, osmotic adjustment, and carbon isotope discrimination. A total of 68 QTLs with a limit of detection score > or =2.5 were detected for the traits evaluated under two water treatments and the two traits calculated from both treatments. The number of QTLs identified for each trait varied from one to 12, indicating that the genome contains multiple genes affecting different traits. Two candidate genes and ten differentially expressed sequences were associated with QTLs for drought tolerance traits.