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Despite the increasing accessibility of high-throughput sequencing, obtaining high-quality genomic data on non-model organisms without proximate well-assembled and annotated genomes remains challenging. Here we describe a workflow that takes advantage of distant genomic resources and ingroup transcriptomes to select and jointly enrich long open reading frames (ORFs) and ultraconserved elements (UCEs) from genomic samples for integrative studies of microevolutionary and macroevolutionary dynamics. This workflow is applied to samples of the African unionid bivalve tribe Coelaturini (Parreysiinae) at basin and continent-wide scales. Our results indicate that ORFs are efficiently captured without prior identification of intron-exon boundaries. The enrichment of UCEs was less successful, but nevertheless produced a substantial dataset. Exploratory continent-wide phylogenetic analyses with ORF supercontigs (>515,000 parsimony informative sites) resulted in a fully resolved phylogeny, the backbone of which was also retrieved with UCEs (>11,000 informative sites), although some branches lack support in the latter case. Variant calling on the exome of Coelaturini from the Malawi Basin produced ~2,000 SNPs per population pair. Nucleotide diversity and population differentiation was low compared to previous estimates in mollusks, but comparable to those in recently diversifying Malawi cichlids and other taxa at an early stage of speciation. Skimming non-specific sequence data obtained for Coelaturini of the Malawi Basin, we reconstructed the maternally-inherited mitogenome, which displays an identical gene order to that of the most recent common ancestor of Unionidae. Overall, our workflow and results provide exciting perspectives for the development of integrative genomic studies on micro- and macroevolutionary dynamics in non-model organisms.

Genes that do not segregate in heterozygotes at Mendelian ratios are a potentially important evolutionary force in natural populations. Although the impacts of segregation distortion are widely appreciated, we have little quantitative understanding about how often these loci arise and fix within lineages. Here, we develop a statistical approach for detecting segregation distorting genes from the comprehensive comparison of whole genome sequence data obtained from bulk gamete versus somatic tissues. Our approach enables estimation of map positions and confidence intervals, and quantification of effect sizes of segregation distorters. We apply our method to the pollen of two interspecific F1 hybrids of Arabidopsis lyrata and A. halleri and we identify three loci across eight chromosomes showing significant evidence of segregation distortion in both pollen samples. Based on this, we estimate that novel segregation distortion elements evolve and achieve high frequencies within lineages at a rate of approximately one per 244,000 years. Furthermore, we estimate that haploid-acting segregation distortion may contribute between 10% and 30% of reduced pollen viability in F1 individuals. Our results indicate haploid acting factors evolve rapidly and dramatically influence segregation in F1 hybrid individuals.

A consensus genetic map for chicory (2n = 2x = 18) was obtained after the integration of molecular marker data of two industrial chicory progenies (K28K59, Rubis118) and one witloof chicory progeny (BR). As a limited number of co-dominant markers was available at the beginning of this work, three different microsatellite-enriched libraries were produced from genomic DNA, resulting in 420, 719 and 1,251 sequences, respectively. The level of informative Simple Sequence Repeat (SSR) sequences from the three libraries ranged from 28 to 40%, thus defining a set of 730 SSR markers available for polymorphism screening. A subset of 81 Sequence-Tagged Sites (STS) developed from EST, cDNA, genes, and non-coding sequences was screened through Single Strand Conformational Polymorphism (SSCP) analysis, leading to 46 polymorphic loci integrated in the genetic maps. Markers were grouped and ordered on 9 homologous Linkage Groups (LG) for each of the three maps: 274 markers in K28K59, 282 markers in Rubis118, 178 markers in BR. Co-linear regions between maps were identified through 193 ‘bridge’ markers that allowed the integration of the 9 homologous LG in a consensus map containing 472 markers and covering 878 cM. Comparison across maps revealed the presence of 4 conserved regions with significant distorted markers, also defined as Segregation Distortion Regions (SDR), affected by gametic or zygotic selection factors. Marker distribution was not always uniform; 6 LG possessed homologous clustered regions in all maps. The consensus map could be the starting point for the identification and the cloning of major genes and QTL in fundamental and applied genetic areas in chicory.

Background Freezing provokes severe yield losses to different fall-sown annual legumes. Understanding the molecular bases of freezing tolerance is of great interest for breeding programs. Medicago truncatula Gaertn. is an annual temperate forage legume that has been chosen as a model species for agronomically and economically important legume crops. The present study aimed to identify positional candidate genes for a major freezing tolerance quantitative trait locus that was previously mapped to M. truncatula chromosome 6 (Mt-FTQTL6) using the LR3 population derived from a cross between the freezing-tolerant accession F83005-5 and the freezing-sensitive accession DZA045-5. Results The confidence interval of Mt-FTQTL6 was narrowed down to the region comprised between markers MTIC153 and NT6054 using recombinant F7 and F8 lines. A bacterial-artificial chromosome (BAC) clone contig map was constructed in an attempt to close the residual assembly gap existing therein. Twenty positional candidate genes including twelve C-repeat binding factor (CBF)/dehydration-responsive element binding factor 1 (DREB1) genes were identified from BAC-derived sequences and whole-genome shotgun sequences (WGS). CBF/DREB1 genes are organized in a tandem array within an approximately 296-Kb region. Eleven CBF/DREB1 genes were isolated and sequenced from F83005-5 and DZA045-5 which revealed high polymorphism among these accessions. Unique features characterizing CBF/DREB1 genes from M. truncatula, such as alternative splicing and large tandem duplication, are elucidated for the first time. Conclusions Overall, twenty genes were identified as potential candidates to explain Mt-FTQTL6 effect. Their future functional characterization will uncover the gene(s) involved in freezing tolerance difference observed between F83005-5 and DZA045-5. Knowledge transfer for breeding improvement of crop legumes is expected. Furthermore, CBF/DREB1 related data will certainly have a large impact on research studies targeting this group of transcriptional activators in M. truncatula and other legume species.

International audience; Cold stress affects plant growth and development. In order to better understand the responses to cold (chilling or freezing tolerance), we used two contrasted pea lines. Following a chilling period, the Champagne line becomes tolerant to frost whereas the Terese line remains sensitive. Four suppression subtractive hybridisation libraries were obtained using mRNAs isolated from pea genotypes Champagne and Terese. Using quantitative polymerase chain reaction (qPCR) performed on 159 genes, 43 and 54 genes were identified as differentially expressed at the initial time point and during the time course study, respectively. Molecular markers were developed from the differentially expressed genes and were genotyped on a population of 164 RILs derived from a cross between Champagne and Terese. We identified 5 candidate genes colocalizing with 3 different frost damage quantitative trait loci (QTL) intervals and a protein quantity locus (PQL) rich region previously reported. This investigation revealed the role of constitutive differences between both genotypes in the cold responses, in particular with genes related to glycine degradation pathway that could confer to Champagne a better frost tolerance. We showed that freezing tolerance involves a decrease of expression of genes related to photosynthesis and the expression of a gene involved in the production of cysteine and methionine that could act as cryoprotectant molecules. Although it remains to be confirmed, this study could also reveal the involvement of the jasmonate pathway in the cold responses, since we observed that two genes related to this pathway were mapped in a frost damage QTL interval and in a PQL rich region interval, respectively.

International audience; A simple, efficient protocol for direct in vitro shoot organogenesis and regeneration was established for three species of Miscanthus including two clones of Miscanthus x giganteus, one clone of M. sinensis and one clone of M. sacchariflorus. Shoots were induced from the axillary nodes of both M. x giganteus and M. sacchariflorus and from apical meristems of both M. sinensis and M. sacchariflorus. A tillering method was used to accelerate shoot proliferation. Shoots were rooted in a wet perlite substrate in pots in the greenhouse. Subsequently, rooted plants were transferred to the field. The genetic uniformity of regenerated plants was evaluated using amplified fragment length polymorphism analysis and compared to that of rhizome-propagated plants. A total of 33,443 fragments were generated, representing 869 markers. There were 21 fragments (0.06 % of the fragments) or 19 markers (2.19 % of the markers) that were polymorphic, and almost all of these were singletons. The three species showed similar polymorphisms. Genetic variability was also found in the rhizome-propagated plants, sometimes at a higher rate than in the in vitro culture, indicating that the genetic uniformity was not altered by the protocol. This protocol may help breeders produce new clones of Miscanthus in the future.

International audience; High-density genetic maps were constructed for loci involved in nuclear male sterility (NMS1-locus) and sporophytic self-incompatibility (S-locus) in chicory (Cichorium intybus L.). The mapping population consisted of 389 F1' individuals derived from a cross between two plants, K28 (male-sterile) and K59 (pollen-fertile), both heterozygous at the S-locus. This F1' mapping population segregated for both male sterility (MS) and strong self-incompatibility (SI) phenotypes. Phenotyping F1' individuals for MS allowed us to map the NMS1-locus to linkage group (LG) 5, while controlled diallel and factorial crosses to identify compatible/incompatible phenotypes mapped the S-locus to LG2. To increase the density of markers around these loci, bulked segregant analysis was used. Bulks and parental plants K28 and K59 were screened using amplified fragment length polymorphism (AFLP) analysis, with a complete set of 256 primer combinations of EcoRI-ANN and MseI-CNN. A total of 31,000 fragments were generated, of which 2,350 showed polymorphism between K59 and K28. Thirteen AFLP markers were identified close to the NMS1-locus and six in the vicinity of the S-locus. From these AFLP markers, eight were transformed into sequence-characterized amplified region (SCAR) markers and of these five showed co-dominant polymorphism. The chromosomal regions containing the NMS1-locus and the S-locus were each confined to a region of 0.8 cM. In addition, we mapped genes encoding proteins similar to S-receptor kinase, the female determinant of sporophytic SI in the Brasicaceae, and also markers in the vicinity of the putative S-locus of sunflower, but none of these genes or markers mapped close to the chicory S-locus.

Genome scans based on anonymous Amplified Fragment Length Polymorphism (AFLP) markers scattered throughout the genome are becoming an increasingly popular approach to study the genetic basis of adaptation and speciation in natural populations. A shortcoming of this approach is that despite its efficiency to detect signatures of selection, it can hardly help pinpoint the specific genomic region(s), gene(s), or mutation(s) targeted by selection. Here, we present two methods to be undertaken after performing an AFLP-based genome scan to easily obtain the sequences of AFLP loci detected as outliers by population genomics approaches. The first one is based on the gel excision of the target AFLP fragment, after simplification of the AFLP fingerprint and separation of the fragments by migration. The second one is a combination of classical AFLP protocol and 454 pyrosequencing.

Legumes were among the first plant species to be domesticated, and accompanied cereals in expansion of agriculture from the Fertile Crescent into diverse environments across the Mediterranean basin, Europe, Central Asia, and the Indian subcontinent. Although several recent studies have outlined the molecular basis for domestication and eco-geographic adaptation in the two main cereals from this region, wheat and barley, similar questions remain largely unexplored in their legume counterparts. Here we identify two major loci controlling differences in photoperiod response between wild and domesticated pea, and show that one of these, HIGH RESPONSE TO PHOTOPERIOD ( HR ), is an ortholog of EARLY FLOWERING 3 ( ELF3 ), a gene involved in circadian clock function. We found that a significant proportion of flowering time variation in global pea germplasm is controlled by HR , with a single, widespread functional variant conferring altered circadian rhythms and the reduced photoperiod response associated with the spring habit. We also present evidence that ELF3 has a similar role in lentil, another major legume crop, with a distinct functional variant contributing to reduced photoperiod response in cultivars widely deployed in short-season environments. Our results identify the factor likely to have permitted the successful prehistoric expansion of legume cultivation to Northern Europe, and define a conserved genetic basis for major adaptive changes in flowering phenology and growth habit in an important crop group.

Genome scans based on anonymous Ampli fi ed Fragment Length Polymorphism (AFLP) markers scattered throughout the genome are becoming an increasingly popular approach to study the genetic basis of adaptation and speciation in natural populations. A shortcoming of this approach is that despite its ef fi ciency to detect signatures of selection, it can hardly help pinpoint the speci fi c genomic region(s), gene(s), or mutation(s) targeted by selection. Here, we present two methods to be undertaken after performing an AFLP-based genome scan to easily obtain the sequences of AFLP loci detected as outliers by population genomics approaches. The fi rst one is based on the gel excision of the target AFLP fragment, after simpli fi cation of the AFLP fi ngerprint and separation of the fragments by migration. The second one is a combination of classical AFLP protocol and 454 pyrosequencing.

Background The Asteraceae represents an important plant family with respect to the numbers of species present in the wild and used by man. Nonetheless, genomic resources for Asteraceae species are relatively underdeveloped, hampering within species genetic studies as well as comparative genomics studies at the family level. So far, six BAC libraries have been described for the main crops of the family, i.e. lettuce and sunflower. Here we present the characterization of BAC libraries of chicory (Cichorium intybus L.) constructed from two genotypes differing in traits related to sexual and vegetative reproduction. Resolving the molecular mechanisms underlying traits controlling the reproductive system of chicory is a key determinant for hybrid development, and more generally will provide new insights into these traits, which are poorly investigated so far at the molecular level in Asteraceae. Findings Two bacterial artificial chromosome (BAC) libraries, CinS2S2 and CinS1S4, were constructed from HindIII-digested high molecular weight DNA of the contrasting genotypes C15 and C30.01, respectively. C15 was hermaphrodite, non-embryogenic, and S2S2 for the S-locus implicated in self-incompatibility, whereas C30.01 was male sterile, embryogenic, and S1S4. The CinS2S2 and CinS1S4 libraries contain 89,088 and 81,408 clones. Mean insert sizes of the CinS2S2 and CinS1S4 clones are 90 and 120 kb, respectively, and provide together a coverage of 12.3 haploid genome equivalents. Contamination with mitochondrial and chloroplast DNA sequences was evaluated with four mitochondrial and four chloroplast specific probes, and was estimated to be 0.024% and 1.00% for the CinS2S2 library, and 0.028% and 2.35% for the CinS1S4 library. Using two single copy genes putatively implicated in somatic embryogenesis, screening of both libraries resulted in detection of 12 and 13 positive clones for each gene, in accordance with expected numbers. Conclusions This indicated that both BAC libraries are valuable tools for molecular studies in chicory, one goal being the positional cloning of the S-locus in this Asteraceae species.

Glutathione S-tranferases (GSTs) are a heterogeneous family of proteins, which perform diverse pivotal catalytic and non-enzymatic functions during plant development and in plant stress responses. Previous studies have shown that a GST activity (EC 2.5.1.18) is closely linked with the precocious phases of somatic embryogenesis in leaf tissues of an interspecific chicory hybrid (Cichorium intybus L. var. sativa × C. endivia L. var. latifolia). In order to learn more about the involvement of this enzyme in this process, in situ-hybridization as well as immunolocalization were performed in parallel. GST-mRNAs and proteins were colocalized in small veins, particularly in young protoxylem cell walls. During cell reactivation, the in situ and protein signals became less intense and were associated with chloroplasts. The GST-mRNAs and corresponding proteins were not always colocalized in the same tissues. While high amounts of transcripts could be detected in multicellular embryos, the proteins were not well labeled. Our results indicated that GSTs belong to a complex anti-oxidant mechanism within the cell, and also at the cell wall level. GSTs presence in reactivated cell and multicellular embryos is discussed in relation to redox cell status.

International audience; Hemoglobins are ancient O2‑binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum x C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.