Emeline Lhuillier

Genome skimming by shotgun sequencing helps resolve the phylogeny of a pantropical tree family.

Whole genome sequencing is helping generate robust phylogenetic hypotheses for a range of taxonomic groups that were previously recalcitrant to classical molecular phylogenetic approaches. As a case study, we performed a shallow shotgun sequencing of eight species in the tropical tree family Chrysobalanaceae to retrieve large fragments of high‐copy number DNA regions and test the potential of these regions for phylogeny reconstruction. We were able to assemble the nuclear ribosomal cluster (nrDNA), the complete plastid genome (ptDNA) and a large fraction of the mitochondrial genome (mtDNA) with approximately 1000×, 450× and 120× sequencing depth respectively. The phylogenetic tree obtained with ptDNA resolved five of the seven internal nodes. In contrast, the tree obtained with mtDNA and nrDNA data were largely unresolved. This study demonstrates that genome skimming is a cost‐effective approach and shows potential in plant molecular systematics within Chrysobalanaceae and other under‐studied groups.

From museums to genomics: old herbarium specimens shed light on a C3 to C4 transition

Collections of specimens held by natural history museums are invaluable material for biodiversity inventory and evolutionary studies, with specimens accumulated over 300 years readily available for sampling. Unfortunately, most museum specimens yield low-quality DNA. Recent advances in sequencing technologies, so called next-generation sequencing, are revolutionizing phylogenetic investigations at a deep level. Here, the Illumina technology (HiSeq) was used on herbarium specimens of Sartidia (subfamily Aristidoideae, Poaceae), a small African-Malagasy grass lineage (six species) characteristic of wooded savannas, which is the C3 sister group of Stipagrostis, an important C4 genus from Africa and SW Asia. Complete chloroplast and nuclear ribosomal sequences were assembled for two Sartidia species, one of which (S. perrieri) is only known from a single specimen collected in Madagascar 100 years ago. Partial sequences of a few single-copy genes encoding phosphoenolpyruvate carboxylases (ppc) and malic enzymes (nadpme) were also assembled. Based on these data, the phylogenetic position of Malagasy Sartidia in the subfamily Aristidoideae was investigated and the biogeographical history of this genus was analysed with full species sampling. The evolutionary history of two genes for C4 photosynthesis (ppc-aL1b and nadpme-IV) in the group was also investigated. The gene encoding the C4 phosphoenolpyruvate caroxylase of Stipagrostis is absent from S. dewinteri suggesting that it is not essential in C3 members of the group, which might have favoured its recruitment into a new metabolic pathway. Altogether, the inclusion of historical museum specimens in phylogenomic analyses of biodiversity opens new avenues for evolutionary studies.

A DNA metabarcoding study of a primate dietary diversity and plasticity across its entire fragmented range.

In tropical regions, most primary ecosystems have been replaced by mosaic landscapes in which species must cope with a large shift in the distribution of their habitat and associated food resources. Primates are particularly vulnerable to habitat modifications. Most species persist in small fragments surrounded by complex human-mediated matrices whose structure and connectivity may strongly influence their dispersal and feeding behavior. Behavioral plasticity appears to be a crucial parameter governing the ability of organisms to exploit the resources offered by new matrix habitats and thus to persist in fragmented habitats. In this study, we were interested in the dietary plasticity of the golden-crowned sifaka (Propithecus tattersalli), an endangered species of lemur, found only in the Daraina region in north-eastern Madagascar. We used a DNA-based approach combining the barcoding concept and Illumina next-generation sequencing to (i) describe the species diet across its entire range and (ii) evaluate the influence of landscape heterogeneity on diet diversity and composition. Faeces from 96 individuals were sampled across the entire species range and their contents were analyzed using the trnL metabarcoding approach. In parallel, we built a large DNA reference database based on a checklist of the plant species of the Daraina region. Our results suggest that golden-crowned sifakas exhibit remarkable dietary diversity with at least 130 plant species belonging to 80 genera and 49 different families. We highlighted an influence of both habitat type and openness on diet composition suggesting a high flexibility of foraging strategies. Moreover, we observed the presence of numerous cultivated and naturalized plants in the faeces of groups living in forest edge areas. Overall, our findings support our initial expectation that P. tattersalli is able to cope with the current level of alteration of the landscape and confirm our previous results on the distribution and the dispersal ability of this species.

Gonad transcriptome analysis of pearl oyster Pinctada margaritifera: identification of potential sex differentiation and sex determining genes

BackgroundBlack pearl farming is based on culture of the blacklip pearl oyster Pinctada margaritifera (Mollusca, lophotrochozoa), a protandrous hermaphrodite species. At first maturation, all individuals are males. The female sex appears progressively from two years old, which represents a limitation for broodstock conditioning for aquaculture production. In marine mollusks displaying hermaphroditic features, data on sexual determinism and differentiation, including the molecular sex determining cascade, are scarce. To increase genomic resources and identify the molecular mechanisms whereby gene expression may act in the sexual dimorphism of P. margaritifera, we performed gonad transcriptome analysis.ResultsThe gonad transcriptome of P. margaritifera was sequenced from several gonadic samples of males and females at different development stages, using a Next-Generation-Sequencing method and RNAseq technology. After Illumina sequencing, assembly and annotation, we obtained 70,147 contigs of which 62.2% shared homologies with existing protein sequences, and 9% showed functional annotation with Gene Ontology terms. Differential expression analysis identified 1,993 differentially expressed contigs between the different categories of gonads. Clustering methods of samples revealed that the sex explained most of the variation in gonad gene expression. K-means clustering of differentially expressed contigs showed 815 and 574 contigs were more expressed in male and female gonads, respectively. The analysis of these contigs revealed the presence of known specific genes coding for proteins involved in sex determinism and/or differentiation, such as dmrt and fem-1 like for males, or foxl2 and vitellogenin for females. The specific gene expression profiles of pmarg-fem1-like, pmarg-dmrt and pmarg-foxl2 in different reproductive stages (undetermined, sexual inversion and regression) suggest that these three genes are potentially involved in the sperm-oocyte switch in P. margaritifera.ConclusionsThe study provides a new transcriptomic tool to study reproduction in hermaphroditic marine mollusks. It identifies sex differentiation and potential sex determining genes in P. margaritifera, a protandrous hermaphrodite species.

SNP discovery and genetic mapping using genotyping by sequencing of whole genome genomic DNA from a pea RIL population

BackgroundProgress in genetics and breeding in pea still suffers from the limited availability of molecular resources. SNP markers that can be identified through affordable sequencing processes, without the need for prior genome reduction or a reference genome to assemble sequencing data would allow the discovery and genetic mapping of thousands of molecular markers. Such an approach could significantly speed up genetic studies and marker assisted breeding for non-model species.ResultsA total of 419,024 SNPs were discovered using HiSeq whole genome sequencing of four pea lines, followed by direct identification of SNP markers without assembly using the discoSnp tool. Subsequent filtering led to the identification of 131,850 highly designable SNPs, polymorphic between at least two of the four pea lines.A subset of 64,754 SNPs was called and genotyped by short read sequencing on a subpopulation of 48 RILs from the cross ‘Baccara’ x ‘PI180693’. This data was used to construct a WGGBS-derived pea genetic map comprising 64,263 markers. This map is collinear with previous pea consensus maps and therefore with the Medicago truncatula genome. Sequencing of four additional pea lines showed that 33 % to 64 % of the mapped SNPs, depending on the pairs of lines considered, are polymorphic and can therefore be useful in other crosses.The subsequent genotyping of a subset of 1000 SNPs, chosen for their mapping positions using a KASP™ assay, showed that almost all generated SNPs are highly designable and that most (95 %) deliver highly qualitative genotyping results. Using rather low sequencing coverages in SNP discovery and in SNP inferring did not hinder the identification of hundreds of thousands of high quality SNPs.ConclusionsThe development and optimization of appropriate tools in SNP discovery and genetic mapping have allowed us to make available a massive new genomic resource in pea. It will be useful for both fine mapping within chosen QTL confidence intervals and marker assisted breeding for important traits in pea improvement.

Fast assembly of the mitochondrial genome of a plant parasitic nematode (Meloidogyne graminicola) using next generation sequencing

Little is known about the variations of nematode mitogenomes (mtDNA). Sequencing a complete mtDNA using a PCR approach remains a challenge due to frequent genome reorganizations and low sequence similarities between divergent nematode lineages. Here, a genome skimming approach based on HiSeq sequencing (shotgun) was used to assemble de novo the first complete mtDNA sequence of a root-knot nematode (Meloidogyne graminicola). An AT-rich genome (84.3%) of 20,030 bp was obtained with a mean sequencing depth superior to 300. Thirty-six genes were identified with a semi-automated approach. A comparison with a gene map of the M. javanica mitochondrial genome indicates that the gene order is conserved within this nematode lineage. However, deep genome rearrangements were observed when comparing with other species of the superfamily Hoplolaimoidea. Repeat elements of 111 bp and 94 bp were found in a long non-coding region of 7.5 kb, as similarly reported in M. javanica and M. hapla. This study points out the power of next generation sequencing to produce complete mitochondrial genomes, even without a reference sequence, and possibly opening new avenues for species/race identification, phylogenetics and population genetics of nematodes.

Mass production of SNP markers in a nonmodel passerine bird through RAD sequencing and contig mapping to the zebra finch genome

Here, we present an adaptation of restriction‐site‐associated DNA sequencing (RAD‐seq) to the Illumina HiSeq2000 technology that we used to produce SNP markers in very large quantities at low cost per unit in the Réunion grey white‐eye (Zosterops borbonicus), a nonmodel passerine bird species with no reference genome. We sequenced a set of six pools of 18–25 individuals using a single sequencing lane. This allowed us to build around 600 000 contigs, among which at least 386 000 could be mapped to the zebra finch (Taeniopygia guttata) genome. This yielded more than 80 000 SNPs that could be mapped unambiguously and are evenly distributed across the genome. Thus, our approach provides a good illustration of the high potential of paired‐end RAD sequencing of pooled DNA samples combined with comparative assembly to the zebra finch genome to build large contigs and characterize vast numbers of informative SNPs in nonmodel passerine bird species in a very efficient and cost‐effective way.

Shotgun assembly of the complete mitochondrial genome of the neotropical cracker butterfly Hamadryas epinome.

Abstract The complete mitochondrial genome of the cracker butterfly Hamadryas epinome (C. Felder and R. Felder, 1867) (Lepidoptera: Nymphalidae: Biblidinae) has been sequenced using a genome-skimming approach on an Illumina Hiseq 2000 platform. The mitochondrial genome of H. epinome was determined to be 15,207 bp long and presents an organization similar to other Ditrysia mitogenomes. A non-coding poly-AT region of uncertain length is present at position 6180.

Sequencing of the mitochondrial genome of the avocado lace bug Pseudacysta perseae (Heteroptera, Tingidae) using a genome skimming approach.

Lace bugs (Tingidae) are a family of phytophagous heteropterans, some of which are important agricultural and forestry pests. They currently comprise around 2500 species distributed worldwide, for which only one mitochondrial genome has been described so far. We sequenced the complete mitochondrial genome and the nuclear ribosomal gene segment of the avocado lace bug Pseudacysta perseae using a genome skimming approach on an Illumina Hiseq 2000 platform. Fifty-four additional heteropteran mitogenomes, including the one of the sycamore lace bug Corythucha ciliata, were retrieved to allow for comparisons and phylogenetic analyses. P. perseae mitochondrial genome was determined to be 15,850 bp long, and presented the typical organisation of insect mitogenomes. The phylogenetic analysis placed P. perseae as a sister to C. ciliata but did not confirm the monophyly of Miroidae including Tingidae. Our results contradicted widely accepted phylogenetic hypothesis, which highlights the limits of analyses based on mitochondrial data only. Shotgun sequencing approaches should provide substantial improvements in harmonizing mitochondrial and nuclear databases.

A novel locus on chromosome 1 underlies the evolution of a melanic plumage polymorphism in a wild songbird

Understanding the mechanisms responsible for phenotypic diversification within and among species ultimately rests with linking naturally occurring mutations to functionally and ecologically significant traits. Colour polymorphisms are of great interest in this context because discrete colour patterns within a population are often controlled by just a few genes in a common environment. We investigated how and why phenotypic diversity arose and persists in the Zosterops borbonicus white-eye of Reunion (Mascarene archipelago), a colour polymorphic songbird in which all highland populations contain individuals belonging to either a brown or a grey plumage morph. Using extensive phenotypic and genomic data, we demonstrate that this melanin-based colour polymorphism is controlled by a single locus on chromosome 1 with two large-effect alleles, which was not previously described as affecting hair or feather colour. Differences between colour morphs appear to rely upon complex cis-regulatory variation that either prevents the synthesis of pheomelanin in grey feathers, or increases its production in brown ones. We used coalescent analyses to show that, from a ‘brown’ ancestral population, the dominant ‘grey’ allele spread quickly once it arose from a new mutation. Since colour morphs are always found in mixture, this implies that the selected allele does not go to fixation, but instead reaches an intermediate frequency, as would be expected under balancing selection.