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

International audienceBackground - Progress 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. Results - A 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. Conclusions - The 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

Extensive Clonality and Strong Differentiation in the Insular Pacific Tree Santalum insulare: Implications for its Conservation

• Background and Aims The impact of evolutionary forces on insular systems is particularly exacerbated by the remoteness of islands, strong founder effects, small population size and the influence of biotic and abiotic factors. Patterns of molecular diversity were analysed in an island system with Santalum insulare, a sandalwood species endemic to eastern Polynesia. The aims were to evaluate clonality and to study the genetic diversity and structure of this species, in order to understand the evolutionary process and to define a conservation strategy

Development and characterization of 24 polymorphic microsatellite loci in two Antirrhinum majus subspecies (Plantaginaceae) using pyrosequencing technology

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

International audienceThe 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

Shotgun assembly of the assassin bug <em>Brontostoma colossus</em> mitochondrial genome (Heteroptera, Reduviidae)

International audienceThe complete mitochondrial genome of the assassin bug Brontostoma colossus (Distant, 1902) (Heteroptera: Reduviidae) has been sequenced using a genome-skimming approach on an Illumina Hiseq 2000 platform. Fifty-four additional heteropteran mitogenomes, including five assassin bug species, were retrieved to allow for comparisons and phylogenetic analyses. The mitochondrial genome of B. colossus was determined to be 16,625 bp long, and consists of 13 protein-coding genes (PCGs), 23 transfer-RNA genes (tRNAs), two ribosomal-RNA genes (rRNAs), and one control region. The nucleotide composition is biased toward adenine and thymine (A + T = 73.4%). Overall, architecture, nucleotide composition and genome asymmetry are similar among all available assassin bug mitogenomes. All PCGs have usual start-codons (Met and Ile). Three T and two TA incomplete termination codons were identified adjacent to tRNAs, which was consistent with the punctuation model for primary transcripts processing followed by 3' polyadenylation of mature mRNA. All tRNAs exhibit the classic clover-leaf secondary structure except for tRNA(Ser)(AcN) in which the DHU arm forms a simple loop. Two notable features are present in the B. colossus mitogenome: (i) a 131 bp duplicated unit including the complete tRNA(Arg) gene, resulting in 23 potentially functional tRNAs in total, and (ii) a 857 bp duplicated region comprising 277 bp of the srRNA gene and 580 bp of the control region. A phylogenetic analysis based on 55 true bug mitogenomes confirmed that B. colossus belongs to Reduviidae, but contradicted a widely accepted hypothesis. This highlights the limits of phylogenetic analyses based on mitochondrial data only

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

Background Black 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. Results The 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. Conclusions The 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

From museums to genomics: old herbarium specimens shed light on a C-3 to C-4 transition

International audienceCollections 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 C-3 sister group of Stipagrostis, an important C-4 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 C-4 photosynthesis (ppc-aL1b and nadpme-IV) in the group was also investigated. The gene encoding the C-4 phosphoenolpyruvate caroxylase of Stipagrostis is absent from S. dewinteri suggesting that it is not essential in C-3 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

Lowering relative humidity level increases epidermal protein deimination and drives human filaggrin breakdown

BACKGROUND: Deimination (also known as citrullination), the conversion of arginine in a protein to citrulline, is catalyzed by a family of enzymes called peptidylarginine deiminases (PADs). Three PADs are expressed in the epidermis, one of their targets being filaggrin. Filaggrin plays a central role in atopic dermatitis and is a key protein for the epidermal barrier. It aggregates keratins and is cross-linked to cornified envelopes. Following its deimination, it is totally degraded to release free amino acids, contributing to the natural moisturizing factor (NMF). The mechanisms controlling this multistep catabolism in human are unknown. OBJECTIVE: To test whether external humidity plays a role, and investigate the molecular mechanisms involved. METHODS: Specimens of reconstructed human epidermis (RHEs) produced in humid or dry conditions ( \u3e 95% or 30-50% relative humidity) were compared. RESULTS: RHEs produced in the dry condition presented structural changes, including a thicker stratum corneum and a larger amount of keratohyalin granules. The transepidermal water loss and the stratum corneum pH were decreased whereas the quantity of NMF was greater. This highly suggested that filaggrin proteolysis was up-regulated. The expression/activity of the proteases involved in filaggrin breakdown did not increase while PAD1 expression and the deimination rate of proteins, including filaggrin, were drastically enhanced. Partial inhibition of PADs with Cl-amidine reversed the effect of dryness on filaggrin breakdown. CONCLUSION: These results demonstrate the importance of external humidity in the control of human filaggrin metabolism, and suggest that deimination plays a major role in this regulation