Mutation detection using ENDO1: Application to disease diagnostics in humans and TILLING and Eco-TILLING in plants

<p>Abstract</p> <p>Background</p> <p>Most enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA by a mismatch-specific endonuclease at mismatch sites and the analysis of the digestion product on a DNA sequencer. Important limitations of these methods are the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in pool of DNA, the cost of the analysis and the ease by which the technique could be implemented in a standard molecular biology laboratory.</p> <p>Results</p> <p>The co-agroinfiltration of ENDO1 and p19 constructs into <it>N. benthamiana </it>leaves allowed high level of transient expression of a mismatch-specific and sensitive endonuclease, ENDO1 from <it>Arabidopsis thaliana</it>. We demonstrate the broad range of uses of the produced enzyme in detection of mutations. In human, we report the diagnosis of the G1691A mutation in <it>Leiden factor-V </it>gene associated with venous thrombosis and the fingerprinting of HIV-1 quasispecies in patients subjected to antiretroviral treatments. In plants, we report the use of ENDO1 system for detection of mutant alleles of <it>Retinoblastoma</it>-<it>related </it>gene by TILLING in <it>Pisum sativum </it>and discovery of natural sequence variations by Eco-TILLING in <it>Arabidopsis thaliana</it>.</p> <p>Conclusion</p> <p>We introduce a cost-effective tool based on a simplified purification protocol of a mismatch-specific and sensitive endonuclease, ENDO1. Especially, we report the successful applications of ENDO1 in mutation diagnostics in humans, fingerprinting of complex population of viruses, and in TILLING and Eco-TILLING in plants.</p

<em>Tendril-less</em> regulates tendril formation in pea leaves

Tendrils are contact-sensitive, filamentous organs that permit climbing plants to tether to their taller neighbors. Tendrilled legume species are grown as field crops, where the tendrils contribute to the physical support of the crop prior to harvest. The homeotic tendril-less (tl) mutation in garden pea (Pisum sativum), identified almost a century ago, transforms tendrils into leaflets. In this study, we used a systematic marker screen of fast neutron–generated tl deletion mutants to identify Tl as a Class I homeodomain leucine zipper (HDZIP) transcription factor. We confirmed the tendril-less phenotype as loss of function by targeting induced local lesions in genomes (TILLING) in garden pea and by analysis of the tendril-less phenotype of the t mutant in sweet pea (Lathyrus odoratus). The conversion of tendrils into leaflets in both mutants demonstrates that the pea tendril is a modified leaflet, inhibited from completing laminar development by Tl. We provide evidence to show that lamina inhibition requires Unifoliata/LEAFY-mediated Tl expression in organs emerging in the distal region of the leaf primordium. Phylogenetic analyses show that Tl is an unusual Class I HDZIP protein and that tendrils evolved either once or twice in Papilionoid legumes. We suggest that tendrils arose in the Fabeae clade of Papilionoid legumes through acquisition of the Tl gene

UTILLdb, a Pisum sativum in silico forward and reverse genetics tool

UTILLdb is a database of phenotypic and sequence information on mutant genes from a reference Pisum sativum EMS-mutant population

EcoTILLING for the identification of allelic variants of melon eIF4E, a factor that controls virus susceptibility

<p>Abstract</p> <p>Background</p> <p>Translation initiation factors of the 4E and 4G protein families mediate resistance to several RNA plant viruses in the natural diversity of crops. Particularly, a single point mutation in melon eukaryotic translation initiation factor 4E (eIF4E) controls resistance to <it>Melon necrotic spot virus </it>(MNSV) in melon. Identification of allelic variants within natural populations by EcoTILLING has become a rapid genotype discovery method.</p> <p>Results</p> <p>A collection of <it>Cucumis </it>spp. was characterised for susceptibility to MNSV and <it>Cucumber vein yellowing virus </it>(CVYV) and used for the implementation of EcoTILLING to identify new allelic variants of <it>eIF4E</it>. A high conservation of <it>eIF4E </it>exonic regions was found, with six polymorphic sites identified out of EcoTILLING 113 accessions. Sequencing of regions surrounding polymorphisms revealed that all of them corresponded to silent nucleotide changes and just one to a non-silent change correlating with MNSV resistance. Except for the MNSV case, no correlation was found between variation of eIF4E and virus resistance, suggesting the implication of different and/or additional genes in previously identified resistance phenotypes. We have also characterized a new allele of <it>eIF4E </it>from <it>Cucumis zeyheri</it>, a wild relative of melon. Functional analyses suggested that this new <it>eIF4E </it>allele might be responsible for resistance to MNSV.</p> <p>Conclusion</p> <p>This study shows the applicability of EcoTILLING in <it>Cucumis </it>spp., but given the conservation of eIF4E, new candidate genes should probably be considered to identify new sources of resistance to plant viruses. Part of the methodology described here could alternatively be used in TILLING experiments that serve to generate new <it>eIF4E </it>alleles.</p

Linking Auxin with Photosynthetic Rate via Leaf Venation

International audienceLand plants lose vast quantities of water to the atmosphere during photosynthetic gas exchange. In angiosperms, a complex network of veins irrigates the leaf, and it is widely held that the density and placement of these veins determines maximum leaf hydraulic capacity and thus maximum photosynthetic rate. This theory is largely based on interspecific comparisons and has never been tested using vein mutants to examine the specific impact of leaf vein morphology on plant water relations. Here we characterize mutants at the Crispoid (Crd) locus in pea (Pisum sativum), which have altered auxin homeostasis and activity in developing leaves, as well as reduced leaf vein density and aberrant placement of free-ending veinlets. This altered vein phenotype in crd mutant plants results in a significant reduction in leaf hydraulic conductance and leaf gas exchange. We find Crispoid to be a member of the YUCCA family of auxin biosynthetic genes. Our results link auxin biosynthesis with maximum photosynthetic rate through leaf venation and substantiate the theory that an increase in the density of leaf veins coupled with their efficient placement can drive increases in leaf photosynthetic capacity

Thermodynamic and Kinetic Modelling of Scales Formation at the Soultz-sous-Forêts Geothermal Power Plant

Geothermal energy has been a subject of great interest since the 1990s in the Upper Rhine Graben (URG), where the first European Enhanced Geothermal System (EGS) pilot site has been developed, in Soultz-sous-Forêts (SsF), France. Several studies have already been conducted on scales occurring at the reinjection side at the geothermal plants located in the URG. It has been observed that the composition of the scales changes as chemical treatment is applied to inhibit metal sulfate. The purpose of this study was to model the scaling phenomenon occurring in the surface pipes and the heat exchangers at the SsF geothermal plant. PhreeqC, a geochemical modelling software, was used to reproduce the scaling observations in the geothermal plant during exploitation. A suitable database was chosen based on the availability of chemical elements, minerals, and gas. A thermodynamic model and a kinetic model were proposed for modelling the scaling phenomenon. The thermodynamic model gave insight on possible minerals precipitated while the kinetic model, after modifying the initial rates equation, produced results that were close to the expected scale composition at the SsF geothermal plant. Additional laboratory studies on the kinetics of the scales are proposed to complement the current model

Hight throughput identification of Pisum sativum mutant lines by TILLING: a tool for crop improvement using either forward of reverse genetics approaches

International audienc

TILLING resources in pea: a valuable tool for functional validation

National audienceSince 2004, two INRA teams (UMR Agroecology Dijon and IJBP, Versailles), both partners of PEAMUST, have created 3 EMS-induced mutant collections in the framework of European or French ANR projects. The genotypes chosen for these collections have specific agronomic traits according to the research subject. For example, the Terese rms3 and -rms4 branched genotypes (Rameau et al., 1997) are well suited to the study of shoot architecture in relation with strigolactone and the lines easy to grow and to backcross in glasshouse conditions. Caméor, an early cultivar with high seed protein content, is the reference genotype for the pea sequencing project. The 336/11 winter pea line with the Hr allele is very sensitive to photoperiod and cold tolerant, which enables the screening of genes contributing to winter pea performance. These 3 collections currently represent a total of 13500 mutated lines. The high-throughput detection of mutations in specific regions of the genome is critical to exploit allelic resources from these collections. Since 2014, mutation detection system has evolved toward NGS technologies and a new one has been implemented in INRA (IPS2) increasing consequently the sensitivity of the detection, allowing a better exploitation of these collections. Spanning the 3 collections mutation rate was assessed ranging from 10 to 40 thousand mutations per genome. As part of the PeaMUST project, about 15 kb of candidate gene sequences were sequenced across 81,000 families. 313 mutations were detected, of which 9% induce a stop codon or a splice defect and 70% induce amino acid changes. These results showed the very high potential of these collections for their exploitation in functional validation or creation of allelic variants. Rameau C, Bodelin C, Cadier D, Grandjean O, Miard F, Murfet IC (1997) New ramosus mutants at loci Rms1, Rms3 and Rms4 resulting from the mutation breeding program at Versailles. Pisum Genet 29: 7–1