SNP mining in C. clementina BAC end sequences; transferability in the Citrus genus (Rutaceae), phylogenetic inferences and perspectives for genetic mapping

<p>Abstract</p> <p>Background</p> <p>With the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers. However, SNP chip data generally suffers from ascertainment biases caused by the SNP discovery and selection process in which a small number of individuals are used as discovery panels. The ongoing International Citrus Genome Consortium sequencing project of the highly heterozygous Clementine and sweet orange genomes will soon result in the release of several hundred thousand SNPs. The primary goals of this study were: (i) to estimate the transferability within the genus <it>Citrus </it>of SNPs discovered from Clementine BACend sequencing (BES), (ii) to estimate bias associated with the very narrow discovery panel, and (iii) to evaluate the usefulness of the Clementine-derived SNP markers for diversity analysis and comparative mapping studies between the different cultivated <it>Citrus </it>species.</p> <p>Results</p> <p>Fifty-four accessions covering the main <it>Citrus </it>species and 52 interspecific hybrids between pummelo and Clementine were genotyped on a GoldenGate array platform using 1,457 SNPs mined from Clementine BES and 37 SNPs identified between and within <it>C. maxima, C. medica, C. reticulata </it>and <it>C. micrantha</it>. Consistent results were obtained from 622 SNP loci. Of these markers, 116 displayed incomplete transferability primarily in <it>C. medica, C. maxima </it>and wild <it>Citrus </it>species. The two primary biases associated with the SNP mining in Clementine were an overestimation of the <it>C. reticulata </it>diversity and an underestimation of the interspecific differentiation. However, the genetic stratification of the gene pool was high, with very frequent significant linkage disequilibrium. Furthermore, the shared intraspecific polymorphism and accession heterozygosity were generally enough to perform interspecific comparative genetic mapping.</p> <p>Conclusions</p> <p>A set of 622 SNP markers providing consistent results was selected. Of the markers mined from Clementine, 80.5% were successfully transferred to the whole <it>Citrus </it>gene pool. Despite the ascertainment biases in relation to the Clementine origin, the SNP data confirm the important stratification of the gene pools around <it>C. maxima, C. medica </it>and <it>C. reticulata </it>as well as previous hypothesis on the origin of secondary species. The implemented SNP marker set will be very useful for comparative genetic mapping in <it>Citrus </it>and genetic association in <it>C. reticulata</it>.</p

The evolution with strain of the stored energy in different texture components of cold-rolled if steel revealed by high resolution X-ray diffraction

During the deformation of low carbon steel by cold-rolling, dislocations are created and stored in grains depending on local crystallographic orientation, deformation, and deformation gradient. Orientation dependent dislocation densities have been estimated from the broadening of X-ray diffraction lines measured on a synchrotron beamline. Different cold-rolling levels (from 30% to 95% thickness reduction) have been considered. It is shown that the present measurements are consistent with the hypothesis of the sole consideration of screw dislocations for the analysis of the data. The presented evolutions show that the dislocation density first increases within the α fiber (={hkl}) and then within the γ fiber (={111}). A comparison with EBSD measurements is done and confirms that the storage of dislocations during the deformation process is orientation dependent and that this dependence is correlated to the cold-rolling level. If we assume that this dislocation density acts as a driving force during recrystallization, these observations can explain the fact that the recrystallization mechanisms are generally different after moderate or large strains

BMC Nephrol

An amendment to this paper has been published and can be accessed via the original article

Discovery and mapping of a new expressed sequence tag-single nucleotide polymorphism and simple sequence repeat panel for large-scale genetic studies and breeding of Theobroma cacao L.

Theobroma cacao is an economically important tree of several tropical countries. Its genetic improvement is essential to provide protection against major diseases and improve chocolate quality. We discovered and mapped new expressed sequence tag-single nucleotide polymorphism (EST-SNP) and simple sequence repeat (SSR) markers and constructed a high-density genetic map. By screening 149 650 ESTs, 5246 SNPs were detected in silico, of which 1536 corresponded to genes with a putative function, while 851 had a clear polymorphic pattern across a collection of genetic resources. In addition, 409 new SSR markers were detected on the Criollo genome. Lastly, 681 new EST-SNPs and 163 new SSRs were added to the pre-existing 418 co-dominant markers to construct a large consensus genetic map. This high-density map and the set of new genetic markers identified in this study are a milestone in cocoa genomics and for marker-assisted breeding. The data are available at http://tropgenedb.cirad.fr

Translational Genomics in Legumes Allowed Placing In Silico 5460 Unigenes on the Pea Functional Map and Identified Candidate Genes in Pisum sativum L.

To identify genes involved in phenotypic traits, translational genomics from highly characterized model plants to poorly characterized crop plants provides a valuable source of markers to saturate a zone of interest as well as functionally characterized candidate genes. In this paper, an integrated view of the pea genetic map was developed. A series of gene markers were mapped and their best reciprocal homologs were identified on M. truncatula, L. japonicus, soybean, and poplar pseudomolecules. Based on the syntenic relationships uncovered between pea and M. truncatula, 5460 pea Unigenes were tentatively placed on the consensus map. A new bioinformatics tool, http://www.thelegumeportal.net/pea_mtr_translational_toolkit, was developed that allows, for any gene sequence, to search its putative position on the pea consensus map and hence to search for candidate genes among neighboring Unigenes. As an example, a promising candidate gene for the hypernodulation mutation nod3 in pea was proposed based on the map position of the likely homolog of Pub1, a M. truncatula gene involved in nodulation regulation. A broader view of pea genome evolution was obtained by revealing syntenic relationships between pea and sequenced genomes. Blocks of synteny were identified which gave new insights into the evolution of chromosome structure in Papillionoids and Eudicots. The power of the translational genomics approach was underlined

Biomedicines

Antibody-mediated rejection (ABMR) is the leading cause of allograft failure in kidney transplantation. Its histological hallmark is represented by lesions of glomerulitis i.e., inflammatory cells within glomeruli. Current therapies for ABMR fail to prevent chronic allograft damage i.e., transplant glomerulopathy, leading to allograft loss. We used laser microdissection of glomeruli from formalin-fixed allograft biopsies combined with mass spectrometry-based proteomics to describe the proteome modification of 11 active and 10 chronic active ABMR cases compared to 8 stable graft controls. Of 1335 detected proteins, 77 were deregulated in glomerulitis compared to stable grafts, particularly involved in cellular stress mediated by interferons type I and II, leukocyte activation and microcirculation remodeling. Three proteins extracted from this protein profile, TYMP, WARS1 and GBP1, showed a consistent overexpression by immunohistochemistry in glomerular endothelial cells that may represent relevant markers of endothelial stress during active ABMR. In transplant glomerulopathy, 137 proteins were deregulated, which favor a complement-mediated mechanism, wound healing processes through coagulation activation and ultimately a remodeling of the glomerular extracellular matrix, as observed by light microscopy. This study brings novel information on glomerular proteomics of ABMR in kidney transplantation, and highlights potential targets of diagnostic and therapeutic interest

The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation

BACKGROUND: Mutations in CALR observed in myeloproliferative neoplasms (MPN) were recently shown to be pathogenic via their interaction with MPL and the subsequent activation of the Janus Kinase - Signal Transducer and Activator of Transcription (JAK-STAT) pathway. However, little is known on the impact of those variant CALR proteins on endoplasmic reticulum (ER) homeostasis. METHODS: The impact of the expression of Wild Type (WT) or mutant CALR on ER homeostasis was assessed by quantifying the expression level of Unfolded Protein Response (UPR) target genes, splicing of X-box Binding Protein 1 (XBP1), and the expression level of endogenous lectins. Pharmacological and molecular (siRNA) screens were used to identify mechanisms involved in CALR mutant proteins degradation. Coimmunoprecipitations were performed to define more precisely actors involved in CALR proteins disposal. RESULTS: We showed that the expression of CALR mutants alters neither ER homeostasis nor the sensitivity of hematopoietic cells towards ER stress-induced apoptosis. In contrast, the expression of CALR variants is generally low because of a combination of secretion and protein degradation mechanisms mostly mediated through the ER-Associated Degradation (ERAD)-proteasome pathway. Moreover, we identified a specific ERAD network involved in the degradation of CALR variants. CONCLUSIONS: We propose that this ERAD network could be considered as a potential therapeutic target for selectively inhibiting CALR mutant-dependent proliferation associated with MPN, and therefore attenuate the associated pathogenic outcomes

Identification des mutations à visée diagnostique et pronostique dans les néoplasies myéloprolifératives et impact sur l'épissage alternatif

Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are a group of Philadelphia-negative myeloproliferative neoplasm (MPN). These diseases share a common mutation, JAK2 V617F, in varying proportions. The mutated JAK2 protein has a constitutive tyrosine kinase activity, implicated in the physiopathology of MPN. This mutation alone does not explain the phenotypic heterogeneity within MPN.High throughput sequencing techniques helped understanding the physiopathology. This work aimed to identify additional mutations in two patient cohorts related to the aggravation risk of the disease. The first one consisted of patients in chronic phase (JAK2 V617F ET and PV), the second consisted in patients with myelofibrosis treated with interferon. Like other studies, we have shown that the number of mutations and the presence of additional mutations are associated with disease progression or with response to treatment. Some identified mutations could influence splicing. The second part of this work aimed at studying the putative impact of the JAK2 V617F mutation, on alternative splicing (AS).We also analyzed global AS profiles in ET. JAK2 exon 14 skipping has been described in NMP patients with or without the JAK2 V617F mutation.This mutation was predicted to alter the binding site of the SRSF6 splice-regulating protein. We observed that exon 14 skipping was an uncommon event in patients, in part related to SR protein expression. In addition, our transcriptomic-wide analysis showed a great heterogeneity between the patients with respect to both gene expression and splicing. This prevented us from identifying any characteristic profile. These results underscore the importance of identifying additional mutations at diagnosis and during follow-up. We have also been able to uncover some alternative transcripts associated with the presence of these mutations.The functional role of these variants remains to be defined.Les néoplasies myéloprolifératives (NMP), non BCR-ABL1, regroupent principalement la polyglobulie de Vaquez (PV), la thrombocytémie essentielle (TE) et la myélofibrose primitive (MFP).Ces pathologies partagent, dans des proportions variables, une mutation commune, la mutation JAK2 V617F. La protéine JAK2 mutée a une activité tyrosine kinase constitutive, impliquée dans le développement de la maladie. Cette mutation, seule, n’explique pas l’hétérogénéité phénotypique au sein des NMP. L’avènement des techniques de séquençage haut débit a permis de mieux appréhender la physiopathologie. Notre travail avait pour objectif l’identification de mutations additionnelles au sein de deux cohortes suivies au long cours en lien avec un risque d’aggravation de la maladie, l’une regroupant des patients en phase chronique (TE et PV JAK2 V617F), la seconde regroupant des patients avec une myélofibrose traitée par interféron. A l’instar d’autres travaux récents, nous avons montré que le nombre de mutations et la présence de mutations additionnelles sont associés à l’évolution de la maladie, voire à la réponse au traitement.Parmi les mutations identifiées, certaines pourraient influencer l’épissage. La deuxième partie de ce travail a donc consisté à étudier l’épissage alternatif en fonction des mutations présentes, et en particulier la mutation JAK2 (V617F) et de manière globale dans les TE. Un saut de l’exon 14 de JAK2 a été décrit chez des patients NMP présentant, ou non, la mutation JAK2 V617F. Cette mutation du gène JAK2 est prédite pour altérer un site de fixation de la protéine SRSF6 régulatrice de l’épissage. Nous observons que le saut de l’exon 14 est un événement peu fréquent chez les patients, modulé en partie par l’expression des protéines SR. L’analyse transcriptomique montre une grande hétérogénéité entre les patients en termes d’expression et d’épissage, ce qui ne nous a pas permis de mettre en évidence de profil caractéristique. Ces résultats soulignent l’importance de l’identification des mutations additionnelles au diagnostic et au cours du suivi.Nous avons pu, en outre, identifier quelques transcrits alternatifs associés à la présence de ces mutations. Le rôle fonctionnel de ces variants reste à définir