311 SSRs and 71 gene based SNPs were developed from mapping population of C. arietinum ICC 4958 × C. reticulatum PI 489777 CMap Visualization Links: ICC 4958 X PI 489777 DOI: doi:10.1007/s00122-010-1265-1

Trabajo presentado en la 3rd Annual Conference of the COST Action Sustain (FA1208), celebrada en Banyuls s/ Mer (France) del 17 al 19 de febrero de 2016.

Background: Previous studies identified microRNAs (miRNAs) and messenger RNAs with significantly different expression between normal pancreas and pancreatic cancer (PDAC) tissues. Due to technological limitations of microarrays and real-time PCR systems these studies focused on a fixed set of targets. Expression of other RNA classes such as long intergenic non-coding RNAs or sno-derived RNAs has rarely been examined in pancreatic cancer. Here, we analysed the coding and non-coding transcriptome of six PDAC and five control tissues using next-generation sequencing.Results: Besides the confirmation of several deregulated mRNAs and miRNAs, miRNAs without previous implication in PDAC were detected: miR-802, miR-2114 or miR-561. SnoRNA-derived RNAs (e.g. sno-HBII-296B) and piR-017061, a piwi-interacting RNA, were found to be differentially expressed between PDAC and control tissues. In silico target analysis of miR-802 revealed potential binding sites in the 3'UTR of TCF4, encoding a transcription factor that controls Wnt signalling genes. Overexpression of miR-802 in MiaPaCa pancreatic cancer cells reduced TCF4 protein levels. Using Massive Analysis of cDNA Ends (MACE) we identified differential expression of 43 lincRNAs, long intergenic non-coding RNAs, e.g. LINC00261 and LINC00152 as well as several natural antisense transcripts like HNF1A-AS1 and AFAP1-AS1. Differential expression was confirmed by qPCR on the mRNA/miRNA/lincRNA level and by immunohistochemistry on the protein level.Conclusions: Here, we report a novel lncRNA, sncRNA and mRNA signature of PDAC. In silico prediction of ncRNA targets allowed for assigning potential functions to differentially regulated RNAs.

Background: MLN2480 is an orally administered, investigational small molecule pan-RAF kinase inhibitor currently in Phase 1 clinical development. Here, we built integrated PK/PD/efficacy models to understand the relationship between MAPK pathway inhibition and efficacy as well as the effect of dose schedule in two melanoma xenograft models, A375 and SKMEL-2. Materials and Methods: We fit a one-compartment PK model to MLN2480 plasma concentration time profiles following a single dose in mice. Tumor pERK levels in xenograft-bearing mice, measured by Western blotting following single (A375) or multiple (SKMEL-2) doses of MLN2480 were fit to a direct or indirect inhibitory Emax model, respectively, to describe the PK/PD relationship between the plasma concentration and %pERK inhibition. Activity in each xenograft was evaluated as the percent growth rate inhibition (%GRI), the percent change between treated and control exponential tumor volume growth rates over the treatment cycle. To connect PK, PD, and efficacy for each xenograft, we simulated the PK and PD profiles over the treatment cycle for each dose group, and estimated the averages of plasma concentration (Cavg) and %pERK inhibition, respectively, using non-compartmental analysis. Results: From a multiple linear regression, schedule-related parameters are not predictive for drug effect demonstrating schedule-independent activity. For a variety of dose schedules, the relationship between total dose and SKMEL-2 xenograft growth rate is approximately linear (R = 0.90, p < 0.0001). A sigmoidal PK/efficacy model captures the relationship between Cavg and %GRI for both xenografts. A375 exhibits greater sensitivity than SKMEL-2 to the same Cavg of MLN2480, with the models predicting tumor regression (Emax = 172% GRI) and stasis (Emax = 102% GRI) as the maximal effects, respectively. Both xenografts have steep, sigmoidal PD/efficacy relationships, which show the same pERK inhibition is associated with greater %GRI in A375, and %GRI saturates with residual pERK (EC90 = 38% in A375, 22% in SKMEL-2). Conclusions: Integrated PK/PD and PK/efficacy modeling for MLN2480 response in A375 and SKMEL-2 xenografts shows a strong relationship between pERK inhibition and preclinical activity, and that maximal effect is achieved without complete inhibition of the MAPK pathway. The findings of pERK inhibition-dependent but not schedule-dependent activity provide translational guidance to clinical dose and schedule selection.

A draft sequence of the staple crop kabuli chickpea, together with resequencing and analysis of 90 additional lines from 10 countries, provides a resource for breeders. Chickpea (Cicer arietinum) is the second most widely grown legume crop after soybean, accounting for a substantial proportion of human dietary nitrogen intake and playing a crucial role in food security in developing countries. We report the ∼738-Mb draft whole genome shotgun sequence of CDC Frontier, a kabuli chickpea variety, which contains an estimated 28,269 genes. Resequencing and analysis of 90 cultivated and wild genotypes from ten countries identifies targets of both breeding-associated genetic sweeps and breeding-associated balancing selection. Candidate genes for disease resistance and agronomic traits are highlighted, including traits that distinguish the two main market classes of cultivated chickpea—desi and kabuli. These data comprise a resource for chickpea improvement through molecular breeding and provide insights into both genome diversity and domestication.

The seed protein of narrow-leafed lupin ( Lupinus angustifolius ) is known for its exceptional functional properties as food ingredient and, thus, represents a resource of vegetable protein for human consumption. To take advantage of this potential for a broad range of food applications the regional R&D network 'PlantsProFood' was set up in Northern Germany. Lupins shall be enhanced as a protein resource for food purposes, like ice cream, sausages, bakery products or pasta. The network consists of four research institutions and ten local companies and aims at processing the value chain from the development of (I) high-yielding varieties, (II) new processing approaches towards (III) innovative and healthy food. Requirements for these efforts are high and stable yields of narrow-leafed lupins. This may be achieved by plant breeding as far as sufficient genetic variability is available. To expand the genetic variability of advanced lupin breeding material an EMS (ethyl methanesulfonate) mutagenesis of cv. 'Boruta' was performed and the offspring screened for novel phenotypes. Phenotypes with a conspicuous novel growth type, such as vigorous growth or high branching compared to the wildtype 'Boruta', were identified and devised to homozygous and stable mutant (M) lines. The yield potential of the M lines was evaluated under field conditions and for some lines the potential was confirmed by significant increase in kernel yields. For genetic analyses, crosses of the respective M lines with genebank accessions as well as with the wildtype 'Boruta' were carried out. Segregation analysis of F2 populations indicated a monogenic-recessive inheritance of novel growth types. Promising M lines are going to be subjected to an analysis of differentially expressed sequences of mutant lines and the wildtype via RNAseq techniques and SNP identification. Currently, selected SNPs are analyzed via high-resolution melt analysis to develop selection tools for marker-assisted selection.

A consensus genetic map of chickpea (Cicer arietinum L.) was constructed by merging linkage maps from 10 different populations, using STMS (Sequence-tagged Microsatellite Sites) as bridging markers. These populations derived from five wide crosses (C. arietinum × Cicer reticulatum) and five narrow crosses (Desi × Kabuli types) were previously used for mapping genes for several agronomic traits such as ascochyta blight, fusarium wilt, rust resistance, seed weight, flowering time and days to flower. The integrated map obtained from wide crosses consists of 555 loci including, among other markers, 135 STMSs and 33 cross-genome markers distributed on eight linkage groups and covers 652.67 cM. The map obtained from narrow crosses comprises 99 STMSs, 3 SCARs, 1 ASAP, fusarium resistance gene, 5 morphological traits as well as RAPD and ISSR markers distributed on eight linkage groups covering 426.99 cM. Comparison between maps from wide and narrow crosses reflects a general coincidence, although some discrepancies are discussed. Medicago truncatula cross-genome markers were BLASTed against the M. truncatula pseudogenome permitting assignments of chickpea linkage groups LGI, II, III, IV, V and VI on Medicago chromosomes 2, 5, 7, 1, 3 and 4, respectively. A marker detectable on Medicago chromosome 4 were also located on LGVIII, This consensus map is an important progress to assist breeders for selecting suitable markers to be used in marker-assisted selection (MAS).

A genetic map composed of 10 linkage groups (LGs) covering 848.1cM was constructed from a recombinant inbred line population from the cross Cicer arietinum (ICCL81001)×Cicer reticulatum (Cr5-9). The lines were genotyped with random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and sequence tagged microsatellite site (STMS) markers, and phenotyped with respect to flowering time, flower colour, growth habit, seed size, seed coat reticulation, seed coat thickness and reaction to infection by two races of fusarium wilt. Segregation distortion was observed for 35% of markers, mainly clustered on LG4, LG7 and two unassigned linkage groups. Rs/rs (seed coat reticulation) together with a quantitative trait locus (QTL) accounted for >50% of the variation for seed coat thickness. Both Hg/hg (growth habit) and a flowering time QTL mapped to LG3A. The STMS marker TA142 was closely linked to this QTL. Two QTL for seed size were detected—one on LG4 linked to STMS markers GAA47 and STMS11, and the other on LG2 linked to STMS TA110. Two tightly linked QTL on LG2 closely linked to STMS marker TA59 controlled the resistance to both races of the pathogen causing fusarium wilt.

As a tool for high-throughput, quantitative gene expression analysis, serial analysis of gene expression (SAGE) is one of the most powerful techniques. However, the short size of tags (14 bp) has hindered the application of SAGE to a vast majority of eukaryotes without sufficient genomic resources, including expressed sequence tag and genome sequences. To overcome this problem, we developed SuperSAGE, which is based on 26-bp tags from complementary DNA (cDNA), using EcoP15I as a tagging enzyme. Because longer cDNA fragments can easily be recovered by 3 $^\prime$ -rapid amplification of cDNA ends (RACE) PCR using primers corresponding to the 26-bp tag sequences in non-model organisms, SuperSAGE allows the identification of novel genes in all eukaryotic organisms, and recommends itself as a useful platform in various fields of biological studies. Here, we present an updated SuperSAGE protocol, which incorporates several modifications and some recommendations to avoid total failure, particularly in the EcoP15I digestion step.

This study aimed at the development of microsatellite markers for Phaseolus vulgaris L. by two techniques: hybridization-based detection, and transfer of microsatellite markers developed for chickpea to the common bean. Small-insert genomic libraries of common bean were screened for simple sequence repeat (SSR)-containing fragments with a set of microsatellite-specific oligonucleotide probes. Twenty-five (GA)10 positive clones detected by a dinucleotide repeat probe were selected for sequencing. Sixteen of 18 primer pairs, complementary to the SSR-flanking regions and tested in eight P. vulgaris L. accessions, are polymorphic at an intra-specific level. Mendelian inheritance of the sequence-tagged microsatellite site (STMS) markers was demonstrated using a set of recombinant inbred lines (RILs) and their parents. A total of 46 chickpea STMS markers of 447 different primer combinations amplified loci in the genome of common bean. Sequencing of amplified products from Phaseolus with these primer combinations demonstrated that the sequence of microsatellite marker TA 176s proved to be significantly similar to transcription factor SCOF1 of soy bean, controlling the response to cold stress, and also to transcription factor zinc finger protein (a Krüppel-like zinc finger protein, or TFIIIA), coordinating the reaction to osmotic stress in Medicago truncatula. Sequence analysis of this site in the two common bean parental lines BAT 477 and DOR 364 revealed that the sequence of BAT 477 was 5 bp longer than the corresponding sequence of DOR 364. Amplification of this fragment in six RILs of common bean detected the existence of sequence polymorphisms between the different lines.

The pantropical genus Vigna (Leguminosae) comprises 7 cultivated species that are adapted to a wide range of extreme agroclimatic conditions. Few data are available on the relationships among these cultivated species or on their importance as sources of resistance against biotic and abiotic stresses. Therefore, we optimized DNA amplification fingerprinting (DAF) to estimate the genetic diversity within, and genetic relationships among, a representative core collection of cowpea, as compared with 16 accessions representing cultivars from 6 Vigna species. A set of 26 primers was selected from 262 tested random primers and used for the characterization of 85 Vigna accessions (6 V. angularis , 4 each of V. mungo and V. radiata , 2 V. umbellata , 1 V. aconitifolia , and 68 V. unguiculata ), with Phaseolus vulgaris subsp. vulgaris as outgroup. A total of 212 polymorphic bands were used for maximum parsimony analysis. Our results clearly distinguished Brazilian from African V. unguiculata genotypes. At the species level, V. angularis was the most related and V. radiata the most divergent species relative to V. unguiculata. DAF markers were also informative at the intraspecific level, detecting a large diversity between cowpea cultivars. The implications of the presented results for cowpea breeding programs are discussed.

Chickpea is a staple protein source in many Asian and Middle Eastern countries. The seeds contain carotenoids such as beta-carotene, cryptoxanthin, lutein and zeaxanthin in amounts above the engineered beta-carotene-containing "golden rice" level. Thus, breeding for high carotenoid concentration in seeds is of nutritional, socio-economic, and economic importance. To study the genetics governing seed carotenoids in chickpea, we studied the relationship between seed weight and concentrations of beta-carotene and lutein by means of high-performance liquid chromatography in segregating progeny from a cross between an Israeli cultivar and wild Cicer reticulatum Ladiz. Seeds of the cross progeny varied with respect to their carotenoid concentration (heritability estimates ranged from 0.5 to 0.9), and a negative genetic correlation was found between mean seed weight and carotenoid concentration in the F-3. To determine the loci responsible for the genetic variation observed, the population was genotyped using 91 sequence tagged microsatellite site markers and two CytP450 markers to generate a genetic map consisting of nine linkage groups and a total length of 344.6 cM. Using quantitative data collected for beta-carotene and lutein concentration and seed weight of the seeds of the F-2 population, we were able to identify quantitative trait loci (QTLs) by interval mapping. At a LOD score of 2, four QTLs for beta-carotene concentration, a single QTL for lutein concentration and three QTLs for seed weight were detected. The results of this investigation may assist in improving the nutritional quality of chickpea.

Sequence-tagged microsatellite site (STMS) and sequence-tagged site (STS) markers linked closely to Fusarium oxysporum f. sp. ciceris race 3 resistance gene in chickpea were identified, and linkage between three wilt resistance genes was elucidated. The resistance to race 3 in chickpea germplasm accession WR-315 was inherited as a single gene, designated foc-3, in 100 F(7) recombinant inbred lines derived from the cross of WR-315 (resistant) x C-104 (susceptible). The foc-3 gene was mapped 0.6 cM from STMS markers TA96 and TA27 and STS marker CS27A. Another STMS marker, TA194, at 14.3 cM, flanked the gene on the other side. Linkage between foc-3 and two other chickpea wilt resistance genes, foc-1 (syn. h(1)) and foc-4, was established. foc-3 was mapped 9.8 cM from foc-1 and 8.7 cM from foc-4, whereas foc-1 and foc-4 are closely linked at 1.1 cM. The identification of closely linked markers to resistance genes will facilitate marker-assisted selection for introgression of the race 3 resistance gene to susceptible chickpea lines.