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Cobos, Maria Jose/0000-0002-8636-1266; Millan, Teresa/0000-0003-1302-1794; Rubio, Josefa/0000-0002-5185-2719; Gil, Juan/0000-0001-7938-442X WOS: 000280780700004 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 x Cicer reticulatum) and five narrow crosses (Desi x 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). European Union (GLIP)European Union (EU) [FOOD-CT-2004-506223] The authors appreciate financial support from the European Union (GLIP FOOD-CT-2004-506223), and T.M. thanks for a GLIP fellowship.

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 Kruppel-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 cool season grain legume of exceptionally high nutritive value and most versatile food use. It is mostly grown under rain fed conditions in arid and semi-arid areas around the world. Despite growing demand and high yield potential, chickpea yield is unstable and productivity is stagnant at unacceptably low levels. Major yield increases could be achieved by development and use of cultivars that resist/tolerate abiotic and biotic stresses. In recent years the wide use of early maturing cultivars that escape drought stress led to significant increases in chickpea productivity. In the Mediterranean region, yield could be increased by shifting the sowing date from spring to winter. However, this is hampered by the sensitivity of the crop to low temperatures and the fungal pathogen Ascochyta rabiei. Drought, pod borer (Helicoverpa spp.) and the fungus Fusarium oxysporum additionally reduce harvests there and in other parts of the world. Tolerance to rising salinity will be a future advantage in many regions. Therefore, chickpea breeding focuses on increasing yield by pyramiding genes for resistance/tolerance to the fungi, to pod borer, salinity, cold and drought into elite germplasm. Progress in breeding necessitates a better understanding of the genetics underlying these traits. Marker-assisted selection (MAS) would allow a better targeting of the desired genes. Genetic mapping in chickpea, for a long time hampered by the little variability in chickpea’s genome, is today facilitated by highly polymorphic, co-dominant microsatellite-based markers. Their application for the genetic mapping of traits led to inter-laboratory comparable maps. This paper reviews the current situation of chickpea genome mapping, tagging of genes for ascochyta blight, fusarium wilt resistance and other traits, and requirements for MAS. Conventional breeding strategies to tolerate/avoid drought and chilling effects at flowering time, essential for changing from spring to winter sowing, are described. Recent approaches and future prospects for functional genomics of chickpea 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.

A population of 131 recombinant inbred lines from a wide cross between chickpea ( Cicer arietinum L., resistant parent) and Cicer reticulatum (susceptible parent) segregating for the closely linked resistances against Fusarium oxysporum f.sp. ciceri races 4 and 5 was used to develop DNA amplification fingerprinting markers linked to both resistance loci. Bulked segregant analysis revealed 19 new markers on linkage group 2 of the genetic map on which the resistance genes are located. Closest linkage (2.0 cM) was observed between marker R-2609-1 and the race 4 resistance locus. Seven other markers flanked this locus in a range from 4.1 to 9.0 cM. These are the most closely linked markers available for this locus up to date. The sequences of the linked markers were highly similar to genes encoding proteins involved in plant pathogen response, such as a PR-5 thaumatin-like protein and an important regulator of the phytoalexin pathway, anthranilate N-hydroxycinnamoyl-benzoyltransferase. Others showed significant alignments to genes encoding housekeeping enzymes such as the MutS2 DNA-mismatch repair protein. In the Arabidopsis genome, similar genes are located on short segments of chromosome 1 and 5, respectively, suggesting synteny between the fusarium resistance gene cluster of chickpea and the corresponding regions in the Arabidopsis genome. Three marker sequences were similar to retrotransposon-derived and/or satellite DNA sequences. The markers developed here provide a starting point for physical mapping and map-based cloning of the fusarium resistance genes and exploration of synteny in this highly interesting region of the chickpea genome.

Resistance of chickpea against the disease caused by the ascomycete Ascochyta rabiei is encoded by two or three quantitative trait loci, QTL1, QTL2 and QTL 3. A total of 94 recombinant inbred lines developed from a wide cross between a resistant chickpea line and a susceptible accession of Cicer reticulatum, a close relative of cultivated chickpea, was used to identify markers closely linked to QTL1 by DNA amplification fingerprinting in combination with bulked segregant analysis. Of 312 random 10mer oligonucleotides, 3 produced five polymorphic bands between the parents and bulks. Two of them were transferred to the population on which the recent genetic map of chickpea is based, and mapped to linkage group 4. These markers, OPS06-1 and OPS03-1, were linked at LOD-scores above 5 to markers UBC733B and UBC181A flanking the major ascochyta resistance locus. OPS06-1 mapped at the peak of the QTL between markers UBC733B (distance 4.1 cM) and UBC181A (distance 9.6 cM), while OPS03-1 mapped 25.1 cM away from marker UBC733B on the other flank of the resistance locus. STMS markers localised on this linkage group were transferred to the population segregating for ascochyta resistance. Three of these markers were closely linked to QTL1. Twelve of 14 STMS markers could be used in both populations. The order of STMS markers was essentially similar in both populations, with differences in map distances between them. The availability of flanking STMS markers for the major resistance locus QTL1 will help to elucidate the complex resistance against different Ascochyta pathotypes in future.

A genetic map was developed for the ascomyceteDidymella rabiei (Kovachevski) v. Arx (anamorph:Ascochyta rabiei Pass. Labr.), the causal agent of Ascochyta blight in chickpea (Cicer arietinum L.). The map was generated with 77 F1 progeny derived from crossing an isolate from the U.S.A. and an isolate from Syria. A total of 232 DAF (DNA Amplification Fingerprinting) primers and 37 STMS (Sequence-Tagged Microsatellite Site) primer pairs were tested for polymorphism between the parental isolates; 50 markers were mapped, 36 DAFs and 14 STMSs. These markers cover 261.4cM in ten linkage groups. Nineteen markers remained unlinked. Significant deviation from the expected 1:1 segregation ratios was observed for only two markers (Prob. of χ2

Resistance gene analogues (RGAs) of Cicer were isolated by different PCR approaches and mapped in an inter-specific cross segregating for fusarium wilt by RFLP and CAPS analysis. Initially, two pairs of degenerate primers targeting sequences encoded at nucleotide-binding sites (NBS), which are conserved in plant disease resistance genes such as RPS2, L6 and N, were selected for amplification. Cloning and sequence analysis of amplified products from C. arietinum DNA revealed eight different RGAs. Additionally, five RGAs were identified after characterisation of the presumptive RGA alleles from C. reticulatum. Therefore, a total of 13 different RGAs were isolated from Cicer and classified through pair-wise comparison into nine distinct classes with sequence similarities below a 68% amino acid identity threshold. Sequence comparison of seven RGA alleles of C. arietinum and C. reticulatum revealed polymorphisms in four RGAs with identical numbers of synonymous and non-synonymous substitutions. An NlaIII site, unique in the RGA-A allele of C. arietinum, was exploited for CAPS analysis. Genomic organisation and map position of the NBS-LRR candidate resistance genes was probed by RFLP analysis. Both single-copy as well as multi-copy sequence families were present for the selected RGAs, which represented eight different classes. Five RGAs were mapped in an inter-specific population segregating for three race-specific Fusarium resistances. All RGAs mapped to four of the previously established eight linkage groups for chickpea. Two NBS-LRR clusters were identified that could not be resolved in our mapping population. One of these clusters, which is characterised by RFLP probe CaRGA-D, mapped to the linkage group harbouring two of three Fusarium resistance genes characterised in the inter-specific population. Our study provides a starting point for the characterisation and genetic mapping of candidate resistance genes in Cicer that is useful for marker-assisted selection and as a pool for resistance genes of Cicer.

An integrated molecular marker map of the chickpea genome was established using 130 recombinant inbred lines from a wide cross between a cultivar resistant to fusarium wilt caused by Fusarium oxysporum Schlecht. emend. Snyd. &. Hans f. sp. ciceri (Padwick) Snyd & Hans, and an accession of Cicer reticulatum (PI 489777), the wild progenitor of chickpea. A total of 354 markers were mapped on the RILs including 118 STMSs, 96 DAFs, 70 AFLPs, 37 ISSRs, 17 RAPDs, eight isozymes, three cDNAs, two SCARs and three loci that confer resistance against different races of fusarium wilt. At a LOD-score of 4.0, 303 markers cover 2077.9 cM in eight large and eight small linkage groups at an average distance of 6.8 cM between markers. Fifty one markers (14.4%) were unlinked. A clustering of markers in central regions of linkage groups was observed. Markers of the same class, except for ISSR and RAPD markers, tended to generate subclusters. Also, genes for resistance to races 4 and 5 of fusarium wilt map to the same linkage group that includes an STMS and a SCAR marker previously shown to be linked to fusarium wilt race 1, indicating a clustering of several fusarium-wilt resistance genes around this locus. Significant deviation from the expected 1 : 1 segregation ratio was observed for 136 markers (38.4%, P

The conservation of 90 microsatellite-flanking sequences from chickpea in 39 accessions of eight annual and 1 accession of a perennial species of the genus Cicer was investigated. All of the primer sequences successfully amplified microsatellites in related species, indicating the conservation of microsatellite-flanking sequences in chickpea’s relatives. However, the degree of conservation of the primer sites varied between species depending on their known phylogenetic relationship to chickpea, ranging from 92.2% in C. reticulatum, chickpea’s closest relative and potential ancestor, down to 50% for C. cuneatum. A phylogenetic tree revealed that chickpea and the other members of its crossability group were more closely related to the perennial C. anatolicum than to other annual species of the genus. Considerable variation in size and number of amplification products between and within species was observed. Sequence analysis of highly divergent amplification products proved that variation is either due to large differences in the number of microsatellite repeats or to the amplification of a locus unrelated to the one amplified from chickpea. Sequence information and bootstrapping using PAUP suggested that STMSs derived from chickpea may be efficiently and reliably used for synteny studies in chickpea’s crossability group, including C. anatolicum. However, care should be taken when applying these markers to other species of the genus. Considering the data presented here and the known historical record, the age of section Monocicer, including chickpea, is estimated to be about 100,000 years.

Phylogenetic relationships within Bromeliaceae were assessed by comparative sequencing of chloroplast trnL(UAA) intronic DNA. Sixty-seven taxa were analyzed, encompassing 62 species, two subspecies and two varieties from 32 bromeliad genera. One representative of Rapateaceae and two of Velloziaceae were used as outgroups. Introns were amplified by the polymerase chain reaction and conserved flanking primers. Amplification products were sequenced directly. Of 700 base pairs total trnL (UAA) intron size, 551 bp could be unambiguously aligned. Sequence divergence among ingroup taxa ranged from zero to 4.6 %. Phylogenetic reconstruction by parsimony analysis of 73 informative base substitutions yielded 1092 most parsimonious trees. The topology of the resulting strict consensus tree was only partially consistent with current classification schemes. Brocchinia and Ayensua grouped close together and formed a well-supported basal clade, suggesting a sister group relationship of both to the remainder of the family. The remaining ingroup species were divided into an unresolved polytomy of three lineages, comprising (1) all members of subfamily Tillandsioideae, (2) the three investigated Hechtia species, and (3) all Bromelioideae and the remaining Pitcairnioideae (except Brocchinia, Ayensua and Hechtia). The latter clade was split into another polytomy of five sublineages, one of which comprised all Bromelioideae. Whereas monophyly of Tillandsioideae and Bromelioideae was supported by trnL intron data, Pitcairnioideae as traditionally circumscribed are clearly polyphyletic. There is evidence that the genera Tillandsia, Vriesea, Guzmania, Abromeitiella, Deuterocohnia, Dyckia, Pitcairnia and Aechmea in the present circumscription are not monophyletic.

A set of 12 randomly selected (TAA)n microsatellite loci of the cultivated chickpea (Cicer arietinum L.) were screened in a worldwide sample comprising 72 landraces, four improved cultivars and two wild species of the primary gene pool (C. reticulatum and C. echinosperum) to determine the level and pattern of polymorphism in these populations. A single fragment was amplified from all the accessions with each of 12 sequence-tagged microsatellite site markers, except for one locus where no fragment was obtained from either of the two wild species. There was a high degree of intraspecific polymorphism at these microsatellite loci, although isozymes, conventional RFLPs and RAPDs show very little or no polymorphism. Overall, the repeat number at a locus (excluding null alleles) ranged from 7 to 42. The average number of alleles per locus was 14.1 and the average genetic diversity was 0.86. Based on the estimates obtained, 11 out of the 12 frequency distributions of alleles at the loci tested can be considered to be non-normal. A significant positive correlation between the average number of repeats (size of the locus) and the amount of variation was observed, indicating that replication slippage may be the molecular mechanism involved in generation of variability at the loci. A comparison between the infinite allele and stepwise mutation models revealed that for 11 out of the 12 loci the number of alleles observed fell in between the values predicted by the two models. Phylogenetic analysis of microsatellite polymorphism in C. arietinum showed no relationship between accession and geographic origin, which is compatible with the recent expansion of this crop throughout the world.

Three major repetitive DNA sequences were isolated from a genomic library of chickpea (Cicer arietinum L.) and characterized with respect to their genomic organization and chromosomal localization. All repetitive elements are genus-specific and mostly located in the AT-rich pericentric heterochromatin. Two families are organized as satellite DNAs with repeat lengths of 162–168 bp (CaSat1) and 100 bp (CaSat2). CaSat1 is mainly located adjacent to the 18S rDNA clusters on chromosomes A and B, whereas CaSat2 is a major component of the pericentric heterochromatin on all chromosomes. The high abundance of these sequences in closely related species of the genus Cicer as well as their variation in structure and copy number among the annual species provide useful tools for taxonomic studies. The retrotransposon-like sequences of the third family (CaRep) display a more complex organization and are represented by two independent sets of clones (CaRep1 and CaRep2) with homology to different regions of Ty3-gypsy-like retrotransposons. They are distributed over the pericentric heterochromatin block on all chromosomes with extensions into euchromatic regions. Conserved structures within different crossability groups of related Cicer species suggest independent amplification or transposition events during the evolution of the annual species of the genus.

The poor definition of variation in the ascochyta blight fungus (Ascochyta rabiei) has historically hindered breeding for resistance to the chickpea (Cicer arietinum L.) blight disease in West Asia and North Africa. We have employed 14 RAPD markers and an oligonucleotide probe complementary to the microsatellite sequence (GATA)4 to construct a genotype-specific DNA fragment profile from periodically sampled Syrian field isolates of this fungus. By using conventional pathogenicity tests and genome analysis with RAPD and microsatellite markers, we demonstrated that the DNA markers distinguish variability within and among the major pathotypes of A. rabiei and resolved each pathotypes into several genotypes. The genetic diversity estimate based on DNA marker analysis within pathotypes was highest for the least-aggressive pathotype (pathotype I), followed by the aggressive (pathotype II) and the most-aggressive pathotype (pathotype III). The pair-wise genetic distance estimated for all the isolates varied from 0.00 to 0.39, indicating a range from a clonal to a diverse relationship. On the basis of genome analysis, and information on the spatial and temporal distribution of the pathogen, a general picture of A. rabiei evolution in Syria is proposed.

The genome of the fungal chickpea pathogen Ascochyta rabiei was screened for polymorphisms by microsatellite-primed PCR. While ethidium-bromide staining of electrophoretically separated amplification products showed only limited polymorphism among 24 Tunisian A. rabiei isolates, Southern hybridization of purified PCR fragments to restriction digests of fungal DNA revealed polymorphic DNA fingerprints. One particular probe that gave rise to a hypervariable single-locus hybridization signal was cloned from the Syrian isolate AA6 and sequenced. It contained a large compound microsatellite harbouring the penta- and decameric repeat units (CATTT)n, (CATTA)n, (CATATCATTT)n and (TATTT)n. We call this locus ArMS1 (Ascochyta rabiei microsatellite 1). Unique flanking sequences were used to design primer pairs for locus- specific microsatellite amplification and direct sequencing of additional ArMS1 alleles from Tunisian and Pakistani isolates. A high level of sequence variation was observed, suggesting that multiple mutational mechanisms have contributed to polymorphism. Hybridization and PCR analyses were performed on the parents and 62 monoascosporic F1 progeny derived from a cross between two different mating types of the fungus. Progeny alleles could be traced back to the parents, with one notable exception, where a longer than expected fragment was observed. Direct sequencing of this new length allele revealed an alteration in the copy number of the TATTT repeat [(TATTT)53 to (TATTT)65], while the remainder of the sequence was unchanged.

DNA fingerprinting with a set of synthetic oligonucleotides complementary to simple repetitive sequences was used to develop molecular markers for Ascochyta rabiei , the most important fungal pathogen of chickpea ( Cicer arietinum ). Two compatible mating type isolates (MatI and MatII) from the U.S. Pacific Northwest with the same low level of aggressivity were compared to highly virulent isolates from the Mediterranean region and Pakistan to find suitable mating partners for the production of a mapping population. After Hinf I or Taq I restriction, electrophoresis and in-gel hybridization with ten different simple repetitive oligonucleotides, all tested single-spored isolates exhibited unique fingerprint patterns. The analysis revealed that the two U.S. mating types share a considerable amount of genetic variability. A total of 77 polymorphic marker bands were detected. A higher number of polymorphic bands (up to 104) was observed between these isolates and those from different geographical regions. The isolates from the Mediterranean region and Pakistan shared a lower degree (between 80 and 90 bands) of detectable genetic diversity. These data permit selection of highly virulent crossing partners for the different mating types with a high degree of detectable polymorphism. A sexual cross was performed to prove the Mendelian segregation of fingerprint bands for future linkage analysis. Additionally, the fingerprint data based on 268 informative characters combined with phenetic and phylogenetic algorithms allow determination of the genetic identity, relatedness and diversity of the different isolates. To confirm the phylogenetic data, two outgroupers Ascochyta fabae and Ascochyta pisi , were included. Results indicate that A. pisi is more closely related to A. rabiei than A. fabae .

DNA fingerprinting with synthetic simple repetitive oligonucleotides such as (CA) 8 or (CAA) 5 detected polymorphisms between various isolates of the ascomycete Mycosphaerella fijiensis, the causal agent of the black Sigatoka disease of Musa. These microsatellite motifs are present at multiple chromosomal locations and in high copy numbers in the Mycosphaerella genome, generating informative fingerprints with low background. Variability exists on a macro- as well as a microgeographical scale: it occurred within one lesion, between lesions of one plant, between plants, cultivars, and geographic locations. Mathematical analysis of the data produced dendrograms that demonstrated the presence of different genetically related groups of Mycosphaerella fijiensis in Nigeria.

Legumes are important food crops worldwide, contributing to more than 33% of human dietary protein. The production of crop legumes is frequently impacted by abiotic and biotic stresses. It is therefore important to identify genes conferring resistance to biotic stresses and tolerance to abiotic stresses that can be used to both understand molecular mechanisms of plant response to the environment and to accelerate crop improvement. Recent advances in genomics offer a range of approaches such as the sequencing of genomes and transcriptomes, gene expression microarray as well as RNA-seq based gene expression profiling, and map-based cloning for the identification and isolation of biotic and abiotic stress-responsive genes in several crop legumes. These candidate stress associated genes should provide insights into the molecular mechanisms of stress tolerance and ultimately help to develop legume varieties with improved stress tolerance and productivity under adverse conditions. This review provides an overview on recent advances in the functional genomics of crop legumes that includes the discovery as well as validation of candidate genes.

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. Copyright © 2013 Nature America, Inc.

We have used random amplified polymorphic DNA (RAPD) analysis to characterize eleven cultivars of the five economically most important yam species grown in Jamaica (Dioscorea alata, D. cayenensis, D. rotundata, D. trifida and D. esculenta). Amplification of genomic DNA samples with nine different arbitrary 10mer primers revealed a total of 338 different band positions, ranging in size from 0.3 to 2.5 kb. RAPD patterns proved to be highly reproducible and somatically stable. While no variation was observed among plants belonging to the same cultivar, a large number of intervarietal and interspecific polymorphisms enabled us to reliably discriminate between all Jamaican cultivars investigated.

The abundance and polymorphism of 38 different simple-sequence repeat motifs was studied in four accessions of cultivated chickpea (Cicer arietinum L.) by in-gel hybridization of synthetic oligonucleotides to genomic DNA digested with 14 different restriction enzymes. Among 38 probes tested, 35 yielded detectable hybridization signals. The abundance and level of polymorphism of the target sequences varied considerably. The probes fell into three broad categories: (1) probes yielding distinct, polymorphic banding patterns; (2) probes yielding distinct, monomorphic banding patterns, and (3) probes yielding blurred patterns, or diffused bands superimposed on a high in lane background. No obvious correlation existed between abundance, fingerprint quality, and the sequence characteristics of a particular motif. Digestion with methyl-sensitive enzymes revealed that simple-sequence motifs are enriched in highly methylated genomic regions. The high level of intraspecific polymorphism detected by oligonucleotide fingerprinting suggests the suitability of simple-sequence repeat probes as molecular markers for genome mapping.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; Common bean (Phaseolus vulgaris L.) is often subject to various environmental constraints including soil salinity and phosphorus deficiency as major limitations for the yield of most grain legumes, especially when the plant growth depends upon N-2 fixation. In order to assess the genetic variation for tolerance to moderate salinity and phosphorus deficiency and identify the related morphological, physiological and genetic traits, 37 common bean recombinant inbred lines (RILs) were inoculated with Rhizobium tropici CIAT899, and grown in a glasshouse with 25 mM NaCl or 75 mu mol P plant(-1) week(-1), compared to optimal nutrient solution in hydroaeroponic culture system. Large genotypic variation in tolerance to P deficiency and salt was found with some RILs being tolerant to both constraints. By contrast some of the RILs showed tolerance to only one constraint while the most sensitive to salinity were also sensitive to P-deficiency. By using 18 microsatellite primer-pairs with six most contrasting RILs, 4 alleles were found to discriminate among the RILs. It is concluded that these genotypes and the microsatellites primers can be used to identify genes involved in salinity and P deficiency tolerance of N-2-dependent legume.

SixBrassica species, known as the “triangle of U”, and four species from related genera were characterized by DNA fingerprinting with simple repetitive oligonucleotide probes. Our results show that CT-, TCC-, and GTG-repeat motifs are equally abundant in the genomes of the sixBrassica species. In contrast, GATA-, GGAT-, and GACA-multimers are unevenly distributed among different species. As judged from the number and strength of hybridization signals, the highest copy number of all three motifs occurs inBrassica nigra, while the lowest is observed inB. oleracea. The abundance of GATA-and GACA-repeats varies in a coordinate way. The amphidiploid genomes ofB. juncea, B. carinata, andB. napus each harbour intermediate amounts of (GATA)4 and (GACA)4-detected repeats as compared to their diploid progenitors, thus supporting the concept of the “U triangle”. GATA-, GACA-, and GGAT-repeats were also abundant inEruca sativa andSinapis arvensis, but not inRaphanus sativus andSinapis alba. These results support the idea thatBrassica nigra is more closely related toSinapis arvensis than to otherBrassica species such asB. rapa andB. oleracea.

The distribution and abundance of simple repetitive sequences complementary to the synthetic oligonucleotides (GACA)4, (GATA)4, (GTG)5 and (CA)8 in the genomes of several cultivars of Beta vulgaris and in the wild beet B. vulgaris ssp. maritima were investigated. Hybridization experiments revealed that all four motifs were present, though at different abundances, in the genomes of all of the investigated beet cultivars. Considerable intraspecific variation of the resulting DNA fingerprints was observed. The extent of polymorphism depends on the oligonucleotide probe. The most informative banding patterns were obtained with the (GATA)4 probe hybridized to HinfI-, HaeIII-, or RsaI-restricted DNA, respectively. DNA fingerprinting with (GATA)4 allowed a clear differentiation of double-haploid breeding lines (DH lines). We demonstrated that the application of oligonucleotide probes for DNA fingerprinting is a sensitive tool for genome diagnosis in cultivated beet.

Yellow Sigatoka caused by the ascomycete Mycosphaerella musicola Leach, is one of the most severe banana diseases worldwide, which spread in most banana growing areas, until Black Sigatoka, a more aggressive disease caused by Mycosphaerella fijiensis, appeared. Because of the highly devastating nature of the latter pathogen, recent research almost exclusively focused on M. fijiensis. To close the gap of knowledge and to study the population structure of M. musicola in Yellow Sigatoka-infested areas, we cloned and characterized a versatile set of 26 polymorphic locus-specific microsatellite markers.

Chickpea (Cicer arietinum L.) is a grain legume of world importance (average annual production approximately 8,500,0001) ranking third after pea and dry bean on the world market (Duke 1981; FAOSTAT 2000). Major production areas are the Indian subcontinent, West Asia and North Africa (WANA), European countries surrounding the Mediterranean basin, and Latin America. In these regions, chickpea belongs to the traditional diet of the people, and serves as animal fodder as well. In recent years chickpea production in the USA, Australia, and Canada has also increased and provides a growing surplus for export. In all these areas, chickpea is an important component of rain-fed, cereal-based intercropping systems.

With the exception of the fact that it is made up of eight different chromosomes, the physical organization of the 738-Mb genome of the important legume crop chickpea (Cicer arietinum L.) is unknown. In an attempt to increase our knowledge of the basic structure of this genome, we determined the map positions of a series of genes involved in plant defence responses (DR) by genetic linkage analysis. Exploiting the sequence data available in GenBank, we selected genes known to be induced in chickpea and other plants by pathogen attack. Gene-specific primers were designed based on conserved regions, and used to detect the corresponding gene sequences in a segregating population derived from an interspecific cross between Cicer arietinum and C. reticulatum. Forty-seven gene-specific markers were integrated into an existing map based on STMS, AFLP, DAF and other anonymous markers. The potential of this approach is discussed.

Numerous molecular markers, linked with traits of agronomic importance in the food legumes, pea, chickpea and lentil, have been identified. Microsatellite markers are being developed and mapped in a collaborative effort between the International Center for Agricultural Research in the Dry Areas (ICARDA) and the University of Frankfurt to overcome the relatively low amount of information that can be derived from the widely used dominant markers in chickpea. Besides mapping and identifying host plant resistance, efforts are being made to characterize the pathogen populations. Once host plant resistance has been identified and mapped, it will be feasible to deploy the relevant resistance genes when shifts occur in the pathogen population. The technology for using these markers in marker-assisted selection (MAS) has been greatly improved. The ability to use MAS to pyramid genes will make this technology an essential tool for legume breeders.

A size-selected genomic library comprising 280,000 colonies and representing approximately 18% of the chickpea genome, was screened for (GA)n, (GAA)n and (TAA)n microsatellite-containing clones, of which 389 were sequenced. The majority (approximately 75%) contained perfect repeats; interrupted, interrupted compound and compound repeats were only present in 6%-9% of cases. (TAA)-microsatellites contained the longest repeats, with unit numbers from 9 to 131. For 218 loci primers could be designed and used for the detection of microsatellite length polymorphisms in six chickpea breeding cultivars, as well as in C. reticulatum and C. echinospermum, wild, intercrossable relatives of chickpea. A total of 174 primer pairs gave interpretable banding patterns, 137 (79%) of which revealed at least two alleles on native polyacrylamide gels. A total of 120 sequence-tagged microsatellite site (STMS) markers were genetically mapped in 90 recombinant inbred lines from an inter-species cross between C. reticulatum and the chickpea cultivar ICC 4958. Markers could be arranged in 11 linkage groups (at a LOD score of 4) covering 613 cM. Clustering as well as random distribution of loci was observed. Segregation of 46 markers (39%) deviated significantly (Por = 0.05) from the expected 1:1 ratio. The majority of these loci (73%) were located in three distinct regions of the genome. The present STMS marker map represents the most advanced co-dominant DNA marker map of the chickpea genome.

During the past decade, the molecular mechanisms of crown gall and hairy root development have been elucidated in considerable detail. It now appears that the genetic ‘colonization’ of plant cells by Agrobacterium evolved by continual adaptation of groups of genes that existed long before the evolution of this plant-microbe association. This is most evident for the signal transduction system leading to vir gene induction, and for the early steps of T-DNA transfer to plant cells which have probably evolved from the bacterial conjugation and protein export machinery. However, the later steps, i.e. nuclear targeting of the T-DNA-protein complex, and integration into the host genome by illegitimate recombination are reminiscent of viral infection, where the T-complex resembles a viral particle. The present article reviews the current knowledge of the molecular basis of crown gall and hairy root tumorigenesis, with some emphasis on the mechanisms of signal exchange between plants and bacteria, as well as of T-DNA excision, transfer, integration and expression.

Brassica napus plants, artificially synthesized through somatic hybridization of B. oleracea and B. campestris protoplasts, were analyzed by oligonucleotide fingerprinting. While the fingerprint patterns of the different hybrid plants looked very much alike, they did not simply represent a combination of the parental patterns. Instead, the absence of parental bands as well as the presence of new bands suggest that elimination and/or rearrangements occurred during or after the fusion of the two genomes. The fingerprints of individual F1 progeny plants of selfed hybrids did not detect major changes. Thus, once formed, the artificially resynthesized amphidiploid B. napus genome appears to be stable. Taken together, our experiments demonstrate the usefulness of oligonucleotide fingerprinting for the characterization of artificial hybrids in the genus Brassica.

The technique of DNA fingerprinting is frequently used for studies of genetic diversity and relatedness in a wide range of organisms. In humans and animals, multilocus fingerprints are mainly applied to paternity and identity test cases, behavioral ecology, and the analysis of population structures. In plants and fungi, the frequent occurrence of “low-variability” fingerprint patterns additionally allows to use multilocus fingerprinting for studying taxonomical problems at an intraspecific level. In the present article, we (1) present an overview of such approaches in a series of plant species, (2) summarize our attempts to estimate genetic relationships within two cultivated plant species, banana and tomato, by band sharing data derived from oligonucleotide fingerprints, and (3) discuss the limitations and potentials of multilocus fingerprinting for the determination of genetic relatedness.

The potential of plant gene technology encompasses a multitude of different techniques ranging from the isolation of useful genes, their characterization and in vitro manipulation to the reintroduction of the modified constructs into target plants, where they are expressed at a rate that alters the phenotype of the plants. Genome analysis, on the other hand, aims at characterizing the genome architecture and function(s).

Nucleosomes were isolated from chromatin of suspension cultured cells of Nicotiana tabacum var. White Burley, which were either habituated or transformed by Agrobacterium tumefaciens, strain T37. Chromatin repeat length in both types of tissue was identical and can be estimated to be 195 ± 10 bp. Using Southern transfer of nucleosomal DNA and hybridization with cloned nick-translated HindIII fragments of pTi C58 we show that the T-DNA originating from the Ti-plasmid of A. tumefaciens is organized in nucleosomes within the chromatin of crown gall tumor cells.