Boulos Chalhoub

Boulos Chalhoub Portrait

+33 1 60 87 45 03

Comparative Genomics:
Organization and Evolution of Plant Genomes (OEPG)
Genomic duplications and polyploidy
Wheat Projects
Cloning of the hexaploid wheat genome into BAC libraries
Identification of the gene-containing islands of the wheat genome.
Effects of polyploidy and domestication on the evolution of the wheat (Triticum and Aegilops) genomes
Organization and Evolution of the Hardness (Ha) locus
Organization and evolution of homologs of resistance genes in the wheat genome
Effect of polyploidy on plant genome biodiversity and evolution
Effect of transposable elements on regulation of gene expression
Drought stress

Introduction to the brassica napus evolution
Preparation of physical resources
Global Comparative Genomics with A. thaliana and Brassica species (I)
Global Comparative Genomics with A. thaliana and Brassica species (II)
Forward comparative genomics: homologs of flavonoid biosynthesis genes
Reverse comparative genomics: characterization and cloning of B. napus genes
Determination of mechanisms (molecular basis) of genomic rearrangements in the B.napus synthetic polyploids
Other collaborative genome projects
OEPG Publications
Funded Project and Consortia

Scientific Career

1990 MSc Degree in Genetics
Ecole Nationale Supérieure Agronomique (ENSA), Toulouse, France
1994 PhD in Biology, Plant Genetics and Pathology
Institut National Polytechnique de Toulouse (INPT), France
(PhD diploma awarded with the French equivalent of summa cum lauda)
Molecular evolution of the genomes of cereal viruses / genetics of resistance of barley (Hordeum vulgare, L.) to the barley yellow dwarf virus (BYDV-PAV).
1995 - 1996 Postdoctoral research scientist I
"Laboratory of Genetics and plant breeding", INRA-Versailles, France.
Subject: Molecular mapping of genes of resistance in peas to the pea seed borne mosaic potyvirus (PSbMV).
1997 Postdoctoral research scientist II
"Laboratory of cell biology", INRA-Versailles, France.
Subject: Activation of the tobacco mobile retrotransposon (Tnt1) and its use in gene tagging and molecular mapping.
1998 - 1999 Project leader in Genomics and plant Biotechnology
Laboratory of Plant Genetcis and Biotechnology, Monsanto SAS, Toury, France
Since Oct 1999 Projects and Group Leader
Group of Organization and evolution of plant genomes, INRA-URGV, France
2005 HDR: Habilitation to direct research diploma
("Habilitation à diriger des recherches") Evry University, France



2014          2013         2012         2012         2010         2009         2008         2007         2006         2005         2004         2003         2001


Meiotic gene evolution: can you teach a new dog new tricks?

Mol Biol Evol. 2014 Apr 1. PMID: 24694832
Lloyd A, Ranoux M, Vautrin S, Glover N, Fourment J, Charif D, Choulet F, Lassalle G, Marande W, Tran J, Granier F, Pingault L, Remay A, Marquis C, Belcram H, Chalhoub B, Feuillet C, Bergès H, Sourdille P, Jenczewski E.

Meiosis, the basis of sex, evolved through iterative gene duplications. To understand whether subsequent duplications have further enriched the core meiotic "tool-kit", we investigated the fate of meiotic gene duplicates following Whole Genome Duplication (WGD), a common occurrence in eukaryotes. We show that meiotic genes return to a single copy more rapidly than genome-wide average in Angiosperms, one of the lineages in which WGD is most vividly exemplified. The rate at which duplicates are lost decreases through time, a tendency that is also observed genome-wide and may thus prove to be a general trend post-WGD. The sharpest decline is observed for the subset of genes mediating meiotic recombination; however, we found no evidence that the presence of these duplicates is counter-selected in two recent polyploid crops selected for fertility. We therefore propose that their loss is passive, highlighting how quickly WGDs are resolved in the absence of selective duplicate retention.

Sixteen cytosolic glutamine synthetase genes identified in the Brassica napus L. genome are differentially regulated depending on nitrogen regimes and leaf senescence.

J Exp Bot. 2014 Feb 24. PMID: 24567494
Orsel M, Moison M, Clouet V, Thomas J, Leprince F, Canoy AS, Just J, Chalhoub B, Masclaux-Daubresse C.

A total of 16 BnaGLN1 genes coding for cytosolic glutamine synthetase isoforms (EC were found in the Brassica napus genome. The total number of BnaGLN1 genes, their phylogenetic relationships, and genetic locations are in agreement with the evolutionary history of Brassica species. Two BnaGLN1.1, two BnaGLN1.2, six BnaGLN1.3, four BnaGLN1.4, and two BnaGLN1.5 genes were found and named according to the standardized nomenclature for the Brassica genus. Gene expression showed conserved responses to nitrogen availability and leaf senescence among the Brassiceae tribe. The BnaGLN1.1 and BnaGLN1.4 families are overexpressed during leaf senescence and in response to nitrogen limitation. The BnaGLN1.2 family is up-regulated under high nitrogen regimes. The members of the BnaGLN1.3 family are not affected by nitrogen availability and are more expressed in stems than in leaves. Expression of the two BnaGLN1.5 genes is almost undetectable in vegetative tissues. Regulations arising from plant interactions with their environment (such as nitrogen resources), final architecture, and therefore sink-source relations in planta, seem to be globally conserved between Arabidopsis and B. napus. Similarities of the coding sequence (CDS) and protein sequences, expression profiles, response to nitrogen availability, and ageing suggest that the roles of the different GLN1 families have been conserved among the Brassiceae tribe. These findings are encouraging the transfer of knowledge from the Arabidopsis model plant to the B. napus crop plant. They are of special interest when considering the role of glutamine synthetase in crop yield and grain quality in maize and wheat.



A Dominant Point Mutation in a RINGv E3 Ubiquitin Ligase Homoeologous Gene Leads to Cleistogamy in Brassica napus.

Plant Cell. 2012 Dec 31 PMID: 23277363
Lu YH, Arnaud D, Belcram H, Falentin C, Rouault P, Piel N, Lucas MO, Just J, Renard M, Delourme R, Chalhoub B.

In the allopolyploid Brassica napus, we obtained a petal-closed flower mutation by ethyl methanesulfonate mutagenesis. Here, we report cloning and characterization of the Bn-CLG1A (CLG for cleistogamy) gene and the Bn-clg1A-1D mutant allele responsible for the cleistogamy phenotype. Bn-CLG1A encodes a RINGv E3 ubiquitin ligase that is highly conserved across eukaryotes. In the Bn-clg1A-1D mutant allele, a C-to-T transition converts a Pro at position 325 to a Leu (P325L), causing a dominant mutation leading to cleistogamy. B. napus and Arabidopsis thaliana plants transformed with a Bn-clg1A-1D allele show cleistogamous flowers, and characterization of these flowers suggests that the Bn-clg1A-1D mutation causes a pronounced negative regulation of cutin biosynthesis or loading and affects elongation or differentiation of petal and sepal cells. This results in an inhibition or a delay of petal development, leading to folded petals. A homoeologous gene (Bn-CLG1C), which shows 99.5% amino acid identity and is also constitutively and equally expressed to the wild-type Bn-CLG1A gene, was also identified. We showed that P325L is not a loss-of-function mutation and did not affect expression of Bn-clg1A-1D or Bn-CLG1C. Our findings suggest that P325L is a gain-of-function semidominant mutation, which led to either hyper- or neofunctionalization of a redundant homoeologous gene.

Prevalence of gene expression additivity in genetically stable wheat allohexaploids.

New Phytol. 2012 Dec 21. doi: 10.1111/nph.12108 PMID: 23278496
Chelaifa H, Chagué V, Chalabi S, Mestiri I, Arnaud D, Deffains D, Lu Y, Belcram H, Huteau V, Chiquet J, Coriton O, Just J, Jahier J, Chalhoub B.

The reprogramming of gene expression appears as the major trend in synthetic and natural allopolyploids where expression of an important proportion of genes was shown to deviate from that of the parents or the average of the parents. In this study, we analyzed gene expression changes in previously reported, highly stable synthetic wheat allohexaploids that combine the D genome of Aegilops tauschii and the AB genome extracted from the natural hexaploid wheat Triticum aestivum. A comprehensive genome-wide analysis of transcriptional changes using the Affymetrix GeneChip Wheat Genome Array was conducted. Prevalence of gene expression additivity was observed where expression does not deviate from the average of the parents for 99.3% of 34 820 expressed transcripts. Moreover, nearly similar expression was observed (for 99.5% of genes) when comparing these synthetic and natural wheat allohexaploids. Such near-complete additivity has never been reported for other allopolyploids and, more interestingly, for other synthetic wheat allohexaploids that differ from the ones studied here by having the natural tetraploid Triticum turgidum as the AB genome progenitor. Our study gave insights into the dynamics of additive gene expression in the highly stable wheat allohexaploids.


Duplication and partitioning in evolution and function of homoeologous Q loci governing domestication characters in polyploid wheat.

Zhang Z, Belcram H, Gornicki P, Charles M, Just J, Huneau C, Magdelenat G, Couloux A, Samain S, Gill BS, Rasmussen JB, Barbe V, Faris JD, Chalhoub B.
Proc Natl Acad Sci U S A. 2011 Oct 31. PMID: 22042872

The Q gene encodes an AP2-like transcription factor that played an important role in domestication of polyploid wheat. The chromosome 5A Q alleles (5AQ and 5Aq) have been well studied, but much less is known about the q alleles on wheat homoeologous chromosomes 5B (5Bq) and 5D (5Dq). We investigated the organization, evolution, and function of the Q/q homoeoalleles in hexaploid wheat (Triticum aestivum L.). Q/q gene sequences are highly conserved within and among the A, B, and D genomes of hexaploid wheat, the A and B genomes of tetraploid wheat, and the A, S, and D genomes of the diploid progenitors, but the intergenic regions of the Q/q locus are highly divergent among homoeologous genomes. Duplication of the q gene 5.8 Mya was likely followed by selective loss of one of the copies from the A genome progenitor and the other copy from the B, D, and S genomes. A recent V(329)-to-I mutation in the A lineage is correlated with the Q phenotype. The 5Bq homoeoalleles became a pseudogene after allotetraploidization. Expression analysis indicated that the homoeoalleles are coregulated in a complex manner. Combined phenotypic and expression analysis indicated that, whereas 5AQ plays a major role in conferring domestication-related traits, 5Dq contributes directly and 5Bq indirectly to suppression of the speltoid phenotype. The evolution of the Q/q loci in polyploid wheat resulted in the hyperfunctionalization of 5AQ, pseudogenization of 5Bq, and subfunctionalization of 5Dq, all contributing to the domestication traits.

The genome of the mesopolyploid crop species Brassica rapa.

The Brassica rapa Genome Sequencing Project Consortium, Wang X, Wang H, Wang J, Sun R, Wu J, Liu S, Bai Y, Mun JH, Bancroft I, Cheng F, Huang S, Li X, Hua W, Wang J, Wang X, Freeling M, Pires JC, Paterson AH, Chalhoub B, Wang B, Hayward A, Sharpe AG, Park BS, Weisshaar B, Liu B, Li B, Liu B, Tong C, Song C, Duran C, Peng C, Geng C, Koh C, Lin C, Edwards D, Mu D, Shen D, Soumpourou E, Li F, Fraser F, Conant G, Lassalle G, King GJ, Bonnema G, Tang H, Wang H, Belcram H, Zhou H, Hirakawa H, Abe H, Guo H, Wang H, Jin H, Parkin IA, Batley J, Kim JS, Just J, Li J, Xu J, Deng J, Kim JA, Li J, Yu J, Meng J, Wang J, Min J, Poulain J, Wang J, Hatakeyama K, Wu K, Wang L, Fang L, Trick M, Links MG, Zhao M, Jin M, Ramchiary N, Drou N, Berkman PJ, Cai Q, Huang Q, Li R, Tabata S, Cheng S, Zhang S, Zhang S, Huang S, Sato S, Sun S, Kwon SJ, Choi SR, Lee TH, Fan W, Zhao X, Tan X, Xu X, Wang Y, Qiu Y, Yin Y, Li Y, Du Y, Liao Y, Lim Y, Narusaka Y, Wang Y, Wang Z, Li Z, Wang Z, Xiong Z, Zhang Z.
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (IVF, CAAS), Beijing, China
Nat Genet. 2011 Aug 28. doi: 10.1038/ng.919. PMID: 21873998

We report the annotation and analysis of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. We modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. We used Arabidopsis thaliana as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the number of members of gene families present in the genome may contribute to the remarkable morphological plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops.

Qualitative and quantitative resistances to leaf rust finely mapped within two nucleotide-binding site leucine-rich repeat (NBS-LRR)-rich genomic regions of chromosome 19 in poplar.

Bresson A, Jorge V, Dowkiw A, Guerin V, Bourgait I, Tuskan GA, Schmutz J, Chalhoub B, Bastien C, Faivre Rampant P.
INRA, UMR1165, UMR INRA/Université de Evry: Unité de Recherche en Génomique Végétale, Centre de Recherche de Versailles-Grignon, Evry Cedex, 91057, France INRA, UR0588, Unité de Recherche Amélioration, Génétique et Physiologie Forestières, Centre de Recherche d'Orléans, Orléans Cedex 2, 45075, France Oak Ridge National Laboratory, PO Box 2008, MS-6422, Bldg. 1062, Rm 215, Oak Ridge, TN 37831-6422, USA Hudson Alpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL 3508-2908, USA

Abstract • R (US) is a major dominant gene controlling quantitative resistance, inherited from Populus trichocarpa, whereas R(1) is a gene governing qualitative resistance, inherited from P. deltoides. • Here, we report a reiterative process of concomitant fine-scale genetic and physical mapping guided by the P. trichocarpa genome sequence. The high-resolution linkage maps were developed using a P. deltoides × P. trichocarpa progeny of 1415 individuals. R(US) and R(1) were mapped in a peritelomeric region of chromosome 19. Markers closely linked to R(US) were used to screen a bacterial artificial chromosome (BAC) library constructed from the P. trichocarpa parent, heterozygous at the locus R(US) . • Two local physical maps were developed, one encompassing the R(US) allele and the other spanning r(US) . The alignment of the two haplophysical maps showed structural differences between haplotypes. The genetic and physical maps were anchored to the genome sequence, revealing genome sequence misassembly. Finally, the R(US) locus was localized within a 0.8-cM interval, whereas R(1) was localized upstream of R(US) within a 1.1-cM interval. • The alignment of the genetic and physical maps with the local reorder of the chromosome 19 sequence indicated that R(US) and R(1) belonged to a genomic region rich in nucleotide-binding site leucine-rich repeat (NBS-LRR) and serine threonine kinase (STK) genes.
© 2011 INRA. New Phytologist © 2011 New Phytologist Trust.

Alteration in expression of hormone-related genes in wild emmer wheat roots associated with drought adaptation mechanisms.

Funct Integr Genomics. 2011 Jun 8. PMID: 21656015
Krugman T, Peleg Z, Quansah L, Chagué V, Korol AB, Nevo E, Saranga Y, Fait A, Chalhoub B, Fahima T.
Department of Evolutionary and Environmental Biology, Institute of Evolution, Faculty of Natural Sciences, University of Haifa, Mt. Carmel, Haifa, 31905, Israel

Transcriptomic and metabolomic profiles were used to unravel drought adaptation mechanisms in wild emmer wheat (Triticum turgidum ssp. dicoccoides), the progenitor of cultivated wheat, by comparing the response to drought stress in roots of genotypes contrasting in drought tolerance. The differences between the drought resistant (R) and drought susceptible (S) genotypes were characterized mainly by shifts in expression of hormone-related genes (e.g., gibberellins, abscisic acid (ABA) and auxin), including biosynthesis, signalling and response; RNA binding; calcium (calmodulin, caleosin and annexin) and phosphatidylinositol signalling, in the R genotype. ABA content in the roots of the R genotype was higher in the well-watered treatment and increased in response to drought, while in the S genotype ABA was invariant. The metabolomic profiling revealed in the R genotype a higher accumulation of tricarboxylic acid cycle intermediates and drought-related metabolites, including glucose, trehalose, proline and glycine. The integration of transcriptomics and metabolomics results indicated that adaptation to drought included efficient regulation and signalling pathways leading to effective bio-energetic processes, carbon metabolism and cell homeostasis. In conclusion, mechanisms of drought tolerance were identified in roots of wild emmer wheat, supporting our previous studies on the potential of this genepool as a valuable source for novel candidate genes to improve drought tolerance in cultivated wheat.

The impact of Ty3-gypsy group LTR retrotransposons Fatima on B-genome specificity of polyploid wheats.

BMC Plant Biol. 2011 Jun 3;11(1):99. PMID: 21635794
Salina EA, Sergeeva EM, Adonina IG, Shcherban AB, Belcram H, Huneau C, Chalhoub B.

BACKGROUND: Transposable elements (TEs) are a rapidly evolving fraction of the eukaryotic genomes and the main contributors to genome plasticity and divergence. Recently, occupation of the A- and D-genomes of allopolyploid wheat by specific TE families was demonstrated. Here, we investigated the impact of the well-represented family of gypsy LTR-retrotransposons, Fatima, on B-genome divergence of allopolyploid wheat using the fluorescent in situ hybridisation (FISH) method and phylogenetic analysis.
RESULTS: FISH analysis of a BAC clone (BAC_2383A24) initially screened with Spelt1 repeats demonstrated its predominant localisation to chromosomes of the B-genome of allopolyploid wheat and its putative diploid progenitor Aegilops speltoides. Analysis of the complete BAC_2383A24 nucleotide sequence (113 605 bp) demonstrated that it contains 55.6% TEs, 0.9% subtelomeric tandem repeats (Spelt1), and five genes. LTR retrotransposons are predominant, representing 50.7% of the total nucleotide sequence. Three elements of the gypsy LTR retrotransposon family Fatima make up 47.2% of all the LTR retrotransposons in this BAC. In situ hybridisation of the Fatima_2383A24-3 subclone suggests that individual representatives of the Fatima family contribute to the majority of the B-genome specific FISH pattern for BAC_2383A24. Phylogenetic analysis of various Fatima elements available from databases in combination with the data on their insertion dates demonstrated that the Fatima elements fall into several groups. One of these groups, containing Fatima_2383A24-3, is more specific to the B-genome and proliferated around 0.5-2.5 MYA, prior to allopolyploid wheat formation.
CONCLUSION: The B-genome specificity of the gypsy-like Fatima, as determined by FISH, is explained to a great degree by the appearance of a genome-specific element within this family for Ae. speltoides. Moreover, its proliferation mainly occurred in this diploid species before it entered into allopolyploidy. Most likely, this scenario of emergence and proliferation of the genome-specific variants of retroelements, mainly in the diploid species, is characteristic of the evolution of all three genomes of hexaploid wheat.

The Ma gene for complete-spectrum resistance to Meloidogyne spp. in Prunus is a TNL with a huge repeated C-terminal post-LRR region.

Plant Physiol. 2011 Apr 11, PMID: 21482634
Claverie M, Dirlewanger E, Bosselut N, Van Ghelder C, Voisin R, Kleinhentz M, Lafargue B, Abad P, Rosso MN, Chalhoub B, Esmenjaud D.

Root-knot nematodes (RKN) Meloidogyne spp. are major polyphagous pests of most crops world-wide and cultivars with durable resistance are urgently needed because of nematicide ban. The Ma gene from the Prunus cerasifera plum confers complete-spectrum, heat-stable and high-level resistance to RKN, which is remarkable in comparison with the Mi-1 gene from tomato, the sole RKN resistance gene cloned. We report here the positional cloning and the functional validation of the Ma locus present at the heterozygous status in the P.2175 accession. High resolution mapping totalling over 3000 segregants reduced the Ma locus interval to a 32-kb cluster of three TIR-NBS-LRR genes (TNL1 to TNL3) including a pseudogene (TNL2) and a truncated gene (TNL3). The sole complete gene in this interval (TNL1) was validated as Ma as it conferred the same complete-spectrum and high-level resistance (as in P.2175) using its genomic sequence and native promoter region in A. rhizogenes transformed hairy roots and composite plants. The full-length cDNA (2048 aa) of Ma is the longest of all R genes cloned to date. Its TNL structure is completed by a huge post-LRR (PL) sequence (1088 aa) comprising five repeated C-terminal PL exons with two conserved motifs. The N-terminal region (213 aa) of the LRR exon is conserved between alleles and contrasts with the high interallelic polymorphisms of its distal region (111 aa) and of PL domains. The Ma gene highlights the importance of these uncharacterized PL domains which may be involved in pathogen recognition through the decoy hypothesis or in nuclear signalling.


Genome-wide gene expression changes in genetically stable synthetic and natural wheat allohexaploids.

New Phytol. 2010 Jun 25. PMID: 20591055
Chagué V, Just J, Mestiri I, Balzergue S, Tanguy AM, Huneau C, Huteau V, Belcram H, Coriton O, Jahier J, Chalhoub B.

Summary *The present study aims to understand regulation of gene expression in synthetic and natural wheat (Triticum aestivum) allohexaploids, that combines the AB genome of Triticum turgidum and the D genome of Aegilops tauschii; and which we have recently characterized as genetically stable. *We conducted a comprehensive genome-wide analysis of gene expression that allowed characterization of the effect of variability of the D genome progenitor, the intergenerational stability as well as the comparison with natural wheat allohexaploid. We used the Affymetrix GeneChip Wheat Genome Array, on which 55 049 transcripts are represented. *Additive expression was shown to represent the majority of expression regulation in the synthetic allohexaploids, where expression for more than c. 93% of transcripts was equal to the mid-parent value measured from a mixture of parental RNA. This leaves c. 2000 (c. 7%) transcripts, in which expression was nonadditive. No global gene expression bias or dominance towards any of the progenitor genomes was observed whereas high intergenerational stability and low effect of the D genome progenitor variability were revealed. *Our study suggests that gene expression regulation in wheat allohexaploids is established early upon allohexaploidization and highly conserved over generations, as demonstrated by the high similarity of expression with natural wheat allohexaploids.

Evolutionary analysis of the CACTA DNA-transposon Caspar across wheat species using sequence comparison and in situ hybridization.

Mol Genet Genomics. 2010 May 29. PMID: 20512353
Sergeeva EM, Salina EA, Adonina IG, Chalhoub B.

Mobile elements constitute a considerable part of the eukaryotic genome. This work is focused on the distribution and evolution of DNA-transposons in the genomes of diploid and allopolyploid Triticeae species and their role in the formation of functionally important chromosomal subtelomeric regions. The Caspar family is among the most abundant of CACTA DNA-transposons in Triticeae. To study the evolution of Caspar-like elements in Triticeae genomes, we analyzed their sequences and distribution in chromosomes by in situ hybridization. In total, 46 Caspar-like elements from the wheat and barley Caspar, Clifford, and Donald families were analyzed after being extracted from databases using the transposase consensus sequence. Sequence alignment and subsequent phylogenetic analyses revealed that the transposase DNA sequences formed three major distinct groups: (1) Clifford, (2) Caspar_Triticinae, and (3) Caspar_Hordeinae. Additionally, in situ hybridization demonstrated that Caspar_Triticinae transposons are predominantly compartmentalized in the subtelomeric chromosomal regions of wheat and its progenitors. Analysis of data suggested that compartmentalization in the subtelomeric chromosomal region was a characteristic feature of all the main groups of Caspar-like elements. Furthermore, a dot plot analysis of the terminal repeats demonstrated that the divergence of these repeats strictly correlated with the divergence of Caspar coding sequences. A clear distinction in the Caspar DNA sequences among the species Triticum/Aegilops (Caspar_Triticinae), Hordeum (Caspar_Hordeinae), and different distributions in individual hexaploid wheat genomes (A/B and D) suggest an independent proliferation of these elements in wheat (or its progenitors) and barley genomes. Thus, Caspar-like transposons can significantly contribute to the formation and differentiation of subtelomeric regions in Triticeae species.

Multilevel regulation and signalling processes associated with adaptation to terminal drought in wild emmer wheat.

Funct Integr Genomics. 2010 Mar 24. PMID: 20333536
Krugman T, Chagué V, Peleg Z, Balzergue S, Just J, Korol AB, Nevo E, Saranga Y, Chalhoub B, Fahima T.

Low water availability is the major environmental factor limiting crop productivity. Transcriptome analysis was used to study terminal drought response in wild emmer wheat, Triticum dicoccoides, genotypes contrasting in their productivity and yield stability under drought stress. A total of 5,892 differentially regulated transcripts were identified between drought and well-watered control and/or between drought resistant (R) and drought susceptible (S) genotypes. Functional enrichment analyses revealed that multilevel regulatory and signalling processes were significantly enriched among the drought-induced transcripts, in particular in the R genotype. Therefore, further analyses were focused on selected 221 uniquely expressed or highly abundant transcripts in the R genotype, as potential candidates for drought resistance genes. Annotation of the 221 genes revealed that 26% of them are involved in multilevel regulation, including: transcriptional regulation, RNA binding, kinase activity and calcium and abscisic acid signalling implicated in stomatal closure. Differential expression patterns were also identified in genes known to be involved in drought adaptation pathways, such as: cell wall adjustment, cuticular wax deposition, lignification, osmoregulation, redox homeostasis, dehydration protection and drought-induced senescence. These results demonstrate the potential of wild emmer wheat as a source for candidate genes for improving drought resistance.

Newly synthesized wheat allohexaploids display progenitor-dependent meiotic stability and aneuploidy but structural genomic additivity.

New Phytol. 2010 Feb 10 PMID: 20149116
Mestiri I, Chagué V, Tanguy AM, Huneau C, Huteau V, Belcram H, Coriton O, Chalhoub B, Jahier J.

Summary *To understand key mechanisms leading to stabilized allopolyploid species, we characterized the meiotic behaviour of wheat allohexaploids in relation to structural and genetic changes. *For that purpose, we analysed first generations of synthetic allohexaploids obtained through interspecific hybridization, followed by spontaneous chromosome doubling, between several genotypes of Triticum turgidum and Aegilops tauschii wheat species, donors of AB and D genomes, respectively. *As expected for these Ph1 (Pairing homoeologous 1) gene-carrying allopolyploids, chromosome pairing at metaphase I of meiosis essentially occurs between homologous chromosomes. However, the different synthetic allohexaploids exhibited progenitor-dependent meiotic irregularities, such as incomplete homologous pairing, resulting in univalent formation and leading to aneuploidy in the subsequent generation. *Stability of the synthetic allohexaploids was shown to depend on the considered genotypes of both AB and D genome progenitors, where few combinations compare to the natural wheat allohexaploid in terms of regularity of meiosis and euploidy. Aneuploidy represents the only structural change observed in these synthetic allohexaploids, as no apparent DNA sequence elimination or rearrangement was observed when analysing euploid plants with molecular markers, developed from expressed sequence tags (ESTs) as well as simple sequence repeat (SSR) and transposable element sequences.

The first meiosis of resynthesized Brassica napus, a genome blender.

New Phytol. 2010 Feb 8. PMID: 20149113
Szadkowski E, Eber F, Huteau V, Lodé M, Huneau C, Belcram H, Coriton O, Manzanares-Dauleux MJ, Delourme R, King GJ, Chalhoub B, Jenczewski E, Chèvre AM.

Summary *Polyploidy promotes the restructuring of merged genomes within initial generations of resynthesized Brassica napus, possibly caused by homoeologous recombination at meiosis. However, little is known about the impact of the first confrontation of two genomes at the first meiosis which could lead to genome exchanges in progeny. Here, we assessed the role of the first meiosis in the genome instability of synthetic B. napus. *We used three different newly resynthesized B. napus plants and established meiotic pairing frequencies for the A and C genomes. We genotyped the three corresponding progenies in a cross to a natural B. napus on the two homoeologous A1 and C1 chromosomes. Pairing at meiosis in a set of progenies with various rearrangements was scored. *Here, we confirmed that the very first meiosis of resynthesized plants of B. napus acts as a genome blender, with many of the meiotic-driven genetic changes transmitted to the progenies, in proportions that depend significantly on the cytoplasm background inherited from the progenitors. *We conclude that the first meiosis generates rearrangements on both genomes and promotes subsequent restructuring in further generations. Our study advances the knowledge on the timing of genetic changes and the mechanisms that may bias their transmission.

Genome sequencing and analysis of the model grass Brachypodium distachyon.

Nature. 2010 Feb 11;463(7282):763-768. PMID: 20148030
The International Brachypodium Initiative;
Principal investigators, Vogel JP, Garvin DF, Mockler TC, Schmutz J, Rokhsar D, Bevan MW;
DNA sequencing and assembly, Barry K, Lucas S, Harmon-Smith M, Lail K, Tice H, Schmutz Leader J, Grimwood J, McKenzie N, Bevan MW;
Pseudomolecule assembly and BAC end sequencing, Huo N, Gu YQ, Lazo GR, Anderson OD, Vogel Leader JP, You FM, Luo MC, Dvorak J, Wright J, Febrer M, Bevan MW, Idziak D, Hasterok R, Garvin DF;
Transcriptome sequencing and analysis, Lindquist E, Wang M, Fox SE, Priest HD, Filichkin SA, Givan SA, Bryant DW, Chang JH, Mockler Leader TC, Wu H, Wu W, Hsia AP, Schnable PS, Kalyanaraman A, Barbazuk B, Michael TP, Hazen SP, Bragg JN, Laudencia-Chingcuanco D, Vogel JP, Garvin DF, Weng Y, McKenzie N, Bevan MW;
Gene analysis and annotation, Haberer G, Spannagl M, Mayer Leader K, Rattei T, Mitros T, Rokhsar D, Lee SJ, Rose JK, Mueller LA, York TL;
Repeats analysis, Wicker Leader T, Buchmann JP, Tanskanen J, Schulman Leader AH, Gundlach H, Wright J, Bevan M, Costa de Oliveira A, da C Maia L, Belknap W, Gu YQ, Jiang N, Lai J, Zhu L, Ma J, Sun C, Pritham E;
Comparative genomics, Salse Leader J, Murat F, Abrouk M, Haberer G, Spannagl M, Mayer K, Bruggmann R, Messing J, You FM, Luo MC, Dvorak J;
Small RNA analysis, Fahlgren N, Fox SE, Sullivan CM, Mockler TC, Carrington JC, Chapman EJ, May GD, Zhai J, Ganssmann M, Guna Ranjan Gurazada S, German M, Meyers BC, Green Leader PJ;
Manual annotation and gene family analysis, Bragg JN, Tyler L, Wu J, Gu YQ, Lazo GR, Laudencia-Chingcuanco D, Thomson J, Vogel Leader JP, Hazen SP, Chen S, Scheller HV, Harholt J, Ulvskov P, Fox SE, Filichkin SA, Fahlgren N, Kimbrel JA, Chang JH, Sullivan CM, Chapman EJ, Carrington JC, Mockler TC, Bartley LE, Cao P, Jung KH, Sharma MK, Vega-Sanchez M, Ronald P, Dardick CD, De Bodt S, Verelst W, Inzé D, Heese M, Schnittger A, Yang X, Kalluri UC, Tuskan GA, Hua Z, Vierstra RD, Garvin DF, Cui Y, Ouyang S, Sun Q, Liu Z, Yilmaz A, Grotewold E, Sibout R, Hematy K, Mouille G, Höfte H, Michael T, Pelloux J, O'Connor D, Schnable J, Rowe S, Harmon F, Cass CL, Sedbrook JC, Byrne ME, Walsh S, Higgins J, Bevan M, Li P, Brutnell T, Unver T, Budak H, Belcram H, Charles M, Chalhoub B, Baxter I.

Three subfamilies of grasses, the Ehrhartoideae, Panicoideae and Pooideae, provide the bulk of human nutrition and are poised to become major sources of renewable energy. Here we describe the genome sequence of the wild grass Brachypodium distachyon (Brachypodium), which is, to our knowledge, the first member of the Pooideae subfamily to be sequenced. Comparison of the Brachypodium, rice and sorghum genomes shows a precise history of genome evolution across a broad diversity of the grasses, and establishes a template for analysis of the large genomes of economically important pooid grasses such as wheat. The high-quality genome sequence, coupled with ease of cultivation and transformation, small size and rapid life cycle, will help Brachypodium reach its potential as an important model system for developing new energy and food crops.


Impact of transposable elements on the organization and function of allopolyploid genomes.

New Phytologist, December 2009, PMID: 20002321
Christian Parisod (1,2) , Karine Alix (3) , Jérémy Just (4) , Maud Petit (1) , Véronique Sarilar (3) , Corinne Mhiri (1) , Malika Ainouche (5) , Boulos Chalhoub (4) and Marie-Angèle Grandbastien (1)
(1) Laboratoire de Biologie Cellulaire, Institut Jean-Pierre Bourgin, INRA, 78026 Versailles, France ;
(2) National Centre for Biosystematics, Natural History Museum, University of Oslo, 0318 Oslo, Norway ;
(3) AgroParisTech, UMR de Génétique Végétale, INRA – Université Paris-Sud – CNRS – AgroParisTech, Ferme du Moulon, 91190 Gif-sur-Yvette, France ;
(4) Unité de Recherches en Génomique Végétale, INRA, 91057 Évry, France ;
(5) Université Rennes 1, UMR 6553 ECOBIO, 35042 Rennes, France

Transposable elements (TEs) represent an important fraction of plant genomes and are likely to play a pivotal role in fuelling genome reorganization and functional changes following allopolyploidization. Various processes associated with allopolyploidy (i.e. genetic redundancy, bottlenecks during the formation of allopolyploids or genome shock following genome merging) may allow accumulation of TE insertions. Our objective in carrying out a survey of the literature and a comparative analysis across different allopolyploid systems is to shed light on the structural, epigenetic and functional modifications driven by TEs during allopolyploidization and subsequent diploidization. The available evidence indicates that TE proliferation in the short or the long term after allopolyploidization may be restricted to a few TEs, in specific polyploid systems. By contrast, data indicate major structural changes in the TE genome fraction immediately after allopolyploidization, mainly through losses of TE sequences as a result of recombination. Emerging evidence also suggests that TEs are targeted by substantial epigenetic changes, which may impact gene expression and genome stability. Furthermore, TEs may directly or indirectly support the evolution of new functionalities in allopolyploids during diploidization. All data stress allopolyploidization as a shock associated with drastic genome reorganization. Mechanisms controlling TEs during allopolyploidization as well as their impact on diploidization are discussed.

Brassica orthologs from BANYULS belong to a small multigene family, which is involved in procyanidin accumulation in the seed.

Planta. 2009 Sep 17. PMID: 19760260
Auger B, Baron C, Lucas MO, Vautrin S, Bergès H, Chalhoub B, Fautrel A, Renard M, Nesi N.
UMR118 Amélioration des Plantes et Biotechnologies Végétales, INRA, Agrocampus Ouest, Université Rennes1, BP 35327, 35653, Le Rheu Cedex, France.


As part of a research programme focused on flavonoid biosynthesis in the seed coat of Brassica napus L. (oilseed rape), orthologs of the BANYULS gene that encoded anthocyanidin reductase were cloned in B. napus as well as in the related species Brassica rapa and Brassica oleracea. B. napus genome contained four functional copies of BAN, two originating from each diploid progenitor. Amino acid sequences were highly conserved between the Brassicaceae including B. napus, B. rapa, B. oleracea as well as the model plant Arabidopsis thaliana. Along the 200 bp in 5' of the ATG codon, Bna.BAN promoters (ProBna.BAN) were conserved with AtANR promoter and contained putative cis-acting elements. In addition, transgenic Arabidopsis and oilseed rape plants carrying the first 230 bp of ProBna.BAN fused to the UidA reporter gene were generated. In the two Brassicaceae backgrounds, ProBna.BAN activity was restricted to the seed coat. In B. napus seed, ProBna.BAN was activated in procyanidin-accumulating cells, namely the innermost layer of the inner integument and the micropyle-chalaza area. At the transcriptional level, the four Bna.BAN genes were expressed in the seed. Laser microdissection assays of the seed integuments showed that Bna.BAN expression was restricted to the inner integument, which was consistent with the activation profile of ProBna.BAN. Finally, Bna.BAN genes were mapped onto oilseed rape genetic maps and potential co-localisations with seed colour quantitative trait loci are discussed.

Isolation and sequence analysis of the wheat B genome subtelomeric DNA.

BMC Genomics. 2009 Sep 5;10(1):414. PMID: 19732459
Salina EA, Sergeeva EM, Adonina IG, Shcherban AB, Afonnikov DA, Belcram H, Huneau C, Chalhoub B.
Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Science, pr. Lavrentieva 10, Novosibirsk, 630090 Russia
UMR INRA 1165 – CNRS 8114 UEVE – Unite de Recherche en Genomique Vegetale (URGV), 2, rue Gaston Cremieux, CP5708, 91057 Evry cedex, France


ABSTRACT: BACKGROUND: Telomeric and subtelomeric regions are essential for genome stability and regular chromosome replication. In this work, we have characterized the wheat BAC (bacterial artificial chromosome) clones containing Spelt1 and Spelt52 sequences, which belong to the subtelomeric repeats of the B/G genomes of wheats and Aegilops species from the section Sitopsis. RESULTS: The BAC library from Triticum aestivum cv. Renan was screened using Spelt1 and Spelt52 as probes. Nine positive clones were isolated; of them, clone 2050O8 was localized mainly to the distal parts of wheat chromosomes by in situ hybridization. The distribution of the other clones indicated the presence of different types of repetitive sequences in BACs. Use of different approaches allowed us to prove that seven of the nine isolated clones belonged to the subtelomeric chromosomal regions. Clone 2050O8 was sequenced and its sequence of 119 737 bp was annotated. It is composed of 33% transposable elements (TEs), 8.2% Spelt52 (namely, the subfamily Spelt52.2) and five non-TE-related genes. DNA transposons are predominant, making up 24.6% of the entire BAC clone, whereas retroelements account for 8.4% of the clone length. The full-length CACTA transposon Caspar covers 11 666 bp, encoding a transposase and CTG-2 proteins, and this transposon accounts for 40% of the DNA transposons. The in situ hybridization data for 2050O8 derived subclones in combination with the BLAST search against wheat mapped ESTs (expressed sequence tags) suggest that clone 2050O8 is located in the terminal bin 4BL-10 (0.95-1.0). Additionally, four of the predicted 2050O8 genes showed significant homology to four putative orthologous rice genes in the distal part of rice chromosome 3S and confirm the synteny to wheat 4BL. CONCLUSIONS: Satellite DNA sequences from the subtelomeric regions of diploid wheat progenitor can be used for selecting the BAC clones from the corresponding regions of hexaploid wheat chromosomes. It has been demonstrated for the first time that Spelt52 sequences were involved in the evolution of terminal regions of common wheat chromosomes. Our research provides new insights into the microcollinearity in the terminal regions of wheat chromosomes 4BL and rice chromosome 3S.

Sixty million years in evolution of soft grain trait in grasses: emergence of the softness locus in the common ancestor of Pooideae and Ehrhartoideae, after their divergence from Panicoideae.

Mol Biol Evol. 2009 Apr 24. PMID: 19395588
Charles M, Tang H, Belcram H, Paterson A, Gornicki P, Chalhoub B.
URGV (UMR INRA 1165 - CNRS 8114 - UEVE) Organization and evolution of Plant Genomes, 91057 Evry Cedex, France.

Together maize, Sorghum, rice and wheat grass (Poaceae) species are the most important cereal crops in the world and exhibit different 'Grain endosperm texture'. This trait has been studied extensively in wheat because of its pivotal role in determining quality of products obtained from wheat grain. Grain Softness protein-1 and Puroindolines A and B (grain storage proteins), encoded by Ha-like genes: Gsp-1, Pina and Pinb, of the Hardness (Ha) locus, are the main determinants of the grain softness/hardness trait in wheat. The origin and evolution of grain endosperm texture in grasses was addressed by comparing genomic sequences of the Ha orthologous region of wheat, Brachypodium, rice and Sorghum. Results show that the Ha-like genes are present in wheat and Brachypodium but are absent from Sorghum bicolor. A truncated remnant of a Ha-like gene is present in rice. Synteny analysis of the genomes of these grass species shows that only one of the paralogous Ha regions, created 70 MYA by whole-genome duplication, contained Ha-like genes. The comparative genome analysis and evolutionary comparison with genes encoding grain reserve proteins of grasses suggest that an ancestral Ha-like gene emerged, as a new member of the prolamin gene family, in a common ancestor of the Pooideae (Triticeae and Brachypoidieae tribes) and Ehrhartoideae (rice), between 60-50 MYA, after their divergence from Panicoideae (Sorghum). It was subsequently lost in Ehrhartoideae. Recurring duplications, deletions and/or truncations occurred independently and appear to characterize Ha-like gene evolution in the grass species. The Ha-like genes gained a new function in Triticeae, such as wheat, underlying the soft grain phenotype. Loss of these genes in some wheat species leads, in turn, to hard endosperm seeds.

New insights into the origin of the B genome of hexaploid wheat:
Evolutionary relationships at the SPA genomic region with the S genome of the diploid relative Aegilops speltoides.

BMC Genomics. 2008 Nov 25;9(1):555. PMID: 19032732
Salse J, Chague V, Bolot S, Magdelenat G, Huneau C, Pont C, Belcram H, Couloux A, Gardais S, Evrard A, Segurens B, Charles M, Ravel C, Samain S, Charmet G, Boudet N, Chalhoub B.
Organisation and Evolution of Plant Genomes (OEPG), UMR INRA 1165 - CNRS 8114
UEVE - Unité de Recherche en Génomique Végétale (URGV). 2, rue Gaston Crémieux,
CP5708, 91057 Evry cedex. France.

Fig. 1

ABSTRACT: BACKGROUND: Several studies suggested that the diploid ancestor of the B genome of tetraploid and hexaploid wheat species belongs to the Sitopsis section, having Aegilops speltoides (SS, 2n=14) as the closest identified relative. However molecular relationships based on genomic sequence comparison, including both coding and non-coding DNA, have never been investigated. In an attempt to clarify these relationships, we compared, in this study, sequences of the Storage Protein Activator (SPA) locus region of the S genome of Ae. speltoides (2n=14) to that of the A, B and D genomes co-resident in the hexaploid wheat species (Triticum aestivum, AABBDD, 2n=42). RESULTS: Four BAC clones, spanning the SPA locus of respectively the A, B, D and S genomes, were isolated and sequenced. Orthologous genomic regions were identified as delimited by shared non-transposable elements and non-coding sequences surrounding the SPA gene and correspond to 35 268, 22 739, 43 397 and 53 919 bp for the A, B, D and S genomes, respectively. Sequence length discrepancies within and outside the SPA orthologous regions are the result of non-shared transposable elements (TE) insertions, all of which inserted after the progenitors of the four genomes divergence. CONCLUSION: On the basis of conserved sequence length as well as identity of the shared non-TE regions and the SPA coding sequence, Ae speltoides appears to be more evolutionary related to the B genome of T. aestivum than the A and D genomes. However, the differential insertions of TEs, none of which are conserved between the two genomes led to the conclusion that the S genome of Ae. speltoides has diverged very early from the progenitor of the B genome which remains to be identified.

Dynamics and differential proliferation of transposable elements during the evolution of the B and A genomes of wheat.

Genetics. 2008 Sep 9. PMID: 18780739
Charles M, Belcram H, Just J, Huneau C, Viollet A, Couloux A, Segurens B, Carter M, Huteau V, Coriton O, Appels R, Samain S, Chalhoub B.

figure 5

Transposable elements (TEs) constitute more than 80% of the wheat genome but their dynamics and contribution to size variation and evolution of wheat genomes (Triticum and Aegilops species) remains unexplored. In this study, 10 genomic regions have been sequenced from wheat chromosome 3B and used to constitute, along with all publicly available genomic sequences of wheat, 1.98 Mb of sequence (from 13 BAC clones) of the wheat B genome and 3.63 Mb of sequence (from 19 BAC clones) of the wheat A genome. Analysis of TEs sequence proportions (%), ratios of complete to truncated copies and estimation of insertion dates of class I retrotransposons showed that specific types of TEs have undergone waves of differential proliferation in the B and A genomes of wheat. While both genomes show similar rates and relatively ancient proliferation periods for the Athila retrotransposons, the Copia retrotransposons proliferated more recently in the A genome whereas Gypsy retrotransposon proliferation is more recent in the B genome. It was possible to estimate for the first time, proliferation periods of the abundant CACTA class II DNA transposons, relative to that of the three main retrotransposon superfamilies. Proliferation of these TEs started prior to and overlaps with that of the Athila retrotransposons in both genomes. However, they proliferated also during same periods as Gypsy and Copia retrotransposons in the A genome, but not in the B genome. As estimated from their insertion dates and confirmed by PCR-based tracing analysis, the majority of differential proliferation of TEs in B and A genomes of wheat (87% and 83% respectively), leading to rapid sequence divergence, occurred prior to the allotetraploidization event that brought them together in T. turgidum and T. aestivum, less than 0.5 million years ago (MYA). More importantly, the allotetraploidization event appears to have neither enhanced nor repressed retrotranspositions. We discussed the apparent proliferation of TEs as resulting from their insertion, removal and/or combinations of both evolutionary forces.

Acc homoeoloci and the evolution of wheat genomes.

Proc Natl Acad Sci U S A. 2008 Jul 3. PMID: 18599450
Chalupska D, Lee HY, Faris JD, Evrard A, Chalhoub B, Haselkorn R, Gornicki P.
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637;

The DNA sequences of wheat Acc-1 and Acc-2 loci, encoding the plastid and cytosolic forms of the enzyme acetyl-CoA carboxylase, were analyzed with a view to understanding the evolution of these genes and the origin of the three genomes in modern hexaploid wheat. Acc-1 and Acc-2 loci from each of the wheats Triticum urartu (A genome), Aegilops tauschii (D genome), Triticum turgidum (AB genome), and Triticum aestivum (ABD genome), as well as two Acc-2-related pseudogenes from T. urartu were sequenced. The 2.3-2.4 Mya divergence time calculated here for the three homoeologous chromosomes, on the basis of coding and intron sequences of the Acc-1 genes, is at the low end of other estimates. Our clock was calibrated by using 60 Mya for the divergence between wheat and maize. On the same time scale, wheat and barley diverged 11.6 Mya, based on sequences of Acc and other genes. The regions flanking the Acc genes are not conserved among the A, B, and D genomes. They are conserved when comparing homoeologous genomes of diploid, tetraploid, and hexaploid wheats. Substitution rates in intergenic regions consisting primarily of repetitive sequences vary substantially along the loci and on average are 3.5-fold higher than the Acc intron substitution rates. The composition of the Acc homoeoloci suggests haplotype divergence exceeding in some cases 0.5 Mya. Such variation might result in a significant overestimate of the time since tetraploid wheat formation, which occurred no more than 0.5 Mya.

Contrasted Microcolinearity and Gene Evolution Within a Homoeologous Region of Wheat and Barley Species.

J Mol Evol. 2008 Feb 15 PMID: 18274696
Chantret N, Salse J, Sabot F, Bellec A, Laubin B, Dubois I, Dossat C, Sourdille P, Joudrier P, Gautier MF, Cattolico L, Beckert M, Sébastien Aubourg,, Weissenbach J, Caboche M, Leroy P, Bernard M, Boulos Chalhoub

We study here the evolution of genes located in the same physical locus using the recently sequenced Ha locus in seven wheat genomes in diploid, tetraploid, and hexaploid species and compared them with barley and rice orthologous regions. We investigated both the conservation of microcolinearity and the molecular evolution of genes, including coding and noncoding sequences. Microcolinearity is restricted to two groups of genes (Unknown gene-2, VAMP, BGGP, Gsp-1, and Unknown gene-8 surrounded by several copies of ATPase), almost conserved in rice and barley, but in a different relative position. Highly conserved genes between wheat and rice run along with genes harboring different copy numbers and highly variable sequences between close wheat genomes. The coding sequence evolution appeared to be submitted to heterogeneous selective pressure and intronic sequences analysis revealed that the molecular clock hypothesis is violated in most cases.


A unified classification system for eukaryotic transposable elements.

Nat Rev Genet. 2007 Dec;8(12):973-82. PMID: 17984973
Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, Flavell A, Leroy P, Morgante M, Panaud O, Paux E, Sanmiguel P, Schulman AH.
Institute of Plant Biology, University Zurich, Zollikerstrasse 107, CH-8008 Zurich, Switzerland.

Our knowledge of the structure and composition of genomes is rapidly progressing in pace with their sequencing. The emerging data show that a significant portion of eukaryotic genomes is composed of transposable elements (TEs). Given the abundance and diversity of TEs and the speed at which large quantities of sequence data are emerging, identification and annotation of TEs presents a significant challenge. Here we propose the first unified hierarchical classification system, designed on the basis of the transposition mechanism, sequence similarities and structural relationships, that can be easily applied by non-experts. The system and nomenclature is kept up to date at the WikiPoson web site.

Homeologous recombination plays a major role in chromosome rearrangements that occur during meiosis of Brassica napus haploids.

Detection of autosyndesis at metaphase I

Genetics. 2007 Feb;175(2):487-503. Epub 2006 Dec 6 PMID: 17151256
Nicolas SD, Le Mignon G, Eber F, Coriton O, Monod H, Clouet V, Huteau V, Lostanlen A, Delourme R, Chalhoub B, Ryder CD, Chevre AM, Jenczewski E. (2007)
UMR INRA-Agrocampus Rennes, Amélioration des Plantes et Biotechnologies Végétales, 35653 Le Rheu, France.

Chromosomal rearrangements can be triggered by recombination between distinct but related regions. Brassica napus (AACC; 2n = 38) is a recent allopolyploid species whose progenitor genomes are widely replicated. In this article, we analyze the extent to which chromosomal rearrangements originate from homeologous recombination during meiosis of haploid B. napus (n = 19) by genotyping progenies of haploid x euploid B. napus with molecular markers. Our study focuses on three pairs of homeologous regions selected for their differing levels of divergence (N1/N11, N3/N13, and N9/N18). We show that a high number of chromosomal rearrangements occur during meiosis of B. napus haploid and are transmitted by first division restitution (FDR)-like unreduced gametes to their progeny; half of the progeny of Darmor-bzh haploids display duplications and/or losses in the chromosomal regions being studied. We demonstrate that half of these rearrangements are due to recombination between regions of primary homeology, which represents a 10- to 100-fold increase compared to the frequency of homeologous recombination measured in euploid lines. Some of the other rearrangements certainly result from recombination between paralogous regions because we observed an average of one to two autosyndetic A-A and/or C-C bivalents at metaphase I of the B. napus haploid. These results are discussed in the context of genome evolution of B. napus.

Generation and Screening of a BAC Library from a Diploid Potato Clone to Unravel Durable Late Blight Resistance on Linkage Group IV.

Int J Plant Genomics. 2007;2007:51421. PMID: 18273389
Hein I, McLean K, Chalhoub B, Bryan GJ.

We describe the construction and screening of a large insert genomic library from the diploid potato clone HB171(13) that has been shown to express durable quantitative field resistance to Phytophthora infestans, the causal agent of potato late blight disease. Integrated genetic mapping of the field resistance quantitative trait locus with markers developed from populations segregating for Rpi-blb3, Rpi-abpt, R2, and R2-like resistance, all located on linkage group IV, has positioned the field resistance QTL within the proximity of this R gene cluster. The library has been successfully screened with resistance gene analogues (RGA) potentially linked to the R gene cluster. Over 30 positive BAC clones were identified and confirmed by PCR and Southern hybridisations to harbour RGA-like sequences. In addition, BAC end sequencing of positive clones has corroborated two BAC clones with a very high level of nucleotide similarity to the RGA probes utilised.


Types and rates of sequence evolution at the high-molecular-weight glutenin locus in hexaploid wheat and its ancestral genomes.

Genetics. 2006 Nov;174(3):1493-504. Epub 2006 Oct 8. PMID: 17028342
Gu YQ, Salse J, Coleman-Derr D, Dupin A, Crossman C, Lazo GR, Huo N, Belcram H, Ravel C, Charmet G, Charles M, Anderson OD, Chalhoub B.(2006)
United States Department of Agriculture-Agricultural Research Service, Western Regional Research Center, Albany, CA 94710, USA.

The Glu-1 locus, encoding the high-molecular-weight glutenin protein subunits, controls bread-making quality in hexaploid wheat (Triticum aestivum) and represents a recently evolved region unique to Triticeae genomes. To understand the molecular evolution of this locus region, three orthologous Glu-1 regions from the three subgenomes of a single hexaploid wheat species were sequenced, totaling 729 kb of sequence. Comparing each Glu-1 region with its corresponding homologous region from the D genome of diploid wheat, Aegilops tauschii, and the A and B genomes of tetraploid wheat, Triticum turgidum, revealed that, in addition to the conservation of microsynteny in the genic regions, sequences in the intergenic regions, composed of blocks of nested retroelements, are also generally conserved, although a few nonshared retroelements that differentiate the homologous Glu-1 regions were detected in each pair of the A and D genomes. Analysis of the indel frequency and the rate of nucleotide substitution, which represent the most frequent types of sequence changes in the Glu-1 regions, demonstrated that the two A genomes are significantly more divergent than the two B genomes, further supporting the hypothesis that hexaploid wheat may have more than one tetraploid ancestor.

Single-nucleotide polymorphism frequency in a set of selected lines of bread wheat (Triticum aestivum L.).

Genome. 2006 Sep;49(9):1131-1139. PMID: 17110993
INRA, UMR1095, Amélioration et Santé des Plantes, 234 avenue du Bézet, Clermont-Ferrand, F-63100 France.
Ravel C, Praud S, Murigneux A, Canaguier A, Sapet F, Samson D, Balfourier F, Dufour P, Chalhoub B, Brunel D, Beckert M, Charmet G. (2006)

Information on single-nucleotide polymorphisms (SNPs) in hexaploid bread wheat is still scarce. The goal of this study was to detect SNPs in wheat and examine their frequency. Twenty-six bread wheat lines from different origins worldwide were used. Specific PCR-products were obtained from 21 genes and directly sequenced. SNPs were discovered from the alignment of these sequences. The overall sequence polymorphism observed in this sample appears to be low; 64 single-base polymorphisms were detected in approximately 21.5 kb (i.e., 1 SNP every 335 bp). The level of polymorphism is highly variable among the different genes studied. Fifty percent of the genes studied contained no sequence polymorphism, whereas most SNPs detected were located in only 2 genes. As expected, taking into account a synthetic line created with a wild Triticum tauschii parent increases the level of polymorphism (101 SNPs; 1 SNP every 212 bp). The detected SNPs are available at"> Data on linkage disequilibrium (LD) are still preliminary. They showed a significant level of LD in the 2 most polymorphic genes. To conclude, the genome size of hexaploid wheat and its low level of polymorphism complicate SNP discovery in this species.

Advanced resources for plant genomics: a BAC library specific for the short arm of wheat chromosome 1B.

Plant J. 2006 Sep;47(6):977-986. PMID: 16911585
Janda J, Safar J, Kubalakova M, Bartos J, Kovarova P, Suchankova P, Pateyron S, Cihalikova J, Sourdille P, Simkova H, Faivre-Rampant P, Hribova E, Bernard M, Lukaszewski A, Dolezel J, Chalhoub B. (2006)
Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská 6, CZ-77200 Olomouc, Czech Republic.

Common wheat (Triticum aestivum L., 2n = 6x = 42) is a polyploid species possessing one of the largest genomes among the cultivated crops (1C is approximately 17 000 Mb). The presence of three homoeologous genomes (A, B and D), and the prevalence of repetitive DNA make sequencing the wheat genome a daunting task. We have developed a novel 'chromosome arm-based' strategy for wheat genome sequencing to simplify this task; this relies on sub-genomic libraries of large DNA inserts. In this paper, we used a di-telosomic line of wheat to isolate six million copies of the short arm of chromosome 1B (1BS) by flow sorting. Chromosomal DNA was partially digested with HindIII and used to construct an arm-specific BAC library. The library consists of 65 280 clones with an average insert size of 82 kb. Almost half of the library (45%) has inserts larger than 100 kb, while 18% of the inserts range in size between 75 and 100 kb, and 37% are shorter than 75 kb. We estimated the chromosome arm coverage to be 14.5-fold, giving a 99.9% probability of identifying a clone corresponding to any sequence on the short arm of 1B. Each chromosome arm in wheat can be flow sorted from an appropriate cytogenetic stock, and we envisage that the availability of chromosome arm-specific BAC resources in wheat will greatly facilitate the development of ready-to-sequence physical maps and map-based gene cloning.

Anchoring a large set of markers onto a BAC library for the development of a draft physical map of the grapevine genome.

Theor Appl Genet. 2006 Jul;113(2):344-56. Epub 2006 May 18. PMID: 16791700
Lamoureux D, Bernole A, Le Clainche I, Tual S, Thareau V, Paillard S, Legeai F, Dossat C, Wincker P, Oswald M, Merdinoglu D, Vignault C, Delrot S, Caboche M, Chalhoub B, Adam-Blondon AF.
UMR INRA-CNRS-UEVE de Recherches en Génomique Végétale, 2 rue Gaston Crémieux, BP5708, 91057 Evry Cedex, France.

Five hundred and six EST-derived markers, 313 SSR markers and 26 BAC end-derived or SCAR markers were anchored by PCR on a subset of a Cabernet Sauvignon BAC library representing six genome equivalents pooled in three dimensions. In parallel, the 12,351 EST clusters of the grapevine UniGene set (build #11) from NCBI were used to design 12,125 primers pairs and perform electronic PCR on 67,543 nonredundant BAC-end sequences. This in silico experiment yielded 1,140 positive results concerning 638 different markers, among which 602 had not been already anchored by PCR. The data obtained will provide an easier access to the regulatory sequences surrounding important genes (represented by ESTs). In total, 1,731 islands of BAC clones (set of overlapping BAC clones containing at least one common marker) were obtained and 226 of them contained at least one genetically mapped anchor. These assigned islands are very useful because they will link the genetic map and the future fingerprint-based physical map and because they allowed us to indirectly place 93 ESTs on the genetic map. The islands containing two or more mapped SSR markers were also used to assess the quality of the integrated genetic map of the grapevine genome.

A hAT superfamily transposase recruited by the cereal grass genome.

Mol Genet Genomics. 2006 Jun;275(6):553-63. Epub 2006 Feb 9. PMID: 16468023
Department of Agronomy and Plant Genetics, University of Minnesota, 411 Borlaug Hall, St. Paul, MN, 55108, USA.
Muehlbauer GJ, Bhau BS, Syed NH, Heinen S, Cho S, Marshall D, Pateyron S, Buisine N, Chalhoub B, Flavell AJ. (2006)

Transposable elements are ubiquitous genomic parasites with an ancient history of coexistence with their hosts. A few cases have emerged recently where these genetic elements have been recruited for normal function in the host organism. We have identified an expressed hobo/Ac/Tam (hAT) family transposase-like gene in cereal grasses which appears to represent such a case. This gene, which we have called gary, is found in one or two copies in barley, two diverged copies in rice and two very similar copies in hexaploid wheat. No gary homologues are found in Arabidopsis. In all three cereal species, an apparently complete 2.5 kb transposase-like open reading frame is present and nucleotide substitution data show evidence for positive selection, yet the predicted gary protein is probably not an active transposase, as judged by the absence of key amino acids required for transposase function. Gary is expressed in wheat and barley spikes and gary cDNA sequences are also found in rice, oat, rye, maize, sorghum and sugarcane. The short inverted terminal repeats, flanked by an eight-nucleotide host sequence duplication, which are characteristic of a hAT transposon are absent. Genetic mapping in barley shows that gary is located on the distal end of the long arm of chromosome 2H. Wheat homologues of gary map to the same approximate location on the wheat group 2 chromosomes by physical bin-mapping and the more closely related of the two rice garys maps to the syntenic location near the bottom of rice chromosome 4. These data suggest that gary has resided in a single genomic location for at least 60 Myr and has lost the ability to transpose, yet expresses a transposase-related protein that is being conserved under host selection. We propose that the gary transposase-like gene has been recruited by the cereal grasses for an unknown function.

Anchoring a large set of markers onto a BAC library for the development of a draft physical map of the grapevine genome.

Theor Appl Genet. 2006 May 18; 113 : 344-356 PMID: 16708229
Full Text pdf Version (Subscribers only)
The results can be accessed here
Lamoureux D, Bernole A, Le Clainche I, Tual, S, Thareau V, Paillard S, Legeai F, Dossat C, Wincker P, Oswald M, Merdinoglu D, Vignault C, Delrot S, Caboche M, Chalhoub B, Adam-Blondon A-F (2006)

Five hundred and six EST-derived markers, 313 SSR markers and 26 BAC end-derived or SCAR markers were anchored by PCR on a subset of a Cabernet Sauvignon BAC library representing six genome equivalents pooled in three dimensions. In parallel, the 12,351 EST clusters of the grapevine UniGene set (build #11) from NCBI were used to design 12,125 primers pairs and perform electronic PCR on 67,543 nonredundant BAC-end sequences. This in silico experiment yielded 1,140 positive results concerning 638 different markers, among which 602 had not been already anchored by PCR. The data obtained will provide an easier access to the regulatory sequences surrounding important genes (represented by ESTs). In total, 1,731 islands of BAC clones (set of overlapping BAC clones containing at least one common marker) were obtained and 226 of them contained at least one genetically mapped anchor. These assigned islands are very useful because they will link the genetic map and the future fingerprint-based physical map and because they allowed us to indirectly place 93 ESTs on the genetic map. The islands containing two or more mapped SSR markers were also used to assess the quality of the integrated genetic map of the grapevine genome.


Updating of transposable element annotations from large wheat genomic sequences reveals diverse activities and gene associations

Mol Genet Genomics. 2005 Sep;274(2):119-30. Epub 2005 Oct 11. PMID: 16034625
Sabot F, Guyot R, Wicker T, Chantret N, Laubin B, Chalhoub B, Leroy P, Sourdille P, Bernard M. (2005)
UMR 1095 INRA/UBP Amélioration et Santé des Plantes, 234 Avenue du Brézet, 63100 Clermont-Ferrand Cedex, France.

Triticeae species (including wheat, barley and rye) have huge and complex genomes due to polyploidization and a high content of transposable elements (TEs). TEs are known to play a major role in the structure and evolutionary dynamics of Triticeae genomes. During the last 5 years, substantial stretches of contiguous genomic sequence from various species of Triticeae have been generated, making it necessary to update and standardize TE annotations and nomenclature. In this study we propose standard procedures for these tasks, based on structure, nucleic acid and protein sequence homologies. We report statistical analyses of TE composition and distribution in large blocks of genomic sequences from wheat and barley. Altogether, 3.8 Mb of wheat sequence available in the databases was analyzed or re-analyzed, and compared with 1.3 Mb of re-annotated genomic sequences from barley. The wheat sequences were relatively gene-rich (one gene per 23.9 kb), although wheat gene-derived sequences represented only 7.8% (159 elements) of the total, while the remainder mainly comprised coding sequences found in TEs (54.7%, 751 elements). Class I elements [mainly long terminal repeat (LTR) retrotransposons] accounted for the major proportion of TEs, in terms of sequence length as well as element number (83.6% and 498, respectively). In addition, we show that the gene-rich sequences of wheat genome A seem to have a higher TE content than those of genomes B and D, or of barley gene-rich sequences. Moreover, among the various TE groups, MITEs were most often associated with genes: 43.1% of MITEs fell into this category. Finally, the TRIM and copia elements were shown to be the most active TEs in the wheat genome. The implications of these results for the evolution of diploid and polyploid wheat species are discussed.

Construction and characterization of BAC libraries from major grapevine cultivars.

Theor Appl Genet. 2005 May;110(8):1363-71. Epub 2005 Apr 16. PMID: 15834699
Adam-Blondon A-F, Bernole A, Faes G, Lamoureux D, Pateyron S, Grando MS, Caboche M, Velasco R, Chalhoub B (2005)
Unité Mixte de Recherches sur les Génomes des Végétaux, INRA, 2 rue Gaston Crémieux, 5708 91 057, Evry Cedex, France.

Genome projects were initiated on grapevine (Vitis vinifera L., 2n=38, genome size 475 Mb) through the successful construction of four bacterial artificial chromosome (BAC) libraries from three major cultivars, Cabernet Sauvignon (Cabernet S), Syrah and two different clones of Pinot Noir (Pinot N). Depending on the library, the genome coverage represented 4.5-14.8 genome equivalents with clones having a mean insert size of 93-158 kb. BAC pools suitable for PCR screening were constructed for two of these BAC libraries [Cabernet S and Pinot N clone (cl) 115] and subsequently used to confirm the genome coverage of both libraries by PCR anchoring of 74 genetic markers sampled from the 19 linkage groups. For ten of these markers, two bands on separate BAC pools were differentiated that could correspond either to different alleles or to a duplication of the locus being studied. Finally, a preliminary assessment of the correspondence between genetic and physical distances was made through the anchoring of all the markers mapped along linkage group 1 of the V. vinifera genetic map. A pair of markers, 2.1 cM apart, anchored the same BAC clones, which allowed us to estimate that 1 cM corresponded in this particular region to a maximum length of 130 kb.

Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region.

Funct Integr Genomics. 2005 Apr;5(2):97-103. Epub 2005 Jan 22. PMID: 15666175
Isidore E, Scherrer B, Bellec A, Budin K, Faivre-Rampant P, Waugh R, Keller B, Caboche M, Feuillet C, Chalhoub B. (2005)
Institute of Plant Biology, University of Zürich, Zollikerstrasse 107, 8008 Zürich, Switzerland.

To isolate genes of interest in plants, it is essential to construct bacterial artificial chromosome (BAC) libraries from specific genotypes. Construction and organisation of BAC libraries is laborious and costly, especially from organisms with large and complex genomes. In the present study, we developed the pooled BAC library strategy that allows rapid and low cost generation and screening of genomic libraries from any genotype of interest. The BAC library is constructed, directly organised into a few pools and screened for BAC clones of interest using PCR and hybridisation steps, without requiring organization into individual clones. As a proof of concept, a pooled BAC library of approximately 177,000 recombinant clones has been constructed from the barley cultivar Cebada Capa that carries the Rph7 leaf rust resistance gene. The library has an average insert size of 140 kb, a coverage of six barley genome equivalents and is organised in 138 pools of about 1,300 clones each. We rapidly established a single contig of six BAC clones spanning 230 kb at the Rph7 locus on chromosome 3HS. The described low-cost cloning strategy is fast and will greatly facilitate direct targeting of genes and large-scale intra- and inter-species comparative genome analysis.

Ancient haplotypes resulting from extensive molecular rearrangements in the wheat A genome have been maintained in species of three different ploidy levels

Genome Res. 2005 Apr;15(4):526-36. PMID: 15805493
Isidore, E., Scherrer, B., Chalhoub, B., Feuillet, C., Keller, B. (2005)
Institute of Plant Biology, University of Zürich, 8008 Zürich, Switzerland.

Plant genomes, in particular grass genomes, evolve very rapidly. The closely related A genomes of diploid, tetraploid, and hexaploid wheat are derived from a common ancestor that lived <3 million years ago and represent a good model to study molecular mechanisms involved in such rapid evolution. We have sequenced and compared physical contigs at the Lr10 locus on chromosome 1AS from diploid (211 kb), tetraploid (187 kb), and hexaploid wheat (154 kb). A maximum of 33% of the sequences were conserved between two species. The sequences from diploid and tetraploid wheat shared all of the genes, including Lr10 and RGA2 and define a first haplotype (H1). The 130-kb intergenic region between Lr10 and RGA2 was conserved in size despite its activity as a hot spot for transposon insertion, which resulted in >70% of sequence divergence. The hexaploid wheat sequence lacks both Lr10 and RGA2 genes and defines a second haplotype, H2, which originated from ancient and extensive rearrangements. These rearrangements included insertions of retroelements and transposons deletions, as well as unequal recombination within elements. Gene disruption in haplotype H2 was caused by a deletion and subsequent large inversion. Gene conservation between H1 haplotypes, as well as conservation of rearrangements at the origin of the H2 haplotype at three different ploidy levels indicate that the two haplotypes are ancient and had a stable gene content during evolution, whereas the intergenic regions evolved rapidly. Polyploidization during wheat evolution had no detectable consequences on the structure and evolution of the two haplotypes.

Molecular basis of evolutionary events that shaped the hardness locus in diploid and polyploid wheat species (triticum and aegilops).

Plant Cell. 2005 Apr;17(4):1033-45. Epub 2005 Mar 4. PMID: 15749759
Chantret N, Salse J, Sabot F, Rahman S, Bellec A, Laubin B, Dubois I, Dossat C, Sourdille P, Joudrier P, Gautier M-F, Cattolico L, Beckert M, Aubourg S, Weissenbach J, Caboche M, Bernard M, Leroy P and Chalhoub B (2005)
Institut National de la Recherche Agronomique-Centre de Cooperation Internationale en Recherche Agronomique pour le Développement, Biotrop, F-34398 Montpellier Cedex 5, France.

The Hardness (Ha) locus controls grain hardness in hexaploid wheat (Triticum aestivum) and its relatives (Triticum and Aegilops species) and represents a classical example of a trait whose variation arose from gene loss after polyploidization. In this study, we investigated the molecular basis of the evolutionary events observed at this locus by comparing corresponding sequences of diploid, tertraploid, and hexaploid wheat species (Triticum and Aegilops). Genomic rearrangements, such as transposable element insertions, genomic deletions, duplications, and inversions, were shown to constitute the major differences when the same genomes (i.e., the A, B, or D genomes) were compared between species of different ploidy levels. The comparative analysis allowed us to determine the extent and sequences of the rearranged regions as well as rearrangement breakpoints and sequence motifs at their boundaries, which suggest rearrangement by illegitimate recombination. Among these genomic rearrangements, the previously reported Pina and Pinb genes loss from the Ha locus of polyploid wheat species was caused by a large genomic deletion that probably occurred independently in the A and B genomes. Moreover, the Ha locus in the D genome of hexaploid wheat (T. aestivum) is 29 kb smaller than in the D genome of its diploid progenitor Ae. tauschii, principally because of transposable element insertions and two large deletions caused by illegitimate recombination. Our data suggest that illegitimate DNA recombination, leading to various genomic rearrangements, constitutes one of the major evolutionary mechanisms in wheat species.

Large intra-specific haplotype variability at the Rph7 locus results from rapid and recent divergence in the barley genome.

Plant Cell. 2005 Feb;17(2):361-74. Epub 2005 Jan 19. PMID: 15659632
Scherrer, B., Isidore, E., Klein, P., Kim, J-S., Bellec, 8. A., Chalhoub, B., Kelle,r B., Feuillet, C. (2005)
Institute of Plant Biology, University of Zürich, 8008 Zürich, Switzerland.

To study genome evolution and diversity in barley (Hordeum vulgare), we have sequenced and compared more than 300 kb of sequence spanning the Rph7 leaf rust disease resistance gene in two barley cultivars. Colinearity was restricted to five genic and two intergenic regions representing <35% of the two sequences. In each interval separating the seven conserved regions, the number and type of repetitive elements were completely different between the two homologous sequences, and a single gene was absent in one cultivar. In both cultivars, the nonconserved regions consisted of approximately 53% repetitive sequences mainly represented by long-terminal repeat retrotransposons that have inserted <1 million years ago. PCR-based analysis of intergenic regions at the Rph7 locus and at three other independent loci in 41 H. vulgare lines indicated large haplotype variability in the cultivated barley gene pool. Together, our data indicate rapid and recent divergence at homologous loci in the genome of H. vulgare, possibly providing the molecular mechanism for the generation of high diversity in the barley gene pool. Finally, comparative analysis of the gene composition in barley, wheat (Triticum aestivum), rice (Oryza sativa), and sorghum (Sorghum bicolor) suggested massive gene movements at the Rph7 locus in the Triticeae lineage.

Stress activation and genomic impact of Tnt1 retrotransposons in Solanaceae.

Cytogenet Genome Res. 2005;110(1-4):229-41. Review. PMID: 16093677
Grandbastien MA, Audeon C, Bonnivard E, Casacuberta JM, Chalhoub B, Costa AP, Le QH, Melayah D, Petit M, Poncet C, Tam SM, Van Sluys MA, Mhiri C.(2005)
Laboratoire de Biologie Cellulaire, INRA, Centre de Versailles, Versailles, France.

Tnt1 elements are a superfamily of LTR-retrotransposons distributed in the Solanaceae plant family and represent good model systems for studying regulatory and evolutionary controls established between hosts and transposable elements. Tnt1 retrotransposons tightly control their activation, by restricting expression to specific conditions. The Tnt1A element, originally discovered in tobacco, is expressed in response to stress, and its activation by microbial factors is followed by amplification, demonstrating that factors of pathogen origin can generate genetic diversity in plants. The Tnt1A promoter has the potential to be activated by various biotic and abiotic stimuli but a number of these are specifically repressed in tobacco and are revealed only when the LTR promoter is placed in a heterologous context. We propose that a tobacco- and stimulus-specific repression has been established in order to minimize activation in conditions that might generate germinal transposition. In addition to tight transcriptional controls, Tnt1A retrotransposons self-regulate their activity through gradual generation of defective copies that have reduced transcriptional activity. Tnt1 retrotransposons found in various Solanaceae species are characterized by a high level of variability in the LTR sequences involved in transcription, and have evolved by gaining new expression patterns, mostly associated with responses to diverse stress conditions. Tnt1A insertions associated with genic regions are initially favored but seem subsequently counter-selected, while insertions in repetitive DNA are maintained. On the other hand, amplification and loss of insertions may result from more brutal occurrences, as suggested by the large restructuring of Tnt1 populations observed in tobacco compared to each of its parental species. The distribution of Tnt1 elements thus appears as a dynamic flux, with amplification counterbalanced by loss of insertions. Tnt1 insertion polymorphisms are too high to reveal species relationships in the Nicotiana genus, but can be used to evaluate species relationships in the Lycopersicon and Capsicum genera. This also demonstrates that the behavior of Tnt1 retrotransposons differs between host species, most probably in correlation to differences in expression conditions and in the evolutionary and environmental history of each host.


Construction of a subgenomic BAC library specific for chromosomes 1D, 4D and 6D of hexaploid wheat.

Theor Appl Genet. 2004 Nov;109(7):1337-45. Epub 2004 Sep 10. PMID: 15365624.
Janda, J., Bartos, J., Safar, J., Kubalakova, M., Valarik, M., Cihalikova, J., Simkova, H., Caboche, M., Sourdille, P., Bernard, M., Chalhoub, B. and Dolezel, J. (2004)
Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská 6, 77200 Olomouc, Czech Republic.

The analysis of the hexaploid wheat genome (Triticum aestivum L., 2 n=6 x=42) is hampered by its large size (16,974 Mb/1C) and presence of three homoeologous genomes (A, B and D). One of the possible strategies is a targeted approach based on subgenomic libraries of large DNA inserts. In this work, we purified by flow cytometry a total of 10(7) of three wheat D-genome chromosomes: 1D, 4D and 6D. Chromosomal DNA was partially digested with HindIII and used to prepare a specific bacterial artificial chromosome (BAC) library. The library (designated as TA-subD) consists of 87,168 clones, with an average insert size of 85 kb. Among these clones, 53% had inserts larger than 100 kb, only 29% of inserts being shorter than 75 kb. The coverage was estimated to be 3.4-fold, giving a 96.5% probability of identifying a clone corresponding to any sequence on the three chromosomes. Specificity for chromosomes 1D, 4D and 6D was confirmed after screening the library pools with single-locus microsatellite markers. The screening indicated that the library was not biased and gave an estimated coverage of sixfold. This is the second report on BAC library construction from flow-sorted plant chromosomes, which confirms that dissecting of the complex wheat genome and preparation of subgenomic BAC libraries is possible. Their availability should facilitate the analysis of wheat genome structure and evolution, development of cytogenetic maps, construction of local physical maps and map-based cloning of agronomically important genes.

A workshop report on wheat genome sequencing: International Genome Research on Wheat (IGROW) consortium.

Genetics. 2004 Oct;168(2):1087-96. PMID: 15514080
Gill, B.S., Appels, R., Botha-Oberholster, A.M., Buell, C.R., Bennetzen, J.F., Chalhoub, B., Chumley, F., Dvorak, J., Iwanaga, M., Keller, B., Li, W., McCombie, R., Ogihara, Y., Quetier, F. and Sasaki, T. (2004)
Plant Pathology Department, Kansas State University, Manhattan, Kansas 66506-5502, USA.

Sponsored by the National Science Foundation and the U.S. Department of Agriculture, a wheat genome sequencing workshop was held November 10-11, 2003, in Washington, DC. It brought together 63 scientists of diverse research interests and institutions, including 45 from the United States and 18 from a dozen foreign countries (see list of participants at The objectives of the workshop were to discuss the status of wheat genomics, obtain feedback from ongoing genome sequencing projects, and develop strategies for sequencing the wheat genome. The purpose of this report is to convey the information discussed at the workshop and provide the basis for an ongoing dialogue, bringing forth comments and suggestions from the genetics community.

High-resolution mapping and chromosome landing at the root-know nematode resistance locus Ma from Myrobalan plum using a large-insert BAC DNA library.

Theor Appl Genet. 2004 Oct;109(6):1318-27. PMID: 15322755
Claverie M., Dirlewanger E., Cosson P., Bosselut N., Lecouls A-C., Voisin R., Kleinhentz M., Lafargue B., Caboche M., Chalhoub B., Esmenjaud, D. (2004).
UMR Interactions Plantes-Microorganismes et Santé Végétale, Equipe de Nématologie, Institut National de la Recherche Agronomique, Sophia Antipolis, France.

The Ma gene for root-knot nematode (RKN)resistance from Myrobalan plum (Prunus cerasifera L.)confers a complete-spectrum and a heat-stable resistance to Meloidogvne spp., conversely to Mi-I from tomato,which has a more restricted spectrum and a reduced efficiency at high temperature. This gene was identified from a perennial self-incompatible near-wild rootstock species and lies in cosegregation with the SCAR marker SCAFLP2 on the Prunus linkage group 7 in a 2.3 cM interval between the SCAR SCAL19 and SSR pchgms6 markers. We initiated a map-based cloning of Ma and report here the strategy that rapidly led to fine mapping and direct chromosome landing at the locus. Three pairs of bulks, totaling 90 individuals from half-sibling progenies derived from the Ma-heterozygous resistant accession P.2175, were constructed using mapping data, and saturation of the Ma region was performed by bulked segregant analysis (BSA) of 320 AFLP primer pair combinations. The closest three AFLP markers were transformed into codominant SCARs or CAPS designatedSCAFLP3, SCAFLP4 and SCAFLP5. By completing the mapping population up to 1,332 offspring from P.2175,Ma and SCAFLP2 were mapped in a 0.8 cM interval between SCAFLP3 and SCAFLP4. A large-insert bacterial artificial chromosome (BAC) DNA library of P.2175,totaling 30,720 clones with a mean insert size of 145 kb and a 14-15x Prunus haploid genome coverage was constructed and used to land on the Ma spanning interval with few BAC clones. As P.2175 is heterozygous for the gene, we constructed the resistant and susceptible physical contigs by PCR screening of the library with codominant markers. Additional microsatellite markers were then designed from BAC subcloning or BAC end sequencing.In the resistant contig, a single 280 kb BAC clone was shown to carry the Ma gene; this BAC contains two flanking markers on each side of the gene as well as two cosegregating markers. These results should allow future cloning of the Ma gene in this perennial species.

Dissecting large and complex genomes: flow sorting and BAC cloning of individual chromosomes from bread wheat.

Plant J. 2004 Sep;39(6):960-8. PMID: 15341637

Safar, J., Bartos, J., Janda, J., Bellec, A., Kubalakova, M., Valarik, M., Pateyron, S., Weiserova, J., Tuskova, R., Cihalikova, J., Vrana, J., Simkova, H., Faivre-Rampant, P., Sourdille, P., Caboche, M., Bernard, M., Dolezel, J., Chalhoub, B. (2004)
Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovska 6, CZ-77200 Olomouc, Czech Republic.

The analysis of the complex genome of common wheat (Triticum aestivum, 2n = 6x = 42, genome formula AABBDD) is hampered by its large size ( approximately 17 000 Mbp) and allohexaploid nature. In order to simplify its analysis, we developed a generic strategy for dissecting such large and complex genomes into individual chromosomes. Chromosome 3B was successfully sorted by flow cytometry and cloned into a bacterial artificial chromosome (BAC), using only 1.8 million chromosomes and an adapted protocol developed for this purpose. The BAC library (designated as TA-3B) consists of 67 968 clones with an average insert size of 103 kb. It represents 6.2 equivalents of chromosome 3B with 100% coverage and 90% specificity as confirmed by genetic markers. This method was validated using other chromosomes and its broad application and usefulness in facilitating wheat genome analysis were demonstrated by target characterization of the chromosome 3B structure through cytogenetic mapping. This report on the successful cloning of flow-sorted chromosomes into BACs marks the integration of flow cytogenetics and genomics and represents a great leap forward in genetics and genomic analysis.

Distribution of the Tnt1 retrotransposon family in the amphidiploid tobacco (Nicotiana tabacum) and its wild Nicotiana relatives

Biological Journal of the Linnean Society Volume 82 Issue 4 Page 639-649, August 2004
Melayah D.1, Yong-Lim K.2, Bonnivard E.1, Chalhoub B.1, Dorlhac de Borne F.3, Mhiri C.1, Leitch A.2, Granbastien M-A.1. (2004).
1 Laboratoire de Biologie Cellulaire, Institut Jean-Pierre Bourgin, INRA—Centre de Versailles, F-78026 Versailles cedex, France
2 School of Biological Sciences, Queen Mary College, University of London, London E1 4NS, UK
3 Institut du Tabac, Altadis, F-24100, Bergerac, France

Transposable elements can generate considerable genetic diversity. Here we examine the distribution of the Tnt1 retrotransposon family in representative species of the genus Nicotiana. We show that multiple Tnt1 insertions are found in all Nicotiana species. However, Tnt1 insertions are too polymorphic to reveal species relationships. This indicates that Tnt1 has amplified rapidly and independently after Nicotiana speciation. We compare patterns of Tnt1 insertion in allotetraploid tobacco (N. tabacum) with those in the diploid species that are most closely related to the progenitors of tobacco, N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). We found no evidence for Tnt1 insertion sites of N. otophora origin in tobacco. Nicotiana sylvestris has a higher Tnt1 content than N. tomentosiformis and the elements are distributed more uniformly across the genome. This is reflected in tobacco where there is a higher Tnt1 content in S-genome chromosomes. However, the total Tnt1 content of tobacco is not the sum of the two modern-day parental species. We also observed tobacco-specific Tnt1 insertions and an absence of tobacco Tnt1 insertion sites in the diploid relatives. These data indicate Tnt1 evolution subsequent to allopolyploidy. We explore the possibility that fast evolution of Tnt1 is associated with 'genomic-shock' arising out of interspecific hybridization and allopolyploidy.

Construction and characterisation of a BAC library for genome analysis of the allotetraploid coffee species (Coffea arabica L).

Theor Appl Genet. 2004 Jun;109(1):225-30. Epub 2004 Mar 2. PMID: 14997299
Noir S., Pateyron S., Combes M-C., Lashermes P., Chalhoub B. (2004).
IRD, GeneTrop, BP 64501, 34394 Montpellier Cedex 5, France.

In order to promote genome research on coffee trees, one of the most important tropical crops, a bacterial artificial chromosome (BAC) library of the coffee allotetraploid species, Coffea arabica, was constructed. The variety IAPAR 59, which is widely distributed in Latin America and exhibits a fair level of resistance to several pathogens, was chosen. High-efficiency BAC cloning of the high molecular weight genomic DNA partially digested by HindIII was achieved. In total, the library contains 88,813 clones with an average insert size of 130 kb, and represents approximately eight C. arabica dihaploid genome equivalents. One original feature of this library is that it can be divided into four sublibraries with mean insert sizes of 96, 130, 183 and 210 kb. Characterisation of the library showed that less than 4.5% of the clones contained organelle DNA. Furthermore, this library is representative and shows good genome coverage, as established by hybridisation screening of high-density filters using a number of nuclear probes distributed across the allotetraploid genome. This Arabica BAC library, the first large-insert DNA library so far constructed for the genus Coffea, is well-suited for many applications in genome research, including physical mapping, map-based cloning, functional and comparative genomics as well as polyploid genome analyses.

Efficient cloning of plant genomes into bacterial artificial chromosome (BAC) libraries with larger and more uniform insert size

Plant Biotechnol J. 2004 May;2(3):181-8. PMID: 17147609
Chalhoub, B., Belcram, H. and Caboche M. (2004)

The construction of bacterial artificial chromosome (BAC) libraries remains relatively complex and laborious, such that any technological improvement is considered to be highly advantageous. In this study, we addressed several aspects that improved the quality and efficiency of cloning of plant genomes into BACs. We set the 'single tube vector' preparation method with no precipitation or gel electrophoresis steps, which resulted in less vector DNA damage and a remarkable two- to threefold higher transformation efficiency compared with other known vector preparation methods. We used a reduced amount of DNA for partial digestion (up to 5 microg), which resulted in less BAC clones with small inserts. We performed electrophoresis in 0.25 x TBE (Tris, boric acid, ethylenediaminetetraacetic acid) buffer instead of 0.5 x TBE, which resulted in larger and more uniformly sized BAC inserts and, surprisingly, a two- to threefold higher transformation efficiency, probably due to less contamination with borate ions. We adopted a triple size selection that resulted in an increased mean insert size of up to 70 kb and a transformation efficiency comparable with that of double size selection. Overall, the improved protocol presented in this study resulted in a five- to sixfold higher cloning efficiency and larger and more uniformly sized BAC inserts. BAC libraries with the desired mean insert size (up to 200 kb) were constructed from several plant species, including hexaploid wheat. The improved protocol will render the construction of BAC libraries more available in plants and will greatly enhance genome analysis, gene mapping and cloning.

A putative Ca 2+ and calmodulin-dependant protein kinase required for bacterial and fungal symbioses.

Science. 2004 Feb 27;303(5662):1361-4. Epub 2004 Feb 12. PMID: 14963335
Lévy J., Bres C., Geurts R., Chalhoub B., Kuilkova O., Duc G., Journet E.P., Ané J. M., Lauber E., Bisseling T., Dénarié J., Rosenberg C., Debellé, F. (2004).
Laboratoire des Interactions Plantes-Microorganismes INRA-CNRS, BP27, 31326 Castanet-Tolosan Cedex, France.

Legumes can enter into symbiotic relationships with both nitrogen-fixing bacteria (rhizobia) and mycorrhizal fungi. Nodulation by rhizobia results from a signal transduction pathway induced in legume roots by rhizobial Nod factors. DMI3, a Medicago truncatula gene that acts immediately downstream of calcium spiking in this signaling pathway and is required for both nodulation and mycorrhizal infection, has high sequence similarity to genes encoding calcium and calmodulin-dependent protein kinases (CCaMKs). This indicates that calcium spiking is likely an essential component of the signaling cascade leading to nodule development and mycorrhizal infection, and sheds light on the biological role of plant CCaMKs.


Construction and characterization of a hexaploid wheat (Triticum aestivum L.) BAC library from the reference germplasm 'chines spring'.

Cereal Res. Comm. 31: 331-338.
Allouis, S.M., Bellec, A., Sharp, R., Faivre Rampant, P., Mortimer, K., Pateyron, S., Foote, T.N., Griffiths, S., Caboche, M. and Chalhoub, B. (2003)
John Innes Centre Genome Laboratory

(Triticum aestivum, cultivar: Chinese Spring)
A BAC library consisting of 1,200,000 clones, constructed using pIndigoBAC5 as vector. This library has an average insert size of 130kb and was made using DNA from the leaves of Triticum aestivum, cultivar Chinese Spring. This library was constructed as part of a joint collaboration between BBSRC (UK) and INRA (France) to develop genomic resources for positional cloning and structural genomics in Wheat.


The mobility of the tobacco Tnt1 retrotransposon correlates with its transcriptional activation by fungal factors.

Plant J. 2001 Oct;28(2):159-68. PMID: 11722759
Melayah D, Bonnivard E, Chalhoub B, Audeon C, Grandbastien MA.
Laboratoire de Biologie Cellulaire, INRA, 78026 Versailles, France.

We have analyzed the stress-induced amplification of the tobacco Tnt1 element, one of the rare active plant retrotransposons. Tnt1 mobility was monitored using the retrotransposon-anchored SSAP strategy that allows the screening of multiple insertion sites of high copy number elements. We have screened for Tnt1 insertion polymorphisms in plants regenerated from mesophyll leaf cells, either via explant culture or via protoplast isolation. The second procedure includes an overnight exposure to fungal extracts known to induce high levels of Tnt1 transcription. Newly transposed Tnt1 copies were detected in nearly 25% of the plants regenerated via protoplast isolation, and in less than 3% of the plants derived from explant culture. These results show that Tnt1 transcription is followed by transposition, and that fungal extracts efficiently activate Tnt1 mobility. Transcription appears to be the key step to controlling Tnt1 amplification, as newly transposed Tnt1 copies show high sequence similarities to the subpopulations of transcribed Tnt1 elements. Our results provide direct evidence that factors of microbial origin are able to induce retrotransposon amplification in plants, and strengthen the hypothesis that stress modulation of transposable elements might play a role in generating host genetic plasticity in response to environmental stresses.

  1. Differential interaction between PAV-like isolates of Barley Yellow Dwarf Virus and barley (Hordeum vulgare L.) genotypes.
    Journal of Phytopathology 142, 189-198.
    # Chalhoub B., Sarrafi A., Beuve M.A., Lapierre H.D. (1994a).
  2. Sequence variability in the genome 3'-terminal region for 10 geographically distinct PAVlike isolates of barley yellow dwarf virus; Analyses of the ORF6 variation.
    Archives of Virology 139, 403-416.
    # Chalhoub B., Kelly L., Robaglia C., Lapierre H.D. (1994b).
  3. Evidence of RNA recombination in the genome 3'-terminal region of PAV-like isolates of barley yellow dwarf virus (BYDV-PAV).
    agronomie 15, 409-413.
    # Chalhoub B., Kelly L., Robaglia C., Lapierre H.D. (1995a).
  4. Diallel analysis for partial resistance of five barley (Hordeum vulgare L.) genotypes to a PAV-like isolate of barley yellow dwarf virus.
    # Chalhoub B., Sarrafi A., Lapierre H.D. (1995b).
    Journal of Genetics and Breeding 49, 31-36.
  5. Partial resistance in the barley (Hordeum vulgare L) cultivar "Chikurin Ibaraki" to two PAV-like isolates of barley yellow dwarf virus: allelic variability at the Yd2 gene locus.
    # Chalhoub B., Sarrafi A., Lapierre H.D. (1995c).
    Plant Breeding 114, 303-307.
  6. Silver staining and reovery of AFLP™ amplification products on large denaturing polyacrylamide gels.
    # Chalhoub B., Thibault S., Laucou V., Rameau C., Höfte H., Cousin R. (1997)
    BioTechniques 22, 216-220.
  7. The mobility of the tobacoo Tnt1 retrotransposon correlates with its transcriptional activation by fungal factors.
    Melayah D., Bonnivard E., Chalhoub B., Audeon C., Granbastien M-A. (2001).
    Plant J 28(2), 159-168. PMID: 11722759
  8. Importance of RNA recombination in luteoviruses evolution.
    # Chalhoub B., Lapierre H.D. (1995)
    Agronomie 15, 393-400.