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

<p>Abstract</p> <p>Background</p> <p>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 <it>gypsy </it>LTR-retrotransposons, <it>Fatima</it>, on B-genome divergence of allopolyploid wheat using the fluorescent <it>in situ </it>hybridisation (FISH) method and phylogenetic analysis.</p> <p>Results</p> <p>FISH analysis of a BAC clone (BAC_2383A24) initially screened with Spelt1 repeats demonstrated its predominant localisation to chromosomes of the B-genome and its putative diploid progenitor <it>Aegilops speltoides </it>in hexaploid (genomic formula, BBAADD) and tetraploid (genomic formula, BBAA) wheats as well as their diploid progenitors. 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 <it>gypsy </it>LTR retrotransposon family <it>Fatima </it>make up 47.2% of all the LTR retrotransposons in this BAC. <it>In situ </it>hybridisation of the <it>Fatima</it>_2383A24-3 subclone suggests that individual representatives of the <it>Fatima </it>family contribute to the majority of the B-genome specific FISH pattern for BAC_2383A24. Phylogenetic analysis of various <it>Fatima </it>elements available from databases in combination with the data on their insertion dates demonstrated that the <it>Fatima </it>elements fall into several groups. One of these groups, containing <it>Fatima</it>_2383A24-3, is more specific to the B-genome and proliferated around 0.5-2.5 MYA, prior to allopolyploid wheat formation.</p> <p>Conclusion</p> <p>The B-genome specificity of the <it>gypsy</it>-like <it>Fatima</it>, as determined by FISH, is explained to a great degree by the appearance of a genome-specific element within this family for <it>Ae. speltoides</it>. Moreover, its proliferation mainly occurred in this diploid species before it entered into allopolyploidy.</p> <p>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.</p

Isolation and sequence analysis of the wheat B genome subtelomeric DNA

<p>Abstract</p> <p>Background</p> <p>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 <it>Aegilops </it>species from the section <it>Sitopsis</it>.</p> <p>Results</p> <p>The BAC library from <it>Triticum aestivum </it>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 <it>in situ </it>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 <it>Caspar </it>covers 11 666 bp, encoding a transposase and CTG-2 proteins, and this transposon accounts for 40% of the DNA transposons. The <it>in situ </it>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.</p> <p>Conclusion</p> <p>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.</p

Molecular analysis of Arabidopsis thaliana transparent testa (tt) genes in Brassica napus

Lotz T, Snowdon R, Horn R, et al. Molecular analysis of Arabidopsis thaliana transparent testa (tt) genes in Brassica napus. In: PLANT BREEDING Opportunities for new cruciferous crops. Rapeseed Congress Proceedings. 2003

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

<p>Abstract</p> <p>Background</p> <p>Several studies suggested that the diploid ancestor of the B genome of tetraploid and hexaploid wheat species belongs to the <it>Sitopsis </it>section, having <it>Aegilops speltoides </it>(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 <it>Ae. speltoides </it>(2n = 14) to that of the A, B and D genomes co-resident in the hexaploid wheat species (<it>Triticum aestivum, AABBDD</it>, 2n = 42).</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>On the basis of conserved sequence length as well as identity of the shared non-TE regions and the SPA coding sequence, <it>Ae speltoides </it>appears to be more evolutionary related to the B genome of <it>T. aestivum </it>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 <it>Ae. speltoides </it>has diverged very early from the progenitor of the B genome which remains to be identified.</p

Organization, evolution and function of the major wheat domestication (Q/q) gene and its homoeologs in polyploid wheat

En 2006, après un siècle d’investigations, le gène majeur de domestication 5AQ, conférant de nombreux caractères comme la non-déhiscence et un battage facile des épillets des blés polyploïdes, a été identifié comme un facteur de transcription homologue au gène Apetala2 d’Arabidopsis. Bien que ceci représente une avancée importante, le rôle des autres homéologues de ce gène, présents dans le blé tétraploïde (Triticum turgidum) et le blé hexaploïde (T. aestivum), reste à élucider. Dans ce contexte mon sujet de recherche porte sur l’appréciation de l’organisation, l’évolution et le fonctionnement du gène majeur de domestication (Q) et de ses homéologues chez les blés polyploïdes. J’ai tout d’abord séquencé et analysé 11 régions génomiques (clones BAC) portant les copies du gène Q/q dans différents génomes de blés polyploïdes et diploïdes; constituant ainsi la plus grande analyse comparative réalisée aujourd’hui chez le blé. Les comparaisons entre les différents génomes et différents niveaux de ploïdie montrent que le gène Q/q est la seule séquence conservée, en commun dans les régions génomiques comparées, et que l’homéologue 5Bq est pseudogénéisé dans les blés hexaploïdes. Les comparaisons montrent que le reste des séquences génomiques sont constituées d’environ 80% d’éléments transposables (TEs) qui sont entièrement différents quand on compare les génomes A, B, D et S entre eux. A l’inverse, les TEs sont relativement mieux conservés entre haplotypes du même génome et continuent leurs dynamiques d’insertions et de délétions différentielles, conduisant à 19 événements de rupture de synténie. Parmi ces événements, j’ai pu identifier le premier Hélitron actif du blé, inséré dans le pseudogène 5Bq du cultivar Renan. La recherche de son origine par comparaison de séquences et l’étude de la variabilité haplotypique m’ont permis de confirmer l’insertion récente et l’origine commune de cet élément au blé sauvage Aegilops ventricosa. Cette espèce a été introgressée dans certaines variétés de blés hexaploïdes. L’analyse fonctionnelle comparant les caractères de domestication ainsi que l’expression et les interactions entre les différents homéologues du gène Q/q dans le blé hexaploïde a été rendue possible par la caractérisation des « lignées de délétion », où une ou plusieurs copies homéologues ont été perdues ou substituées. J’ai pu ainsi caractériser l’hyper-fonctionalisation de l’homéologue 5AQ, et la sous-fonctionalisation des homéologues 5Dq et plus étonnamment 5Bq, pseudogénéisé ; les trois homéologues contribuent aux caractères de domestication et se régulent entre eux. Les comparaisons précises des séquences des allèles 5AQ et 5Aq pour plusieurs génotypes domestiqués et sauvages m’ont permis d’identifier une mutation SNP associée, dans le site d’adressage d’un micro RNA (miR172). L’utilisation d’une technique RACE-PCR semi-quantitative montre que la mutation dans l’allèle 5AQ conduit à moins d’ARNm clivés par les miR172 et donc à sa plus forte expression ; comparée à celle de l’allèle 5Aq. Ceci suggère un rôle des miR172 dans la régulation des différents homéologues du gène Q/q.In 2006, and after a century of investigations, the major domestication gene in polyploid wheat (5AQ), involved in non free threshing and spike easy beating, among many other traits, has been identified as a homolog of Apetala2 gene of Arabidopsis. While this represents an important breakthrough, nothing was yet known about the role of other homoeologs of the Q/q gene present in tetraploid (Triticum turgidum) and hexaploid (T. aestivum) wheat. In this context, my PhD thesis consists in characterizing organization, evolution and function of the major wheat domestication (Q/q) gene and its homoeologs in polyploid wheat. I realized first comparative sequencing and analysis of 11 genomic regions (BAC clones) spanning the Q/q gene homolog’s in different hexaploid, tetraploid and diploid wheat; constituting the most important comparative analysis done for this group of species. Comparisons show that only Q/q gene homologs are conserved in different genomes and across different ploidy levels and that the 5Bq homoeolog is pseudogenized in hexaploid wheat. The remaining genomic sequences, constituted of ~80% of transposable elements (TEs) are completely different when comparing A, B, D and S genomes between each others. On the contrary, TEs are more conserved between different haplotypes of a same genome and continue their active insertion and deletion dynamic, leading to 19 identified synteny breaks. Among these, I identified the first active Hélitron in wheat inserted into the 5Bq pseudogene of a hexaploid wheat cv. Renan. The Hélitron insertion was subsequently retraced as recently occurring whereas it could have been originated from the wild wheat Aegilops ventricosa which has been introgressed into hexaploid wheat. Functional analysis comparing phenotype, domestication traits, expression and interaction between different Q/q homoeologs was rendered possible using series of “deletion lines”, where one or several homoeologs were deleted. This allows determining the hyper-functionalization of 5AQ and the subfunctionalization of 5Dq and more interestingly the subfunctionalization of the pseudogene 5Bq. All three homoeologs were shown to contribute to the domestication traits and regulate each others.Precise sequence comparison of 5AQ and 5Aq alleles from different domesticated and wild genotypes allow identification of a SNP mutation, associated with domestication, in the target site of a micro RNA (miR172). Using an adapted semi-quantitative RACE-PCR, I showed that the mutation leads to less cleaved mRNA of the 5AQ gene by the miR172 and consequently its higher expression than the 5Aq allele. This also suggests a general role of miR172 in regulating the different homoeologs of the Q/q gene

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

18 ref. doi: 10.1111/j.1467-7652.2004.00065.xInternational audienceThe 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 µg), which resulted in less BAC clones with small inserts. We performed electrophoresis in 0.25 × TBE (Tris, boric acid, ethylenediaminetetraacetic acid) buffer instead of 0.5 × 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

Use of multiparental inbred populations to determine allelic relationships of molecular markers

International audienc

Use of multiparental inbred populations to determine allelic relationships of molecular markers

International audienc