Cytogenetics in the Study of Chromosomal Rearrangement during Wheat Evolution and Breeding

Cytogenetic methods such as chromosome banding and in situ hybridization remain relevant in the post-genomic era, especially for allopolyploid species where genome duplication in some cases makes it difficult to assess the reorganization of chromosomes during evolution. In this review, we give a brief description of cytogenetic methods for the analysis of homoeological chromosomes in cereals. Emphasis is placed on the development of methods for the study of polyploid wheat and its progenitors and on tandem repeats and retrotransposons as markers to evaluate chromosome reorganization throughout evolution and breeding. The most effective cytological probes used for the identification of chromosomes in wheat and Triticeae species by fluorescence and genomic in situ hybridization are described. Particular attention is paid to ribosomal genes used as markers in phylogenetic studies and for chromosome identification. Utility of these cytogenetic methods in the evaluation of breeding lines is demonstrated. A strategy for cytological analysis of wheat hybrids according to the degree of relationships between the species involved in crosses is also discussed

Relationship between homoeologous regulatory and structural genes in allopolyploid genome – A case study in bread wheat

<p>Abstract</p> <p>Background</p> <p>The patterns of expression of homoeologous genes in hexaploid bread wheat have been intensively studied in recent years, but the interaction between structural genes and their homoeologous regulatory genes remained unclear. The question was as to whether, in an allopolyploid, this interaction is genome-specific, or whether regulation cuts across genomes. The aim of the present study was cloning, sequence analysis, mapping and expression analysis of <it>F3H </it>(flavanone 3-hydroxylase – one of the key enzymes in the plant flavonoid biosynthesis pathway) homoeologues in bread wheat and study of the interaction between <it>F3H </it>and their regulatory genes homoeologues – <it>Rc </it>(red coleoptiles).</p> <p>Results</p> <p>PCR-based cloning of <it>F3H </it>sequences from hexaploid bread wheat (<it>Triticum aestivum </it>L.), a wild tetraploid wheat (<it>T. timopheevii</it>) and their putative diploid progenitors was employed to localize, physically map and analyse the expression of four distinct bread wheat <it>F3H </it>copies. Three of these form a homoeologous set, mapping to the chromosomes of homoeologous group 2; they are highly similar to one another at the structural and functional levels. However, the fourth copy is less homologous, and was not expressed in anthocyanin pigmented coleoptiles. The presence of dominant alleles at the <it>Rc-1 </it>homoeologous loci, which are responsible for anthocyanin pigmentation in the coleoptile, was correlated with <it>F3H </it>expression in pigmented coleoptiles. Each dominant <it>Rc-1 </it>allele affected the expression of the three <it>F3H </it>homoeologues equally, but the level of <it>F3H </it>expression was dependent on the identity of the dominant <it>Rc-1 </it>allele present. Thus, the homoeologous <it>Rc-1 </it>genes contribute more to functional divergence than do the structural <it>F3H </it>genes.</p> <p>Conclusion</p> <p>The lack of any genome-specific relationship between <it>F3H-1 </it>and <it>Rc-1 </it>implies an integrative evolutionary process among the three diploid genomes, following the formation of hexaploid wheat. Regulatory genes probably contribute more to the functional divergence between the wheat genomes than do the structural genes themselves. This is in line with the growing consensus which suggests that although heritable morphological traits are determined by the expression of structural genes, it is the regulatory genes which are the prime determinants of allelic identity.</p

Dormant non-culturable Mycobacterium tuberculosis retains stable low-abundant mRNA

BACKGROUND: Dormant Mycobacterium tuberculosis bacilli are believed to play an important role in latent tuberculosis infection. Previously, we have demonstrated that cultivation of M. tuberculosis in K(+)-deficient medium resulted in generation of dormant cells. These bacilli were non-culturable on solid media (a key feature of dormant M. tuberculosis in vivo) and characterized by low metabolism and tolerance to anti-tuberculosis drugs. The dormant bacteria demonstrated a high potential to reactivation after K(+) reintroduction even after prolonged persistence under rifampicin. In this work, we studied the transcriptome and stability of transcripts in persisting dormant bacilli under arrest of mRNA de novo synthesis. RESULTS: RNA-seq-based analysis of the dormant non-culturable population obtained under rifampicin exposure revealed a 30–50-fold decrease of the total mRNA level, indicating global transcriptional repression. However, the analysis of persisting transcripts displayed a cohort of mRNA molecules coding for biosynthetic enzymes, proteins involved in adaptation and repair processes, detoxification, and control of transcription initiation. This ‘dormant transcriptome’ demonstrated considerable stability during M. tuberculosis persistence and mRNA de novo synthesis arrest. On the contrary, several small non-coding RNAs showed increased abundance on dormancy. Interestingly, M. tuberculosis entry into dormancy was accompanied by the cleavage of 23S ribosomal RNA at a specific point located outside the ribosome catalytic center. CONCLUSIONS: Dormant non-culturable M. tuberculosis bacilli are characterized by a global transcriptional repression. At the same time, the dormant bacilli retain low-abundant mRNAs, which are considerably stable during in vitro persistence, reflecting their readiness for translation upon early resuscitation steps. Increased abundance of non-coding RNAs on dormancy may indicate their role in the entry into and maintenance of M. tuberculosis dormant non-culturable state. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-2197-6) contains supplementary material, which is available to authorized users

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

Construction by dielectrophoresis of microbial aggregates for the study of bacterial cell dormancy

A study of the effect of aggregate size on the resuscitation of dormant M. smegmatis was conducted by constructing cell aggregates with defined sizes and shapes using dielectrophoresis and monitoring the resuscitation process under controlled laboratorial conditions in a long-term cell feeding system. Differently sized cell aggregates were created on the surface of indium tin oxide coated microelectrodes, their heights and shapes controlled by the strength of the induced electric field and the shape of the microelectrodes. Both two-dimensional (ring-patterned) and three-dimensional cell aggregates were produced. The cell aggregates were maintained under sterile conditions at 37 °C for up to 14 days by continuously flushing Sauton’s medium through the chamber. Resuscitation of dormant M. smegmatis was evaluated by the production of the fluorescent dye 5-cyano-2,3-ditolyltetrazolium chloride. The results confirm that the resuscitation of dormant M. smegmatis is triggered by the accumulation of a resuscitation promoting factor inside the aggregates by diffusion limitation

Finding of the Low Molecular Weight Inhibitors of Resuscitation Promoting Factor Enzymatic and Resuscitation Activity

Background: Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new antituberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs. Methodology/Principal Findings: Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC50 1–7 mg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC50. The candidate compounds suppressed resuscitation of dormant (‘‘non-culturable’’) cells of M. smegmatis at 1 mg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 mg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations. Conclusions/Significance: NPT are the first example of low molecular weight compounds that inhibit the enzymatic an

Genome-Wide Association Study in BRCA1 Mutation Carriers Identifies Novel Loci Associated with Breast and Ovarian Cancer Risk

BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7×10-8, HR = 1.14, 95% CI: 1.09-1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4×10-8, HR = 1.27, 95% CI: 1.17-1.38) and 4q32.3 (rs4691139, P = 3.4×10-8, HR = 1.20, 95% CI: 1.17-1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific associat