The nature and organization of satellite DNAs in Petunia hybrida, related, and ancestral genomes

IntroductionThe garden petunia, Petunia hybrida (Solanaceae) is a fertile, diploid, annual hybrid species (2n=14) originating from P. axillaris and P. inflata 200 years ago. To understand the recent evolution of the P. hybrida genome, we examined tandemly repeated or satellite sequences using bioinformatic and molecular cytogenetic analysis.MethodsRaw reads from available genomic assemblies and survey sequences of P. axillaris N (PaxiN), P. inflata S6, (PinfS6), P. hybrida (PhybR27) and the here sequenced P. parodii S7 (PparS7) were used for graph and k-mer based cluster analysis of TAREAN and RepeatExplorer. Analysis of repeat specific monomer lengths and sequence heterogeneity of the major tandem repeat families with more than 0.01% genome proportion were complemented by fluorescent in situ hybridization (FISH) using consensus sequences as probes to chromosomes of all four species.ResultsSeven repeat families, PSAT1, PSAT3, PSAT4, PSAT5 PSAT6, PSAT7 and PSAT8, shared high consensus sequence similarity and organisation between the four genomes. Additionally, many degenerate copies were present. FISH in P. hybrida and in the three wild petunias confirmed the bioinformatics data and gave corresponding signals on all or some chromosomes. PSAT1 is located at the ends of all chromosomes except the 45S rDNA bearing short arms of chromosomes II and III, and we classify it as a telomere associated sequence (TAS). It is the most abundant satellite repeat with over 300,000 copies, 0.2% of the genomes. PSAT3 and the variant PSAT7 are located adjacent to the centromere or mid-arm of one to three chromosome pairs. PSAT5 has a strong signal at the end of the short arm of chromosome III in P. axillaris and P.inflata, while in P. hybrida additional interstitial sites were present. PSAT6 is located at the centromeres of chromosomes II and III. PSAT4 and PSAT8 were found with only short arrays.DiscussionThese results demonstrate that (i) repeat families occupy distinct niches within chromosomes, (ii) they differ in the copy number, cluster organization and homogenization events, and that (iii) the recent genome hybridization in breeding P. hybrida preserved the chromosomal position of repeats but affected the copy number of repetitive DNA

Integration of Banana Streak Badnavirus into theMusaGenome: Molecular and Cytogenetic Evidence

AbstractBreeding and tissue culture of certain cultivars of bananas (Musa) have led to high levels of banana streak badnavirus (BSV) infection in progeny from symptomless parents. BSV DNA hybridized to genomic DNA of one such parent, Obino l'Ewai, suggesting integration of viral sequences. Sequencing of clones of Obino l'Ewai genomic DNA revealed an interface between BSV andMusasequences and a complex BSV integrant.In situhybridization revealed two different BSV sequence locations in Obino l'Ewai chromosomes and a complex arrangement of BSV andMusasequences was shown by probing stretched DNA fibers. This is the first report of integrated sequences that possibly lead to a plant pararetrovirus episomal infection by a mechanism differing markedly from animal retroviral systems

Flow cytometry-based determination of ploidy from dried leaf specimens in genomically complex collections of the tropical forage grass Urochloa

Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. We aimed to develop an optimized approach to determine ploidy of germplasm collection of this tropical forage grass group using dried leaf material, including approaches to collect, dry and preserve plant samples for flow cytometry analysis. Our methods enable robust identification of ploidy levels (coefficient of variation of G0/G1 peaks, CV, typically <5%). Ploidy of some 348 forage grass accessions (ploidy range from 2x to 9x), from international genetic resource collections, showing variation in basic chromosome numbers and reproduction modes (apomixis and sexual), were determined using our defined standard protocol. Two major Urochloa agamic complexes are used in the current breeding programs at CIAT and EMBRAPA: the ’brizantha’ and ’humidicola’ agamic complexes are variable, with multiple ploidy levels. Some U. brizantha accessions have odd level of ploidy (5x), and the relative differences in fluorescence values of the peak positions between adjacent cytotypes is reduced, thus more precise examination of this species is required. Ploidy measurement of U. humidicola revealed aneuploidy

Repetitive DNA sequences in Crocus vernus Hill (Iridaceae): The genomic organization and distribution of dispersed elements in the genus Crocus and its allies.

Abstract: Eight clones of repetitive DNA were isolated from Crocus vernus Hill. The genomic organization of the clones was analyzed by in situ hybridization to C. vernus and Southern hybridization to a range of Crocus and other species. Seven clones were used for in situ hybridization. Sequence analysis showed that all eight clones were nonhomologous, and thus represented eight different sequence-families. In situ hybridization showed that six were dispersed in high copy numbers on all chromosomes of the C. vernus genome, whereas one was localized proximal to the secondary constriction, at the NOR (nucleolar organizer region) and was not further analyzed, as it was considered part of the 18S–25S rDNA repeat. Except for short palindromes, none of the sequences showed notable internal structures. Clone pCvKB4 showed homology to the reverse transcriptase gene of Ty1-copia-like retrotransposons; the others showed no homology to known sequences. When used as probes for Southern hybridization, four showed a ladder of 3–4 bands superimposed by irregular patterns, indicating organization in short tandem arrays. Each clone had a unique distribution among Crocus species (12–16 species analyzed with each clone) and six species of Iridaceae, Liliaceae, and Amaryllidaceae; all seven investigated sequences were Iridaceae specific and four were Crocus specific. The species distribution of these seven clones showed notable discrepancies with the taxonomic subdivision of the genus at the subgenus, section, and series levels. The results suggest that the phylogeny and taxonomic structure of the genus Crocus might need reconsideration. The analysis of repetitive DNA as a major and rapidly evolving part of the genome could contribute to the study of species relationships and evolution

The locatization of mitochondrial sequences to chromosomal DNA in orthopterans.

There is growing evidence that the integration of mitochondrial DNA sequences into nuclear and chloroplast genomes of higher organisms may be widespread rather than exceptional. We report the localization of 18S–25S rDNA and mitochondrial DNA sequences to meiotic chromosomes of several orthopteran species using in situ hybridisation. The cytochrome oxidase I (COI) sequence localizes to the centromeric and two telomeric regions of the eight bivalents of Chorthippus parallelus, the telomeric regions in Schistocerca gregaria and is present throughout the genome of Italopodisma sp. (Orthoptera: Acrididae). The control region of the mitochondrion and COI localize to similar chromosomal regions in S. gregaria. These data explain sequencing data that are inconsistent with the COI sequence being solely mitochondrial. The different nuclear locations of mtDNA in the different genera studied suggest that grasshopper mtDNA-like sequences have been inserted into the nuclear genome more than once in Acridid history, and there may have been different mechanisms involved when these events occurred in each of these species

The activity of nucleolar organizing chromosomes in multigeneric F1 hybrids involving wheat, tricale, and tritrdeum.

The nucleolar activity in interspecific hybrids between tritordeum, wheat, and triticale was studied using the silver salt – nylon technique. rRNA genes from the major NORs from wheat (chromosome pairs 1B and 6B) and Hordeum chilense (chromosome pairs 5Hch and 6Hch) were active in genomic constitutions AABBHchHch, AABBHch, and AABBDHch. The rye chromosome 1R NOR suppression found in triticale and in wheat × rye F1 hybrids is also observed in trigeneric hybrids, genome constitution AABBRHch, derived from crosses between triticale and tritordeum. As expected, the maximum number of NORs per metaphase cell is coincident with the maximum number of nucleoli per interphase cell. Occasional extra nucleolar activity was observed, probably owing to minor sites of wheat NORs, since no other sites of active or inactive rRNA genes were detected in H. chilense. However, it is notable that the two H. chilense loci and two major rDNA loci of wheat origin are expressed together

CENH3 morphogenesis reveals dynamic centromere associations during synaptonemal complex formation and the progression through male meiosis in hexaploid wheat.

During meiosis, centromeres in some species undergo a series of associations, but the processes and progression to homologous pairing is still a matter of debate. Here, we aimed to correlate meiotic centromere dynamics and early telomere behaviour to the progression of synapotonemal complex (SC) construction in hexaploid wheat (2n=42) by triple immunolabelling of CENH3 protein marking functional centromeres, and SC proteins ASY1 (unpaired lateral elements) and ZYP1 (central elements in synapsed chromosomes). We show that single or multiple centromere associations formed in meiotic interphase undergo a progressive polarisation (clustering) at the nuclear periphery in early leptotene, leading to formation of the telomere bouquet. Critically, immunolabelling shows the dynamics of these presynaptic centromere associations and a structural reorganisation of the centromeric chromatin coinciding with key events of synapsis initiation from the subtelomeric regions. As short stretches of subtelomeric synapsis emerged at early zygotene, centromere clusters lost their strong polarization, gradually resolving as individual centromeres indicated by more than 21 CENH3 foci associated with unpaired lateral elements. Only following this centromere depolarisation were homologous chromosome arms connected, as observed by the alignment and fusion of interstitial ZYP1 loci elongating at zygotene so synapsis at centromeres is a continuation of the interstitial synapsis. Our results thus reveal that centromere associations are a component of the timing and progression of chromosome synapsis, and the gradual release of the individual centromeres from the clusters correlates with the elongation of interstitial synapsis between the corresponding homologues

Introgression of rye chromatin on chromosome 2D in the Portuguese wheat landrace 'Barbela'.

Abstract: The old Portuguese wheat landrace aggregate known as ‘Barbela’ shows good productivity under the lowfertility conditions often associated with acid soils. The use of genomic rye DNA, in combination with 45S rDNA and the repetitive sequences dpTa1 and pSc119.2 as probes, in two sequential in situ hybridization steps enabled the identification of all chromosomes in the ‘Barbela’ wheat lines and the detection of the introgression of rye-origin chromatin onto wheat chromosome arm 2DL in two of the lines. Amplification of microsatellite loci using published primer pairs showed that the distal segment of wheat chromosome 2DL, which was involved in the rye translocation, was deleted. The identification and characterization of small recombinant chromosome segments in wheat–rye lines may allow their use in plant breeding programmes. Their presence in farmer-maintained material demonstrates the importance of maintaining, characterizing, and collecting landrace material before valuable genetic combinations are lost as uniform commercial crops are introduced

Least-squares methods for identifying biochemical regulatory networks from noisy measurements.

Background: We consider the problem of identifying the dynamic interactions in biochemical networks from noisy experimental data. Typically, approaches for solving this problem make use of an estimation algorithm such as the well-known linear Least-Squares (LS) estimation technique. We demonstrate that when time-series measurements are corrupted by white noise and/or drift noise, more accurate and reliable identification of network interactions can be achieved by employing an estimation algorithm known as Constrained Total Least Squares (CTLS). The Total Least Squares (TLS) technique is a generalised least squares method to solve an overdetermined set of equations whose coefficients are noisy. The CTLS is a natural extension of TLS to the case where the noise components of the coefficients are correlated, as is usually the case with timeseries measurements of concentrations and expression profiles in gene networks. Results: The superior performance of the CTLS method in identifying network interactions is demonstrated on three examples: a genetic network containing four genes, a network describing p53 activity and mdm2 messenger RNA interactions, and a recently proposed kinetic model for interleukin (IL)-6 and (IL)-12b messenger RNA expression as a function of ATF3 and NF-κB promoter binding. For the first example, the CTLS significantly reduces the errors in the estimation of the Jacobian for the gene network. For the second, the CTLS reduces the errors from the measurements that are corrupted by white noise and the effect of neglected kinetics. For the third, it allows the correct identification, from noisy data, of the negative regulation of (IL)-6 and (IL)-12b by ATF3. Conclusion: The significant improvements in performance demonstrated by the CTLS method under the wide range of conditions tested here, including different levels and types of measurement noise and different numbers of data points, suggests that its application will enable more accurate and reliable identification and modelling of biochemical networks