Comparative Analysis of Tandem Repeats from Hundreds of Species Reveals Unique Insights into Centromere Evolution

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data. The assumption that the most abundant tandem repeat is the centromere DNA was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and in length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond ~50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution, including the appearance of higher order repeat structures in which several polymorphic monomers make up a larger repeating unit. While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animals and plants. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes

Exploring the Diversity of Plant DNA Viruses and Their Satellites Using Vector-Enabled Metagenomics on Whiteflies

Current knowledge of plant virus diversity is biased towards agents of visible and economically important diseases. Less is known about viruses that have not caused major diseases in crops, or viruses from native vegetation, which are a reservoir of biodiversity that can contribute to viral emergence. Discovery of these plant viruses is hindered by the traditional approach of sampling individual symptomatic plants. Since many damaging plant viruses are transmitted by insect vectors, we have developed “vector-enabled metagenomics” (VEM) to investigate the diversity of plant viruses. VEM involves sampling of insect vectors (in this case, whiteflies) from plants, followed by purification of viral particles and metagenomic sequencing. The VEM approach exploits the natural ability of highly mobile adult whiteflies to integrate viruses from many plants over time and space, and leverages the capability of metagenomics for discovering novel viruses. This study utilized VEM to describe the DNA viral community from whiteflies (Bemisia tabaci) collected from two important agricultural regions in Florida, USA. VEM successfully characterized the active and abundant viruses that produce disease symptoms in crops, as well as the less abundant viruses infecting adjacent native vegetation. PCR assays designed from the metagenomic sequences enabled the complete sequencing of four novel begomovirus genome components, as well as the first discovery of plant virus satellites in North America. One of the novel begomoviruses was subsequently identified in symptomatic Chenopodium ambrosiodes from the same field site, validating VEM as an effective method for proactive monitoring of plant viruses without a priori knowledge of the pathogens. This study demonstrates the power of VEM for describing the circulating viral community in a given region, which will enhance our understanding of plant viral diversity, and facilitate emerging plant virus surveillance and management of viral diseases

Diversity and phylogeography of begomovirus-associated beta satellites of okra in India

<p>Abstract</p> <p>Background</p> <p>Okra (<it>Abelmoschus esculentus</it>; family <it>Malvaceae</it>) is grown in temperate as well as subtropical regions of the world, both for human consumption as a vegetable and for industrial uses. Okra yields are affected by the diseases caused by phyopathogenic viruses. India is the largest producer of okra and in this region a major biotic constraint to production are viruses of the genus <it>Begomovirus</it>. Begomoviruses affecting okra across the Old World are associated with specific, symptom modulating satellites (beta satellites). We describe a comprehensive analysis of the diversity of beta satellites associated with okra in India.</p> <p>Results</p> <p>The full-length sequences of 36 beta satellites, isolated from okra exhibiting typical begomovirus symptoms (leaf curl and yellow vein), were determined. The sequences segregated in to four groups. Two groups correspond to the beta satellites Okra leaf curl beta satellite (OLCuB) and Bhendi yellow vein beta satellite (BYVB) that have previously been identified in okra from the sub-continent. One sequence was distinct from all other, previously isolated beta satellites and represents a new species for which we propose the name Bhendi yellow vein India beta satellite (BYVIB). This new beta satellite was nevertheless closely related to BYVB and OLCuB. Most surprising was the identification of Croton yellow vein mosaic beta satellite (CroYVMB) in okra; a beta satellite not previously identified in a malvaceous plant species. The okra beta satellites were shown to have distinct geographic host ranges with BYVB occurring across India whereas OLCuB was only identified in northwestern India. Okra infections with CroYVMB were only identified across the northern and eastern central regions of India. A more detailed analysis of the sequences showed that OLCuB, BYVB and BYVIB share highest identity with respect βC1 gene. βC1 is the only gene encoded by beta satellites, the product of which is the major pathogenicity determinant of begomovirus-beta satellite complexes and is involved in overcoming host defenses based on RNAi.</p> <p>Conclusion</p> <p>The diversity of beta satellites in okra across the sub-continent is higher than previously realized and is higher than for any other malvaceous plant species so far analyzed. The beta satellites identified in okra show geographic segregation, which has implications for the development and introduction of resistant okra varieties. However, the finding that the βC1 gene of the major okra beta satellites (OLCuB, BYVB and BYVIB) share high sequence identity and provides a possible avenue to achieve a broad spectrum resistance.</p

Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription

Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy

Statistical analysis on detecting recombination sites in DNA-beta satellites associated with the old world geminiviruses

Although an exchange of genetic information by recombination plays an important role in the evolution of viruses, it is not clear how it generates diversity. {\it Geminiviruses} are plant viruses which have ambisense single-stranded circular DNA genomes and one of the most economically important plant viruses in agricultural production. Small circular single-stranded DNA satellites, termed DNA-$\beta$, have recently been found associated with some geminivirus infections. In this paper we analyze a satellite molecule DNA-$\beta$ of geminiviruses for recombination events using phylogenetic and statistical analysis and we find that one strain from ToLCMaB has a recombination pattern and is possibly recombinant molecule between two strains from two species, PaLCuB-[IN:Chi:05] (major parent) and ToLCB-[IN:CP:04] (minor parent).Comment: 8 figures and 2 tables. To appear in Frontiers in Systems Biolog

MiIP: The Monomer Identification and Isolation Program

Repetitive elements within genomic DNA are both functionally and evolutionarily informative. Discovering these sequences ab initio is computationally challenging, compounded by the fact that selection on these repeats is often relaxed; thus sequence identity between repetitive elements can vary significantly. Here we present a new application, the Monomer Identification and Isolation Program (MiIP), which provides functionality to both search for a particular repeat as well as discover repetitive elements within a larger genomic sequence. To compare MiIP’s performance with other repeat detection tools, analysis was conducted for synthetic sequences as well as several α21-II clones and HC21 BAC sequences. The primary benefit of MiIP is the fact that it is a single tool capable of searching for both known monomeric sequences as well as discovering the occurrence of repeats ab initio, per the user’s required sensitivity of the search. Furthermore, the report functionality helps easily facilitate subsequent phylogenetic analysis

A study of the Southern African begomovirus pathosystem : determining the diversity of whitefly transmitted geminiviruses (WTG) infecting indigenous plants in South Africa

Abstrract : With an ever increasing world population, there is growing pressure on agricultural production to keep up with the demand for food, fuel and fiber products. Agricultural intensification has resulted in the conversion of approximately one-half of the Earth’s terrestrial surface to agricultural crops and range-land. This has significantly changed the natural landscape around agricultural borders, with the development of agro-ecological interface’ between wild ecosystems and crop or pasture lands. These areas are populated with crop plants, weeds of crop systems and non-crop plants that consist of both native and introduced plants. One very important aspect of this change in landscape, is the influence of microroganisms, particularly plant viruses, that move across the interface between crop and wild plants (virus reservoirs) and significantly impact agricultural production. Due to the potential role of these essentially unknown or poorly studied plant viruses in crop diseases and yield loss, they should be a focus of study. The aim of this project, was to characterize the circular viral DNA viral diversity in infecting indigenous or non-cultivated plant species in various cropping systems in South Africa, using a metagenomics approach. A survey was carried out in 2014 and 2015 in three Provinces (Limpopo, Mphumalanga and KwaZulu-Natal) of South Africa. Samples were collected from a total of 230 plants growing alongside cultivated crops and screened for viral infection. The viral diversity in these samples were explored using viral amplification by polymerase chain reaction (PCR), rolling circle amplification (RCA), RCA/restriction fragment length polymorphisms) (RCA/RFLP) and the results extended by next generation sequencing (NGS). Initial screening of the plants by PCR, using universal/degenerate begomovirus primers, found that 24% of the non-cultivated plants tested positive for possible begomovirus infection. To further reveal the full circular, viral diversity of the collected samples, they were screened by RCA/RFLP, followed by NGS for virus identification. The use of the RCA/RFLP was found to be a rapid, reproducible method to screen a large number of plant samples for viral biodiversity. Because of the high cost of NGS, a combined strategy was used, where initially RCA positive samples were pooled, and sequenced in one NGS run (NGS 2014, Pool-1 and 2), and after further screening by PCR, the plant samples suspected to contain circular viral molecules, were sequencing in individual NGS runs (NGS 2015). The pooled NGS, strategy was found to be an effective method for viral discovery, as it did allow the detection and characterization of both previously known viruses and previously unknown, novel virus genomes (Table 4.2). The drawback of the pooling-strategy was that for most viral contigs, the full viral genome could not be assembled and, post-NGS, it required a lengthy follow up procedure to trace the original plant samples, where the viral molecules originated from. 4 The data provided by the individual NGS runs (NGS 2015) allowed for the identification of both previously known viruses (four strains or variants of Tomato Curly Stunt Virus), and previously unknown, novel virus species (one monopartite and two bipartite begomoviruses, two betasatellite molecules and one genomovirus) in four different, uncultivated host species (Table 4.3). Previous studies have shown the widespread presence of ToCSV throughout South Africa and indeed, four ToCSV genomes were recovered in this study, both from tomato plants and weed species. The two ToCSV genomes recovered from the weeds species, Malvastrum coromandelianum and Acalypha indica, represent new natural host report for this virus and should be targeted for removal as they are acting as begomovirus reservoir in these cropping areas. The novel begomovirus species identified in this study, include one monopartite begomovirus, named Malvastrum curly stunt virus (MalCSV), that was identified in M. coromandelianum, along with an isolate of Tomato leaf curl Togo betasatellite (ToLCTGB) and one bipartite begomovirus, named Sida corlifolia golden mosaic virus (SiCGMV) that was identified in S. corlifolia, along with a novel betasatellite that was termed Sida corlifolia yellow mosaic betasatellite (SiCYMB).The MalCSV was identified from a symptomless host, but the SiCGMV and SiCYMB viral molecules were detected in a weed plant displaying symptoms of viral infection, i.e. bright yellow and green mosaic coloration of the apical leaves. The SiCGMV isolate displayed unique ‘NW-like’ genetic features and phylogenetic analysis grouped SiCGMV with other Corchoroviruses, a subgroup of whitefly transmitted viruses, genetically distinct, and basal to all other begomoviruses. These Old World (OW) viruses, with unique New Word (NW) genetic features, are likely due to distinct evolutionary histories or genetic isolation in their host species. It also lends support to the idea that NW begomoviruses may have originated in the OW and were subsequently disseminated to the NW, and that Corchoviruses may be a remnant of such NW begomoviruses that once populated the OW. Furthermore, this study provided the first reports of beta-satellite molecules in South Africa. A new bipartite begomovirus was identified in Phaseolus vulgaris (bean), for which the name Phaseolus vulgaris begomovirus was proposed. Phylogenetic analysis grouped this viral isolate with ScCBV, a member of the‘legumoviruses’ group. Begomoviruses infecting legumes often cluster phylogenetically as a group between the OW and NW begomoviruses and have collectively been referred to as ‘legumoviruses’. No symptoms were observed on the bean plant at the time of collection, thus further investigation into the biological characteristics and possible economic impact of this newly identified virus is required...M.Sc. (Biochemistry