Publications by authors named "Gernot G Presting"

25 Publications

  • Page 1 of 1

Gapless assembly of maize chromosomes using long-read technologies.

Genome Biol 2020 05 20;21(1):121. Epub 2020 May 20.

Department of Genetics, University of Georgia, Athens, GA, 30602, USA.

Creating gapless telomere-to-telomere assemblies of complex genomes is one of the ultimate challenges in genomics. We use two independent assemblies and an optical map-based merging pipeline to produce a maize genome (B73-Ab10) composed of 63 contigs and a contig N50 of 162 Mb. This genome includes gapless assemblies of chromosome 3 (236 Mb) and chromosome 9 (162 Mb), and 53 Mb of the Ab10 meiotic drive haplotype. The data also reveal the internal structure of seven centromeres and five heterochromatic knobs, showing that the major tandem repeat arrays (CentC, knob180, and TR-1) are discontinuous and frequently interspersed with retroelements.
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http://dx.doi.org/10.1186/s13059-020-02029-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238635PMC
May 2020

Centromeric retrotransposons and centromere function.

Curr Opin Genet Dev 2018 04 27;49:79-84. Epub 2018 Mar 27.

University of Hawaii, United States. Electronic address:

The centromeric DNA of most multicellular eukaryotes consists of tandem repeats (TR) that bind centromere-specific proteins and act as a substrate for the efficient repair of frequent double-stranded DNA breaks. Some retrotransposons target active centromeres during integration with such specificity that they can be used to deduce current and historic centromere positions. The roles of transposons in centromere function remain incompletely understood but appear to include maintaining centromere size and increasing the repeat content of neocentromeres that lack TR. Retrotransposons are known to give rise to TR. Centromere-targeting elements thus have the potential to replace centromeric TR essentially in situ, providing a mechanism to explain the centromere paradox, that is, the presence of unrelated centromeric TRs in closely related species.
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http://dx.doi.org/10.1016/j.gde.2018.03.004DOI Listing
April 2018

Improved maize reference genome with single-molecule technologies.

Nature 2017 06 12;546(7659):524-527. Epub 2017 Jun 12.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.
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http://dx.doi.org/10.1038/nature22971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052699PMC
June 2017

High Quality Maize Centromere 10 Sequence Reveals Evidence of Frequent Recombination Events.

Front Plant Sci 2016 23;7:308. Epub 2016 Mar 23.

Department of Molecular Biosciences and Bioengineering, University of Hawaíi at Mānoa Honolulu, HI, USA.

The ancestral centromeres of maize contain long stretches of the tandemly arranged CentC repeat. The abundance of tandem DNA repeats and centromeric retrotransposons (CR) has presented a significant challenge to completely assembling centromeres using traditional sequencing methods. Here, we report a nearly complete assembly of the 1.85 Mb maize centromere 10 from inbred B73 using PacBio technology and BACs from the reference genome project. The error rates estimated from overlapping BAC sequences are 7 × 10(-6) and 5 × 10(-5) for mismatches and indels, respectively. The number of gaps in the region covered by the reassembly was reduced from 140 in the reference genome to three. Three expressed genes are located between 92 and 477 kb from the inferred ancestral CentC cluster, which lies within the region of highest centromeric repeat density. The improved assembly increased the count of full-length CR from 5 to 55 and revealed a 22.7 kb segmental duplication that occurred approximately 121,000 years ago. Our analysis provides evidence of frequent recombination events in the form of partial retrotransposons, deletions within retrotransposons, chimeric retrotransposons, segmental duplications including higher order CentC repeats, a deleted CentC monomer, centromere-proximal inversions, and insertion of mitochondrial sequences. Double-strand DNA break (DSB) repair is the most plausible mechanism for these events and may be the major driver of centromere repeat evolution and diversity. In many cases examined here, DSB repair appears to be mediated by microhomology, suggesting that tandem repeats may have evolved to efficiently repair frequent DSBs in centromeres.
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http://dx.doi.org/10.3389/fpls.2016.00308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806543PMC
April 2016

Inbreeding drives maize centromere evolution.

Proc Natl Acad Sci U S A 2016 Feb 8;113(8):E987-96. Epub 2016 Feb 8.

Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI 96822

Functional centromeres, the chromosomal sites of spindle attachment during cell division, are marked epigenetically by the centromere-specific histone H3 variant cenH3 and typically contain long stretches of centromere-specific tandem DNA repeats (∼1.8 Mb in maize). In 23 inbreds of domesticated maize chosen to represent the genetic diversity of maize germplasm, partial or nearly complete loss of the tandem DNA repeat CentC precedes 57 independent cenH3 relocation events that result in neocentromere formation. Chromosomal regions with newly acquired cenH3 are colonized by the centromere-specific retrotransposon CR2 at a rate that would result in centromere-sized CR2 clusters in 20,000-95,000 y. Three lines of evidence indicate that CentC loss is linked to inbreeding, including (i) CEN10 of temperate lineages, presumed to have experienced a genetic bottleneck, contain less CentC than their tropical relatives; (ii) strong selection for centromere-linked genes in domesticated maize reduced diversity at seven of the ten maize centromeres to only one or two postdomestication haplotypes; and (iii) the centromere with the largest number of haplotypes in domesticated maize (CEN7) has the highest CentC levels in nearly all domesticated lines. Rare recombinations introduced one (CEN2) or more (CEN5) alternate CEN haplotypes while retaining a single haplotype at domestication loci linked to these centromeres. Taken together, this evidence strongly suggests that inbreeding, favored by postdomestication selection for centromere-linked genes affecting key domestication or agricultural traits, drives replacement of the tandem centromere repeats in maize and other crop plants. Similar forces may act during speciation in natural systems.
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http://dx.doi.org/10.1073/pnas.1522008113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776452PMC
February 2016

The Hawaiian freshwater algae biodiversity survey (2009-2014): systematic and biogeographic trends with an emphasis on the macroalgae.

BMC Ecol 2014 Oct 25;14:28. Epub 2014 Oct 25.

Background: A remarkable range of environmental conditions is present in the Hawaiian Islands due to their gradients of elevation, rainfall and island age. Despite being well known as a location for the study of evolutionary processes and island biogeography, little is known about the composition of the non-marine algal flora of the archipelago, its degree of endemism, or affinities with other floras. We conducted a biodiversity survey of the non-marine macroalgae of the six largest main Hawaiian Islands using molecular and microscopic assessment techniques. We aimed to evaluate whether endemism or cosmopolitanism better explain freshwater algal distribution patterns, and provide a baseline data set for monitoring future biodiversity changes in the Hawaiian Islands.

Results: 1,786 aquatic and terrestrial habitats and 1,407 distinct collections of non-marine macroalgae were collected from the islands of Kauai, Oahu, Molokai, Maui, Lanai and Hawaii from the years 2009-2014. Targeted habitats included streams, wet walls, high elevation bogs, taro fields, ditches and flumes, lakes/reservoirs, cave walls and terrestrial areas. Sites that lacked freshwater macroalgae were typically terrestrial or wet wall habitats that were sampled for diatoms and other microalgae. Approximately 50% of the identifications were of green algae, with lesser proportions of diatoms, red algae, cyanobacteria, xanthophytes and euglenoids. 898 DNA sequences were generated representing eight different markers, which enabled an assessment of the number of taxonomic entities for genera collected as part of the survey. Forty-four well-characterized taxa were assessed for global distribution patterns. This analysis revealed no clear biogeographic affinities of the flora, with 27.3% characterized as "cosmopolitan", 11.4% "endemic", and 61.3% as intermediate.

Conclusions: The Hawaiian freshwater algal biodiversity survey represents the first comprehensive effort to characterize the non-marine algae of a tropical region in the world using both morphological and molecular tools. Survey data were entered in the Hawaiian Freshwater Algal Database, which serves as a digital repository of photographs and micrographs, georeferenced localities and DNA sequence data. These analyses yielded an updated checklist of the non-marine macroalgae of the Hawaiian Islands, and revealed varied biogeographic affinities of the flora that are likely a product of both natural and anthropogenic dispersal.
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http://dx.doi.org/10.1186/s12898-014-0028-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222836PMC
October 2014

Evolution of centromeric retrotransposons in grasses.

Genome Biol Evol 2014 May 9;6(6):1335-52. Epub 2014 May 9.

Department of Molecular Biosciences and Bioengineering, University of Hawaii, Mānoa

Centromeric retrotransposons (CRs) constitute a family of plant retroelements, some of which have the ability to target their insertion almost exclusively to the functional centromeres. Our exhaustive analysis of CR family members in four grass genomes revealed not only horizontal transfer (HT) of CR elements between the oryzoid and panicoid grass lineages but also their subsequent recombination with endogenous elements that in some cases created prolific recombinants in foxtail millet and sorghum. HT events are easily identifiable only in cases where host genome divergence significantly predates HT, thus documented HT events likely represent only a fraction of the total. If the more difficult to detect ancient HT events occurred at frequencies similar to those observable in present day grasses, the extant long terminal repeat retrotransposons represent the mosaic products of HT and recombination that are optimized for retrotransposition in their host genomes. This complicates not only phylogenetic analysis but also the establishment of a meaningful retrotransposon nomenclature, which we have nevertheless attempted to implement here. In contrast to the plant-centric naming convention used currently for CR elements, we classify elements primarily based on their phylogenetic relationships regardless of host plant, using the exhaustively studied maize elements assigned to six different subfamilies as a standard. The CR2 subfamily is the most widely distributed of the six CR subfamilies discovered in grass genomes to date and thus the most likely to play a functional role at grass centromeres.
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http://dx.doi.org/10.1093/gbe/evu096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4079200PMC
May 2014

Tandem repeats derived from centromeric retrotransposons.

BMC Genomics 2013 Mar 4;14:142. Epub 2013 Mar 4.

Background: Tandem repeats are ubiquitous and abundant in higher eukaryotic genomes and constitute, along with transposable elements, much of DNA underlying centromeres and other heterochromatic domains. In maize, centromeric satellite repeat (CentC) and centromeric retrotransposons (CR), a class of Ty3/gypsy retrotransposons, are enriched at centromeres. Some satellite repeats have homology to retrotransposons and several mechanisms have been proposed to explain the expansion, contraction as well as homogenization of tandem repeats. However, the origin and evolution of tandem repeat loci remain largely unknown.

Results: CRM1TR and CRM4TR are novel tandem repeats that we show to be entirely derived from CR elements belonging to two different subfamilies, CRM1 and CRM4. Although these tandem repeats clearly originated in at least two separate events, they are derived from similar regions of their respective parent element, namely the long terminal repeat (LTR) and untranslated region (UTR). The 5' ends of the monomer repeat units of CRM1TR and CRM4TR map to different locations within their respective LTRs, while their 3' ends map to the same relative position within a conserved region of their UTRs. Based on the insertion times of heterologous retrotransposons that have inserted into these tandem repeats, amplification of the repeats is estimated to have begun at least ~4 (CRM1TR) and ~1 (CRM4TR) million years ago. Distinct CRM1TR sequence variants occupy the two CRM1TR loci, indicating that there is little or no movement of repeats between loci, even though they are separated by only ~1.4 Mb.

Conclusions: The discovery of two novel retrotransposon derived tandem repeats supports the conclusions from earlier studies that retrotransposons can give rise to tandem repeats in eukaryotic genomes. Analysis of monomers from two different CRM1TR loci shows that gene conversion is the major cause of sequence variation. We propose that successive intrastrand deletions generated the initial repeat structure, and gene conversions increased the size of each tandem repeat locus.
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http://dx.doi.org/10.1186/1471-2164-14-142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3648361PMC
March 2013

The Hawaiian Freshwater Algal Database (HfwADB): a laboratory LIMS and online biodiversity resource.

BMC Ecol 2012 Oct 25;12:22. Epub 2012 Oct 25.

Department of Botany, University of Hawaii at Manoa, 3190 Maile Way, Honolulu, Hawaii 96822, USA.

Background: Biodiversity databases serve the important role of highlighting species-level diversity from defined geographical regions. Databases that are specially designed to accommodate the types of data gathered during regional surveys are valuable in allowing full data access and display to researchers not directly involved with the project, while serving as a Laboratory Information Management System (LIMS). The Hawaiian Freshwater Algal Database, or HfwADB, was modified from the Hawaiian Algal Database to showcase non-marine algal specimens collected from the Hawaiian Archipelago by accommodating the additional level of organization required for samples including multiple species.

Description: The Hawaiian Freshwater Algal Database is a comprehensive and searchable database containing photographs and micrographs of samples and collection sites, geo-referenced collecting information, taxonomic data and standardized DNA sequence data. All data for individual samples are linked through unique 10-digit accession numbers ("Isolate Accession"), the first five of which correspond to the collection site ("Environmental Accession"). Users can search online for sample information by accession number, various levels of taxonomy, habitat or collection site. HfwADB is hosted at the University of Hawaii, and was made publicly accessible in October 2011. At the present time the database houses data for over 2,825 samples of non-marine algae from 1,786 collection sites from the Hawaiian Archipelago. These samples include cyanobacteria, red and green algae and diatoms, as well as lesser representation from some other algal lineages.

Conclusions: HfwADB is a digital repository that acts as a Laboratory Information Management System for Hawaiian non-marine algal data. Users can interact with the repository through the web to view relevant habitat data (including geo-referenced collection locations) and download images of collection sites, specimen photographs and micrographs, and DNA sequences. It is publicly available at http://algae.manoa.hawaii.edu/hfwadb/.
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http://dx.doi.org/10.1186/1472-6785-12-22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3526539PMC
October 2012

Retrotransposon insertion targeting: a mechanism for homogenization of centromere sequences on nonhomologous chromosomes.

Genes Dev 2012 Apr;26(7):638-40

Division of Biological Sciences, University of Missouri at Columbia, Columbia, Missouri 65211, USA.

The centromeres of most eukaryotic organisms consist of highly repetitive arrays that are similar across nonhomologous chromosomes. These sequences evolve rapidly, thus posing a mystery as to how such arrays can be homogenized. Recent work in species in which centromere-enriched retrotransposons occur indicates that these elements preferentially insert into the centromeric regions. In two different Arabidopsis species, a related element was recognized in which the specificity for such targeting was altered. These observations provide a partial explanation for how homogenization of centromere DNA sequences occurs.
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http://dx.doi.org/10.1101/gad.191049.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3323874PMC
April 2012

Classification of plant associated bacteria using RIF, a computationally derived DNA marker.

PLoS One 2011 Apr 21;6(4):e18496. Epub 2011 Apr 21.

Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.

A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018496PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3080875PMC
April 2011

Epigenetic aspects of centromere function in plants.

Curr Opin Plant Biol 2011 Apr 14;14(2):217-22. Epub 2011 Mar 14.

Division of Biological Sciences, University of Missouri, Columbia, MO 65211, USA.

Centromeres were once thought to be boring structures on the chromosome involved with transmission through mitosis and meiosis. Recent data from a wide spectrum of organisms reveal an epigenetic component to centromere specification in that they can become inactive easily or form over unique DNA as neocentromeres. However, the constancy of centromere repeats at primary constrictions in most species, the fact that these repeats are transcribed and incorporated into the kinetochore, and the phenomenon of reactivation of formerly inactive centromeres at the same chromosomal sites suggests some type of role of DNA sequence or configuration in establishing the site of kinetochores. Here we present evidence for epigenetic and structural aspects involved with centromere activity in plants.
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http://dx.doi.org/10.1016/j.pbi.2011.02.004DOI Listing
April 2011

Distinct influences of tandem repeats and retrotransposons on CENH3 nucleosome positioning.

Epigenetics Chromatin 2011 Feb 25;4. Epub 2011 Feb 25.

Department of Plant Biology, University of Georgia, Athens, Georgia, USA.

Background: Unique structural characteristics of centromere chromatin enable it to support assembly of the kinetochore and its associated tensions. The histone H3 variant CENH3 (centromeric histone H3) is viewed as the key element of centromere chromatin and its interaction with centromere DNA is epigenetic in that its localization to centromeres is not sequence-dependent.

Results: In order to investigate what influence the DNA sequence exerts on CENH3 chromatin structure, we examined CENH3 nucleosome footprints on maize centromere DNA. We found a predominant average nucleosome spacing pattern of roughly 190-bp intervals, which was also the dominant arrangement for nucleosomes genome-wide. For CENH3-containing nucleosomes, distinct modes of nucleosome positioning were evident within that general spacing constraint. Over arrays of the major ~156-bp centromeric satellite sequence (tandem repeat) CentC, nucleosomes were not positioned in register with CentC monomers but in conformity with a striking ~10-bp periodicity of AA/TT dimers within the sequence. In contrast, nucleosomes on a class of centromeric retrotransposon (CRM2) lacked a detectable AA/TT periodicity but exhibited tightly phased positioning.

Conclusions: These data support a model in which general chromatin factors independent of both DNA sequence and CENH3 enforce roughly uniform centromeric nucleosome spacing while allowing flexibility in the mode in which nucleosomes are positioned. In the case of tandem repeat DNA, the natural bending effects related to AA/TT periodicity produce an energetically-favourable arrangement consistent with conformationally rigid nucleosomes and stable chromatin at centromeres.
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http://dx.doi.org/10.1186/1756-8935-4-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3053214PMC
February 2011

The Hawaiian Rhodophyta Biodiversity Survey (2006-2010): a summary of principal findings.

BMC Plant Biol 2010 Nov 22;10:258. Epub 2010 Nov 22.

Botany Department, 3190 Maile Way, University of Hawaii, Honolulu, HI 96822, USA.

Background: The Hawaiian red algal flora is diverse, isolated, and well studied from a morphological and anatomical perspective, making it an excellent candidate for assessment using a combination of traditional taxonomic and molecular approaches. Acquiring and making these biodiversity data freely available in a timely manner ensures that other researchers can incorporate these baseline findings into phylogeographic studies of Hawaiian red algae or red algae found in other locations.

Results: A total of 1,946 accessions are represented in the collections from 305 different geographical locations in the Hawaiian archipelago. These accessions represent 24 orders, 49 families, 152 genera and 252 species/subspecific taxa of red algae. One order of red algae (the Rhodachlyales) was recognized in Hawaii for the first time and 196 new island distributional records were determined from the survey collections. One family and four genera are reported for the first time from Hawaii, and multiple species descriptions are in progress for newly discovered taxa. A total of 2,418 sequences were generated for Hawaiian red algae in the course of this study--915 for the nuclear LSU marker, 864 for the plastidial UPA marker, and 639 for the mitochondrial COI marker. These baseline molecular data are presented as neighbor-joining trees to illustrate degrees of divergence within and among taxa. The LSU marker was typically most conserved, followed by UPA and COI. Phylogenetic analysis of a set of concatenated LSU, UPA and COI sequences recovered a tree that broadly resembled the current understanding of florideophyte red algal relationships, but bootstrap support was largely absent above the ordinal level. Phylogeographic trends are reported here for some common taxa within the Hawaiian Islands and include examples of those with, as well as without, intraspecific variation.

Conclusions: The UPA and COI markers were determined to be the most useful of the three and are recommended for inclusion in future algal biodiversity surveys. Molecular data for the survey provide the most extensive assessment of Hawaiian red algal diversity and, in combination with the morphological/anatomical and distributional data collected as part of the project, provide a solid baseline data set for future studies of the flora. The data are freely available via the Hawaiian Algal Database (HADB), which was designed and constructed to accommodate the results of the project. We present the first DNA sequence reference collection for a tropical Pacific seaweed flora, whose value extends beyond Hawaii since many Hawaiian taxa are shared with other tropical areas.
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http://dx.doi.org/10.1186/1471-2229-10-258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012605PMC
November 2010

Widespread gene conversion in centromere cores.

PLoS Biol 2010 Mar 9;8(3):e1000327. Epub 2010 Mar 9.

Department of Plant Biology, University of Georgia, Athens, Georgia, United States of America.

Centromeres are the most dynamic regions of the genome, yet they are typified by little or no crossing over, making it difficult to explain the origin of this diversity. To address this question, we developed a novel CENH3 ChIP display method that maps kinetochore footprints over transposon-rich areas of centromere cores. A high level of polymorphism made it possible to map a total of 238 within-centromere markers using maize recombinant inbred lines. Over half of the markers were shown to interact directly with kinetochores (CENH3) by chromatin immunoprecipitation. Although classical crossing over is fully suppressed across CENH3 domains, two gene conversion events (i.e., non-crossover marker exchanges) were identified in a mapping population. A population genetic analysis of 53 diverse inbreds suggests that historical gene conversion is widespread in maize centromeres, occurring at a rate >1x10(-5)/marker/generation. We conclude that gene conversion accelerates centromere evolution by facilitating sequence exchange among chromosomes.
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http://dx.doi.org/10.1371/journal.pbio.1000327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2834711PMC
March 2010

JunctionViewer: customizable annotation software for repeat-rich genomic regions.

BMC Bioinformatics 2010 Jan 12;11:23. Epub 2010 Jan 12.

Department of Molecular Biosciences and Bioengineering, University of Hawai'i at Mânoa, Honolulu, HI 96822, USA.

Background: Repeat-rich regions such as centromeres receive less attention than their gene-rich euchromatic counterparts because the former are difficult to assemble and analyze. Our objectives were to 1) map all ten centromeres onto the maize genetic map and 2) characterize the sequence features of maize centromeres, each of which spans several megabases of highly repetitive DNA. Repetitive sequences can be mapped using special molecular markers that are based on PCR with primers designed from two unique "repeat junctions". Efficient screening of large amounts of maize genome sequence data for repeat junctions, as well as key centromere sequence features required the development of specific annotation software.

Results: We developed JunctionViewer to automate the process of identifying and differentiating closely related centromere repeats and repeat junctions, and to generate graphical displays of these and other features within centromeric sequences. JunctionViewer generates NCBI BLAST, WU-BLAST, cross_match and MUMmer alignments, and displays the optimal alignments and additional annotation data as concise graphical representations that can be viewed directly through the graphical interface or as PostScript output.This software enabled us to quickly characterize millions of nucleotides of newly sequenced DNA ranging in size from single reads to assembled BACs and megabase-sized pseudochromosome regions. It expedited the process of generating repeat junction markers that were subsequently used to anchor all 10 centromeres to the maize map. It also enabled us to efficiently identify key features in large genomic regions, providing insight into the arrangement and evolution of maize centromeric DNA.

Conclusions: JunctionViewer will be useful to scientists who wish to automatically generate concise graphical summaries of repeat sequences. It is particularly valuable for those needing to efficiently identify unique repeat junctions. The scalability and ability to customize homology search parameters for different classes of closely related repeat sequences make this software ideal for recurring annotation (e.g., genome projects that are in progress) of genomic regions that contain well-defined repeats, such as those in centromeres. Although originally customized for maize centromere sequence, we anticipate this software to facilitate the analysis of centromere and other repeat-rich regions in other organisms.
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http://dx.doi.org/10.1186/1471-2105-11-23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824676PMC
January 2010

The B73 maize genome: complexity, diversity, and dynamics.

Science 2009 Nov;326(5956):1112-5

Center for Plant Genomics, Iowa State University, Ames, IA 50011, USA.

We report an improved draft nucleotide sequence of the 2.3-gigabase genome of maize, an important crop plant and model for biological research. Over 32,000 genes were predicted, of which 99.8% were placed on reference chromosomes. Nearly 85% of the genome is composed of hundreds of families of transposable elements, dispersed nonuniformly across the genome. These were responsible for the capture and amplification of numerous gene fragments and affect the composition, sizes, and positions of centromeres. We also report on the correlation of methylation-poor regions with Mu transposon insertions and recombination, and copy number variants with insertions and/or deletions, as well as how uneven gene losses between duplicated regions were involved in returning an ancient allotetraploid to a genetically diploid state. These analyses inform and set the stage for further investigations to improve our understanding of the domestication and agricultural improvements of maize.
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http://dx.doi.org/10.1126/science.1178534DOI Listing
November 2009

Maize centromere structure and evolution: sequence analysis of centromeres 2 and 5 reveals dynamic Loci shaped primarily by retrotransposons.

PLoS Genet 2009 Nov 20;5(11):e1000743. Epub 2009 Nov 20.

Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, Hawaii, USA.

We describe a comprehensive and general approach for mapping centromeres and present a detailed characterization of two maize centromeres. Centromeres are difficult to map and analyze because they consist primarily of repetitive DNA sequences, which in maize are the tandem satellite repeat CentC and interspersed centromeric retrotransposons of maize (CRM). Centromeres are defined epigenetically by the centromeric histone H3 variant, CENH3. Using novel markers derived from centromere repeats, we have mapped all ten centromeres onto the physical and genetic maps of maize. We were able to completely traverse centromeres 2 and 5, confirm physical maps by fluorescence in situ hybridization (FISH), and delineate their functional regions by chromatin immunoprecipitation (ChIP) with anti-CENH3 antibody followed by pyrosequencing. These two centromeres differ substantially in size, apparent CENH3 density, and arrangement of centromeric repeats; and they are larger than the rice centromeres characterized to date. Furthermore, centromere 5 consists of two distinct CENH3 domains that are separated by several megabases. Succession of centromere repeat classes is evidenced by the fact that elements belonging to the recently active recombinant subgroups of CRM1 colonize the present day centromeres, while elements of the ancestral subgroups are also found in the flanking regions. Using abundant CRM and non-CRM retrotransposons that inserted in and near these two centromeres to create a historical record of centromere location, we show that maize centromeres are fluid genomic regions whose borders are heavily influenced by the interplay of retrotransposons and epigenetic marks. Furthermore, we propose that CRMs may be involved in removal of centromeric DNA (specifically CentC), invasion of centromeres by non-CRM retrotransposons, and local repositioning of the CENH3.
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http://dx.doi.org/10.1371/journal.pgen.1000743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776974PMC
November 2009

The Hawaiian Algal Database: a laboratory LIMS and online resource for biodiversity data.

BMC Plant Biol 2009 Sep 4;9:117. Epub 2009 Sep 4.

Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, Hawaii 96822, USA.

Background: Organization and presentation of biodiversity data is greatly facilitated by databases that are specially designed to allow easy data entry and organized data display. Such databases also have the capacity to serve as Laboratory Information Management Systems (LIMS). The Hawaiian Algal Database was designed to showcase specimens collected from the Hawaiian Archipelago, enabling users around the world to compare their specimens with our photographs and DNA sequence data, and to provide lab personnel with an organizational tool for storing various biodiversity data types.

Description: We describe the Hawaiian Algal Database, a comprehensive and searchable database containing photographs and micrographs, geo-referenced collecting information, taxonomic checklists and standardized DNA sequence data. All data for individual samples are linked through unique accession numbers. Users can search online for sample information by accession number, numerous levels of taxonomy, or collection site. At the present time the database contains data representing over 2,000 samples of marine, freshwater and terrestrial algae from the Hawaiian Archipelago. These samples are primarily red algae, although other taxa are being added.

Conclusion: The Hawaiian Algal Database is a digital repository for Hawaiian algal samples and acts as a LIMS for the laboratory. Users can make use of the online search tool to view and download specimen photographs and micrographs, DNA sequences and relevant habitat data, including georeferenced collecting locations. It is publicly available at http://algae.manoa.hawaii.edu.
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http://dx.doi.org/10.1186/1471-2229-9-117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2746215PMC
September 2009

Sustained retrotransposition is mediated by nucleotide deletions and interelement recombinations.

Proc Natl Acad Sci U S A 2008 Oct 1;105(40):15470-4. Epub 2008 Oct 1.

Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI 96822, USA.

The term "C-value paradox" was coined by C. A. Thomas, Jr. in 1971 [Thomas CA (1971) Ann Rev Genetics 5:237-256] to describe the initially puzzling lack of correlation between an organism's genome size and its morphological complexity. Polyploidy and the expansion of repetitive DNA, primarily transposable elements, are two mechanisms that have since been found to account for this differential. While the inactivation of retrotransposons by methylation and their removal from the genome by illegitimate recombination have been well documented, the cause of the apparently periodic bursts of retrotranposon expansion is as yet unknown. We show that the expansion of the CRM1 retrotransposon subfamily in the ancient allotetraploid crop plant corn is linked to the repeated formation of novel recombinant elements derived from two parental retrotransposon genotypes, which may have been brought together during the hybridization of two sympatric species that make up the present day corn genome, thus revealing a unique mechanism linking polyploidy and retrotransposition.
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http://dx.doi.org/10.1073/pnas.0805694105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2563092PMC
October 2008

Application of universally amplifying plastid primers to environmental sampling of a stream periphyton community.

Mol Ecol Resour 2008 Sep 28;8(5):1011-4. Epub 2008 Jun 28.

Department of Botany, Department of Molecular Biosciences and Bioengineering, 1955 East-West Road, University of Hawai'i, Honolulu, HI 96822, USA.

To demonstrate the utility of universal plastid primers for probing of environmental samples, we extracted DNA from a tropical stream periphyton community and created two environmental clone libraries. We demonstrate the recovery of DNA sequences corresponding to the major groups of algae observed microscopically in the sample, illustrating the utility of these primers for analysis of environmental samples. Using a touchdown polymerase chain reaction technique, almost 99% of recovered sequences correspond to plastid-containing or cyanobacterial taxa, which allows algae to be targeted to the almost complete exclusion of noncyanobacterial prokaryotes and nonplastid-containing eukaryotes.
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http://dx.doi.org/10.1111/j.1755-0998.2008.02138.xDOI Listing
September 2008

Centromeric retrotransposon lineages predate the maize/rice divergence and differ in abundance and activity.

Mol Genet Genomics 2008 Feb 14;279(2):133-47. Epub 2007 Nov 14.

Department of Molecular Biosciences and Bioengineering, University of Hawaii, 1955 East-West Road, Honolulu, HI 96822, USA.

Centromeric retrotransposons (CR) are located almost exclusively at the centromeres of plant chromosomes. Analysis of the emerging Zea mays inbred B73 genome sequence revealed two novel subfamilies of CR elements of maize (CRM), bringing the total number of known CRM subfamilies to four. Orthologous subfamilies of each of these CRM subfamilies were discovered in the rice lineage, and the orthologous relationships were demonstrated with extensive phylogenetic analyses. The much higher number of CRs in maize versus Oryza sativa is due primarily to the recent expansion of the CRM1 subfamily in maize. At least one incomplete copy of a CRM1 homolog was found in O. sativa ssp. indica and O. officinalis, but no member of this subfamily could be detected in the finished O. sativa ssp. japonica genome, implying loss of this prolific subfamily in that subspecies. CRM2 and CRM3, as well as the corresponding rice subfamilies, have been recently active but are present in low numbers. CRM3 is a full-length element related to the non-autonomous CentA, which is the first described CRM. The oldest subfamily (CRM4), as well as its rice counterpart, appears to contain only inactive members that are not located in currently active centromeres. The abundance of active CR elements is correlated with chromosome size in the three plant genomes for which high quality genomic sequence is available, and the emerging picture of CR elements is one in which different subfamilies are active at different evolutionary times. We propose a model by which CR elements might influence chromosome and genome size.
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http://dx.doi.org/10.1007/s00438-007-0302-5DOI Listing
February 2008

Precise centromere mapping using a combination of repeat junction markers and chromatin immunoprecipitation-polymerase chain reaction.

Genetics 2006 Oct 1;174(2):1057-61. Epub 2006 Sep 1.

Department of Plant Biology, University of Georgia, Georgia 30602, USA.

Centromeres are difficult to map even in species where genetic resolution is excellent. Here we show that junctions between repeats provide reliable single-copy markers for recombinant inbred mapping within centromeres and pericentromeric heterochromatin. Repeat junction mapping was combined with anti-CENH3-mediated ChIP to provide a definitive map position for maize centromere 8.
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http://dx.doi.org/10.1534/genetics.106.060467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1602074PMC
October 2006

Analysis of papaya BAC end sequences reveals first insights into the organization of a fruit tree genome.

Mol Genet Genomics 2006 Jul 16;276(1):1-12. Epub 2006 May 16.

Department of Molecular Biosciences and Bioengineering, University of Hawaii, 1955 East-West Road, Agricultural Sciences Building Room 218, Honolulu, HI, 96822, USA.

Papaya (Carica papaya L.) is a major tree fruit crop of tropical and subtropical regions with an estimated genome size of 372 Mbp. We present the analysis of 4.7% of the papaya genome based on BAC end sequences (BESs) representing 17 million high-quality bases. Microsatellites discovered in 5,452 BESs and flanking primer sequences are available to papaya breeding programs at http://www.genomics.hawaii.edu/papaya/BES . Sixteen percent of BESs contain plant repeat elements, the vast majority (83.3%) of which are class I retrotransposons. Several novel papaya-specific repeats were identified. Approximately 19.1% of the BESs have homology to Arabidopsis cDNA. Increasing numbers of completely sequenced plant genomes and BES projects enable novel approaches to comparative plant genomics. Paired BESs of Carica, Arabidopsis, Populus, Brassica and Lycopersicon were mapped onto the completed genomes of Arabidopsis and Populus. In general the level of microsynteny was highest between closely related organisms. However, papaya revealed a higher degree of apparent synteny with the more distantly related poplar than with the more closely related Arabidopsis. This, as well as significant colinearity observed between peach and poplar genome sequences, support recent observations of frequent genome rearrangements in the Arabidopsis lineage and suggest that the poplar genome sequence may be more useful for elucidating the papaya and other rosid genomes. These insights will play a critical role in selecting species and sequencing strategies that will optimally represent crop genomes in sequence databases.
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http://dx.doi.org/10.1007/s00438-006-0122-zDOI Listing
July 2006

Mapping multiple co-sequenced T-DNA integration sites within the Arabidopsis genome.

Bioinformatics 2003 Mar;19(5):579-86

Torrey Mesa Research Institute, 3115 Merryfield Row, San Diego, CA 92121, USA.

Motivation: Insertion mutagenesis, using transgenes or endogenous transposons, is a popular method for generating null mutations (knockouts) in model organisms. Insertions are mapped to specific genes by amplifying (via TAIL-PCR) and sequencing genomic regions flanking the inserted DNA. The presence of multiple TAIL-PCR templates in one sequencing reaction results in chimeric sequence of intermittently low quality. Standard processing of this sequence by applying Phred quality requirements results in loss of informative sequence, whereas not trimming low-quality sequence causes inclusion of low-complexity homopolymers from the ends of sequence runs. Accurate mapping of the flanking sequences is complicated by the presence of gene families.

Results: Methods for extracting informative regions from sequence traces obtained by sequencing multiple TAIL-PCR fragments in a single reaction are described. The completely sequenced Arabidopsis genome was used to identify informative TAIL-PCR sequence regions. Methods were devised to define and select high quality matches and precisely map each insert to the correct genome location. These methods were used to analyze sequence of TAIL-PCR-amplified flanking regions of the inserts from individual plants in a T-DNA-mutagenized population of Arabidopsis thaliana, and are applicable to similar situations where a reference genome can be used to extract information from poor-quality sequence.
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http://dx.doi.org/10.1093/bioinformatics/btg049DOI Listing
March 2003
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