JunctionViewer: customizable annotation software for repeat-rich genomic regions

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

The Cotton Centromere Contains a Ty3-gypsy-like LTR Retroelement

The centromere is a repeat-rich structure essential for chromosome segregation; with the long-term aim of understanding centromere structure and function, we set out to identify cotton centromere sequences. To isolate centromere-associated sequences from cotton, (Gossypium hirsutum) we surveyed tandem and dispersed repetitive DNA in the genus. Centromere-associated elements in other plants include tandem repeats and, in some cases, centromere-specific retroelements. Examination of cotton genomic survey sequences for tandem repeats yielded sequences that did not localize to the centromere. However, among the repetitive sequences we also identified a gypsy-like LTR retrotransposon (Centromere Retroelement Gossypium, CRG) that localizes to the centromere region of all chromosomes in domestic upland cotton, Gossypium hirsutum, the major commercially grown cotton. The location of the functional centromere was confirmed by immunostaining with antiserum to the centromere-specific histone CENH3, which co-localizes with CRG hybridization on metaphase mitotic chromosomes. G. hirsutum is an allotetraploid composed of A and D genomes and CRG is also present in the centromere regions of other AD cotton species. Furthermore, FISH and genomic dot blot hybridization revealed that CRG is found in D-genome diploid cotton species, but not in A-genome diploid species, indicating that this retroelement may have invaded the A-genome centromeres during allopolyploid formation and amplified during evolutionary history. CRG is also found in other diploid Gossypium species, including B and E2 genome species, but not in the C, E1, F, and G genome species tested. Isolation of this centromere-specific retrotransposon from Gossypium provides a probe for further understanding of centromere structure, and a tool for future engineering of centromere mini-chromosomes in this important crop species

Mu Transposon Insertion Sites and Meiotic Recombination Events Co-Localize with Epigenetic Marks for Open Chromatin across the Maize Genome

The Mu transposon system of maize is highly active, with each of the ∼50–100 copies transposing on average once each generation. The approximately one dozen distinct Mu transposons contain highly similar ∼215 bp terminal inverted repeats (TIRs) and generate 9-bp target site duplications (TSDs) upon insertion. Using a novel genome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via 454 technology. 94% of ∼965,000 reads carried Mu TIRs, demonstrating the specificity of this strategy. Among these TIRs, 21 novel Mu TIRs were discovered, revealing additional complexity of the Mu transposon system. The distribution of >40,000 non-redundant Mu insertion sites was strikingly non-uniform, such that rates increased in proportion to distance from the centromere. An identified putative Mu transposase binding consensus site does not explain this non-uniformity. An integrated genetic map containing more than 10,000 genetic markers was constructed and aligned to the sequence of the maize reference genome. Recombination rates (cM/Mb) are also strikingly non-uniform, with rates increasing in proportion to distance from the centromere. Mu insertion site frequencies are strongly correlated with recombination rates. Gene density does not fully explain the chromosomal distribution of Mu insertion and recombination sites, because pronounced preferences for the distal portion of chromosome are still observed even after accounting for gene density. The similarity of the distributions of Mu insertions and meiotic recombination sites suggests that common features, such as chromatin structure, are involved in site selection for both Mu insertion and meiotic recombination. The finding that Mu insertions and meiotic recombination sites both concentrate in genomic regions marked with epigenetic marks of open chromatin provides support for the hypothesis that open chromatin enhances rates of both Mu insertion and meiotic recombination

Maize Inbreds Exhibit High Levels of Copy Number Variation (CNV) and Presence/Absence Variation (PAV) in Genome Content

Following the domestication of maize over the past ∼10,000 years, breeders have exploited the extensive genetic diversity of this species to mold its phenotype to meet human needs. The extent of structural variation, including copy number variation (CNV) and presence/absence variation (PAV), which are thought to contribute to the extraordinary phenotypic diversity and plasticity of this important crop, have not been elucidated. Whole-genome, array-based, comparative genomic hybridization (CGH) revealed a level of structural diversity between the inbred lines B73 and Mo17 that is unprecedented among higher eukaryotes. A detailed analysis of altered segments of DNA conservatively estimates that there are several hundred CNV sequences among the two genotypes, as well as several thousand PAV sequences that are present in B73 but not Mo17. Haplotype-specific PAVs contain hundreds of single-copy, expressed genes that may contribute to heterosis and to the extraordinary phenotypic diversity of this important crop

Repeat Composition of CenH3-chromatin and H3K9me2-marked heterochromatin in Sugar Beet (Beta vulgaris)

Kowar T, Zakrzewski F, Macas J, et al. Repeat Composition of CenH3-chromatin and H3K9me2-marked heterochromatin in Sugar Beet (Beta vulgaris). BMC Plant Biology. 2016;16(1): 120.Background Sugar beet (Beta vulgaris) is an important crop of temperate climate zones, which provides nearly 30 % of the world’s annual sugar needs. From the total genome size of 758 Mb, only 567 Mb were incorporated in the recently published genome sequence, due to the fact that regions with high repetitive DNA contents (e.g. satellite DNAs) are only partially included. Therefore, to fill these gaps and to gain information about the repeat composition of centromeres and heterochromatic regions, we performed chromatin immunoprecipitation followed by sequencing (ChIP-Seq) using antibodies against the centromere-specific histone H3 variant of sugar beet (CenH3) and the heterochromatic mark of dimethylated lysine 9 of histone H3 (H3K9me2). Results ChIP-Seq analysis revealed that active centromeres containing CenH3 consist of the satellite pBV and the Ty3-gypsy retrotransposon Beetle7, while heterochromatin marked by H3K9me2 exhibits heterogeneity in repeat composition. H3K9me2 was mainly associated with the satellite family pEV, the Ty1-copia retrotransposon family Cotzilla and the DNA transposon superfamily of the En/Spm type. In members of the section Beta within the genus Beta, immunostaining using the CenH3 antibody was successful, indicating that orthologous CenH3 proteins are present in closely related species within this section. Conclusions The identification of repetitive genome portions by ChIP-Seq experiments complemented the sugar beet reference sequence by providing insights into the repeat composition of poorly characterized CenH3-chromatin and H3K9me2-heterochromatin. Therefore, our work provides the basis for future research and application concerning the sugar beet centromere and repeat rich heterochromatic regions characterized by the presence of H3K9me2

Demarcation of informative chromosomes in tropical sweet corn inbred lines using microsatellite DNA markers

A study of genetic variation among 10 pairs of chromosomes extracted from 13 tropical sweet corn inbred lines, using 99 microsatellite markers, revealed a wide range of genetic diversity. Allelic richness and the number of effective alleles per chromosome ranged from 2.78 to 4.33 and 1.96 to 3.47, respectively, with respective mean values of 3.62 and 2.73. According to the Shannon's information index (I) and Nei's gene diversity coefficient (Nei), Chromosome 10 was the most informative chromosome (I = 1.311 and Nei = 0.703), while Chromosome 2 possessed the least (I = 0.762 and Nei = 0.456). Based on linkage disequilibrium (LD) measurements for loci less than 50 cM apart on the same chromosome, all loci on Chromosomes 1, 6 and 7 were in equilibrium. Even so, there was a high proportion of genetic variation in Chromosomes 4, 5, 8, 9 and 10, thereby revealing their appropriateness for use in the genetic diversity investigations among tropical sweet corn lines. Chromosome 4, with the highest number of loci in linkage disequilibrium, was considered the best for marker-phenotype association and QTL mapping, followed by Chromosomes 5, 8, 9 and 10