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1.  Genomic Characterization of Large Heterochromatic Gaps in the Human Genome Assembly 
PLoS Computational Biology  2014;10(5):e1003628.
The largest gaps in the human genome assembly correspond to multi-megabase heterochromatic regions composed primarily of two related families of tandem repeats, Human Satellites 2 and 3 (HSat2,3). The abundance of repetitive DNA in these regions challenges standard mapping and assembly algorithms, and as a result, the sequence composition and potential biological functions of these regions remain largely unexplored. Furthermore, existing genomic tools designed to predict consensus-based descriptions of repeat families cannot be readily applied to complex satellite repeats such as HSat2,3, which lack a consistent repeat unit reference sequence. Here we present an alignment-free method to characterize complex satellites using whole-genome shotgun read datasets. Utilizing this approach, we classify HSat2,3 sequences into fourteen subfamilies and predict their chromosomal distributions, resulting in a comprehensive satellite reference database to further enable genomic studies of heterochromatic regions. We also identify 1.3 Mb of non-repetitive sequence interspersed with HSat2,3 across 17 unmapped assembly scaffolds, including eight annotated gene predictions. Finally, we apply our satellite reference database to high-throughput sequence data from 396 males to estimate array size variation of the predominant HSat3 array on the Y chromosome, confirming that satellite array sizes can vary between individuals over an order of magnitude (7 to 98 Mb) and further demonstrating that array sizes are distributed differently within distinct Y haplogroups. In summary, we present a novel framework for generating initial reference databases for unassembled genomic regions enriched with complex satellite DNA, and we further demonstrate the utility of these reference databases for studying patterns of sequence variation within human populations.
Author Summary
At least 5–10% of the human genome remains unassembled, unmapped, and poorly characterized. The reference assembly annotates these missing regions as multi-megabase heterochromatic gaps, found primarily near centromeres and on the short arms of the acrocentric chromosomes. This missing fraction of the genome consists predominantly of long arrays of near-identical tandem repeats called satellite DNA. Due to the repetitive nature of satellite DNA, sequence assembly algorithms cannot uniquely align overlapping sequence reads, and thus satellite-rich domains have been omitted from the reference assembly and from most genome-wide studies of variation and function. Existing methods for analyzing some satellite DNAs cannot be easily extended to a large portion of satellites whose repeat structures are complex and largely uncharacterized, such as Human Satellites 2 and 3 (HSat2,3). Here we characterize HSat2,3 using a novel approach that does not depend on having a well-defined repeat structure. By classifying genome-wide HSat2,3 sequences into subfamilies and localizing them to chromosomes, we have generated an initial HSat2,3 genomic reference, which serves as a critical foundation for future studies of variation and function in these regions. This approach should be generally applicable to other classes of satellite DNA, in both the human genome and other complex genomes.
doi:10.1371/journal.pcbi.1003628
PMCID: PMC4022460  PMID: 24831296
2.  Gene duplication and paleopolyploidy in soybean and the implications for whole genome sequencing 
BMC Genomics  2007;8:330.
Background
Soybean, Glycine max (L.) Merr., is a well documented paleopolyploid. What remains relatively under characterized is the level of sequence identity in retained homeologous regions of the genome. Recently, the Department of Energy Joint Genome Institute and United States Department of Agriculture jointly announced the sequencing of the soybean genome. One of the initial concerns is to what extent sequence identity in homeologous regions would have on whole genome shotgun sequence assembly.
Results
Seventeen BACs representing ~2.03 Mb were sequenced as representative potential homeologous regions from the soybean genome. Genetic mapping of each BAC shows that 11 of the 20 chromosomes are represented. Sequence comparisons between homeologous BACs shows that the soybean genome is a mosaic of retained paleopolyploid regions. Some regions appear to be highly conserved while other regions have diverged significantly. Large-scale "batch" reassembly of all 17 BACs combined showed that even the most homeologous BACs with upwards of 95% sequence identity resolve into their respective homeologous sequences. Potential assembly errors were generated by tandemly duplicated pentatricopeptide repeat containing genes and long simple sequence repeats. Analysis of a whole-genome shotgun assembly of 80,000 randomly chosen JGI-DOE sequence traces reveals some new soybean-specific repeat sequences.
Conclusion
This analysis investigated both the structure of the paleopolyploid soybean genome and the potential effects retained homeology will have on assembling the whole genome shotgun sequence. Based upon these results, homeologous regions similar to those characterized here will not cause major assembly issues.
doi:10.1186/1471-2164-8-330
PMCID: PMC2077340  PMID: 17880721
3.  Intergenic Locations of Rice Centromeric Chromatin 
PLoS Biology  2008;6(11):e286.
Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. Plant and animal centromeres are typically located in megabase-sized arrays of tandem satellite repeats, making their precise mapping difficult. However, some rice centromeres are largely embedded in nonsatellite DNA, providing an excellent model to study centromere structure and evolution. We used chromatin immunoprecipitation and 454 sequencing to define the boundaries of nine of the 12 centromeres of rice. Centromere regions from chromosomes 8 and 9 were found to share synteny, most likely reflecting an ancient genome duplication. For four centromeres, we mapped discrete subdomains of binding by the centromeric histone variant CENH3. These subdomains were depleted in both intact and nonfunctional genes relative to interspersed subdomains lacking CENH3. The intergenic location of rice centromeric chromatin resembles the situation for human neocentromeres and supports a model of the evolution of centromeres from gene-poor regions.
Author Summary
Before a cell divides, its chromosomes must be duplicated and then separated to provide each daughter cell with an identical genome copy. To accomplish this separation, the cell-division apparatus attaches to structures on the chromosomes called centromeres. Most plant and animal centromeres contain highly repetitive DNA sequences and specific proteins such as CENH3; however, it is not known which of the many repeats bind CENH3. Some rice centromeres, however, consist largely of single-copy DNA, providing a tractable model for investigating CENH3-binding patterns. Using modern DNA sequencing technology and an antibody to CENH3, we were able to find which sequences in the rice genome are bound by CENH3. We uncovered evidence that one centromere, Cen8, which has lost much of its repetitive content through a rearrangement within the last approximately 5 million years, is derived from a highly repetitive centromeric region that was duplicated along with the rest of the genome 50–70 million years ago. We also found that CENH3 is bound discontinuously in centromeric subdomains that have fewer genes than subdomains lacking CENH3. These results suggest, not only that centromeres evolve in gene-poor regions, but also how centromeres might evolve from single-copy to repetitive sequences.
A key centromere protein is found to bind discontinuously to subdomains of centromeres that are depleted in genes, suggesting that centromeres evolve in gene-poor regions.
doi:10.1371/journal.pbio.0060286
PMCID: PMC2586382  PMID: 19067486
4.  Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly 
BMC Genomics  2015;16(1):4.
Background
The genome of the melon (Cucumis melo L.) double-haploid line DHL92 was recently sequenced, with 87.5 and 80.8% of the scaffold assembly anchored and oriented to the 12 linkage groups, respectively. However, insufficient marker coverage and a lack of recombination left several large, gene rich scaffolds unanchored, and some anchored scaffolds unoriented. To improve the anchoring and orientation of the melon genome assembly, we used resequencing data between the parental lines of DHL92 to develop a new set of SNP markers from unanchored scaffolds.
Results
A high-resolution genetic map composed of 580 SNPs was used to anchor 354.8 Mb of sequence, contained in 141 scaffolds (average size 2.5 Mb) and corresponding to 98.2% of the scaffold assembly, to the 12 melon chromosomes. Over 325.4 Mb (90%) of the assembly was oriented. The genetic map revealed regions of segregation distortion favoring SC alleles as well as recombination suppression regions coinciding with putative centromere, 45S, and 5S rDNA sites. New chromosome-scale pseudomolecules were created by incorporating to the previous v3.5 version an additional 38.3 Mb of anchored sequence representing 1,837 predicted genes contained in 55 scaffolds. Using fluorescent in situ hybridization (FISH) with BACs that produced chromosome-specific signals, melon chromosomes that correspond to the twelve linkage groups were identified, and a standardized karyotype of melon inbred line T111 was developed.
Conclusions
By utilizing resequencing data and targeted SNP selection combined with a large F2 mapping population, we significantly improved the quantity of anchored and oriented melon scaffold genome assembly. Using genome information combined with FISH mapping provided the first cytogenetic map of an inodorus melon type. With these results it was possible to make inferences on melon chromosome structure by relating zones of recombination suppression to centromeres and 45S and 5S heterochromatic regions. This study represents the first steps towards the integration of the high-resolution genetic and cytogenetic maps with the genomic sequence in melon that will provide more information on genome organization and allow for the improvement of the melon genome draft sequence.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-014-1196-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-014-1196-3
PMCID: PMC4316794  PMID: 25612459
Melon; SNP; Genome; Scaffold; Pseudomolecules; FISH; Karyotype
5.  Tandemly repeated DNA families in the mouse genome 
BMC Genomics  2011;12:531.
Background
Functional and morphological studies of tandem DNA repeats, that combine high portion of most genomes, are mostly limited due to the incomplete characterization of these genome elements. We report here a genome wide analysis of the large tandem repeats (TR) found in the mouse genome assemblies.
Results
Using a bioinformatics approach, we identified large TR with array size more than 3 kb in two mouse whole genome shotgun (WGS) assemblies. Large TR were classified based on sequence similarity, chromosome position, monomer length, array variability, and GC content; we identified four superfamilies, eight families, and 62 subfamilies - including 60 not previously described. 1) The superfamily of centromeric minor satellite is only found in the unassembled part of the reference genome. 2) The pericentromeric major satellite is the most abundant superfamily and reveals high order repeat structure. 3) Transposable elements related superfamily contains two families. 4) The superfamily of heterogeneous tandem repeats includes four families. One family is found only in the WGS, while two families represent tandem repeats with either single or multi locus location. Despite multi locus location, TRPC-21A-MM is placed into a separated family due to its abundance, strictly pericentromeric location, and resemblance to big human satellites.
To confirm our data, we next performed in situ hybridization with three repeats from distinct families. TRPC-21A-MM probe hybridized to chromosomes 3 and 17, multi locus TR-22A-MM probe hybridized to ten chromosomes, and single locus TR-54B-MM probe hybridized with the long loops that emerge from chromosome ends. In addition to in silico predicted several extra-chromosomes were positive for TR by in situ analysis, potentially indicating inaccurate genome assembly of the heterochromatic genome regions.
Conclusions
Chromosome-specific TR had been predicted for mouse but no reliable cytogenetic probes were available before. We report new analysis that identified in silico and confirmed in situ 3/17 chromosome-specific probe TRPC-21-MM. Thus, the new classification had proven to be useful tool for continuation of genome study, while annotated TR can be the valuable source of cytogenetic probes for chromosome recognition.
doi:10.1186/1471-2164-12-531
PMCID: PMC3218096  PMID: 22035034
6.  The Mitochondrial Genome of Soybean Reveals Complex Genome Structures and Gene Evolution at Intercellular and Phylogenetic Levels 
PLoS ONE  2013;8(2):e56502.
Determining mitochondrial genomes is important for elucidating vital activities of seed plants. Mitochondrial genomes are specific to each plant species because of their variable size, complex structures and patterns of gene losses and gains during evolution. This complexity has made research on the soybean mitochondrial genome difficult compared with its nuclear and chloroplast genomes. The present study helps to solve a 30-year mystery regarding the most complex mitochondrial genome structure, showing that pairwise rearrangements among the many large repeats may produce an enriched molecular pool of 760 circles in seed plants. The soybean mitochondrial genome harbors 58 genes of known function in addition to 52 predicted open reading frames of unknown function. The genome contains sequences of multiple identifiable origins, including 6.8 kb and 7.1 kb DNA fragments that have been transferred from the nuclear and chloroplast genomes, respectively, and some horizontal DNA transfers. The soybean mitochondrial genome has lost 16 genes, including nine protein-coding genes and seven tRNA genes; however, it has acquired five chloroplast-derived genes during evolution. Four tRNA genes, common among the three genomes, are derived from the chloroplast. Sizeable DNA transfers to the nucleus, with pericentromeric regions as hotspots, are observed, including DNA transfers of 125.0 kb and 151.6 kb identified unambiguously from the soybean mitochondrial and chloroplast genomes, respectively. The soybean nuclear genome has acquired five genes from its mitochondrial genome. These results provide biological insights into the mitochondrial genome of seed plants, and are especially helpful for deciphering vital activities in soybean.
doi:10.1371/journal.pone.0056502
PMCID: PMC3576410  PMID: 23431381
7.  An improved genome release (version Mt4.0) for the model legume Medicago truncatula 
BMC Genomics  2014;15:312.
Background
Medicago truncatula, a close relative of alfalfa, is a preeminent model for studying nitrogen fixation, symbiosis, and legume genomics. The Medicago sequencing project began in 2003 with the goal to decipher sequences originated from the euchromatic portion of the genome. The initial sequencing approach was based on a BAC tiling path, culminating in a BAC-based assembly (Mt3.5) as well as an in-depth analysis of the genome published in 2011.
Results
Here we describe a further improved and refined version of the M. truncatula genome (Mt4.0) based on de novo whole genome shotgun assembly of a majority of Illumina and 454 reads using ALLPATHS-LG. The ALLPATHS-LG scaffolds were anchored onto the pseudomolecules on the basis of alignments to both the optical map and the genotyping-by-sequencing (GBS) map. The Mt4.0 pseudomolecules encompass ~360 Mb of actual sequences spanning 390 Mb of which ~330 Mb align perfectly with the optical map, presenting a drastic improvement over the BAC-based Mt3.5 which only contained 70% sequences (~250 Mb) of the current version. Most of the sequences and genes that previously resided on the unanchored portion of Mt3.5 have now been incorporated into the Mt4.0 pseudomolecules, with the exception of ~28 Mb of unplaced sequences. With regard to gene annotation, the genome has been re-annotated through our gene prediction pipeline, which integrates EST, RNA-seq, protein and gene prediction evidences. A total of 50,894 genes (31,661 high confidence and 19,233 low confidence) are included in Mt4.0 which overlapped with ~82% of the gene loci annotated in Mt3.5. Of the remaining genes, 14% of the Mt3.5 genes have been deprecated to an “unsupported” status and 4% are absent from the Mt4.0 predictions.
Conclusions
Mt4.0 and its associated resources, such as genome browsers, BLAST-able datasets and gene information pages, can be found on the JCVI Medicago web site (http://www.jcvi.org/medicago). The assembly and annotation has been deposited in GenBank (BioProject: PRJNA10791). The heavily curated chromosomal sequences and associated gene models of Medicago will serve as a better reference for legume biology and comparative genomics.
doi:10.1186/1471-2164-15-312
PMCID: PMC4234490  PMID: 24767513
Medicago; Legume; Genome assembly; Gene annotation; Optical map
8.  High-throughput SNP discovery through deep resequencing of a reduced representation library to anchor and orient scaffolds in the soybean whole genome sequence 
BMC Genomics  2010;11:38.
Background
The Soybean Consensus Map 4.0 facilitated the anchoring of 95.6% of the soybean whole genome sequence developed by the Joint Genome Institute, Department of Energy, but its marker density was only sufficient to properly orient 66% of the sequence scaffolds. The discovery and genetic mapping of more single nucleotide polymorphism (SNP) markers were needed to anchor and orient the remaining genome sequence. To that end, next generation sequencing and high-throughput genotyping were combined to obtain a much higher resolution genetic map that could be used to anchor and orient most of the remaining sequence and to help validate the integrity of the existing scaffold builds.
Results
A total of 7,108 to 25,047 predicted SNPs were discovered using a reduced representation library that was subsequently sequenced by the Illumina sequence-by-synthesis method on the clonal single molecule array platform. Using multiple SNP prediction methods, the validation rate of these SNPs ranged from 79% to 92.5%. A high resolution genetic map using 444 recombinant inbred lines was created with 1,790 SNP markers. Of the 1,790 mapped SNP markers, 1,240 markers had been selectively chosen to target existing unanchored or un-oriented sequence scaffolds, thereby increasing the amount of anchored sequence to 97%.
Conclusion
We have demonstrated how next generation sequencing was combined with high-throughput SNP detection assays to quickly discover large numbers of SNPs. Those SNPs were then used to create a high resolution genetic map that assisted in the assembly of scaffolds from the 8× whole genome shotgun sequences into pseudomolecules corresponding to chromosomes of the organism.
doi:10.1186/1471-2164-11-38
PMCID: PMC2817691  PMID: 20078886
9.  Development and Evaluation of SoySNP50K, a High-Density Genotyping Array for Soybean 
PLoS ONE  2013;8(1):e54985.
The objective of this research was to identify single nucleotide polymorphisms (SNPs) and to develop an Illumina Infinium BeadChip that contained over 50,000 SNPs from soybean (Glycine max L. Merr.). A total of 498,921,777 reads 35–45bp in length were obtained from DNA sequence analysis of reduced representation libraries from several soybean accessions which included six cultivated and two wild soybean (G. soja Sieb. et Zucc.) genotypes. These reads were mapped to the soybean whole genome sequence and 209,903 SNPs were identified. After applying several filters, a total of 146,161 of the 209,903 SNPs were determined to be ideal candidates for Illumina Infinium II BeadChip design. To equalize the distance between selected SNPs, increase assay success rate, and minimize the number of SNPs with low minor allele frequency, an iteration algorithm based on a selection index was developed and used to select 60,800 SNPs for Infinium BeadChip design. Of the 60,800 SNPs, 50,701 were targeted to euchromatic regions and 10,000 to heterochromatic regions of the 20 soybean chromosomes. In addition, 99 SNPs were targeted to unanchored sequence scaffolds. Of the 60,800 SNPs, a total of 52,041 passed Illumina’s manufacturing phase to produce the SoySNP50K iSelect BeadChip. Validation of the SoySNP50K chip with 96 landrace genotypes, 96 elite cultivars and 96 wild soybean accessions showed that 47,337 SNPs were polymorphic and generated successful SNP allele calls. In addition, 40,841 of the 47,337 SNPs (86%) had minor allele frequencies ≥10% among the landraces, elite cultivars and the wild soybean accessions. A total of 620 and 42 candidate regions which may be associated with domestication and recent selection were identified, respectively. The SoySNP50K iSelect SNP beadchip will be a powerful tool for characterizing soybean genetic diversity and linkage disequilibrium, and for constructing high resolution linkage maps to improve the soybean whole genome sequence assembly.
doi:10.1371/journal.pone.0054985
PMCID: PMC3555945  PMID: 23372807
10.  Global repeat discovery and estimation of genomic copy number in a large, complex genome using a high-throughput 454 sequence survey 
BMC Genomics  2007;8:132.
Background
Extensive computational and database tools are available to mine genomic and genetic databases for model organisms, but little genomic data is available for many species of ecological or agricultural significance, especially those with large genomes. Genome surveys using conventional sequencing techniques are powerful, particularly for detecting sequences present in many copies per genome. However these methods are time-consuming and have potential drawbacks. High throughput 454 sequencing provides an alternative method by which much information can be gained quickly and cheaply from high-coverage surveys of genomic DNA.
Results
We sequenced 78 million base-pairs of randomly sheared soybean DNA which passed our quality criteria. Computational analysis of the survey sequences provided global information on the abundant repetitive sequences in soybean. The sequence was used to determine the copy number across regions of large genomic clones or contigs and discover higher-order structures within satellite repeats. We have created an annotated, online database of sequences present in multiple copies in the soybean genome. The low bias of pyrosequencing against repeat sequences is demonstrated by the overall composition of the survey data, which matches well with past estimates of repetitive DNA content obtained by DNA re-association kinetics (Cot analysis).
Conclusion
This approach provides a potential aid to conventional or shotgun genome assembly, by allowing rapid assessment of copy number in any clone or clone-end sequence. In addition, we show that partial sequencing can provide access to partial protein-coding sequences.
doi:10.1186/1471-2164-8-132
PMCID: PMC1894642  PMID: 17524145
11.  Nucleosomes Shape DNA Polymorphism and Divergence 
PLoS Genetics  2014;10(7):e1004457.
An estimated 80% of genomic DNA in eukaryotes is packaged as nucleosomes, which, together with the remaining interstitial linker regions, generate higher order chromatin structures [1]. Nucleosome sequences isolated from diverse organisms exhibit ∼10 bp periodic variations in AA, TT and GC dinucleotide frequencies. These sequence elements generate intrinsically curved DNA and help establish the histone-DNA interface. We investigated an important unanswered question concerning the interplay between chromatin organization and genome evolution: do the DNA sequence preferences inherent to the highly conserved histone core exert detectable natural selection on genomic divergence and polymorphism? To address this hypothesis, we isolated nucleosomal DNA sequences from Drosophila melanogaster embryos and examined the underlying genomic variation within and between species. We found that divergence along the D. melanogaster lineage is periodic across nucleosome regions with base changes following preferred nucleotides, providing new evidence for systematic evolutionary forces in the generation and maintenance of nucleosome-associated dinucleotide periodicities. Further, Single Nucleotide Polymorphism (SNP) frequency spectra show striking periodicities across nucleosomal regions, paralleling divergence patterns. Preferred alleles occur at higher frequencies in natural populations, consistent with a central role for natural selection. These patterns are stronger for nucleosomes in introns than in intergenic regions, suggesting selection is stronger in transcribed regions where nucleosomes undergo more displacement, remodeling and functional modification. In addition, we observe a large-scale (∼180 bp) periodic enrichment of AA/TT dinucleotides associated with nucleosome occupancy, while GC dinucleotide frequency peaks in linker regions. Divergence and polymorphism data also support a role for natural selection in the generation and maintenance of these super-nucleosomal patterns. Our results demonstrate that nucleosome-associated sequence periodicities are under selective pressure, implying that structural interactions between nucleosomes and DNA sequence shape sequence evolution, particularly in introns.
Author Summary
In eukaryotic cells, the majority of DNA is packaged in nucleosomes comprised of ∼147 bp of DNA wound tightly around the highly conserved histone octamer. Nucleosomal DNA from diverse organisms shows an anti-correlated ∼10 bp periodicity of AT-rich and GC-rich dinucleotides. These sequence features influence DNA bending and shape, facilitating structural interactions. We asked whether natural selection mediated through the periodic sequence preferences of nucleosomes shapes the evolution of non-protein-coding regions of D. melanogaster by examining the inter- and intra-species genomic variation relative to these fundamental chromatin building blocks. The sequence changes across nucleosome-bound regions on the melanogaster lineage mirror the observed nucleosome dinucleotide periodicities. Importantly, we show that the frequencies of polymorphisms in natural populations vary across these regions, paralleling divergence, with higher frequencies of preferred alleles. These patterns are most evident for intronic regions and indicate that non-protein coding regions are evolving toward sequences that facilitate the canonical association with the histone core. This result is consistent with the hypothesis that interactions between DNA and the core have systematic impacts on function that are subject to natural selection and are not solely due to mutational bias. These ubiquitous interactions with the histone core partially account for the evolutionary constraint observed in unannotated genomic regions, and may drive broad changes in base composition.
doi:10.1371/journal.pgen.1004457
PMCID: PMC4081404  PMID: 24991813
12.  Epigenetic Remodeling of Meiotic Crossover Frequency in Arabidopsis thaliana DNA Methyltransferase Mutants 
PLoS Genetics  2012;8(8):e1002844.
Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1) mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes.
Author Summary
The majority of eukaryotes reproduce via a specialized cell division called meiosis, which generates gametes with half the number of chromosomes. During meiosis, homologous chromosomes pair and undergo a process of reciprocal exchange, called crossing-over (CO), which generates new combinations of genetic variation. The relative chance of a CO occurring is variable along the chromosome, for example COs are suppressed in the centromeric regions that attach to the spindle during chromosome segregation. These patterns correlate with domains of epigenetic organization along chromosomes, including methylation of the DNA and histones. DNA methylation occurs most densely in the centromeric regions of Arabidopsis thaliana chromosomes, where it is required for transcriptional suppression of repeated sequences. We demonstrate that mutants that lose DNA methylation (met1) show epigenetic remodeling of crossover frequencies, with increases in the centromeric regions and compensatory changes in the chromosome arms, though the total number of crossovers remains the same. As crossover numbers and distributions are subject to homeostatic mechanisms, we propose that these drive crossover remodeling in met1 in response to epigenetic change in the centromeric regions. Together these data demonstrate how domains of epigenetic organization are important for shaping patterns of crossover frequency along eukaryotic chromosomes.
doi:10.1371/journal.pgen.1002844
PMCID: PMC3410864  PMID: 22876192
13.  Histone Modifications within the Human X Centromere Region 
PLoS ONE  2009;4(8):e6602.
Human centromeres are multi-megabase regions of highly ordered arrays of alpha satellite DNA that are separated from chromosome arms by unordered alpha satellite monomers and other repetitive elements. Complexities in assembling such large repetitive regions have limited detailed studies of centromeric chromatin organization. However, a genomic map of the human X centromere has provided new opportunities to explore genomic architecture of a complex locus. We used ChIP to examine the distribution of modified histones within centromere regions of multiple X chromosomes. Methylation of H3 at lysine 4 coincided with DXZ1 higher order alpha satellite, the site of CENP-A localization. Heterochromatic histone modifications were distributed across the 400–500 kb pericentromeric regions. The large arrays of alpha satellite and gamma satellite DNA were enriched for both euchromatic and heterochromatic modifications, implying that some pericentromeric repeats have multiple chromatin characteristics. Partial truncation of the X centromere resulted in reduction in the size of the CENP-A/Cenp-A domain and increased heterochromatic modifications in the flanking pericentromere. Although the deletion removed ∼1/3 of centromeric DNA, the ratio of CENP-A to alpha satellite array size was maintained in the same proportion, suggesting that a limited, but defined linear region of the centromeric DNA is necessary for kinetochore assembly. Our results indicate that the human X centromere contains multiple types of chromatin, is organized similarly to smaller eukaryotic centromeres, and responds to structural changes by expanding or contracting domains.
doi:10.1371/journal.pone.0006602
PMCID: PMC2719913  PMID: 19672304
14.  A Single Molecule Scaffold for the Maize Genome 
PLoS Genetics  2009;5(11):e1000711.
About 85% of the maize genome consists of highly repetitive sequences that are interspersed by low-copy, gene-coding sequences. The maize community has dealt with this genomic complexity by the construction of an integrated genetic and physical map (iMap), but this resource alone was not sufficient for ensuring the quality of the current sequence build. For this purpose, we constructed a genome-wide, high-resolution optical map of the maize inbred line B73 genome containing >91,000 restriction sites (averaging 1 site/∼23 kb) accrued from mapping genomic DNA molecules. Our optical map comprises 66 contigs, averaging 31.88 Mb in size and spanning 91.5% (2,103.93 Mb/∼2,300 Mb) of the maize genome. A new algorithm was created that considered both optical map and unfinished BAC sequence data for placing 60/66 (2,032.42 Mb) optical map contigs onto the maize iMap. The alignment of optical maps against numerous data sources yielded comprehensive results that proved revealing and productive. For example, gaps were uncovered and characterized within the iMap, the FPC (fingerprinted contigs) map, and the chromosome-wide pseudomolecules. Such alignments also suggested amended placements of FPC contigs on the maize genetic map and proactively guided the assembly of chromosome-wide pseudomolecules, especially within complex genomic regions. Lastly, we think that the full integration of B73 optical maps with the maize iMap would greatly facilitate maize sequence finishing efforts that would make it a valuable reference for comparative studies among cereals, or other maize inbred lines and cultivars.
Author Summary
The maize genome contains abundant repeats interspersed by low-copy, gene-coding sequences that make it a challenge to sequence; consequently, current BAC sequence assemblies average 11 contigs per clone. The iMap deals with such complexity by the judicious integration of IBM genetic and B73 physical maps, but the B73 genome structure could differ from the IBM population because of genetic recombination and subsequent rearrangements. Accordingly, we report a genome-wide, high-resolution optical map of maize B73 genome that was constructed from the direct analysis of genomic DNA molecules without using genetic markers. The integration of optical and iMap resources with comparisons to FPC maps enabled a uniquely comprehensive and scalable assessment of a given BAC's sequence assembly, its placement within a FPC contig, and the location of this FPC contig within a chromosome-wide pseudomolecule. As such, the overall utility of the maize optical map for the validation of sequence assemblies has been significant and demonstrates the inherent advantages of single molecule platforms. Construction of the maize optical map represents the first physical map of a eukaryotic genome larger than 400 Mb that was created de novo from individual genomic DNA molecules.
doi:10.1371/journal.pgen.1000711
PMCID: PMC2774507  PMID: 19936062
15.  Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases 
BMC Plant Biology  2010;10:243.
Background
Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship.
Results
We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an isoflavone synthase gene) is co-expressed with several genes encoding isoflavonoid-related metabolic enzymes. We then focused on nodulation-induced P450s and found that CYP728H1 was co-expressed with the genes involved in phenylpropanoid metabolism. Similarly, CYP736A34 was highly co-expressed with lipoxygenase, lectin and CYP83D1, all of which are involved in root and nodule development.
Conclusions
The genome scale analysis of P450s in soybean reveals many unique features of these important enzymes in this crop although the functions of most of them are largely unknown. Gene co-expression analysis proves to be a useful tool to infer the function of uncharacterized genes. Our work presented here could provide important leads toward functional genomics studies of soybean P450s and their regulatory network through the integration of reverse genetics, biochemistry, and metabolic profiling tools. The identification of nodule-specific P450s and their further exploitation may help us to better understand the intriguing process of soybean and rhizobium interaction.
doi:10.1186/1471-2229-10-243
PMCID: PMC3095325  PMID: 21062474
16.  Primary analysis of repeat elements of the Asian seabass (Lates calcarifer) transcriptome and genome 
Frontiers in Genetics  2014;5:223.
As part of our Asian seabass genome project, we are generating an inventory of repeat elements in the genome and transcriptome. The karyotype showed a diploid number of 2n = 24 chromosomes with a variable number of B-chromosomes. The transcriptome and genome of Asian seabass were searched for repetitive elements with experimental and bioinformatics tools. Six different types of repeats constituting 8–14% of the genome were characterized. Repetitive elements were clustered in the pericentromeric heterochromatin of all chromosomes, but some of them were preferentially accumulated in pretelomeric and pericentromeric regions of several chromosomes pairs and have chromosomes specific arrangement. From the dispersed class of fish-specific non-LTR retrotransposon elements Rex1 and MAUI-like repeats were analyzed. They were wide-spread both in the genome and transcriptome, accumulated on the pericentromeric and peritelomeric areas of all chromosomes. Every analyzed repeat was represented in the Asian seabass transcriptome, some showed differential expression between the gonads. The other group of repeats analyzed belongs to the rRNA multigene family. FISH signal for 5S rDNA was located on a single pair of chromosomes, whereas that for 18S rDNA was found on two pairs. A BAC-derived contig containing rDNA was sequenced and assembled into a scaffold containing incomplete fragments of 18S rDNA. Their assembly and chromosomal position revealed that this part of Asian seabass genome is extremely rich in repeats containing evolutionarily conserved and novel sequences. In summary, transcriptome assemblies and cDNA data are suitable for the identification of repetitive DNA from unknown genomes and for comparative investigation of conserved elements between teleosts and other vertebrates.
doi:10.3389/fgene.2014.00223
PMCID: PMC4110674  PMID: 25120555
repeated DNA; Asian seabass; transcriptome; rDNA; chromosomes
17.  Evolutionary and comparative analyses of the soybean genome 
Breeding Science  2012;61(5):437-444.
The soybean genome assembly has been available since the end of 2008. Significant features of the genome include large, gene-poor, repeat-dense pericentromeric regions, spanning roughly 57% of the genome sequence; a relatively large genome size of ~1.15 billion bases; remnants of a genome duplication that occurred ~13 million years ago (Mya); and fainter remnants of older polyploidies that occurred ~58 Mya and >130 Mya. The genome sequence has been used to identify the genetic basis for numerous traits, including disease resistance, nutritional characteristics, and developmental features. The genome sequence has provided a scaffold for placement of many genomic feature elements, both from within soybean and from related species. These may be accessed at several websites, including http://www.phytozome.net, http://soybase.org, http://comparative-legumes.org, and http://www.legumebase.brc.miyazaki-u.ac.jp. The taxonomic position of soybean in the Phaseoleae tribe of the legumes means that there are approximately two dozen other beans and relatives that have undergone independent domestication, and which may have traits that will be useful for transfer to soybean. Methods of translating information between species in the Phaseoleae range from design of markers for marker assisted selection, to transformation with Agrobacterium or with other experimental transformation methods.
doi:10.1270/jsbbs.61.437
PMCID: PMC3406793  PMID: 23136483
Glycine max; soybean; legume evolution; polyploidy; SoyBase; Legume Information System; Legumebase; Phytozome
18.  ChromaSig: A Probabilistic Approach to Finding Common Chromatin Signatures in the Human Genome 
PLoS Computational Biology  2008;4(10):e1000201.
Computational methods to identify functional genomic elements using genetic information have been very successful in determining gene structure and in identifying a handful of cis-regulatory elements. But the vast majority of regulatory elements have yet to be discovered, and it has become increasingly apparent that their discovery will not come from using genetic information alone. Recently, high-throughput technologies have enabled the creation of information-rich epigenetic maps, most notably for histone modifications. However, tools that search for functional elements using this epigenetic information have been lacking. Here, we describe an unsupervised learning method called ChromaSig to find, in an unbiased fashion, commonly occurring chromatin signatures in both tiling microarray and sequencing data. Applying this algorithm to nine chromatin marks across a 1% sampling of the human genome in HeLa cells, we recover eight clusters of distinct chromatin signatures, five of which correspond to known patterns associated with transcriptional promoters and enhancers. Interestingly, we observe that the distinct chromatin signatures found at enhancers mark distinct functional classes of enhancers in terms of transcription factor and coactivator binding. In addition, we identify three clusters of novel chromatin signatures that contain evolutionarily conserved sequences and potential cis-regulatory elements. Applying ChromaSig to a panel of 21 chromatin marks mapped genomewide by ChIP-Seq reveals 16 classes of genomic elements marked by distinct chromatin signatures. Interestingly, four classes containing enrichment for repressive histone modifications appear to be locally heterochromatic sites and are enriched in quickly evolving regions of the genome. The utility of this approach in uncovering novel, functionally significant genomic elements will aid future efforts of genome annotation via chromatin modifications.
Author Summary
The DNA in eukaryotes is packaged by histones. Interestingly, histones can be marked by a variety of posttranslational modifications, and it has been hypothesized that distinct combinations of histone modifications mark at distinct functional regions of the genome. The study of histone modifications has been aided by the development of high-throughput techniques to map a wide assortment of histone modifications on a global scale. However, because much of our current understanding of the human genome is concentrated on promoters, most studies have only examined histone modifications at these well-defined sites, ignoring the vast majority of the genome. To aid in the discovery of functional elements outside of these well-annotated loci, we develop an unbiased method that searches for commonly occurring histone modification patterns on a global scale without using any annotation information. This method recovers known patterns associated with transcriptional enhancers and promoters. Supporting the histone code hypothesis, we discover that the different functional activities of enhancers are closely associated with the presence of different histone modification patterns. We also discover several novel patterns that likely contain other potential regulatory elements. As the availability of large-scale histone modification data increases, the ability of methods such as the one presented here to concisely describe commonly occurring chromatin signatures, thereby abstracting away irrelevant or redundant data, will become increasingly more critical.
doi:10.1371/journal.pcbi.1000201
PMCID: PMC2556089  PMID: 18927605
19.  The Genome Sequence of the Fungal Pathogen Fusarium virguliforme That Causes Sudden Death Syndrome in Soybean 
PLoS ONE  2014;9(1):e81832.
Fusarium virguliforme causes sudden death syndrome (SDS) of soybean, a disease of serious concern throughout most of the soybean producing regions of the world. Despite the global importance, little is known about the pathogenesis mechanisms of F. virguliforme. Thus, we applied Next-Generation DNA Sequencing to reveal the draft F. virguliforme genome sequence and identified putative pathogenicity genes to facilitate discovering the mechanisms used by the pathogen to cause this disease.
Methodology/Principal Findings
We have generated the draft genome sequence of F. virguliforme by conducting whole-genome shotgun sequencing on a 454 GS-FLX Titanium sequencer. Initially, single-end reads of a 400-bp shotgun library were assembled using the PCAP program. Paired end sequences from 3 and 20 Kb DNA fragments and approximately 100 Kb inserts of 1,400 BAC clones were used to generate the assembled genome. The assembled genome sequence was 51 Mb. The N50 scaffold number was 11 with an N50 Scaffold length of 1,263 Kb. The AUGUSTUS gene prediction program predicted 14,845 putative genes, which were annotated with Pfam and GO databases. Gene distributions were uniform in all but one of the major scaffolds. Phylogenic analyses revealed that F. virguliforme was closely related to the pea pathogen, Nectria haematococca. Of the 14,845 F. virguliforme genes, 11,043 were conserved among five Fusarium species: F. virguliforme, F. graminearum, F. verticillioides, F. oxysporum and N. haematococca; and 1,332 F. virguliforme-specific genes, which may include pathogenicity genes. Additionally, searches for candidate F. virguliforme pathogenicity genes using gene sequences of the pathogen-host interaction database identified 358 genes.
Conclusions
The F. virguliforme genome sequence and putative pathogenicity genes presented here will facilitate identification of pathogenicity mechanisms involved in SDS development. Together, these resources will expedite our efforts towards discovering pathogenicity mechanisms in F. virguliforme. This will ultimately lead to improvement of SDS resistance in soybean.
doi:10.1371/journal.pone.0081832
PMCID: PMC3891557  PMID: 24454689
20.  Rice pseudomolecule-anchored cross-species DNA sequence alignments indicate regional genomic variation in expressed sequence conservation 
BMC Genomics  2007;8:283.
Background
Various methods have been developed to explore inter-genomic relationships among plant species. Here, we present a sequence similarity analysis based upon comparison of transcript-assembly and methylation-filtered databases from five plant species and physically anchored rice coding sequences.
Results
A comparison of the frequency of sequence alignments, determined by MegaBLAST, between rice coding sequences in TIGR pseudomolecules and annotations vs 4.0 and comprehensive transcript-assembly and methylation-filtered databases from Lolium perenne (ryegrass), Zea mays (maize), Hordeum vulgare (barley), Glycine max (soybean) and Arabidopsis thaliana (thale cress) was undertaken. Each rice pseudomolecule was divided into 10 segments, each containing 10% of the functionally annotated, expressed genes. This indicated a correlation between relative segment position in the rice genome and numbers of alignments with all the queried monocot and dicot plant databases. Colour-coded moving windows of 100 functionally annotated, expressed genes along each pseudomolecule were used to generate 'heat-maps'. These revealed consistent intra- and inter-pseudomolecule variation in the relative concentrations of significant alignments with the tested plant databases. Analysis of the annotations and derived putative expression patterns of rice genes from 'hot-spots' and 'cold-spots' within the heat maps indicated possible functional differences. A similar comparison relating to ancestral duplications of the rice genome indicated that duplications were often associated with 'hot-spots'.
Conclusion
Physical positions of expressed genes in the rice genome are correlated with the degree of conservation of similar sequences in the transcriptomes of other plant species. This relative conservation is associated with the distribution of different sized gene families and segmentally duplicated loci and may have functional and evolutionary implications.
doi:10.1186/1471-2164-8-283
PMCID: PMC2041955  PMID: 17708759
21.  Novel Gene Acquisition on Carnivore Y Chromosomes 
PLoS Genetics  2006;2(3):e43.
Despite its importance in harboring genes critical for spermatogenesis and male-specific functions, the Y chromosome has been largely excluded as a priority in recent mammalian genome sequencing projects. Only the human and chimpanzee Y chromosomes have been well characterized at the sequence level. This is primarily due to the presumed low overall gene content and highly repetitive nature of the Y chromosome and the ensuing difficulties using a shotgun sequence approach for assembly. Here we used direct cDNA selection to isolate and evaluate the extent of novel Y chromosome gene acquisition in the genome of the domestic cat, a species from a different mammalian superorder than human, chimpanzee, and mouse (currently being sequenced). We discovered four novel Y chromosome genes that do not have functional copies in the finished human male-specific region of the Y or on other mammalian Y chromosomes explored thus far. Two genes are derived from putative autosomal progenitors, and the other two have X chromosome homologs from different evolutionary strata. All four genes were shown to be multicopy and expressed predominantly or exclusively in testes, suggesting that their duplication and specialization for testis function were selected for because they enhance spermatogenesis. Two of these genes have testis-expressed, Y-borne copies in the dog genome as well. The absence of the four newly described genes on other characterized mammalian Y chromosomes demonstrates the gene novelty on this chromosome between mammalian orders, suggesting it harbors many lineage-specific genes that may go undetected by traditional comparative genomic approaches. Specific plans to identify the male-specific genes encoded in the Y chromosome of mammals should be a priority.
Synopsis
Y chromosomes are typically gene poor and enriched with repetitive elements, making them difficult to sequence by standard methods. Hence, the Y chromosome gene repertoire in mammalian species other than human has not been explored until very recently. Here the authors used a directed approach to isolate Y chromosome genes of the domestic cat, an evolutionary divergent species from human and mouse. They found that the feline Y chromosome harbors its own unique set of genes that are expressed specifically in the testes, presumably where they play an important role in spermatogenesis. Paralleling the discoveries seen from the full human Y chromosome sequence, the feline Y chromosome has acquired and remodeled some genes from autosomes, while other genes have a shared ancestry with the X chromosome. However, none of the four new genes are found on the Y chromosomes of human or mouse, although two are shared with the canine Y chromosome. This work highlights the Y chromosome as a source of potential gene novelty in different species and suggests that more directed efforts at characterizing this hitherto understudied chromosome will further enrich our understanding of the types of genes found there and the roles they may play in mammalian spermatogenesis.
doi:10.1371/journal.pgen.0020043
PMCID: PMC1420679  PMID: 16596168
22.  Pigeonpea genomics initiative (PGI): an international effort to improve crop productivity of pigeonpea (Cajanus cajan L.) 
Molecular Breeding  2009;26(3):393-408.
Pigeonpea (Cajanus cajan), an important food legume crop in the semi-arid regions of the world and the second most important pulse crop in India, has an average crop productivity of 780 kg/ha. The relatively low crop yields may be attributed to non-availability of improved cultivars, poor crop husbandry and exposure to a number of biotic and abiotic stresses in pigeonpea growing regions. Narrow genetic diversity in cultivated germplasm has further hampered the effective utilization of conventional breeding as well as development and utilization of genomic tools, resulting in pigeonpea being often referred to as an ‘orphan crop legume’. To enable genomics-assisted breeding in this crop, the pigeonpea genomics initiative (PGI) was initiated in late 2006 with funding from Indian Council of Agricultural Research under the umbrella of Indo-US agricultural knowledge initiative, which was further expanded with financial support from the US National Science Foundation’s Plant Genome Research Program and the Generation Challenge Program. As a result of the PGI, the last 3 years have witnessed significant progress in development of both genetic as well as genomic resources in this crop through effective collaborations and coordination of genomics activities across several institutes and countries. For instance, 25 mapping populations segregating for a number of biotic and abiotic stresses have been developed or are under development. An 11X-genome coverage bacterial artificial chromosome (BAC) library comprising of 69,120 clones have been developed of which 50,000 clones were end sequenced to generate 87,590 BAC-end sequences (BESs). About 10,000 expressed sequence tags (ESTs) from Sanger sequencing and ca. 2 million short ESTs by 454/FLX sequencing have been generated. A variety of molecular markers have been developed from BESs, microsatellite or simple sequence repeat (SSR)-enriched libraries and mining of ESTs and genomic amplicon sequencing. Of about 21,000 SSRs identified, 6,698 SSRs are under analysis along with 670 orthologous genes using a GoldenGate SNP (single nucleotide polymorphism) genotyping platform, with large scale SNP discovery using Solexa, a next generation sequencing technology, is in progress. Similarly a diversity array technology array comprising of ca. 15,000 features has been developed. In addition, >600 unique nucleotide binding site (NBS) domain containing members of the NBS-leucine rich repeat disease resistance homologs were cloned in pigeonpea; 960 BACs containing these sequences were identified by filter hybridization, BES physical maps developed using high information content fingerprinting. To enrich the genomic resources further, sequenced soybean genome is being analyzed to establish the anchor points between pigeonpea and soybean genomes. In addition, Solexa sequencing is being used to explore the feasibility of generating whole genome sequence. In summary, the collaborative efforts of several research groups under the umbrella of PGI are making significant progress in improving molecular tools in pigeonpea and should significantly benefit pigeonpea genetics and breeding. As these efforts come to fruition, and expanded (depending on funding), pigeonpea would move from an ‘orphan legume crop’ to one where genomics-assisted breeding approaches for a sustainable crop improvement are routine.
doi:10.1007/s11032-009-9327-2
PMCID: PMC2948155  PMID: 20976284
Molecular markers; Genetic mapping; Trait mapping; Genomics; Next generation sequencing; Gene discovery; Crop improvement
23.  Using Microsatellites to Understand the Physical Distribution of Recombination on Soybean Chromosomes 
PLoS ONE  2011;6(7):e22306.
Soybean is a major crop that is an important source of oil and proteins. A number of genetic linkage maps have been developed in soybean. Specifically, hundreds of simple sequence repeat (SSR) markers have been developed and mapped. Recent sequencing of the soybean genome resulted in the generation of vast amounts of genetic information. The objectives of this investigation were to use SSR markers in developing a connection between genetic and physical maps and to determine the physical distribution of recombination on soybean chromosomes. A total of 2,188 SSRs were used for sequence-based physical localization on soybean chromosomes. Linkage information was used from different maps to create an integrated genetic map. Comparison of the integrated genetic linkage maps and sequence based physical maps revealed that the distal 25% of each chromosome was the most marker-dense, containing an average of 47.4% of the SSR markers and 50.2% of the genes. The proximal 25% of each chromosome contained only 7.4% of the markers and 6.7% of the genes. At the whole genome level, the marker density and gene density showed a high correlation (R2) of 0.64 and 0.83, respectively with the physical distance from the centromere. Recombination followed a similar pattern with comparisons indicating that recombination is high in telomeric regions, though the correlation between crossover frequency and distance from the centromeres is low (R2 = 0.21). Most of the centromeric regions were low in recombination. The crossover frequency for the entire soybean genome was 7.2%, with extremes much higher and lower than average. The number of recombination hotspots varied from 1 to 12 per chromosome. A high correlation of 0.83 between the distribution of SSR markers and genes suggested close association of SSRs with genes. The knowledge of distribution of recombination on chromosomes may be applied in characterizing and targeting genes.
doi:10.1371/journal.pone.0022306
PMCID: PMC3140510  PMID: 21799819
24.  Organization and Evolution of Primate Centromeric DNA from Whole-Genome Shotgun Sequence Data 
PLoS Computational Biology  2007;3(9):e181.
The major DNA constituent of primate centromeres is alpha satellite DNA. As much as 2%–5% of sequence generated as part of primate genome sequencing projects consists of this material, which is fragmented or not assembled as part of published genome sequences due to its highly repetitive nature. Here, we develop computational methods to rapidly recover and categorize alpha-satellite sequences from previously uncharacterized whole-genome shotgun sequence data. We present an algorithm to computationally predict potential higher-order array structure based on paired-end sequence data and then experimentally validate its organization and distribution by experimental analyses. Using whole-genome shotgun data from the human, chimpanzee, and macaque genomes, we examine the phylogenetic relationship of these sequences and provide further support for a model for their evolution and mutation over the last 25 million years. Our results confirm fundamental differences in the dispersal and evolution of centromeric satellites in the Old World monkey and ape lineages of evolution.
Author Summary
Centromeric DNA has been described as the last frontier of genomic sequencing; such regions are typically poorly assembled during the whole-genome shotgun sequence assembly process due to their repetitive complexity. This paper develops a computational algorithm to systematically extract data regarding primate centromeric DNA structure and organization from that ∼5% of sequence that is not included as part of standard genome sequence assemblies. Using this computational approach, we identify and reconstruct published human higher-order alpha satellite arrays and discover new families in human, chimpanzee, and Old World monkeys. Experimental validation confirms the utility of this computational approach to understanding the centromere organization of other nonhuman primates. An evolutionary analysis in diverse primate genomes supports fundamental differences in the structure and organization of centromere DNA between ape and Old World monkey lineages. The ability to extract meaningful biological data from random shotgun sequence data helps to fill an important void in large-scale sequencing of primate genomes, with implications for other genome sequencing projects.
doi:10.1371/journal.pcbi.0030181
PMCID: PMC1994983  PMID: 17907796
25.  Development of a pooled probe method for locating small gene families in a physical map of soybean using stress related paralogues and a BAC minimum tile path 
Plant Methods  2006;2:20.
Background
Genome analysis of soybean (Glycine max L.) has been complicated by its paleo-autopolyploid nature and conserved homeologous regions. Landmarks of expressed sequence tags (ESTs) located within a minimum tile path (MTP) of contiguous (contig) bacterial artificial chromosome (BAC) clones or radiation hybrid set can identify stress and defense related gene rich regions in the genome. A physical map of about 2,800 contigs and MTPs of 8,064 BAC clones encompass the soybean genome. That genome is being sequenced by whole genome shotgun methods so that reliable estimates of gene family size and gene locations will provide a useful tool for finishing. The aims here were to develop methods to anchor plant defense- and stress-related gene paralogues on the MTP derived from the soybean physical map, to identify gene rich regions and to correlate those with QTL for disease resistance.
Results
The probes included 143 ESTs from a root library selected by subtractive hybridization from a multiply disease resistant soybean cultivar 'Forrest' 14 days after inoculation with Fusarium solani f. sp. glycines (F. virguliforme). Another 166 probes were chosen from a root EST library (Gm-r1021) prepared from a non-inoculated soybean cultivar 'Williams 82' based on their homology to the known defense and stress related genes. Twelve and thirteen pooled EST probes were hybridized to high-density colony arrays of MTP BAC clones from the cv. 'Forrest' genome. The EST pools located 613 paralogues for 201 of the 309 probes used (range 1–13 per functional probe). One hundred BAC clones contained more than one kind of paralogue. Many more BACs (246) contained a single paralogue of one of the 201 probes detectable gene families. ESTs were anchored on soybean linkage groups A1, B1, C2, E, D1a+Q, G, I, M, H, and O.
Conclusion
Estimates of gene family sizes were more similar to those made by Southern hybridization than by bioinformatics inferences from EST collections. When compared to Arabidopsis thaliana there were more 2 and 4 member paralogue families reflecting the diploidized-tetraploid nature of the soybean genome. However there were fewer families with 5 or more genes and the same number of single genes. Therefore the method can identify evolutionary patterns such as massively extensive selective gene loss or rapid divergence to regenerate the unique genes in some families.
doi:10.1186/1746-4811-2-20
PMCID: PMC1716159  PMID: 17156445

Results 1-25 (946809)