[show abstract][hide abstract] ABSTRACT: Caldicellulosiruptor bescii is an anaerobic thermophilic bacterium of special interest for use in the consolidated bioprocessing of plant biomass to biofuels. In the course of experiments to engineer pyruvate metabolism in C. bescii, we isolated a mutant of C. bescii that contained an insertion in the L-lactate dehydrogenase gene (ldh). PCR amplification and sequencing of the ldh gene from this mutant revealed a 1,609-bp insertion that contained a single open reading frame of 479 amino acids (1,440 bp) annotated as a hypothetical protein with unknown function. The ORF is flanked by an 8-base direct repeat sequence. Bioinformatic analysis indicated that this ORF is part of a novel transposable element, ISCbe4, which is only intact in the genus Caldicellulosiruptor, but has ancient relatives that are present in degraded (and previously unrecognized) forms across many bacterial and archaeal clades.
Journal of Industrial Microbiology 10/2013; · 1.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the OMAP (www.OMAP.org) BAC resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2 and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed BIBAC libraries for the maize inbred B73 and the sorghum land race Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven ZMAP BAC/BIBAC libraries have average insert sizes ranging from 92kb to 148kb, organellar DNA from 0.17% to 2.3%, empty vector rates between 0.35% and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.
[show abstract][hide abstract] ABSTRACT: Resistance Gene Homologues (RGHs) were isolated from the switchgrass variety Alamo by a combination of PCR and expressed sequence tag (EST) database mining. Fifty-eight RGHs were isolated by PCR and 295 RGHs were identified in 424,545 switchgrass ESTs. Four NBS-LRR RGHs were selected to investigate RGH haplotypic diversity in seven switchgrass varieties chosen for their representation of a broad range of the switchgrass germplasm. Lowland and upland ecotypes were found to be less similar, even from nearby populations, than were more distant populations with similar growth environments. A majority (83.5%) of the variability in these four RGHs was found to be attributable to the within-population component. The difference in nucleotide diversity between and within populations was observed to be small, while this diversity is maintained to similar degrees at both population and ecotype levels. The results also revealed that the analyzed RGHs were under positive selection in the studied switchgrass accessions. Intragenic recombination was detected in switchgrass RGHs, thereby demonstrating an active genetic process that has the potential to generate new resistance genes with new specificities that might act against newly-arising pathogen races.
[show abstract][hide abstract] ABSTRACT: Pearl millet is one of the most important subsistence crops grown in India and sub-Saharan Africa. In many cereal crops, reduced height is a key trait for enhancing yield, and dwarf mutants have been extensively used in breeding to reduce yield loss due to lodging under intense management. In pearl millet, the recessive dwarfing gene has been deployed widely in commercial germplasm grown in India, the United States, and Australia. Despite its importance, very little research has gone into determining the identity of the gene. We used comparative information, genetic mapping in two F populations representing a total of some 1500 progeny, and haplotype analysis of three tall and three dwarf inbred lines to delineate the region by two genetic markers that, in sorghum, define a region of 410 kb with 40 annotated genes. One of the sorghum genes annotated within this region is , which encodes a P-glycoprotein involved in auxin transport. This gene had previously been shown to underlie the economically important dwarf mutation in sorghum. The cosegregation of with the phenotype, its differential expression in the tall inbred ICMP 451 and the dwarf inbred Tift 23DB, and the similar phenotype of stacked lower internodes in the sorghum and pearl millet mutants suggest that is a likely candidate for .
[show abstract][hide abstract] ABSTRACT: The microsatellite library was developed for Greenhouse Whitefly (Trialeurodes vaporariorum). The citation of this journal include all research studies published at this particular issue of the journal. Our study could be found at:
[show abstract][hide abstract] ABSTRACT: Phrynops geoffroanus is a freshwater turtle species, with a wide distribution in South America, living in many types of habitats, including polluted urban rivers. However, the lack of knowledge hinders broader approaches to various ecological and evolutionary aspects of this species. In this study eight polymorphic microsatellite markers were isolated and characterized in 48 individuals from two natural populations. In the Piracicaba river population, the number of alleles for the eight loci ranged from 4 to 15, whereas the observed and expected heterozygosities per locus varied from 0.29 to 0.87 and from 0.31 to 0.90, respectively. In Piracicamirim stream population, the number of alleles ranged from 4 to 13, and the observed and expected heterozygosities per locus varied from 0.46 to 0.75 and from 0.53 to 0.88, respectively. These microsatellites provide efficient genetic markers for population structure, species relationships and phylogeography studies.
[show abstract][hide abstract] ABSTRACT: Although centromere function is highly conserved in eukaryotes, centromere sequences are highly variable. Only a few centromeres have been sequenced in higher eukaryotes because of their repetitive nature, thus hindering study of their structure and evolution. Conserved single-copy sequences in pericentromeres (CSCPs) of sorghum and maize were found to be diagnostic characteristics of adjacent centromeres. By analyzing comparative map data and CSCP sequences of sorghum, maize, and rice, the major evolutionary events related to centromere dynamics were discovered for the maize lineage after its divergence from a common ancestor with sorghum. (i) Remnants of ancient CSCP regions were found for the 10 lost ancestral centromeres, indicating that two ancient homeologous chromosome pairs did not contribute any centromeres to the current maize genome, whereas two other pairs contributed both of their centromeres. (ii) Five cases of long-distance, intrachromosome movement of CSCPs were detected in the retained centromeres, with inversion the major process involved. (iii) The 12 major chromosomal rearrangements that led to maize chromosome number reduction from 20 to 10 were uncovered. (iv) In addition to whole chromosome insertion near (but not always into) other centromeres, translocation and fusion were found to be important mechanisms underlying grass chromosome number reduction. (v) Comparison of chromosome structures confirms the polyploid event that led to the tetraploid ancestor of modern maize.
Proceedings of the National Academy of Sciences 11/2012; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The draft genome of pear (Pyrus bretschneideri) using a combination of BAC-by-BAC and next generation sequencing is reported. A 512.0 Mb sequence corresponding to 97.1% of the estimated genome size of this highly heterozygous species is assembled with 194x coverage. High-density genetic maps comprising of 2,005 SNP markers anchored 75.5% of the sequence to all 17 chromosomes. The pear genome encodes 42,812 protein-coding genes, and of these, ~28.5% encode multiple isoforms. High quality of the assembly and annotation is assessed and confirmed using Sanger-derived BAC sequences along with transcriptome sequences of different tissues. Repetitive sequences of 271.9 Mb in length, accounting for 53.1% of the pear genome, are identified. Simulation of eudicots to the ancestor of Rosaceae has re-constructed nine ancestral chromosomes. Pear and apple have diverged from each other ~5.4 to 21.5 MYA, and a recent whole-genome duplication (WGD) event must have occurred 30-45 MYA prior to their divergence, but following divergence from strawberry. When compared with the apple genome sequence, size differences between apple and pear genomes are confirmed mainly due to presence of repetitive sequences predominantly contributed by transposable elements (TEs), while genic regions are similar in both species. Genes critical for self-incompatibility, lignified stone cells (a unique feature of pear fruit), sorbitol metabolism and volatile compounds of fruit have also been identified. Multiple candidate SFB genes appear as tandem repeats in the S-locus region of pear; while, lignin synthesis-related gene family expansion and highly expressed gene families of HCT, C3'H, and CCOMT contribute to high accumulation of both G-lignin and S-lignin. Expansion of S6PDH, SDH, and SOT along with evolutionary relationships of pear and apple have demonstrated their divergence from a common ancestor. Moreover, α-linolenic acid metabolism is a key pathway for aroma in pear fruit.
[show abstract][hide abstract] ABSTRACT: Tef (Eragrostis tef) is a major cereal crop in Ethiopia. Lodging is the primary constraint to increasing productivity in this allotetraploid species, accounting for losses of ~15-45% in yield each year. As a first step toward identifying semi-dwarf varieties that might have improved lodging resistance, an ~6X fosmid library was constructed and used to identify both homoeologues of the dw3 semi-dwarfing gene of Sorghum bicolor. An EMS mutagenized population, consisting of ~21,210 tef plants, was planted and leaf materials were collected into 23 superpools. Two dwarfing candidate genes, homoeologues of dw3 of sorghum and rht1 of wheat, were sequenced directly from each superpool with 454 technology, and 120 candidate mutations were identified. Out of ten candidates tested, six independent mutations were validated by Sanger sequencing, including two predicted detrimental mutations in both dw3 homoeologues with a potential to improve lodging resistance in tef through further breeding. This study demonstrates that high throughput sequencing can identify potentially valuable mutations in under-studied plant species like tef, and has provided mutant lines that can now be combined and tested in breeding programs for improved lodging resistance.
[show abstract][hide abstract] ABSTRACT: Animal heterotrimeric G proteins are activated by guanine nucleotide exchange factors (GEF), typically seven transmembrane receptors that trigger GDP release and subsequent GTP binding. In contrast, the Arabidopsis thaliana G protein (AtGPA1) rapidly activates itself without a GEF and is instead regulated by a seven transmembrane Regulator of G protein Signaling (7TM-RGS) protein that promotes GTP hydrolysis to reset the inactive (GDP-bound) state. It is not known if this unusual activation is a major and constraining part of the evolutionary history of G signaling in eukaryotes. In particular, it is not known if this is an ancestral form or if this mechanism is maintained, and therefore constrained, within the plant kingdom. To determine if this mode of signal regulation is conserved throughout the plant kingdom, we analyzed available plant genomes for G protein signaling components, and we purified individually the plant components encoded in an informative set of plant genomes in order to determine their activation properties in vitro. While the subunits of the heterotrimeric G protein complex are encoded in vascular plant genomes, the 7TM-RGS genes were lost in all investigated grasses. Despite the absence of a Gα-inactivating protein in grasses, all vascular plant Gα proteins examined rapidly released GDP without a receptor and slowly hydrolyzed GTP, indicating that these Gα are self-activating. We showed further that a single amino acid substitution found naturally in grass Gα proteins reduced the Gα-RGS interaction, and this amino acid substitution occurred before the loss of the RGS gene in the grass lineage. Like grasses, non-vascular plants also appear to lack RGS proteins. However, unlike grasses, one representative non-vascular plant Gα showed rapid GTP hydrolysis, likely compensating for the loss of the RGS gene. Our findings, the loss of a regulatory gene and the retention of the "self-activating" trait, indicate the existence of divergent Gα regulatory mechanisms in the plant kingdom. In the grasses, purifying selection on the regulatory gene was lost after the physical decoupling of the RGS protein and its cognate Gα partner. More broadly these findings show extreme divergence in Gα activation and regulation that played a critical role in the evolution of G protein signaling pathways.
[show abstract][hide abstract] ABSTRACT: We generated a high-quality reference genome sequence for foxtail millet (Setaria italica). The ∼400-Mb assembly covers ∼80% of the genome and >95% of the gene space. The assembly was anchored to a 992-locus genetic map and was annotated by comparison with >1.3 million expressed sequence tag reads. We produced more than 580 million RNA-Seq reads to facilitate expression analyses. We also sequenced Setaria viridis, the ancestral wild relative of S. italica, and identified regions of differential single-nucleotide polymorphism density, distribution of transposable elements, small RNA content, chromosomal rearrangement and segregation distortion. The genus Setaria includes natural and cultivated species that demonstrate a wide capacity for adaptation. The genetic basis of this adaptation was investigated by comparing five sequenced grass genomes. We also used the diploid Setaria genome to evaluate the ongoing genome assembly of a related polyploid, switchgrass (Panicum virgatum).
[show abstract][hide abstract] ABSTRACT: Eragrostis tef (Zucc.), a member of the Chloridoideae subfamily of grasses, is one of the most important food crops in Ethiopia. Lodging is the most important production problem in tef. The rht1 and sd1 dwarfing genes have been useful for improving lodging resistance in wheat and rice, respectively, in what has been known as the "Green Revolution." All homologs of rht1 and sd1 were cloned and sequenced from 31 tef accessions collected from across Ethiopia. The allotetraploid tef genome was found to carry two rht1 homologs. From sequence variation between these two putative homologs, an approximate ancestral divergence date of 6.4 million years ago was calculated for the two genomes within tef. Three sd1 homologs were identified in tef, with unknown orthologous/paralogous relationships. The genetic diversity in the 31 studied accessions was organized into a relatively small number of haplotypes (2-4) for four of these genes, whereas one rht1 homeologue exhibited 10 haplotypes. A low level of nucleotide diversity was observed at all loci. Linkage disequilibrium analysis demonstrated strong linkage disequilibrium, extending the length of the five genes investigated (2-4 kb), with no significant decline. There was no significant correlation between haplotypes of any of these genes and their recorded site of origin.
[show abstract][hide abstract] ABSTRACT: A salient feature of genomes of higher organisms is the birth and death of gene copies. An example is the alpha prolamin genes, which encode seed storage proteins in grasses (Poaceae) and represent a medium-size gene family. To better understand the mechanism, extent, and pace of gene amplification, we compared prolamin gene copies in the genomes of two different tribes in the Panicoideae, the Paniceae and the Andropogoneae. We identified alpha prolamin (setarin) gene copies in the diploid foxtail millet (Paniceae) genome (490 Mb) and compared them with orthologous regions in diploid sorghum (730 Mb) and ancient allotetraploid maize (2,300 Mb) (Andropogoneae). Because sequenced genomes of other subfamilies of Poaceae like rice (389 Mb) (Ehrhartoideae) and Brachypodium (272 Mb) (Pooideae) do not have alpha prolamin genes, their collinear regions can serve as "empty" reference sites. A pattern emerged, where genes were copied and inserted into other chromosomal locations followed by additional tandem duplications (clusters). We observed both recent (species-specific) insertion events and older ones that are shared by these tribes. Many older copies were deleted by unequal crossing over of flanking sequences or damaged by truncations. However, some remain intact with active and inactive alleles. These results indicate that genomes reflect only a snapshot of the gene content of a species and are far less static than conventional genetics has suggested. Nucleotide substitution rates for active alpha prolamins genes were twice as high as for low copy number beta, gamma, and delta prolamin genes, suggesting that gene amplification accelerates the pace of divergence.
Molecular Biology and Evolution 02/2012; 29(2):861-71. · 10.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (Panicum virgatum L.) ubiquitin genes (PvUbi1 and PvUbi2) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. PvUbi1 and PvUbi2 promoters) fused to the uidA coding region (GUS) and tested for transient and stable expression in a variety of plant species and tissues.
PvUbi1 consists of 607 bp containing cis-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. PvUbi2 consists of 692 bp containing cis-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. PvUbi1 and PvUbi2 were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, PvUbi1 and PvUbi2 promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV 35S, 2x35S, ZmUbi1, and OsAct1 promoters. GUS staining following stable transformation in rice demonstrated that the PvUbi1 and PvUbi2 promoters drove expression in all examined tissues. When stably transformed into tobacco (Nicotiana tabacum), the PvUbi2+3 and PvUbi2+9 promoter fusion variants showed expression in vascular and reproductive tissues.
The PvUbi1 and PvUbi2 promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.
[show abstract][hide abstract] ABSTRACT: Date palm is one of the most economically important woody crops cultivated in the Middle East and North Africa and is a good candidate for improving agricultural yields in arid environments. Nonetheless, long generation times (5-8 years) and dioecy (separate male and female trees) have complicated its cultivation and genetic analysis. To address these issues, we assembled a draft genome for a Khalas variety female date palm, the first publicly available resource of its type for a member of the order Arecales. The ∼380 Mb sequence, spanning mainly gene-rich regions, includes >25,000 gene models and is predicted to cover ∼90% of genes and ∼60% of the genome. Sequencing of eight other cultivars, including females of the Deglet Noor and Medjool varieties and their backcrossed males, identified >3.5 million polymorphic sites, including >10,000 genic copy number variations. A small subset of these polymorphisms can distinguish multiple varieties. We identified a region of the genome linked to gender and found evidence that date palm employs an XY system of gender inheritance.
[show abstract][hide abstract] ABSTRACT: Vascular plants appeared ~410 million years ago, then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes. We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first nonseed vascular plant genome reported. By comparing gene content in evolutionarily diverse taxa, we found that the transition from a gametophyte- to a sporophyte-dominated life cycle required far fewer new genes than the transition from a nonseed vascular to a flowering plant, whereas secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in posttranscriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the trans-acting small interfering RNA pathway, and extensive RNA editing of organellar genes.
[show abstract][hide abstract] ABSTRACT: Transposable elements (TEs) are massively abundant and unstable in all plant genomes, but are mostly silent because of epigenetic suppression. Because all known epigenetic pathways act on all TEs, it is likely that the specialized epigenetic regulation of regular host genes (RHGs) was co-opted from this ubiquitous need for the silencing of TEs and viruses. With their internally repetitive and rearranging structures, and the acquisition of fragments of RHGs, the expression of TEs commonly makes antisense RNAs for both TE genes and RHGs. These antisense RNAs, particularly from heterochromatic reservoirs of 'zombie' TEs that are rearranged to form variously internally repetitive structures, may be advantageous because their induction will help rapidly suppress active TEs of the same family. RHG fragments within rapidly rearranging TEs may also provide the raw material for the ongoing generation of miRNA genes. TE gene expression is regulated by both environmental and developmental signals, and insertions can place nearby RHGs under the regulation (both standard and epigenetic) of the TE. The ubiquity of TEs, their frequent preferential association with RHGs, and their ability to be programmed by epigenetic signals all indicate that RGHs have nearly unlimited access to novel regulatory cassettes to assist plant adaptation.
Current opinion in plant biology 03/2011; 14(2):156-61. · 10.33 Impact Factor