Unknown Genome Research Articles

FluoMEP: A new genotyping method combining the advantages of randomly amplified polymorphic DNA and amplified fragment length polymorphism

PCR-based identification of differences between two unknown genomes often requires complex manipulation of the templates prior to amplification and/or gel electrophoretic separation of a large number of samples with manual methods. Here, we describe a new genotyping method, called fluorescent motif enhanced polymorphism (fluoMEP). The fluoMEP method is based on random amplified polymorphic DNA (RAPD) assay, but combines the advantages of the large collection of unlabelled 10mer primers (ca. 5000) from commercial sources and the power of the automated CE devices used for the detection of amplified fragment length polymorphism (AFLP) patterns. The link between these two components is provided by a fluorescently labeled "common primer" that is used in a two-primer PCR together with an unlabeled RAPD primer. By using the same "common primer" and a series of RAPD primers, DNA templates can be screened quickly and effectively for polymorphisms. Our manuscript describes the optimization of the method and its characterization on different templates. We demonstrate by using several different approaches that the addition of the "common primer" to the PCR changes the profile of amplified fragments, allowing for screening various parts of the genome with the same set of unlabeled primers. We also present an in silico analysis of the genomic localization of fragments amplified by a RAPD primer with two different "common primers" and alone.

Phi29-based amplification of small genomes

based on theisothermal reaction of the enzyme Phi29 and randomprimers, is one of the most popular methods currentlyused in genomic research [1–3]. However, there is onemajor drawback limiting applicability of this technique incertain Welds such as forensics, microbiology, and envi-ronmental monitoring. The WGA method is hardly appli-cable to small genomes and/or degraded DNA, especiallyif the number of genome copies is approaching 100–1000molecules. Here, we describe a simple procedure in whichWGA ampliWes 1fg of the linear pUC19 DNA su Ycientlyfor hundreds of downstream (i.e., post-WGA) PCR reac-tions. Thus, the ampliWcation of limited quantities ofother unknown small genomes or genomic fragmentsshould be possible as well.It was observed previously that self-priming of degener-ate 6-mers might compete with the priming of the templateDNA, especially if the quantity and/or length of the DNAtemplate are small [2–5]. However, resolving self-primingproblem did not remove performance limitations of thePhi29-based WGA procedure [5]. For example, in our labo-ratory, 300 copies of a single copy gene could be WGAampliWed in the context of human DNA but not as an iso-lated plasmid DNA. It seemed that the technical bottleneckwas due to a low hybridization of the degenerate 6-mers tothe template, suggesting a better WGA performance withan equivalent number of copies of longer templates orembedded within longer templates, thereby favoring ahigher number of priming events to occur. Therefore, wedecided to ligate a small template, such as pUC19 of2686bp, with a long carrier DNA, such as poly(dA–dT) orhuman DNA, before performing Phi29-based WGA.We compared WGA performance of serial dilutions of(i)

Gene expression analysis using a modified HiCEP method applicable to prokaryotes: A study of the response of Rhodococcus to isoniazid and ethambutol

We established a novel method to analyze the gene expression of prokaryotes by modifying and optimizing the HiCEP (high coverage gene expression analysis) method, which was originally developed for eukaryotic gene expression profiling. Following the removal of abundant rRNA, the mRNA of prokaryotic cells was enzymatically polyadenylated and subjected to HiCEP analysis. This method was highly reproducible due to selective PCR that was performed by using adaptor specific primers. We confirmed induction of tipA and induction or suppression of cspA, which are genes that are obtained from distinctive actinomycetes and responded to thiostrepton and temperature stress, respectively. Further, we applied this method to explore the gene expression profile of Rhodococcus erythropolis in response to drugs that inhibit cell wall synthetic pathways, and we were able to identify 35 upregulated genes. Among these genes, we confirmed the upregulation of 22 genes by using RT-PCR (reverse transcriptase-polymerase chain reaction). Some of these genes are involved in the synthesis of mycolic acid and arabinogalactan, suggesting a cell response to drug treatment by regulation of the genes involved in cell wall synthesis. This method could prove to be a useful technique for gene expression analysis of prokaryotes, particularly nonmodel strains with unknown genome sequences.

Molecular characterisation of patients with subtelomeric 22q abnormalities using chromosome specific array-based comparative genomic hybridisation

The 22q13 deletion syndrome is associated with global developmental delay, absent or delayed speech, and generalised hypotonia. In this study, the size and nature of 22q13 deletions (n=9) were studied in detail by high-resolution chromosome specific array-based comparative genomic hybridisation (array CGH). The deletion sizes varied considerably between the different patients, that is, the largest deletion spanning 8.4 Mb with the breakpoint mapping to 22q13.2 and the smallest deletion spanning 3.3 Mb with the breakpoint mapping to 22q13.31. In one case, a unique subtelomeric 3.9 Mb deletion associated with a 2.0 Mb duplication of 22q13 was observed, adding to a growing number of similar cases identified for other chromosome ends. Remarkably, this patient had signs suggestive of retinitis pigmentosa, which has never been reported before in the 22q13 deletion syndrome. The identification of two pairs of recurrent proximal breakpoints on 22q13 suggests that these specific regions may be prone to recombination, due to yet unknown genome architectural features. In addition to the copy number changes on 22q13, a duplication of approximately 330 kb on 22q11.1 was observed and shown to be a genetic large-scale copy number variation without clinical consequences. The current study failed to reveal relationships between the clinical features and the deletion sizes. Global developmental delay and absent or severely delayed speech were observed in all patients, whereas hypotonia was present in 89% of the cases (8/9). This study underscores the utility of array CGH for characterising the size and nature of subtelomeric deletions, such as monosomy 22q13, and underlines the considerable variability in deletion size in the 22q13 deletion syndrome regardless of the clinical phenotype.

Orphan transcripts in Arabidopsis thaliana: identification of several hundred previously unrecognized genes

Expressed sequence tags (ESTs) represent a huge resource for the discovery of previously unknown genetic information and functional genome assignment. In this study we screened a collection of 178 292 ESTs from Arabidopsis thaliana by testing them against previously annotated genes of the Arabidopsis genome. We identified several hundreds of new transcripts that match the Arabidopsis genome at so far unassigned loci. The transcriptional activity of these loci was independently confirmed by comparison with the Salk Whole Genome Array Data. To a large extent, the newly identified transcriptionally active genomic regions do not encode 'classic' proteins, but instead generate non-coding RNAs and/or small peptide-coding RNAs of presently unknown biological function. More than 560 transcripts identified in this study are not represented by the Affymetrix GeneChip arrays currently widely used for expression profiling in A. thaliana. Our data strongly support the hypothesis that numerous previously unknown genes exist in the Arabidopsis genome.

Viral discovery and sequence recovery using DNA microarrays.

Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.

PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry.

A number of different approaches have been described to identify proteins from tandem mass spectrometry (MS/MS) data. The most common approaches rely on the available databases to match experimental MS/MS data. These methods suffer from several drawbacks and cannot be used for the identification of proteins from unknown genomes. In this communication, we describe a new de novo sequencing software package, PEAKS, to extract amino acid sequence information without the use of databases. PEAKS uses a new model and a new algorithm to efficiently compute the best peptide sequences whose fragment ions can best interpret the peaks in the MS/MS spectrum. The output of the software gives amino acid sequences with confidence scores for the entire sequences, as well as an additional novel positional scoring scheme for portions of the sequences. The performance of PEAKS is compared with Lutefisk, a well-known de novo sequencing software, using quadrupole-time-of-flight (Q-TOF) data obtained for several tryptic peptides from standard proteins.

Nanoelectrospray tandem mass spectrometry and sequence similarity searching for identification of proteins from organisms with unknown genomes.

AbstractRecent developments in technology and instrumentation have made mass spectrometry the method of choice for identification of proteins in a sequence database [reviewed in refs. (1–4)].Regardless of what mass spectrometric technology is applied, it is ultimately required that acquired mass spectra be accurately matched to a protein sequence from the corresponding database entry. KeywordsTryptic PeptideTandem Mass SpectrumCandidate SequenceAutomate InterpretationTrypsin Cleavage SiteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Adaptation of Perennial Triticeae to the Eastern Central Great Plains

The tribe Triticeae contains over 250 perennial species that are components of grasslands in the temperate and sub-arctic regions of the world and includes some of the world's most valuable forage and rangeland species. Many of these species had not been evaluated previously in the Central Great Plains, USA. A subset of the germplasm of the tribe Triticeae which included over 100 accessions of 55 different species was evaluated in a replicated, space-planted trial in eastern Nebraska during 1994-1996 to determine the survival and forage productivity of the accessions. The evaluated accessions were representative of perennial Triticeae genera and genomes. Perennial grasses of the Triticeae are based on the P, St, H, Ns, E, W, Y genomes and an unknown Xm genome(s). Triticeae that survived and had acceptable forage yields during the period of the trial were the Agropyron's-crested wheatgrasses (PP and PPPP genomes), Psathyrostachys-Russian wildryes (NsNs genomes), Thinopyron's-intermediate and tall wheatgrasses (EEEEStSt and EEEEEEStSt genomes), some Elymus (StStHH genomes), several Leymus (NsNsXmXm genomes), and Pascopyrum-western wheatgrass (StStHHNsNsXmXm genomes). Several Leymus species had not been evaluated previously in this region but showed considerable potential and merit additional evaluation, including L. chinensis, L. akmolinensi, L. racemosus, L. sabulosus, and L. secalinus. Species with only the H genome (Hordeum) and St genome (Pseudoroegneria) were not adapted to the region because of poor survival or low productivity. The study provides an example of how the rapidly emerging field of genomics can have practical applications to grasslands and rangelands. DOI:10.2458/azu_jrm_v54i6_vogel2

Adaptation of perennial triticeae to the eastern Central Great Plains

The tribe Triticeae contains over 250 perennial species that are components of grasslands in the temperate and sub-arctic regions of the world and includes some of the world's most valuable forage and rangeland species. Many of these species had not been evaluated previously in the Central Great Plains, USA. A subset of the germplasm of the tribe Triticeae which included over 100 accessions of 55 different species was evaluated in a replicated, space-planted trial in eastern Nebraska during 1994-1996 to determine the survival and forage productivity of the accessions. The evaluated accessions were representative of perennial Triticeae genera and genomes. Perennial grasses of the Triticeae are based on the P, St, H, Ns, E, W, Y genomes and an unknown Xm genome(s). Triticeae that survived and had acceptable forage yields during the period of the trial were the Agropyron's-crested wheatgrasses (PP and PPPP genomes), Psathyrostachys-Russian wildryes (NsNs genomes), Thinopyron's-intermediate and tall wheatgrasses (EEEEStSt and EEEEEEStSt genomes), some Elymus (StStHH genomes), several Leymus (NsNsXmXm genomes), and Pascopyrum-western wheatgrass (StStHHNsNsXmXm genomes). Several Leymus species had not been evaluated previously in this region but showed considerable potential and merit additional evaluation, including L. chinensis, L. akmolinensi, L. racemosus, L. sabulosus, and L. secalinus. Species with only the H genome (Hordeum) and St genome (Pseudoroegneria) were not adapted to the region because of poor survival or low productivity. The study provides an example of how the rapidly emerging field of genomics can have practical applications to grasslands and rangelands.

Trigenomic origin of the hexaploid Psammopyrum athericum (Triticeae: Poaceae) revealed by in-situ hybridization.

The genomic constitution of the hexaploid Psammopyrum athericum was studied with in-situ DNA hybridization using both genomic DNA and isolated cloned sequences as probes. A genomic probe from Thinopyrum bessarabicum (E genome) hybridized successfully to 14 chromosomes of Ps. athericum and a probe from Festucopsis serpentinii (L genome) hybridized to another 14 chromosomes. The remaining chromosomes did not hybridize, apart from in the centromeric region, to any of the genomic probes used. It is thus proposed that Ps. athericum contains the genomes E, L and X where X stands for a so-far unknown genome. Psammopyrum athericum has three pairs of pTa71 sites and approximately 30 pSc119:2 sites. The origin of the third genome will be a matter for further research using genomic and genome-specific probes.

Empty chateaux

Empty chateaux

Genetic relationships among Stylosanthes species revealed by RFLP and STS analyses

Genetic relationships between 6 unclassified taxa and 24 known species of the genus Stylosanthes were investigated by RFLP and STS analyses. This allowed the diploid taxa used in this study to be classified into nine basal (genome) groups. Representative species in these groups included ’S. seabrana’/S. hamata (Group A), S. viscosa (Group B), S. humilis (Group C), S. macrocephala/S. bracteata (Group D), S. pilosa (Group E), S. leiocarpa (Group F), S. guianensis (Group G), S. tomentosa (Group H) and S. calcicola (Group I). Polyploid taxa used were grouped into five classes based on their putative genomic structures. These are AABB for S. scabra, S. aff. scabra, S. sericeiceps, S. aff. hamata and S.tuberculata; AACC for S. mexicana, S. subsericea, S. sundaica and S. sp.A; DDEE for S. capitata; AAFF for S. sympodialis; and AABBXX for S. erecta, with XX representing an unknown genome. Of the 6 unclassified taxa, three were diploids and 3 tetraploids. Of the 3 diploids, the genome of S. sp. was markedly distinct from those of all other diploids analysed in this study, with that of S. leiocarpa being the closest. The genome of S. sp.B was similar to that of S. humilis, with an average dissimilarity value of 15% between them. The genome of S. aff. viscosa was very similar to that of S. viscosa. Genetic variation between these 2 taxa was not larger than that within each of the 2 taxa. Of the 3 tetraploids, the genomic structure of S. sp.A was similar to those of S. mexicana, S. sundaica and S. subsericea, and the genomic structures of S. aff. scabra and S. aff. hamata were similar to those of S. scabra and S. sericeiceps.

Presence of papillomavirus-like DNA sequences in cutaneous fibropapillomas of the goat udder

Papillomatous lesions were isolated from the mammary skin of goats and examined for evidence of papillomavirus (PV) infection by various criteria, including gross morphology, histology and DNA hybridization. Although some lesions showed gross papillomatous morphological and histological features similar to those caused by papillomavirus in other species, no viral particles were detected. Reverse slot hybridization revealed cross-hybridization between DNA extracted from goat mammary papillomas and human papillomaviruses (HPV). Southern blot, using ovine papillomavirus (OPV) and bovine papillomavirus type 5 (BPV 5) DNA probes under conditions of reduced stringency (Tm −40°C), detected homologous sequences in 40% of the biopsies. DNA fragments corresponding probably to a monomeric form (7000–8000 bp) of an unknown papillomavirus genome were detected. This study provides evidence for the existence of papillomavirus-like sequences in caprine mammary papillomas and suggests that a papillomavirus is likely to be involved in the development of precancerous lesions of goat mammary skin.

The origins of the genomes of Triticum biunciale, t. ovatum, t. neglectum, t. columnare, and t. rectum (poaceae) based on variation in repeated nucleotide sequences

The origins of the genomes of allotetraploid species Triticum biunciale, T. ovatum, T. neglectum, and T. columnare, and allohexaploid T. rectum were investigated by examining the presence of specific restriction fragments of repeated nucleotide sequences in DNAs of the polyploid species. The restriction fragments were detectable either in a single diploid Triticum species (unique characters) or a group of diploid species (unique shared characters). The analysis showed that Triticum biunciale and T. ovatum are closely related. In both species, one pair of genomes is closely related to the genome of T. umbellulatum and the other is a modified genome of T. comosum. The same genome formula, UUM°M°, is proposed for T. biunciale and T. ovatum. Potential reasons for the modification of the M° genome are discussed. Triticum neglectum and T. columnare are also closely related to each other and have the same genomes. They share the U genome with T. biunciale and T. ovatum, but their second pair of genomes is unrelated to the M° genome. No relationship was found of this genome to a genome of any extant diploid species of Triticum or any phylogenetic lineage leading to the extant diploid species. This unknown genome is designated X'.∗∗∗ The proposed genome formula for T. neglectum and T. columnare is UUX'X'∗∗∗. Hexaploid T. rectum originated from hybridization of one of the tetraploid species with the formula UUX'X', likely T. neglectum, with T. uniaristatum (genome N), and its genome formula is UUX'X'NN.

The Origins of the Genomes of Triticum biunciale, T. ovatum, T. neglectum, T. columnare, and T. rectum (Poaceae) Based on Variation in Repeated Nucleotide Sequences

The origins of the genomes of allotetraploid species Triticum biunciale, T. ovatum, T. neglectum, and T. columnare, and allohexaploid T. rectum were investigated by examining the presence of specific restriction fragments of repeated nucleotide sequences in DNAs of the polyploid species. The restriction fragments were detectable either in a single diploid Triticum species (unique characters) or a group of diploid species (unique shared characters). The analysis showed that Triticum biunciale and T. ovatum are closely related. In both species, one pair of genomes is closely related to the genome of T. umbellulatum and the other is a modified genome of T. comosum. The same genome formula, UUM°M°, is proposed for T. biunciale and T. ovatum. Potential reasons for the modification of the M° genome are discussed. Triticum neglectum and T. columnare are also closely related to each other and have the same genomes. They share the U genome with T. biunciale and T. ovatum, but their second pair of genomes is unrelated to the M° genome. No relationship was found of this genome to a genome of any extant diploid species of Triticum or any phylogenetic lineage leading to the extant diploid species. This unknown genome is designated X'.∗∗∗ The proposed genome formula for T. neglectum and T. columnare is UUX'X'∗∗∗. Hexaploid T. rectum originated from hybridization of one of the tetraploid species with the formula UUX'X', likely T. neglectum, with T. uniaristatum (genome N), and its genome formula is UUX'X'NN.

Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.

From mutation mapping to phenotype cloning.

From mutation mapping to phenotype cloning.

The detection and estimation of linkage in polyploids using single-dose restriction fragments

Restriction fragment length polymorphism (RFLP) linkage maps have been constructed in several major diploid crops. However, construction of RFLP maps directly in polyploids has lagged behind for several reasons: (1) there are a large number of possible genotypes for each DNA probe expected in a segregating population, and these genotypes cannot always be identified readily by their banding phenotypes; and (2) the genome constitutions (allopolyploidy versus autopolyploidy) in many high polyploids are not clearly understood. We present here an analysis of these problems and propose a general method for mapping polyploids based on segregation of single-dose restriction fragments (SDRFS). SDRFs segregate 1:1 (presence: absence) in gametes of heterozygous plants. Hypothetical allopolyploid and autopolyploid species with four ploidy levels of 2n = 4x, 6x, 8x, and 10x, are used to illustrate the procedures for identifying SDRFs, detecting linkages among SDRFs, and distinguishing allopolyploid versus autopolyploids from polyploids of unknown genome constitution. Family size required, probability of linkage, and attributes of different mapping populations are discussed. We estimate that a population size of 75 is required to identify SDRFs with 98% level of confidence for the four ploidy levels. This population size is also adequate for detecting and estimating linkages in the coupling phase for both allopolyploids and autopolyploids, but linkages in the repulsion phase can be estimated only in allopolyploids. For autopolyploids, it is impractical to estimate meaningful linkages in repulsion because very large family sizes (>750) are required. For high-level polyploids of unknown genome constitution, the ratio between the number of detected repulsion versus coupling linkages may provide a crude measurement of preferential chromosome pairing, which can be used to distinguish allopolyploidy from autopolyploidy. To create a mapping population, one parent (P1) should have high heterozygosity to ensure a high frequency of SDRFs, and the second parent (P2) should have a low level of heterozygosity to increase the probability of detecting polymorphic fragments. This condition could be satisfied by choosing outcrossed hybrids as one parental type and inbreds, haploids, or doubled haploids as the other parental type.

Homology of chromosomes of the X genomes in common and Uruguayan dallisgrass, Paspalum dilatatum

Two biotypes of dallisgrass, Paspalum dilatatum Poir., designated common and Uruguayan, have chromosome numbers and genome formulas of 2n = 5x = 50 (IIJJX) and 2n = 6x = 60 (IIJJXX), respectively. The relationship between the X genomes in these two biotypes is unknown, and each was arbitrarily assigned the letter X to designate an unknown genome. This study was undertaken to determine the relationship between the X genomes in these two biotypes. Because both biotypes are apomicts and cannot be crossed, a sexual intraspecific F1 hybrid (2n = 45) between sexual yellow-anthered (2n = 4x = 40; IIJJ) and common dallisgrass biotypes was crossed with Uruguayan dallisgrass. This F1 hybrid has complete sets of the I and J genomes but only 5 of the 10 chromosomes of the X genome from common dallisgrass. Two hybrids were recovered. One had 52 and the other had 53 chromosomes, which associated at metaphase I as 22 bivalents + 8 univalents and 23 bivalents + 7 univalents, respectively. Twenty bivalents represent pairing of members of the I and J genomes, and those in excess of 20 represent pairing between members of the two X genomes. The remaining members of the X genome from the Uruguayan biotype were present as univalents at metaphase I. This demonstrates that those chromosomes of the X genome from the common biotype that were present are homologous to members of the X genome of the Uruguayan biotype. Both hybrids are aposporous facultative apomicts with some sterility.Key words: meiosis, intraspecific hybridization, chromosome pairing, genome relations, apomixis.