Systematic overrepresentation of DNA termini and underrepresentation of subterminal regions among sequencing templates prepared from hydrodynamically sheared linear DNA molecules

<p>Abstract</p> <p>Background</p> <p>Analysis of fungal genome sequence assemblies reveals that telomeres are poorly represented even though telomeric reads tend to be superabundant. We surmised that the problem might lie in the DNA shearing conditions used to create clone libraries for genome sequencing.</p> <p>Results</p> <p>A shotgun strategy was used to sequence and assemble circular and linear cosmid DNAs sheared using conditions typical for a genome project. The DNA sheared in circular form assembled into a single sequence contig. However, the linearized cosmid produced an incomplete assembly because the two DNA termini, though greatly overrepresented in the clone library used for sequencing, were separated from neighboring sequences by gaps of ~1.4 and 1.8 kb. These gap sizes were reduced, but not eliminated, by shearing the linear cosmid into smaller fragments. Mapping of shearing breakpoints revealed a paucity of breaks in the subterminal regions of the linearized cosmid and also near chromosome ends of the fungus <it>Neurospora crassa</it>.</p> <p>Conclusion</p> <p>Together, our data indicate that the ends of linear DNA molecules are recalcitrant to hydrodynamic shearing. We propose that this causes DNA termini to be overrepresented in the resulting fragment population but ultimately prevents their incorporation into sequence assemblies.</p

Restriction maps of telomeres from Magnaporthe grisea

At least five genetic maps exist for the rice blast fungus, Magnaporthe grisea (Romao and Hamer 1991 Proc. Natl. Acad. Sci. USA 89:5316-5320; Sweigard et al. 1993 in Genetic Maps, O\u27Brien ed. Cold Spring Harbor Press pp. 3.112-3.115; Skinner et al. 1993 Theor. Appl. Genet. 87:545-557; Hayashi and Naito 1993 in Abstracts for International Symposium on Rice Blast Disease, Madison, WI; and Tharreau et al. 1994 in Abstracts of 7th meeting of International Program on Rice Biotechnology, Bali, Indonesia). We have been integrating three of these maps by placing markers used in other laboratories on the map constructed in our laboratory (Skinner et al.. 1993, ibid.). The integrated map will be a primary reference map for this fungus and the Guy11 and 2539 parents used to generate the mapping progeny will become standard strains. Guy11 is a natural isolate; therefore this map provides information on the genome organization of a wild, rice-infecting isolate

Two complete mitochondrial genomes from Praticolella mexicana Perez, 2011 (Polygyridae) and gene order evolution in Helicoidea (Mollusca, Gastropoda)

Helicoidea is a diverse group of land snails with a global distribution. While much is known regarding the relationships of helicoid taxa, comparatively little is known about the evolution of the mitochondrial genome in the superfamily. We sequenced two complete mitochondrial genomes from Praticolella mexicana Perez, 2011 representing the first such data from the helicoid family Polygyridae, and used them in an evolutionary analysis of mitogenomic gene order. We found the mitochondrial genome of P. mexicana to be 14,008 bp in size, possessing the typical 37 metazoan genes. Multiple alternate stop codons are used, as are incomplete stop codons. Mitogenome size and nucleotide content is consistent with other helicoid species. Our analysis of gene order suggested that Helicoidea has undergone four mitochondrial rearrangements in the past. Two rearrangements were limited to tRNA genes only, and two involved protein coding gene

Two Complete Mitochondrial Genomes from \u3cem\u3ePraticolella mexicana\u3c/em\u3e Perez, 2011 (Polygyridae) and Gene Order Evolution in Helicoidea (Mollusca, Gastropoda)

Helicoidea is a diverse group of land snails with a global distribution. While much is known regarding the relationships of helicoid taxa, comparatively little is known about the evolution of the mitochondrial genome in the superfamily. We sequenced two complete mitochondrial genomes from Praticolella mexicana Perez, 2011 representing the first such data from the helicoid family Polygyridae, and used them in an evolutionary analysis of mitogenomic gene order. We found the mitochondrial genome of P. mexicana to be 14,008 bp in size, possessing the typical 37 metazoan genes. Multiple alternate stop codons are used, as are incomplete stop codons. Mitogenome size and nucleotide content is consistent with other helicoid species. Our analysis of gene order suggested that Helicoidea has undergone four mitochondrial rearrangements in the past. Two rearrangements were limited to tRNA genes only, and two involved protein coding genes

Two complete mitochondrial genomes from Praticolella mexicana Perez, 2011 (Polygyridae) and gene order evolution in Helicoidea (Mollusca, Gastropoda)

Helicoidea is a diverse group of land snails with a global distribution. While much is known regarding the relationships of helicoid taxa, comparatively little is known about the evolution of the mitochondrial genome in the superfamily. We sequenced two complete mitochondrial genomes from Praticolella mexicana Perez, 2011 representing the first such data from the helicoid family Polygyridae, and used them in an evolutionary analysis of mitogenomic gene order. We found the mitochondrial genome of P. mexicana to be 14,008 bp in size, possessing the typical 37 metazoan genes. Multiple alternate stop codons are used, as are incomplete stop codons. Mitogenome size and nucleotide content is consistent with other helicoid species. Our analysis of gene order suggested that Helicoidea has undergone four mitochondrial rearrangements in the past. Two rearrangements were limited to tRNA genes only, and two involved protein coding gene

Chromosome-End Knockoff Strategy to Reshape Alkaloid Profiles of a Fungal Endophyte

Molecular genetic techniques to precisely eliminate genes in asexual filamentous fungi require the introduction of a marker gene into the target genome. We developed a novel strategy to eliminate genes or gene clusters located in subterminal regions of chromosomes, and then eliminate the marker gene and vector backbone used in the transformation procedure. Because many toxin gene clusters are subterminal, this method is particularly suited to generating nontoxic fungal strains. We tested this technique on Epichloë coenophiala, a seed-transmissible symbiotic fungus (endophyte) of the important forage grass, tall fescue (Lolium arundinaceum). The endophyte is necessary for maximal productivity and sustainability of this grass but can produce ergot alkaloids such as ergovaline, which are toxic to livestock. The genome sequence of E. coenophiala strain e19 revealed two paralogous ergot alkaloid biosynthesis gene clusters, designated EAS1 and EAS2. EAS1 was apparently subterminal, and the lpsB copy in EAS2 had a frame-shift mutation. We designed a vector with a fungal-active hygromycin phosphotransferase gene (hph), an lpsA1 gene fragment for homologous recombination at the telomere-distal end of EAS1, and a telomere repeat array positioned to drive spontaneous loss of hph and other vector sequences, and to stabilize the new chromosome end. We transformed E. coenophiala with this vector, then selected “knockoff” endophyte strains, confirmed by genome sequencing to lack 162 kb of a chromosome end including most of EAS1, and also to lack vector sequences. These ∆EAS1 knockoff strains produced no detectable ergovaline, whereas complementation with functional lpsB restored ergovaline production

Genome Sequence Variation in the Constricta Strain Dramatically Alters the Protein Interaction and Localization Map of \u3cem\u3ePotato Yellow Dwarf Virus\u3c/em\u3e

The genome sequence of the constricta strain of Potato yellow dwarf virus (CYDV) was determined to be 12 792 nt long and organized into seven ORFs with the gene order 3′-N-X-P-Y-M-G-L-5′, which encodes the nucleocapsid, phospho, movement, matrix, glyco, and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. Cloned ORFs for each gene, except L, were used to construct a protein interaction and localization map (PILM) for this virus, which shares greater than 80 % amino acid similarity in all ORFs except X and P with the sanguinolenta strain of this species (SYDV). Protein localization patterns and interactions unique to each viral strain were identified, resulting in strain-specific PILMs. Localization of CYDV and SYDV proteins in virus-infected cells mapped subcellular loci likely to be sites of replication, morphogenesis and movement

Identification of the aggregation pheromone of the invasive guam strain of coconut rhinoceros beetle, Oryctes rhinoceros, and determination of stereochemistry

The coconut rhinoceros beetle, Oryctes rhinoceros (CRB), is a major pest of coconut and oil palm that has been effectively controlled by the Oryctes rhinoceros nudivirus for over 30 years. A new haplotype, CRB-G, that is not controlled by the virus appeared in Guam in 2007 and has since spread to other countries in the Region. There has been much research on alternative methods to control CRB-G, and there have been reports that it does not respond to the well-established aggregation pheromone of CRB. We found that the male CRB-G beetles produce ethyl 4-methyloctanoate and 4-methyloctanoic acid in 4:1 ratio, essentially as reported for CRB previously by Hallett et al. (1995). The enantiomers of these compounds were synthesised by enzymatic resolution and both the male-produced compounds were shown to be (R)-enantiomers. Hallet et al. (1995) reported that CRB produced the (S)-enantiomers on the basis of field studies, but re-examination of the pheromone produced by CRB beetles confirmed that they also produced the (R)-enantiomers. Electroantennogram (EAG) responses to natural volatile collections from Oryctes and to the synthetic compounds indicated that both male and female beetles respond to the ester but not to the acid. EAG responses were recorded to both enantiomers, but responses to the ethyl (R)-4-methyloctanoate were consistently greater than those to the (S)-enantiomer. In field testing in Papua New Guinea, ethyl (R)-4-methyloctanoate was attractive to both male and female CRB-G beetles and significantly more attractive than the (S)-enantiomer. The racemic ester was as attractive as the (R)-enantiomer, and addition of (R)-4-methyloctanoic acid gave a marginal increase in attractiveness of the lure. Thus CRB-G beetles produce the same pheromone as CRB, although the enantiomeric composition of this was previously wrongly assigned. Both male and female CRB-G are attracted by racemic ethyl 4-methyloctanoate in the field, so that the same lures can be used for monitoring and control of CRB-G as for CRB

Identification of components of the aggregation pheromone of the Guam strain of coconut rhinoceros beetle, Oryctes rhinoceros, and determination of stereochemistry

The coconut rhinoceros beetle, Oryctes rhinoceros (Linnaeus 1758) (Coleoptera: Scarabaeidae: Dynastinae) (CRB), is endemic to tropical Asia where it damages both coconut and oil palm. A new invasion by CRB occurred on Guam in 2007 and eradication attempts failed using commonly applied Oryctes rhinoceros nudivirus (OrNV) isolates. This and subsequent invasive outbreaks were found to have been caused by a previously unrecognized haplotype, CRB-G, which appeared to be tolerant to OrNV. The male-produced aggregation pheromone of the endemic, susceptible strain of O. rhinoceros (CRB-S) was previously identified as ethyl 4-methyloctanoate. Following reports from growers that commercial lures containing this compound were not attractive to CRB-G, the aim of this work was to identify the pheromone of CRB-G. Initial collections of volatiles from virgin male and female CRB-G adults from the Solomon Islands failed to show any male- or female-specific compounds as candidate pheromone components. Only after five months were significant quantities of ethyl 4-methyloctanoate and 4-methyloctanoic acid produced by males but not by females. No other male-specific compounds could be detected, in particular methyl 4-methyloctanoate, 4-methyl-1-octanol, or 4-methyl-1-octyl acetate, compounds identified in volatiles from some other species of Oryctes. Ethyl 4-methyloctanoate elicited a strong electroantennogram response from both male and female CRB-G, but these other compounds, including 4-methyloctanoic acid, did not. The enantiomers of ethyl 4-methyloctanoate and 4-methyloctanoic acid were conveniently prepared by enzymatic resolution of the commercially-available acid, and the enantiomers of the acid, but not the ester, could be separated by gas chromatography on an enantioselective cyclodextrin phase. Using this approach, both ethyl 4-methyloctanoate and 4-methyloctanoic acid produced by male CRB-G were shown to be exclusively the (R)-enantiomers whereas previous reports had suggested male O. rhinoceros produced the (S)-enantiomers. However, re-examination of the ester and acid produced by male CRB-S from Papua New Guinea showed that these were also the (R)-enantiomers. In field trapping experiments carried out in the Solomon Islands, both racemic and ethyl (R)-4-methyloctanoate were highly attractive to both male and female CRB-G beetles. The (S)-enantiomer and the corresponding acids were only weakly attractive. The addition of racemic 4-methyloctanoic acid to ethyl 4-methyloctanoate did significantly increase attractiveness, but the addition of (R)- or (S)-4-methyloctanoic acid to the corresponding ethyl esters did not. Possible reasons for the difference in assignment of configuration of the components of the CRB pheromone are discussed along with the practical implications of these results

A Comparative Genomic Analysis of Putative Pathogenicity Genes in the Host-Specific Sibling Species \u3cem\u3eColletotrichum graminicola\u3c/em\u3e and \u3cem\u3eColletotrichum sublineola\u3c/em\u3e

Background: Colletotrichum graminicola and C. sublineola cause anthracnose leaf and stalk diseases of maize and sorghum, respectively. In spite of their close evolutionary relationship, the two species are completely host-specific. Host specificity is often attributed to pathogen virulence factors, including specialized secondary metabolites (SSM), and small-secreted protein (SSP) effectors. Genes relevant to these categories were manually annotated in two co-occurring, contemporaneous strains of C. graminicola and C. sublineola. A comparative genomic and phylogenetic analysis was performed to address the evolutionary relationships among these and other divergent gene families in the two strains. Results: Inoculation of maize with C. sublineola, or of sorghum with C. graminicola, resulted in rapid plant cell death at, or just after, the point of penetration. The two fungal genomes were very similar. More than 50% of the assemblies could be directly aligned, and more than 80% of the gene models were syntenous. More than 90% of the predicted proteins had orthologs in both species. Genes lacking orthologs in the other species (non-conserved genes) included many predicted to encode SSM-associated proteins and SSPs. Other common groups of non-conserved proteins included transporters, transcription factors, and CAZymes. Only 32 SSP genes appeared to be specific to C. graminicola, and 21 to C. sublineola. None of the SSM-associated genes were lineage-specific. Two different strains of C. graminicola, and three strains of C. sublineola, differed in no more than 1% percent of gene sequences from one another. Conclusions: Efficient non-host recognition of C. sublineola by maize, and of C. graminicola by sorghum, was observed in epidermal cells as a rapid deployment of visible resistance responses and plant cell death. Numerous non-conserved SSP and SSM-associated predicted proteins that could play a role in this non-host recognition were identified. Additional categories of genes that were also highly divergent suggested an important role for co-evolutionary adaptation to specific host environmental factors, in addition to aspects of initial recognition, in host specificity. This work provides a foundation for future functional studies aimed at clarifying the roles of these proteins, and the possibility of manipulating them to improve management of these two economically important diseases