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
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
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.
Telomeres and subtelomere regions have vital roles in cellular homeostasis and can facilitate niche adaptation. However, information on telomere/subtelomere structure is still limited to a small ...number of organisms. Prior to initiation of this project, the Neurospora crassa genome assembly contained only 3 of the 14 telomeres. The missing telomeres were identified through bioinformatic mining of raw sequence data from the genome project and from clones in new cosmid and plasmid libraries. Their chromosomal locations were assigned on the basis of paired-end read information and/or by RFLP mapping. One telomere is attached to the ribosomal repeat array. The remaining chromosome ends have atypical structures in that they lack distinct subtelomere domains or other sequence features that are associated with telomeres in other organisms. Many of the chromosome ends terminate in highly AT-rich sequences that appear to be products of repeat-induced point mutation, although most are not currently repeated sequences. Several chromosome termini in the standard Oak Ridge wild-type strain were compared to their counterparts in an exotic wild type, Mauriceville. This revealed that the sequences immediately adjacent to the telomeres are usually genome specific. Finally, despite the absence of many features typically found in the telomere regions of other organisms, the Neurospora chromosome termini still retain the dynamic nature that is characteristic of chromosome ends.
The avrCO39 gene conferring avirulence toward rice cultivar CO39 was previously mapped to chromosome 1 of Magnaporthe grisea between cosegregating markers CH5-120H and 1.2H and marker 5-10-F. In the ...present study, this region of the chromosome was physically mapped using RecA-mediated Achilles' cleavage. Cleavage of genomic DNA sequences within CH5-120H and 5-10-F liberated a 610-kb restriction fragment, representing the physical distance between these markers. Chromosome walking was initiated from both markers but was curtailed due to the presence of repetitive DNA sequences and the absence of overlapping clones in cosmid libraries representing several genome equivalents. These obstacles were overcome by directly subcloning the target region after release by Achilles' cleavage and a contig spanning avrCO39 was thus assembled. Transformation of two cosmids into a virulent recipient strain conferred a cultivar-specific avirulence phenotype thus confirming the cloning of avrCO39. Meiotic crossover points were unevenly distributed across this chromosomal region and were clustered around the avrCO39 locus. A 14-fold variation in the relationship between genetic and physical distance was measured over the avrCO39 chromosomal region. Thus the poor correlation of physical to genetic distance previously observed in M. grisea appears to be manifested over relatively short distances.
Magnaporthaceae is a family of ascomycetes that includes three fungi of great economic importance: Magnaporthe oryzae, Gaeumannomyces graminis var. tritici, and Magnaporthe poae. These three fungi ...cause widespread disease and loss in cereal and grass crops, including rice blast disease (M. oryzae), take-all disease in wheat and other grasses (G. graminis), and summer patch disease in turf grasses (M. poae). Here, we present the finished genome sequence for M. oryzae and draft sequences for M. poae and G. graminis var. tritici. We used multiple technologies to sequence and annotate the genomes of M. oryzae, M. poae, and G. graminis var. tritici. The M. oryzae genome is now finished to seven chromosomes whereas M. poae and G. graminis var. tritici are sequenced to 40.0× and 25.0× coverage respectively. Gene models were developed by the use of multiple computational techniques and further supported by RNAseq data. In addition, we performed preliminary analysis of genome architecture and repetitive element DNA.
Telomere-linked RecQ helicase (TLH) genes have been identified in several fungi, where they occur as small gene families with each member copy residing within ~10 kb of a telomere. Here we describe ...the characterization of all 11 TLH gene copies in the reference strain of the fungus Magnaporthe oryzae. A consensus gene prediction revealed that the previously reported TLH1 gene is actually a mutated copy, and the full-length gene is almost two times longer. Only four full-length TLH genes were present in the strain that was analyzed, with the remaining copies containing premature stops caused by point mutations, indels, transposon insertions, and a telomere truncation. Interestingly, all of the TLH gene copies possessed numerous mutations indicative of the action of the repeat-induced point mutation process. However, there was evidence of purifying selection indicating maintenance of gene function. Alignment of full-length proteins from M. oryzae, Schizosaccharomyces pombe and M. anisopliae revealed the presence of a novel, highly conserved protein motif which suggests that the telomere-linked helicases have different functions and/or substrates to the RecQ helicases encoded by “internal” genes.
Telomeres are the sequences that form the ends of eukaryotic chromosomes and are essential structures that confer genome stability and guide chromosome behavior. In addition, the terminal regions of ...the chromosomes tend to house genes with predicted roles in ecological adaptation. Unfortunately, however, most fungal genome assemblies contain very few telomeres and, therefore, the identities of genes residing near the chromosome ends are often unknown. In an effort to develop a complete understanding of the organization and gene content of chromosome ends in a number of fungi, we developed efficient methods for the identification and targeted cloning of telomeres. This chapter describes the basic steps and shows exemplary results from the targeted cloning of Epichloë festucae telomeres.
We describe here the analysis of random T-DNA insertions that were generated as part of a large-scale insertional mutagenesis project for
Magnaporthe oryzae. Chromosomal regions flanking T-DNA ...insertions were rescued by inverse PCR, sequenced and used to search the
M. oryzae genome assembly. Among the 175 insertions for which at least one flank was rescued, 137 had integrated in single-copy regions of the genome, 17 were in repeated sequences, one had no match to the genome, and the remainder were unassigned due to illegitimate T-DNA integration events. These included in order of abundance: head-to-tail tandem insertions, right border excision failures, left border excision failures and insertion of one T-DNA into another. The left borders of the T-DNA were frequently truncated and inserted in sequences with micro-homology to the left terminus. By contrast the right borders were less prone to degradation and appeared to have been integrated in a homology-independent manner. Gross genome rearrangements rarely occurred when the T-DNAs integrated in single-copy regions, although most insertions did cause small deletions at the target site. Significant insertion bias was detected, with promoters receiving two times more T-DNA hits than expected, and open reading frames receiving three times fewer. In addition, we found that the distribution of T-DNA inserts among the
M. oryzae chromosomes was not random. The implications of these findings with regard to saturation mutagenesis of the
M. oryzae genome are discussed.
Gray leaf spot of perennial ryegrass (Lolium perenne) is a severe foliar disease caused by the ascomycete fungus Magnaporthe oryzae (formerly known as Magnaporthe grisea). Control of gray leaf spot ...is completely dependent on the use of fungicides because currently available perennial ryegrass cultivars lack genetic resistance to this disease. M. oryzae isolates from perennial ryegrass (prg) were unable to cause disease on rice cultivars CO39 and 51583, and instead triggered a hypersensitive response. Southern hybridization analysis of DNA from over 50 gray leaf spot isolates revealed that all of them contain sequences corresponding to AVR1-CO39, a host specificity gene that confers avirulence to rice cultivar CO39, which carries the corresponding resistance gene Pi-CO39(t). There was also an almost complete lack of restriction site polymorphism at the avirulence locus. Cloning and sequencing of the AVR1-CO39 gene (AVR1-CO39superscript Lp) from 16 different gray leaf spot isolates revealed just two point mutations, both of which were located upstream of the predicted open reading frame. When an AVR1-CO39superscript Lp gene copy was transferred into ML33, a rice pathogenic isolate that is highly virulent to rice cultivar CO39, the transformants were unable to cause disease on CO39 but retained their virulence to 51583, a rice cultivar that lacks Pi-CO39(t). These data demonstrate that the AVR1-CO39 gene in the gray leaf spot pathogens is functional, and suggest that interaction of AVR1-CO39superscript Lp and Pi-CO39(t) is responsible, at least in part, for the host specificity expressed on CO39. This indicates that it may be possible to use the Pi-CO39(t) resistance gene as part of a transgenic strategy to complement the current deficiency of gray leaf spot resistance in prg. Furthermore, our data indicate that, if Pi-CO39(t) can function in prg, the resistance provided should be broadly effective against a large proportion of the gray leaf spot pathogen population.
A genetic map including three avirulence (Avr) genes, AvrPik, AvrPiz, and AvrPiz-t, was constructed in a genetic cross of two rice field isolates, 84R-62B and Y93-245c-2. The chromosomal locations of ...the Avr genes were determined by using selected markers to probe Southern blots of the parental chromosomes that had been separated by contour-clamped homogenous electric fields electrophoresis. Electrophoretic karyotyping showed that both parental isolates 84R-62B and Y93-245c-2 contained seven chromosomes greater than 3.5 megabases (Mb) in size and 84R-62B possessed a small chromosome of approximately equal to 1.6 Mb. The linkage groups containing AvrPiz and AvrPiz-t were assigned to chromosomes 3 and 7, respectively. Some markers from the linkage group that contained AvrPik hybridized with chromosome 1 and the 1.6-Mb chromosome, yet all of the cloned RAPD markers that were closely linked to AvrPik hybridized exclusively to the 1.6-Mb chromosome in 84R-62B, the parent that possesses AvrPik. Thus, we conclude that AvrPik is located on the 1.6-Mb chromosome in 84R-62B.
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