Genome organization of Magnaporthe grisea: integration of genetic maps, clustering of transposable elements and identification of genome duplications and rearrangements

 A high-density genetic map of the rice blast fungus Magnaporthe grisea (Guy11×2539) was constructed by adding 87 cosmid-derived RFLP markers to previously generated maps. The new map consists of 203 markers representing 132 independently segregating loci and spans approximately 900 cM with an average resolution of 4.5 cM. Mapping of 33 cosmid probes from the genetic map generated by Sweigard et al. has allowed the integration of two M. grisea maps. The integrated map showed that the linear order of markers along all seven chromosomes in both maps is in good agreement. Thirty of eighty seven markers were derived from cosmid clones that contained the retrotransposon MAGGY (M. grisea gypsy element). Mapping of single-copy DNA sequences associated with the MAGGY cosmids indicated that MAGGY elements are scattered throughout the fungal genome. In eight cases, the probes associated with MAGGY elements showed abnormal segregation patterns. This suggests that MAGGY may be involved in genomic rearrangements. Two RFLP probes linked to MAGGY elements, and another flanking other repetitive DNA elements, identified sequences that were duplicated in the Guy11 genome. Most of the MAGGY cosmids also contained other classes of repetitive DNA suggesting that repetitive DNA sequences tend to cluster in the M. grisea genome. Received: 17 February 1997 / Accepted: 21 February 1997     

Pot2, an inverted repeat transposon from the rice blast fungus Magnaporthe grisea

We report the cloning and characterisation of Pot2, a putative transposable element from Magnaporthe grisea. The element is 1857 by in size, has 43-bp perfect terminal inverted repeats (TIRs) and 16-bp direct repeats within the TIRs. A large open reading frame, potentially coding for a transposase-like protein, was identified. This putative protein coding region showed extensive identity to that of Fott, a transposable element from another phytopathogenic fungus, Fusarium oxysporum. Pot2, like the transposable elements Tc1 and Mariner of Caenorhabditis elegans and Drosophila, respectively, duplicates the dinucleotide TA at the target insertion site. Sequence analysis of DNA flanking 12 Pot2 elements revealed similarity to the consensus insertion sequence of Tct. Pot2 is present at a copy number of approximately 100 per haploid genome and represents one of the major repetitive DNAs shared by both rice and non-rice pathogens of M. grisea.     

Pot2, an inverted repeat transposon from the rice blast fungus Magnaporthe grisea

We report the cloning and characterisation of Pot2, a putative transposable element from Magnaporthe grisea. The element is 1857 by in size, has 43-bp perfect terminal inverted repeats (TIRs) and 16-bp direct repeats within the TIRs. A large open reading frame, potentially coding for a transposase-like protein, was identified. This putative protein coding region showed extensive identity to that of Fott, a transposable element from another phytopathogenic fungus, Fusarium oxysporum. Pot2, like the transposable elements Tc1 and Mariner of Caenorhabditis elegans and Drosophila, respectively, duplicates the dinucleotide TA at the target insertion site. Sequence analysis of DNA flanking 12 Pot2 elements revealed similarity to the consensus insertion sequence of Tct. Pot2 is present at a copy number of approximately 100 per haploid genome and represents one of the major repetitive DNAs shared by both rice and non-rice pathogens of M. grisea.     

A genetic map of potato (Solanum tuberosum) integrating molecular markers,including transposons,and classical markers

A genetic map of potato (Solanum tuberosum L.) integrating molecular markers with morphological and isozyme markers was constructed using a backcross population of 67 diploid potato plants. A general method for map construction is described that differs from previous methods employed in potato and other outbreeding plants. First, separate maps for the female and male parents were constructed. The female map contained 132 markers, whereas the male map contained 138 markers. Second, on the basis of the markers in common the two integrated parental maps were combined into one with the computer programme JoinMap. This combined map consisted of 175 molecular markers, 10 morphological markers and 8 isozyme markers. Ninety-two of the molecular markers were derived from DNA sequences flanking either T-DNA inserts in potato or reintegrated maize transposable elements originating from these T-DNA constructs. Clusters of distorted segregation were found on chromosomes 1,2,8 and 11 for the male parent and chromosome 5 for both parents. The total length of the combined map is 1120 cM.     

Genetic Structure of Pyricularia grisea (Cooke) Sacc. Isolates from Italian Paddy Fields

Rice blast disease, caused by the fungus Pyricularia grisea (Cooke) Sacc., is responsible for considerable damages in rice crops in Italy and in other parts of the world. This study was conducted in order to investigate the genetic structure of a P. grisea population in the Po area, the largest rice area in Italy. Rice leaves showing blast symptoms were collected in three successive years (1998–2000) and 43 P. grisea monoconidial culture samples were isolated from infected rice leaves. Fungal DNAs were obtained from mycelia. Moreover, six additional P. grisea DNA samples representative for the five characterized European lineages were also investigated. All 49 DNAs were fingerprinted using the Pot2‐based repetitive polymerase chain reaction specific for the blast pathogen. Unweighted pair‐group method with arithmetic averages cluster analysis shows the presence of three main Italian lineages. Within lineages, similarity was higher than 80%. Samples representative of the three of five known European lineages grouped within these three Italian lineages confirming the presence of three European lineages in Italy. Furthermore, cluster analysis shows the presence of two new haplotypes never found before in the Italian lineage.     

Molecular mapping of two cultivar-specific avirulence genes in the rice blast fungus <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

Rice blast, caused by the fungus Magnaporthe grisea, is a globally important disease of rice that causes annual yield losses. The segregation of genes controlling the virulence of M. grisea on rice was studied to establish the genetic basis of cultivar specificity in the interaction of rice and M. grisea. The segregation of avirulence and virulence was studied in 87 M. grisea F₁ progeny isolates from a cross of two isolates, Guy11 and JS153, using resistance-gene-differential rice cultivars. The segregation ratio indicated that avirulence and virulence in the rice cultivars Aichi–asahi and K59, respectively, are controlled by single major genes. Genetic analyses of backcrosses and full-sib crosses in these populations were also performed. The χ² test of goodness-of-fitness for a 1:1 ratio indicated that one dominant gene controls avirulence in Aichi-asahi and K59 in this population. Based on the resistance reactions of rice differential lines harboring known resistance genes to the parental isolates, two genetically independent avirulence genes, AVR–Pit and AVR–Pia, were identified. Genetic linkage analysis showed that the SSR marker m355–356 is closely linked to AVR–Pit, on the telomere of chromosome 1 at a distance of approximately 2.3 cM. The RAPD marker S487, which was converted to a sequence-characterized amplified region (SCAR) marker, was found to be closely linked to AVR–Pia, on the chromosome 7 telomere at a distance of 3.5 cM. These molecular markers will facilitate the positional cloning of the two AVR genes, and can be applied to molecular-marker-assisted studies of M. grisea populations.     

Electrophoretic karyotype of <Emphasis Type="Italic">Flammulina velutipes</Emphasis> and its variation among monokaryotic progenies

 The karyotype of Flammulina velutipes (Curt. : Fr.) Sing. was investigated using contour-clamped homogeneous electric fields (CHEF) gel electrophoresis. A parental dikaryotic stock, JA, was resolved into at least eight chromosomal DNA bands ranging from 1.4- to 4.9-megabase (Mb) pairs. Overall, little size variation was found among monokaryotic strains with a few major exceptions. Among 13 monokaryotic progenies examined, 11 strains were resolved into at least eight chromosomal DNA bands in a manner similar to the parent dikaryon, whereas the other 2 were resolved into at least seven chromosomes lacking the 2.1-Mb chromosome possessed in the former. A slightly larger size variation was found in a chromosome carrying ribosomal DNA. An estimated haploid genome size of this stock was 24.0 Mb or more. Received: October 11, 2001 / Accepted: November 11, 2002 Acknowledgments We thank Professor T. Morinaga, Hiroshima Prefectural University, and Dr. T. Arima for their technical advice regarding CHEF gel electrophoresis. Correspondence to:E. Tanesaka     

Inheritance of dsRNAs in the rice blast fungus, Magnaporthe grisea

The 1.6 and 1.8 kbp dsRNAs have been found in the rice blast fungus, Magnaporthe grisea strain MG01. These dsRNA molecules are located in cytoplasm of the fungal cells and maintained stably during vegetative growth. Three crosses between dsRNA free and dsRNA containing strains including a parental cross, sib-mating and back cross were made to follow the inheritance of dsRNAs during sexual reproduction. Approximately 10% of ascospore progenies (11 out of 105) contained dsRNAs from all three crosses. These data indicate that dsRNAs of M. grisea are inherited at a low frequency and not in a Mendelian fashion.     

Host Species-specific Repetitive DNA Sequence in the Genome of Magnaporthe grisea,the Rice Blast Fungus

We cloned a repetitive sequence to show RFLPs in the genome of Magnaporthe grisea, a fungal pathogen responsible for rice blast. As the sequence was 0.8 kb in length and dispersed in the genome, it was named MGSR1 (for Magnaporthe grisea short repeat 1). MGSR1 was conserved highly in the genome of rice pathogens, but poorly in the genome of pathogens of grasses other than rice. And the RFLPs, displayed with the sequence, could distinguish between clonal lineages in rice-pathogenic isolates. The nucleotide sequence showed the presence of an internal promoter of RNA polymerase III, a 3?-poly(T), and an 8-bp direct repeat in it.     

TheMagnaporthe grisea DNA fingerprinting probe MGR586 contains the 3′ end of an inverted repeat transposon

TheMagnaporthe grisea repeat (MGR) sequence MGR586 has been widely used for population studies of the rice blast fungus, and has enabled classification of the fungal population into hundreds of genetic lineages. While studying the distribution of MGR586 sequences in strains ofM. grisea, we discovered that the plasmid probe pCB586 contains a significant amount of single-copy DNA. To define precisely the boundary of the repetitive DNA in pCB586, this plasmid and four cosmid clones containing MGR586 were sequenced. Only 740 bp of one end of the 2.6-bp insert in the pCB586 plasmid was common to all clones. DNA sequence analysis of cosmid DNA revealed that all the cosmids contained common sequences beyond the cloning site in pCB586, indicating that the repetitive DNA in the fingerprinting clone is part of a larger element. The entire repetitive element was sequenced and found to resemble an inverted repeat transposon. This putative transposon is 1.86 kb in length and has perfect terminal repeats of 42 bp, which themselves contain direct repeats of 16 bp. The internal region of the transposon possesses one open reading frame which shows similarity at the peptide level to the Pot2 transposon fromM. grisea and Fot1 fromFusarium oxysporum. Hybridization studies using the entire element as a probe revealed that some strains ofM. grisea, whose DNA hybridized to the pCB586 probe, entirely lacked MGR586 transposon sequences.     

Identification and fine mapping of <Emphasis Type="Italic">Pi33</Emphasis>, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene <Emphasis Type="Italic">ACE1</Emphasis>

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) × Azucena (susceptible) and Azucena × Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F₂ populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.Communicated by D.J. Mackill     

<Emphasis Type="Italic">Pearl</Emphasis>, a Novel Family of Putative Transposable Elements in Bivalve Mollusks

Abstract While genome sequencing projects have discovered numerous types of transposable elements in diverse eukaryotes, there are many taxa of ecological and evolutionary significance that have received little attention, such as the molluscan class Bivalvia. Examination of a 0.7-MB genomic sequence database from the cupped oyster Crassostrea virginica revealed the presence of a common interspersed element, CvA. CvA possesses subterminal inverted repeats, a tandemly repeated core element, a tetranucleotide microsatellite region, and the ability to form stable secondary structures. Three other less abundant repetitive elements with a similar structure but little sequence similarity were also found in C. virginica. Ana-1, a repetitive element with similar features, was discovered in the blood ark Anadara trapezia by probing a genomic library with a dimeric repeat element contained in intron 2 of a minor globin gene in that species. All of these elements are flanked by the dinucleotide AA, a putative target-site duplication. They exhibit structural similarity to the sea urchin Tsp family and Drosophila SGM insertion sequences; in addition, they possess regions of sequence similarity to satellite DNA from several bivalve species. We suggest that the Crassostrea repetitive elements and Ana-1 are members of a new MITE-like family of nonautonomous transposable elements, named pearl. Pearl is the first putative nonautonomous DNA transposon to be identified in the phylum Mollusca.     

The 5S ribosomal RNA gene clusters in Tetrahymena thermophila: strain differences, chromosomal localization, and loss during micronuclear ageing

Summary The organization of the 5S genes in the genome of Tetrahymena thermophila was examined in various strains, with germinal ageing, and the 5S gene clusters were mapped to the MIC chromosomes. When MIC or MAC DNA is cut with the restriction enzyme EcoRI, electrophoresed, blotted, and probed with a 5S rDNA probe, the banding patterns represent the clusters of the 5S rRNA genes as well as flanking regions. The use of long gels and 60 h of electrophoresis at 10 mA permitted resolution of some 30–35 5S gene clusters on fragments ranging in size from 30-2 kb (bottom of gel). The majority of the 5S gene clusters were found in both MIC and MAC genomes, a few being MIC limited and a few MAC limited. The relative copy number of 5S genes in each cluster was determined by integrating densitometric tracings made from autoradiograms. The total number of copies in the MAC was found to be 33% greater than in the MIC. When different inbred strains were examined, the majority of the 5S gene clusters were found to be conserved, with a few strain-specific clusters observed. Nine nullisomic strains missing both copies of one or more MIC chromosomes were used to map the 5S gene clusters. The clusters were distributed non-randomly to four of the five MIC chromosomes, with 17 of them localized to chromosome 1. A deletion map of chromosome 1 was constructed using various deletion strains. Some of these deletion strains included B strain clones which had been in continuous culture for 15 years. Losses of 5S gene clusters in these ageing MIC could be attributed to deletions of particular chromosomes. The chromosomal distribution of the 5S gene clusters in Tetrahymena is unlike that found for the well-studied eukaryotes, Drosophila and Xenopus.     

Genetic mapping with dispersed repeated sequences in the rice blast fungus: mapping the SMO locus

Summary The SMO genetic locus in strains of the fungus Magnaporthe grisea that infect weeping lovegrass, directs the formation of correct cell shapes in asexual spores, infection structures, and asci. We have identified and characterized a Smo^– strain of M. grisea that infects rice. The smo mutation in this strain segregates as a single gene mutation and is allelic to previously identified smo alleles. A marked reduction in pathogenicity co-segregates with the Smo^– phenotype, suggesting that the SMO locus plays a role in rice pathogenicity. A family of dispersed repeated DNA sequences, called MGR, have been discovered in the nuclear DNA of M. grisea rice pathogens. Genetic crosses between Smo^– rice pathogens and Smo⁺ non-rice pathogens were used to follow the segregation of the SMO locus and individual MGR sequences. Using DNA blot analysis with cloned MGR hybridization probes, we mapped the SMO locus to a chromosomal region flanked by two closely linked MGR sequences. We demonstrated that the copy number of MGR sequences could be reduced in subsequent crosses to non-rice pathogens of M. grisea, and that new MGR sequences did not occur following meiosis indicating that these sequences are stable in the genome. We conclude that restriction fragment polymorphism mapping with cloned MGR sequences as hybridization probes is an effective way to map genes in the rice blast fungus.     

MAGGY, a retrotransposon in the genome of the rice blast fungusMagnaporthe grisea

Full-length copies of a previously described repetitive DNA sequence (CH2-8) were isolated from the genome of theMagnaporthe grisea strain 2539. One copy of the complete element was sequenced and found to resemble agypsy-like LTR retrotransposon. We named this element MAGGY (MAGnaporthe GYpsy-like element). MAGGY contains two internal ORFs putatively encoding Gag, Pol and Env-like proteins which are similar to peptides encoded by retroelements identified in other filamentous fungi. MAGGY was found to be widely distributed amongM. grisea isolates from geographically dispersed locations and different hosts. It was present in high copy number in the genomes of all nine rice-pathogenic isolates examined. By contrast,M. grisea strains isolated from other Gramineae were found to possess varying copy numbers of MAGGY and in some cases the element was completely absent. The wide distribution of MAGGY suggests that this element invaded the genome ofM. grisea prior to the evolution of rice-specific form(s). It may since have been horizontally transmitted to other sub-specific groups. One copy of MAGGY, corresponding to the element we sequenced, was located at identical locations in the genomes of geographically dispersed strains, suggesting that this copy of the element is a relatively ancient insertion.     

High-resolution mapping, cloning and molecular characterization of the Pi-k h gene of rice, which confers resistance to Magnaporthe grisea

In order to understand the molecular mechanisms involved in the gene-for-gene type of pathogen resistance, high-resolution genetic and physical mapping of resistance loci is required to facilitate map-based cloning of resistance genes. Here, we report the molecular mapping and cloning of a dominant gene (Pi-k ^h ) present in the rice line Tetep, which is associated with resistance to rice blast disease caused by Magnaporthe grisea. This gene is effective against M. grisea populations prevalent in the Northwestern Himalayan region of India. Using 178 sequence tagged microsatellite, sequence-tagged site, expressed sequence tag and simple sequence repeat (SSR) markers to genotype a population of 208 F₂ individuals, we mapped the Pi-k ^h gene between two SSR markers (TRS26 and TRS33) which are 0.7 and 0.5 cM away, respectively, and can be used in marker-assisted-selection for blast-resistant rice cultivars. We used the markers to identify the homologous region in the genomic sequence of Oryza sativa cv. Nipponbare, and a physical map consisting of two overlapping bacterial artificial chromosome and P1 artificial chromosome clones was assembled, spanning a region of 143,537 bp on the long arm of chromosome 11. Using bioinformatic analyses, we then identified a candidate blast-resistance gene in the region, and cloned the homologous sequence from Tetep. The putative Pi-k ^h gene cloned from Tetep is 1.5 kbp long with a single ORF, and belongs to the nucleotide binding site-leucine rich repeat class of disease resistance genes. Structural and expression analysis of the Pi-k ^h gene revealed that its expression is pathogen inducible.     

A repetitive DNA element,associated with telomeric sequences in Drosophila melanogaster,contains open reading frames

He-T sequences are a complex repetitive family of DNA sequences in Drosophila that are associated with telomeric regions, pericentromeric heterochromatin, and the Y chromosome. A component of the He-T family containing open reading frames (ORFs) is described. These ORF-containing elements within the He-T family are designated T-elements, since hybridization in situ with the polytene salivary gland chromosomes results in detectable signal exclusively at the chromosome tips. One T-element that has been sequenced includes ORFs of 1,428 and 1,614 bp. The ORFs are overlapping but one nucleotide out of frame with respect to each other. The longer ORF contains cysteine-histidine motifs strongly resembling nucleic acid binding domains of gag-like proteins, and the overall organization of the T-element ORFs is reminiscent of LINE elements. The T-elements are transcribed and appear to be conserved in Drosophila species related to D. melanogaster. The results suggest that T-elements may play a role in the structure and/or function of telomeres.by W. Hennig     

A full saturated linkage map of<Emphasis Type="Italic"> Picea abies</Emphasis> including AFLP,SSR, ESTP, 5S rDNA and morphological markers

Based on an F₁ progeny of 73 individuals, two parental maps were constructed according to the double pseudo-test cross strategy. The paternal map contained 16 linkage groups for a total genetic length of 1,792 cM. The maternal map covered 1,920 cM, and consisted of 12 linkage groups. These parental maps were then integrated using 66 intercross markers. The resulting consensus map covered 2,035 cM and included 755 markers (661 AFLPs, 74 SSRs, 18 ESTPs, the 5S rDNA and the early cone formation trait) on 12 linkage groups, reflecting the haploid number of chromosomes of Picea abies. The average spacing between two adjacent markers was 2.6 cM. The presence of 39 of the SSR and/or ESTP markers from this consensus map on other published maps of different Picea and Pinus species allowed us to establish partial linkage group homologies across three P. abies maps (up to five common markers per linkage group). This first saturated linkage map of P. abies could be therefore used as a support for developing comparative genome mapping in conifers.Communicated by O. Savolainen     

Genomic organization of repetitive DNAs in the cichlid fish <Emphasis Type="Italic">Astronotus ocellatus</Emphasis>

To contribute to the knowledge of fish genomes, we identified and characterized by means of nucleotide sequencing and physical chromosome mapping, three classes of repetitive DNAs in the genome of the South American cichlid fish Astronotus ocellatus. The first class corresponds to a satellite DNA family (AoSat) that shares similarity with a centromeric satellite DNA of the pufferfish Tetraodon nigroviridis. The second repetitive DNA class (AoRex3) is related to the retrotransposon Rex3, which is widely distributed among teleost fishes. The last repetitive element (AoLINE) shows a high similarity to the CR1-like LINE element of other teleosts. The three isolated repetitive elements are clustered in the centromeric heterochromatin of all chromosomes of the complement. The repetitive sequences are not randomly distributed in the genome, suggesting a pattern of compartmentalization on chromosomes.