The impact of genome defense on mobile elements in <Emphasis Type="Italic">Microbotryum</Emphasis>

Repeat induced point mutation (RIP), a mechanism causing hypermutation of repetitive DNA sequences in fungi, has been described as a ‘genome defense’ which functions to inactivate mobile elements and inhibit their deleterious effects on genome stability. Here we address the interactions between RIP and transposable elements in the Microbotryum violaceum species complex. Ten strains of M. violaceum, most of which belong to different species of the fungus, were all found to contain intragenomic populations of copia-like retrotransposons. Intragenomic DNA sequence variation among the copia-like elements was analyzed for evidence of RIP. Among species with RIP, there was no significant correlation between the frequency of RIP-induced mutations and inferred transposition rate based on diversity. Two strains of M. violaceum, from two different plant species but belonging to the same fungal lineage, contained copia-like elements with very low diversity, as would result from a high transposition rate, and these were also unique in showing no evidence of the hypermutation patterns indicative of the RIP genome defense. In this species, evidence of RIP was also absent from a Class II helitron-like transposable element. However, unexpectedly the absolute repetitive element load was lower than in other strains.     

Analysis of the chromocenter DNA composition in polytene chromosomes of <Emphasis Type="Italic">Drosophila orena</Emphasis> ovarian nurse cells

Chromocenter DNA fragments of polytene chromosomes of Drosophila orena ovarian nurse cells were cloned from a region-specific library (Dore1) in a plasmid vector to yield 133 clones. A total of 76 clones were selected and sequenced. The total length of the sequenced fragments was 23940 bp. Analysis with several software packages revealed various repetitive sequences among the fragments of the Dore1 library, including mobile genetic elements (25 fragments homologous to various LTR retrotransposons, five fragments homologous to LINEs, three fragments homologous to Helitrons, one fragment homologous to Polinton, and one fragment homologous to the mini-me non-LTR retrotransposon), four minisatellites, a satellite (SAR_DM), the (TATATG)n simple sequence repeat, and a low-complexity T-rich repeat. Sequences homologous to protein-coding genes were also found in the Dore1 library. Various repetitive DNA sequences and gene homologs were identified as conserved sequences of pericentric heterochromatin of polytene chromosomes of ovarian nurse cells in nine species of the melanogaster species subgroup.     

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     

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.     

Dynamic linkage relationships to the mating‐type locus in automictic fungi of the genus Microbotryum

Regions of the chromosomes determining mating compatibility in some fungi, including Microbotryum lychnidis‐dioicae and Neurospora tetrasperma, exhibit suppressed recombination similar to sex chromosomes in plants and animals, and recent studies have sought to apply basic theories of sex chromosome evolution to fungi. A phylogeny of the MTL1 locus in Microbotryum indicates that it has become part of the nonrecombining regions of the mating‐type chromosomes in multiple independent events, and that recombination may have been subsequently restored in some cases. This illustrates that fungal mating‐type chromosomes can exhibit linkage relationship that are quite dynamic, adding to the list of similarities to animal or plant sex chromosomes. However, fungi such as M. lychnidis‐dioicae and N. tetrasperma exhibit an automictic mating system, for which an alternate theoretical framework exists to explain the evolution of linkage with the mating‐type locus. This study encourages further comparative studies among fungi to evaluate the role of mating systems in determining the evolution of fungal mating‐type chromosomes.     

Multicolor FISH mapping of the dioecious model plant,<Emphasis Type="Italic"> Silene latifolia</Emphasis>

Silene latifolia is a key plant model in the study of sex determination and sex chromosome evolution. Current studies have been based on genetic mapping of the sequences linked to sex chromosomes with analysis of their characters and relative positions on the X and Y chromosomes. Until recently, very few DNA sequences have been physically mapped to the sex chromosomes of S. latifolia. We have carried out multicolor fluorescent in situ hybridization (FISH) analysis of S. latifolia chromosomes based on the presence and intensity of FISH signals on individual chromosomes. We have generated new markers by constructing and screening a sample bacterial artificial chromosome (BAC) library for appropriate FISH probes. Five newly isolated BAC clones yielded discrete signals on the chromosomes: two were specific for one autosome pair and three hybridized preferentially to the sex chromosomes. We present the FISH hybridization patterns of these five BAC inserts together with previously described repetitive sequences (X-43.1, 25S rDNA and 5S rDNA) and use them to analyze the S. latifolia karyotype. The autosomes of S. latifolia are difficult to distinguish based on their relative arm lengths. Using one BAC insert and the three repetitive sequences, we have constructed a standard FISH karyotype that can be used to distinguish all autosome pairs. We also analyze the hybridization patterns of these sequences on the sex chromosomes and discuss the utility of the karyotype mapping strategy presented to study sex chromosome evolution and Y chromosome degeneration.Communicated by J.S. Heslop-Harrison     

Localization of BAC clones on mitotic chromosomes of <Emphasis Type="Italic">Musa acuminata</Emphasis> using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization

A bacterial artificial chromosome (BAC) library of banana (Musa acuminata) was used to select BAC clones that carry low amounts of repetitive DNA sequences and could be suitable as probes for fluorescence in situ hybridization (FISH) on mitotic metaphase chromosomes. Out of eighty randomly selected BAC clones, only one clone gave a single-locus signal on chromosomes of M. acuminata cv. Calcutta 4. The clone localized on a chromosome pair that carries a cluster of 5S rRNA genes. The remaining BAC clones gave dispersed FISH signals throughout the genome and/or failed to produce any signal. In order to avoid the excessive hybridization of repetitive DNA sequences, we subcloned nineteen BAC clones and selected their ‘low-copy’ subclones. Out of them, one subclone gave specific signal in secondary constriction on one chromosome pair; three subclones were localized into centromeric and peri-centromeric regions of all chromosomes. Other subclones were either localized throughout the banana genome or their use did not result in visible FISH signals. The nucleotide sequence analysis revealed that subclones, which localized on different regions of all chromosomes, contained short fragments of various repetitive DNA sequences. The chromosome-specific BAC clone identified in this work increases the number of useful cytogenetic markers for Musa.     

Genome organization of Magnaporthe grisea: genetic map,electrophoretic karyotype,and occurrence of repeated DNAs

A genetic map of Magnaporthe grisea (anamorph=Pyricularia oryzae and P. grisea), the causal agent of rice blast disease, was generated from segregation data utilizing 97 RFLP markers, two isoenzyme loci and the mating type locus among progeny of a cross between parental strains Guy 11 and 2539. Of the seven chromosomes of M. Grisea, three were resolved by contour-clamped homogeneous electric field (CHEF) electrophoresis, while the remaining four migrated as two doublet bands. By utilizing differences between CHEF mobilities of unresolved chromosomes from the parental strains, Southern analysis with selected markers allowed the chromosomal assignment of all linkage groups. A small translocation involving 1 marker was found in the parental strains used to produce the segregating population from which the map was constructed. Nine classes of repetitive DNA elements were found in the genome of a fungal isolate pathogenic to rice. These occurred only a few times or not at all in the genomes of isolates showing reduced virulence on rice. One repetitive DNA was shown to have structural similarity to the Alu sequences found in primates, a sequence similarity to the copia-like elements of Drosophila, and peptide similarity to transposable elements found in Drosophila, other fungi, and higher plants.The mention of a trademark, proprietary product, or vendor anywhere in this paper does not constitute a guarantee or warranty of the product by the USDA-ARS and does not imply its approval to the exclusion of other products or vendors that also may be suitable     

Genetic and physical maps around the sex-determining <Emphasis Type="Italic">M</Emphasis>-locus of the dioecious plant asparagus

Asparagus officinalis L. is a dioecious plant. A region called the M-locus located on a pair of homomorphic sex chromosomes controls the sexual dimorphism in asparagus. The aim of this work was to clone the region determining sex in asparagus from its position in the genome. The structure of the region encompassing M should be investigated and compared to the sex-determining regions in other dioecious model species. To establish an improved basis for physical mapping, a high-resolution genetic map was enriched with AFLP markers closely linked to the target locus by carrying out a bulked segregant analysis. By screening a BAC library with AFLP- and STS-markers followed by chromosome walking, a physical map with eight contigs could be established. However, the gaps between the contigs could not be closed due to a plethora of repetitive elements. Surprisingly, two of the contigs on one side of the M-locus did not overlap although they have been established with two markers, which mapped in a distance as low as 0.25 cM flanking the sex locus. Thus, the clustering of the markers indicates a reduced recombination frequency within the M-region. On the opposite side of the M-locus, a contig was mapped in a distance of 0.38 cM. Four closely linked BAC clones were partially sequenced and 64 putative ORFs were identified. Interestingly, only 25% of the ORFs showed sequence similarity to known proteins and ESTs. In addition, an accumulation of repetitive sequences and a low gene density was revealed in the sex-determining region of asparagus. Molecular cytogenetic and sequence analysis of BACs flanking the M-locus indicate that the BACs contain highly repetitive sequences that localize to centromeric and pericentromeric locations on all asparagus chromosomes, which hindered the localization of the M-locus to the single pair of sex chromosomes. We speculate that dioecious Silene, papaya and Asparagus species may represent three stages in the evolution of XX, XY sex determination systems. Given that asparagus still rarely produces hermaphroditic flowers and has homomorphic sex chromosomes, this species may be an ideal system to further investigates early sex chromosome evolution and the origins of dioecy.     

Retrotransposon populations of <Emphasis Type="Italic">Vicia</Emphasis> species with varying genome size

The (non-LTR) LINE and Ty3-gypsy-type LTR retrotransposon populations of three Vicia species that differ in genome size (Vicia faba, Vicia melanops and Vicia sativa) have been characterised. In each species the LINE retrotransposons comprise a complex, very heterogeneous set of sequences, while the Ty3-gypsy elements are much more homogeneous. Copy numbers of all three retrotransposon groups (Ty1-copia, Ty3-gypsy and LINE) in these species have been estimated by random genomic sequencing and Southern hybridisation analysis. The Ty3-gypsy elements are extremely numerous in all species, accounting for 18–35% of their genomes. The Ty1-copia group elements are somewhat less abundant and LINE elements are present in still lower amounts. Collectively, 20–45% of the genomes of these three Vicia species are comprised of retrotransposons. These data show that the three retrotransposon groups have proliferated to different extents in members of the Vicia genus and high proliferation has been associated with homogenisation of the retrotransposon population.Electronic Supplementary Material Supplementary material is available for this article at .     

Comparison of the Z and W sex chromosomal architectures in elegant crested tinamou (<Emphasis Type="Italic">Eudromia elegans</Emphasis>) and ostrich (<Emphasis Type="Italic">Struthio camelus</Emphasis>) and the process of sex chromosome differentiation in palaeognathous birds

To clarify the process of avian sex chromosome differentiation in palaeognathous birds, we performed molecular and cytogenetic characterization of W chromosome-specific repetitive DNA sequences for elegant crested tinamou (Eudromia elegans, Tinamiformes) and constructed comparative cytogenetic maps of the Z and W chromosomes with nine chicken Z-linked gene homologues for E. elegans and ostrich (Struthio camelus, Struthioniformes). A novel family of W-specific repetitive sequences isolated from E. elegans was found to be composed of guanine- and cytosine-rich 293-bp elements that were tandemly arrayed in the genome as satellite DNA. No nucleotide sequence homologies were found for the Struthioniformes and neognathous birds. The comparative cytogenetic maps of the Z and W chromosomes of E. elegans and S. camelus revealed that there are partial deletions in the proximal regions of the W chromosomes in the two species, and the W chromosome is more differentiated in E. elegans than in S. camelus. These results suggest that a deletion firstly occurred in the proximal region close to the centromere of the acrocentric proto-W chromosome and advanced toward the distal region. In E. elegans, the W-specific repeated sequence elements were amplified site-specifically after deletion of a large part of the W chromosome occurred.     

Sex-specific SCAR markers in the dioecious plant<Emphasis Type="Italic"> Rumex nivalis</Emphasis> (Polygonaceae) and implications for the evolution of sex chromosomes

We developed SCAR primers based on isolated and sequenced male-specific fragments as identified in an AFLP analysis of the dioecious plant Rumex nivalis. PCR amplification using these primers on females and males resulted in fragments exclusively present in males. Co-amplification of the nuclear rDNA internal transcribed spacer 2 together with the male-specific fragment was applied as an internal control for successful PCR reactions to avoid false-negative sex scoring. With a length of about 164 bp, the AFLP fragment was of a similar size as the tandemly arranged, repetitive sequences of 180 bp located on the Y chromosomes of Rumex acetosa. The genetic distances between the Y-chromosomal sequences of R. nivalis and R. acetosa, both members of the section Acetosa, were substantial. We found intra-individual divergence among cloned sequences of the male-specific fragment in R. nivalis. The patterns of interspecific and intra-individual sequence variation found are in accordance with proposed modes of the evolution of sex chromosomes. Y chromosomes possibly arose only once in the genus Rumex and consist mainly of heterochromatic DNA. Due to the almost complete absence of selection on them, Y chromosomes are likely to accumulate large numbers of mutations.Communicated by R. Hagemann     

The landscape of transposable elements in the finished genome of the fungal wheat pathogen Mycosphaerella graminicola

Background

In addition to gene identification and annotation, repetitive sequence analysis has become an integral part of genome sequencing projects. Identification of repeats is important not only because it improves gene prediction, but also because of the role that repetitive sequences play in determining the structure and evolution of genes and genomes. Several methods using different repeat-finding strategies are available for whole-genome repeat sequence analysis. Four independent approaches were used to identify and characterize the repetitive fraction of the Mycosphaerella graminicola (synonym Zymoseptoria tritici) genome. This ascomycete fungus is a wheat pathogen and its finished genome comprises 21 chromosomes, eight of which can be lost with no obvious effects on fitness so are dispensable.

Results

Using a combination of four repeat-finding methods, at least 17% of the M. graminicola genome was estimated to be repetitive. Class I transposable elements, that amplify via an RNA intermediate, account for about 70% of the total repetitive content in the M. graminicola genome. The dispensable chromosomes had a higher percentage of repetitive elements as compared to the core chromosomes. Distribution of repeats across the chromosomes also varied, with at least six chromosomes showing a non-random distribution of repetitive elements. Repeat families showed transition mutations and a CpA → TpA dinucleotide bias, indicating the presence of a repeat-induced point mutation (RIP)-like mechanism in M. graminicola. One gene family and two repeat families specific to subtelomeres also were identified in the M. graminicola genome. A total of 78 putative clusters of nested elements was found in the M. graminicola genome. Several genes with putative roles in pathogenicity were found associated with these nested repeat clusters. This analysis of the transposable element content in the finished M. graminicola genome resulted in a thorough and highly curated database of repetitive sequences.

Conclusions

This comprehensive analysis will serve as a scaffold to address additional biological questions regarding the origin and fate of transposable elements in fungi. Future analyses of the distribution of repetitive sequences in M. graminicola also will be able to provide insights into the association of repeats with genes and their potential role in gene and genome evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1132) contains supplementary material, which is available to authorized users.     

Isolation of TC/AG Repeat Microsatellite Sequences for Fingerprinting Rice Blast Fungus and Their Possible Horizontal Transfer to Plant Species

Genome fingerprinting has been a major role in characterization of population structure and analysis of the variability in phytopathogenic fungi. In order to characterize Korean rice blast fungal isolates, the genomic DNAs were digested with Alu I endonuclease and subsequent PCR amplifications using random decamer primers with combinations of microsatellite primers had been carried out. This Alu-Inter SSR technique revealed high polymorphism among the Korean blast fungal isolates. Then, fragments from the Alu-Inter SSR analysis were isolated to be used as probes in Southern hybridization, which also revealed high polymorphism between isolates to distinguish individuals. The sequences of the isolated fragments contained TC/AG tandem repeats interspersed with a 30 bp direct repeat. In gel blot analysis, the isolated TC/AG repeat microsatellite sequences were proved to be useful for characterizing the isolates in blast fungi in addition to the conventional MGR (Magnaporthe grisea repeat) probes. One interesting point was that the rice blast fungus derived TC/AG repeat microsatellite sequences were abundant in non-rice blast fungi and plant species, but not in other fungi and yeasts. A discussion on the possible horizontal gene transfer between phytopathogenic fungi and host plants is presented.     

Molecular structure of a novel gypsy-Ty3-like retrotransposon (Kabuki) and nested retrotransposable elements on the W chromosome of the silkworm Bombyx mori

We previously characterized a female-specific randomly amplified polymorphic DNA (RAPD), designated W-Kabuki, derived from the W chromosome of the silkworm, Bombyx mori. To further analyze the W chromosome of B. mori, we obtained a lambda phage clone which contains the W-Kabuki RAPD sequence and sequenced the 18.1-kb DNA insert. We found that this DNA comprises a nested structure of at least seven elements; three retrotransposons, two retroposons, one functionally unknown insertion, and one Bombyx repetitive sequence. The non-LTR retrotransposon BMC1, the retroposon Bm1, a functionally unknown inserted DNA (FUI), and a copia-like LTR retrotransposon (Yokozuna) are themselves inserted into a novel gypsy-Ty3-like LTR retrotransposon, named Kabuki. Furthermore, this Kabuki element is itself inserted into another copy of Bm1. The BMC1 and Yokozuna elements inserted in the Kabuki sequence are intact. Moreover, the Kabuki element is largely intact. These results suggest that many retrotransposable elements have accumulated on the W chromosome, and these elements are expected to evolve more slowly than those on other chromosomes. Received: 7 October 1999 / Accepted: 14 April 2000     

Repeat-induced point mutation and the population structure of transposable elements in Microbotryum violaceum

Repeat-induced point mutation (RIP) is a genome defense in fungi that hypermutates repetitive DNA and is suggested to limit the accumulation of transposable elements. The genome of Microbotryum violaceum has a high density of transposable elements compared to other fungi, but there is also evidence of RIP activity. This is the first report of RIP in a basidiomycete and was obtained by sequencing multiple copies of the integrase gene of a copia-type transposable element and the helicase gene of a Helitron-type element. In M. violaceum, the targets for RIP mutations are the cytosine residues of TCG trinucleotide combinations. Although RIP is a linkage-dependent process that tends to increase the variation among repetitive sequences, a chromosome-specific substructuring was observed in the transposable element population. The observed chromosome-specific patterns are not consistent with RIP, but rather suggest an effect of gene conversion, which is also a linkage-dependent process but results in a homogenization of repeated sequences. Particular sequences were found more widely distributed within the genome than expected by chance and may reflect the recently active variants. Therefore, sequence variation of transposable elements in M. violaceum appears to be driven by selection for transposition ability in combination with the context-specific forces of the RIP and gene conversion.     

A new long terminal repeat (LTR) sequence allows to identify J genome from J<Superscript>S</Superscript> and St genomes of <Emphasis Type="Italic">Thinopyrum intermedium</Emphasis>

A repetitive sequence of 491 bp, named pMD232-500, was isolated from S. cereale cv. Kustro using wheat SSR marker Xgwm232. GenBank BLAST search revealed that the sequence of pMD232-500 was highly similar to a part of retrotransposon Nusif-1. Fluorescence in situ hybridization (FISH) analysis using pMD232-500 as probe indicated that only 14 Thinopyrum intermedium chromosomes and all the chromosomes of S. cereale cv. Kustro bear FISH signals, however, no FISH signals were observed on Dasypyrum villosum chromosomes. In addition, the FISH signals were distributed on whole arms except their terminal regions. Further genomic in situ hybridization (GISH) analysis using genomic DNA from Pseudoroegneria spicata indicated that the 14 Th. intermedium chromosomes bearing FISH signals should belong to J genome. Thereafter, the repetitive elements pMD232-500 showed the unambiguous features of genomic constitution of Th. intermedium. In addition, the results in the present study have indicated the similarity of genomes from Th. intermedium and S. cereale.     

Characterization of Repetitive DNA Elements in Arabidopsis

We have applied computational methods to the available database and identified several families of repetitive DNA elements in the Arabidopsis thaliana genome. While some of the elements have features expected of either miniature inverted-repeat transposable elements (MITEs) or retrotransposons, the most abundant class of repetitive elements, the AthE1 family, is structurally related to neither. The AthE1 family members are defined by conserved 5′ and 3′ sequences, but these terminal sequences do not represent either inverted or direct repeats. AthE1 family members with greater than 98% identity are easily identified on different Arabidopsis chromosomes. Similar to nonautonomous DNA-based transposon families, the AthE1 family contains members in which the conserved terminal domains flank unrelated sequences. The primary utility of characterizing repetitive sequences is in defining, at least in part, the evolutionary architecture of specific Arabidopsis loci. The repetitive elements described here make up approximately 1% of the available Arabidopsis thaliana genomic sequence. Received: 13 October 1998 / Accepted: 30 December 1998     

Distribution of heterochromatin on the mitotic chromosomes of Musca domestica L. in relation to the activity of male-determining factors

In the housefly, male sex is determined by a dominant factor, M, located either on the Y, on the X, or on any of the five autosomes. M factors on autosome I and on fragments of the Y chromosome show incomplete expressivity, whereas M factors on the other autosomes are fully expressive. To test whether these differences might be caused by heterochromatin-dependent position effects, we studied the distribution of heterochromatin on the mitotic chromosomes by C-banding and by fluorescence in situ hybridization of DNA fragments amplified from microdissected mitotic chromosomes. Our results show a correlation between the chromosomal position of M and the strength of its male-determining activity: weakly masculinizing M factors are exclusively located on chromosomes with extensive heterochromatic regions, i.e., on autosome I and on the Y chromosome. The Y is known to contain at least two copies of the M factor, which ensures a strong masculinizing effect despite the heterochromatic environment. The heterochromatic regions of the sex chromosomes consist of repetitive sequences that are unique to the X and the Y, whereas their euchromatic parts contain sequences that are ubiquitously found in the euchromatin of all chromosomes of the complement. Received: 20 February 1998; in revised form: 11 May 1998 / Accepted: 23 May 1998