marY2N, a LINE-like non-long terminal repeat (non-LTR) retroelement from the ectomycorrhizal homobasidiomycete Tricholoma matsutake.

A LINE-like non-LTR retroelement designated marY2N was cloned from the ectomycorrhizal homobasidiomycete Tricholoma matsutake. marY2N has open reading frames that correspond to gag and pol, and a putative promoter and consensus sequences common to those of the mutators from fruit flies. While it is common to T. matsutake and Tricholoma magnivelare, marY2N does not reside in any other species of Tricholoma tested.     

Intra- and inter-specific variations in the copy number of two types of retrotransposons from the ectomycorrhizal basidiomycete Tricholoma matsutake

To explore intra- and inter-specific variations of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces the fruit body matsutake, we carried out real-time PCR analysis based on two types of retrotransposons, one designated marY1, which resembles a retrovirus carrying the long terminal repeat (LTR) and the other marY2N, which resembles mRNA carrying the polyadenylated tail. Calculation based on the average genome size of homobasidiomycetes (34 Mbp) shows that ca. 5.5% of the total genome of T. matsutake isolated from Asia is made up of these retrotransposons, whereas they occupy ca. 1.4% in the isolates from Morocco, ca. 0.8% in isolates from Mexico, and ca. 0.5% in Tricholoma magnivelare, the species which produces American matsutake. Other Tricholoma spp. that produce fruit bodies similar to those of T. matsutake, such as T. bakamatsutake, T. fulvocastaneum, and T. robustum, carry them in the region less than 0.05% of their total genome. Copy number of LTR of marY1 is consistently and markedly higher than that of the coding regions of marY1 and marY2N. Data suggest that retrotransposons are deeply involved in evolution of the ectomycorrhizal symbiont.     

Mobile DNA distributions refine the phylogeny of “matsutake” mushrooms, Tricholoma sect. Caligata

“Matsutake” mushrooms are formed by several species of Tricholoma sect. Caligata distributed across the northern hemisphere. A phylogenetic analysis of matsutake based on virtually neutral mutations in DNA sequences resolved robust relationships among Tricholoma anatolicum, Tricholoma bakamatsutake, Tricholoma magnivelare, Tricholoma matsutake, and Tricholoma sp. from Mexico (=Tricholoma sp. Mex). However, relationships among these matsutake and other species, such as Tricholoma caligatum and Tricholoma fulvocastaneum, were ambiguous. We, therefore, analyzed genomic copy numbers of σ _marY1 , marY1, and marY2N retrotransposons by comparing them with the single-copy mobile DNA megB1 using real-time polymerase chain reaction (PCR) to clarify matsutake phylogeny. We also examined types of megB1-associated domains, composed of a number of poly (A) and poly (T) reminiscent of RNA-derived DNA elements among these species. Both datasets resolved two distinct groups, one composed of T. bakamatsutake, T. fulvocastaneum, and T. caligatum that could have diverged earlier and the other comprising T. magnivelare, Tricholoma sp. Mex, T. anatolicum, and T. matsutake that could have evolved later. In the first group, T. caligatum was the closest to the second group, followed by T. fulvocastaneum and T. bakamatsutake. Within the second group, T. magnivelare was clearly differentiated from the other species. The data suggest that matsutake underwent substantial evolution between the first group, mostly composed of Fagaceae symbionts, and the second group, comprised only of Pinaceae symbionts, but diverged little within each groups. Mobile DNA markers could be useful in resolving difficult phylogenies due to, for example, closely spaced speciation events.     

sigma marY1, the LTR of the gypsy-type retroelement marY1 from the basidiomycete tricholoma matsutake, allows multicopy DNA integration in Lentinula edodes

sigma marY1 is the LTR of the retroelement marY1 from the homobasidiomycete Tricholoma matsutake. Upon integration through transformation, pLC1-hph carrying a sigma marY1 derivative, sigma* marY1, conferred the hygromycin-resistant phenotype stronger than the vector without sigma* marY1 on Lentinula edodes. Based on the densitometric analysis after Southern hybridization, a copy number of the former construct integrated in the genome is much higher than that of the latter. We conclude that sigma marY1 allows multicopy DNA integration and will be useful in the genetic research on this fungal group.     

The long terminal repeat (LTR) sequence of marY1, a retroelement from the ectomycorrhizal homobasidiomycete Tricholoma matsutake, is highly conserved in various higher fungi.

marY1 is an LTR-retroelement from the homobasidiomycete Tricholoma matsutake. Nucleotide sequences that correspond to the putative U3-R region and the R-U5 region of marY1 are highly conserved in various higher fungi. Data suggest that the LTR sequence of marY1 originated early in the evolution of higher fungi and has become widely distributed. Therefore, it may be useful for the construction of an LTR-mediated transformation system in basidiomycetes.     

Highly polymorphic DNA markers to specify strains of the ectomycorrhizal basidiomycete Tricholoma matsutake based on σ marY1 , the long terminal repeat of gypsy-type retroelement marY1

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies—matsutake—in Pinus sp. forest. Here we report that PCR with outward facing primers designed based on sequences comprising _marY1, the long terminal repeat of the gypsy-type retroelement marY1, specifies strains of T. matsutake. PCR with a primer based on the 22-bp sequence conserved at the 5-end of _marY1 conferred 73 reliable bands overall whose profiles depend upon strains of T. matsutake and T. magnivelare, the latter known as American matsutake. This PCR system gave no detectable band in any other species of Tricholoma tested, including T. bakamatsutake and T. fulvocastaneum, symbionts closely related to T. matsutake, as well as a host plant, Pinus densiflora. Similarly, PCR with a set of primers based on 26-bp and 28-bp sequences at bp 48–73 and bp 281–308 of _marY1, internal regions that are mutated in a variant of marY1, conferred 90 reliable bands only in strains of T. matsutake. Theoretically, PCR with the 22-bp primer would allow generation of 2⁷³, or 9.4×10²¹, types of polymorphism, and PCR with a combination of 26- and 28-bp primers, 2⁹⁰, or 1.2×10²⁷ types. The probability of falsely specifying two different isolates as the same strain is <1/10²¹. While polymorphisms conferred by the primer based on the 5 end of _marY1 rather exhibit genetic conservation of a group of T. matsutake, those resulting from primers based on the internal sequences more clearly demonstrate intra-specific diversification. Both systems revealed that T. matsutake is divergent within the species. Ectomycorrhizas formed between P. densiflora and T. matsutake were identified by the PCR systems developed in the present study. This method, using _marY1 as a genetic marker, is useful in analyzing the diversity of T. matsutake, monitoring the behavior of individual mycorrhizas, and specifying the ecological background of fruit bodies traded in markets.     

Retrotransposon Sequence Variation in Four Asexual Plant Species

Transposable elements (TEs) can be viewed as genetic parasites that persist in populations due to their capacity for increase in copy number and the inefficacy of selection against them. A corollary of this hypothesis is that TEs are more likely to spread within sexual populations and be eliminated or inactivated within asexual populations. While previous work with animals has shown that asexual taxa may contain less TE diversity than sexual taxa, comparable work with plants has been lacking. Here we report the results of a study of Ty1/copia, Ty3/gypsy, and LINE-like retroelement diversity in four asexual plant species. Retroelement-like sequences, with a high degree of conservation both within and between species, were isolated from all four species. The sequences correspond to several previously annotated retroelement subfamilies. They also exhibit a pattern of nucleotide substitution characterized by an excess of synonymous substitutions, suggestive of a history of purifying selection. These findings were compared with retroelement sequence evolution in sexual plant taxa. One likely explanation for the discovery of conserved TE sequences in the genomes of these asexual taxa is simply that asexuality within these taxa evolved relatively recently, such that the loss and breakdown of TEs is not yet detectable through analysis of sequence diversity. This explanation is examined by conducting stochastic simulation of TE evolution and by using published information to infer rough estimates of the ages of asexual taxa. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Dmitri Petrov     

Identification of ectomycorrhizae formed betweenTricholoma matsutake andPinus densiflora by polymerase chain reaction (PCR) targeting retroelement coding regions

We previously reported that conservation and diversification of repetitive sequences carrying motifs of retroposons have occurred inTricholoma matsutake and related ectomycorrhizal basidiomycetes through their evolution. Here we report that the polymerase chain reaction using primers designed to amplify retroelement coding regions specified ectomycorrhizae formed betweenT. matsutake andPinus densiflora.     

Characterization of a Tc1-like transposable element in zebrafish (Danio rerio)

We have characterized Tdr1, a family of Tc1-like transposable elements found in the genome of zebrafish (Danio rerio). The copy number and distribution of the sequence in the zebrafish genome have been determined, and by these criteria Tdr1 can be classified as a moderately repetitive, interspersed element. Examination of the sequences and structures of several copies of Tdr1 revealed that a particular deletion derivative, 1250 by long, of the transposon has been amplified to become the dominant form of Tdr1. The deletion in these elements encompasses sequences encoding the N-terminal portion of the putative Tdr1 transposase. Sequences corresponding to the deleted region were also detected, and thus allowed prediction of the nucleotide sequence of a hypothetical full-length element. Well conserved segments of Tc1-like transposons were found in the flanking regions of known fish genes, suggesting that these elements have a long evolutionary history in piscine genomes. Tdr1 elements have long, 208 by inverted repeats, with a short DNA motif repeated four times at the termini of the inverted repeats. Although different from that of the prototype C. elegans transposon Tc1, this inverted repeat structure is shared by transposable elements from salmonid fish species and two Drosophila species. We propose that these transposons form a subgroup within the Tc1-like family. Comparison of Tc1-like transposons supports the hypothesis that the transposase genes and their flanking sequences have been shaped by independent evolutionary constraints. Although Tc1-like sequences are present in the genomes of several strains of zebrafish and in salmonid fishes, these sequences are not conserved in the genus Danio, thus raising the possibility that these elements can be exploited for gene tagging and genome mapping.     

Cloning and characterization of piggyBac- like elements in lepidopteran insects

PiggyBac-like elements (PLE) are widespread in variety of organisms, however, few of them are active or have an intact transposon structure. To further define the distribution PLEs in Lepidoptera, where the original active piggyBac IFP2 was discovered, and potentially isolate new functional elements, a survey for PLEs by PCR amplification and Southern dot blots was performed. Two new PLEs, AyPLE and AaPLE, were successfully isolated from the noctuid species, Agrotis ypsilon and Argyrogramma agnate, respectively. These elements were found to be closely related to each other by sequence similarity, and by sharing the same 16 bp inverted terminal repeat sequences. The AyPLE1.1 and AaPLE1.1 elements are structurally intact having characteristic TTAA target site duplications, inverted terminal repeats and intact open reading frames encoding putative transposases with the presumed piggyBac DDD domains, which are features consistent with autonomous functional transposons. Phylogenetic analysis revealed that AyPLE1.1 and AaPLE1.1 cluster with another noctuid species element, HaPLE1.1, suggesting a common ancestor for the three types of PLEs. This contributes to our understanding of the distribution and evolution of piggyBac in Lepidoptera.     

Genetic diversity of SIRE-1 retroelements in annual and perennial glycine species revealed using SSAP

Sequence Specific Amplification Polymorphisms (SSAP) were used to measure the distribution and structure of SIRE-1 retroelement populations in annual and perennial Glycine species. For SSAP analysis, primers corresponding to a region immediately upstream of the 3’LTR of the soybean retroelement SIRE-1 were chosen. Analysis reveals that SIRE-1 is present throughout the Glycine genus and shows that the annual species have similar SIRE-1 populations whilst the perennial species have much more distinct and diverse populations. The high number of species-specific subgroups suggest that SIRE-1 has been active and evolving independently in each species during the course of Glycine evolution.     

Isolation and characterization of RARE-1, a Ty1/copia-like retrotransposon domesticated in the genome of Rhizophora apiculata

Long terminal repeat (LTR) retrotransposons are predominant mobile elements that play important roles in plant genome evolution. Here, we isolated the first putative complete Ty1/copia-like retrotransposon of 6303 bp in mangrove Rhizophora apiculata, named RARE-1. RARE-1 was homologous to the soybean retroelement 1 (SORE-1) and exhibited abundant cis-regulatory motifs involved in various stress responses in its LTRs. Using the sequence-specific amplification polymorphism (S-SAP) technique, we obtained a total of 112 bands for two R. apiculata populations from Hainan, China and Ranong, Thailand. The Hainan population showed slightly higher S-SAP polymorphism but fewer unique bands than the Ranong population. Moreover, the Hainan population also had significantly more copies of RARE-1 than the Ranong population as revealed by quantitative real-time PCR (qPCR). Our results suggested that RARE-1 might have been domesticated in the R. apiculata genome, as a result of the long-term evolution of mangroves under the extreme environment.     

Phylogenetic evidence for Ty1-copia-like endogenous retroviruses in plant genomes

SIRE-1 is a multi-copy, Ty1-copia-like retroelement family found in the genome of Glycine max. A sequenced SIRE-1 genomic copy has an uninterrupted ORF that can be translated into a gag-pol polyprotein, followed by an unprecedented second ORF whose conceptual translation yielded a theoretical protein predicted to possess many of the same secondary structural elements found in mammalian retroviral envelope proteins. Similar, but clearly pseudogenic, envelope-like sequences were recovered from conceptual translations of 10 Arabidopsis Gen-Bank accessions. All were associated with identifiable Ty1-copia-like retroelements. Phylogenetic analysis of the adjacent ribonuclease H regions from these sequences and three similarly endowed elements, two from maize and one from tomato, indicate that the 14 elements constitute a monophyletic group distinct from several closely related plant Ty1-copia-like elements in which polis immediately followed by a downstream LTR. The conservation of identifiable env-like gene features suggests that these plant elements are endogenous retroviruses whose ancestors were acquired from animal vectors. The finding that the env and env-less retroelements identified in this study form distinct lineages does not support the hypothesis that horizontal transmission of retrotransposons is sponsored by ancestral infectious retroviruses that subsequently lost all traces of env genes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evolutionary analysis of the CACTA DNA-transposon Caspar across wheat species using sequence comparison and in situ hybridization

Mobile elements constitute a considerable part of the eukaryotic genome. This work is focused on the distribution and evolution of DNA-transposons in the genomes of diploid and allopolyploid Triticeae species and their role in the formation of functionally important chromosomal subtelomeric regions. The Caspar family is among the most abundant of CACTA DNA-transposons in Triticeae. To study the evolution of Caspar-like elements in Triticeae genomes, we analyzed their sequences and distribution in chromosomes by in situ hybridization. In total, 46 Caspar-like elements from the wheat and barley Caspar, Clifford, and Donald families were analyzed after being extracted from databases using the transposase consensus sequence. Sequence alignment and subsequent phylogenetic analyses revealed that the transposase DNA sequences formed three major distinct groups: (1) Clifford, (2) Caspar_Triticinae, and (3) Caspar_Hordeinae. Additionally, in situ hybridization demonstrated that Caspar_Triticinae transposons are predominantly compartmentalized in the subtelomeric chromosomal regions of wheat and its progenitors. Analysis of data suggested that compartmentalization in the subtelomeric chromosomal region was a characteristic feature of all the main groups of Caspar-like elements. Furthermore, a dot plot analysis of the terminal repeats demonstrated that the divergence of these repeats strictly correlated with the divergence of Caspar coding sequences. A clear distinction in the Caspar DNA sequences among the species Triticum/Aegilops (Caspar_Triticinae), Hordeum (Caspar_Hordeinae), and different distributions in individual hexaploid wheat genomes (A/B and D) suggest an independent proliferation of these elements in wheat (or its progenitors) and barley genomes. Thus, Caspar-like transposons can significantly contribute to the formation and differentiation of subtelomeric regions in Triticeae species.     

A lineage‐specific centromere retrotransposon in Oryza brachyantha

Most eukaryotic centromeres contain large quantities of repetitive DNA, such as satellite repeats and retrotransposons. Unlike most transposons in plant genomes, the centromeric retrotransposon (CR) family is conserved over long evolutionary periods among a majority of the grass species. CR elements are highly concentrated in centromeres, and are likely to play a role in centromere function. In order to study centromere evolution in the Oryza (rice) genus, we sequenced the orthologous region to centromere 8 of Oryza sativa from a related species, Oryza brachyantha. We found that O. brachyantha does not have the canonical CRR (CR of rice) found in the centromeres of all other Oryza species. Instead, a new Ty3‐gypsy (Metaviridae) retroelement (FRetro3) was found to colonize the centromeres of this species. This retroelement is found in high copy numbers in the O. brachyantha genome, but not in other Oryza genomes, and based on the dating of long terminal repeats (LTRs) of FRetro3 it was amplified in the genome in the last few million years. Interestingly, there is a high level of removal of FRetro3 based on solo‐LTRs to full‐length elements, and this rapid turnover may have played a role in the replacement of the canonical CRR with the new element by active deletion. Comparison with previously described ChIP cloning data revealed that FRetro3 is found in CENH3‐associated chromatin sequences. Thus, within a single lineage of the Oryza genus, the canonical component of grass centromeres has been replaced with a new retrotransposon that has all the hallmarks of a centromeric retroelement.     

Easel, a gypsy LTR-retrotransposon in the Salmonidae

Despite the close similarities between retroviruses and the gypsy/Ty3 group of LTR-retrotransposons their host ranges are largely distinct: the retroviruses are found only in vertebrates, whereas the gypsy LTR-retrotransposons are almost exclusively restricted to invertebrates, plants and fungi. Here we report the amplification by PCR, and characterisation, of one of the first LTR-retrotransposons to be discovered in vertebrates - in several members of the piscine family Salmonidae. Phylogenetic analysis of this retroelement, termed easel, indicates that it is probably a phylogeneticaly basal member of the gypsy group of LTR-retrotransposons and occurs in some of the same species from which retroviruses have previously been isolated. Thus some members of the Salmonidae are the first organisms known to harbour both retroviral branch elements and the gypsy LTR-retrotransposon branch elements. This creates an overlap in the host ranges of the two retroelement families.