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.     

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.     

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.     

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.     

Expression of marY1, a gypsy-type LTR-retroelement from the ectomycorrhizal homobasidiomycete Tricholoma matsutake, in the budding yeast Saccharomyces cerevisiae

marY1 is a gypsy-type LTR-retroelement present in the genome of the ectomycorrhizal homobasidiomycete Tricholoma matsutake. We document here that a marY1-lacZ gene fusion was expressed in the budding yeast Saccharomyces cerevisiae. The finding strongly suggests that marY1 is activated by trans-regulatory factors common to higher fungi, and may be useful for the development of new recombinant systems in ectomycorrhizal fungi and homobasidiomycetes.     

Acquisition of endonuclease specificity during evolution of L1 retrotransposon

L1 is the most proliferative autonomous retroelement that comprises about 20% of mammalian genomes. Why L1s have proliferated so extensively in mammalian genomes is an important yet unsolved question. L1 copies are amplified via retrotransposition, in which the DNA cleavage specificity by the L1-encoded endonuclease (EN) primarily dictates sites of insertion. Whereas mammalian L1s show target preference for 5'-TTAAAA-3', other L1-like elements exhibit various degrees of target specificity. To gain insights on diversification of the EN specificity during L1 evolution, ENs of zebrafish L1 elements were analyzed here. We revealed that they form 3 discrete clades, M, F, and Tx1, which is in stark contrast to a single L1 clade in mammalian species. Interestingly, zebrafish clade M elements cluster as a sister group of mammalian L1s and show target-site preference for 5'-TTAAAA-3'. In contrast, elements of the clade F, the immediate outgroup of the clade M, show little specificity. We identified certain clade-specific amino acid residues in EN, many of which are located in the cleft that recognizes the substrate, suggesting that these amino acid alterations have generated 2 types of ENs with different substrate specificities. The distribution pattern of the 3 clades suggests a possibility that the acquisition of target specificity by the L1 ENs improved the L1 fitness under the circumstances in mammalian hosts.     

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.     

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.     

Retroelement genome painting: cytological visualization of retroelement expansions in the genera Zea and Tripsacum

Divergence of abundant genomic elements among the Zea and Tripsacum genera was examined cytologically and a tool kit established for subsequent studies. The LTR regions from the CRM, Huck, Grande, Prem1, Prem2/Ji, Opie, Cinful-1, and Tekay retroelement families were used as FISH probes on mitotic chromosome spreads from a "trispecies" hybrid containing chromosomes from each of three species: Zea mays (2n = 20), Z. diploperennis (2n = 20), and Tripsacum dactyloides (2n = 36). Except for Tekay, which painted both Zea and Tripsacum chromosomes with nearly equal intensity, the retroelement probes hybridized strongly to the Zea chromosomes, allowing them to be distinguished from those of Tripsacum. Huck and Grande hybridized more intensely to maize than to Z. diploperennis chromosomes. Tripsacum genomic clones containing retroelement sequences were isolated that specifically paint Tripsacum chromosomes. The retroelement paints proved effective for distinguishing different genomes in interspecific hybrids and visualizing alien chromatin from T. dactyloides introgressed into maize lines. Other FISH probes (180-bp knob, TR-1, 5S, NOR, Cent4, CentC, rp1, rp3, and alpha-ZeinA) could be simultaneously visualized with the retroelement probes, emphasizing the value of the retroelement probes for cytogenetic studies of Zea and Tripsacum.     

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.     

A molecular phylogenomic analysis of the ILR1-like family of IAA amidohydrolase genes

The ILR1-like family of hydrolase genes was initially isolated in Arabidopsis thaliana and is thought to help regulate levels of free indole-3-acetic-acid.We have investigated how this family has evolved in dicotyledon, monocotyledon and gymnosperm species by employing the GenBank and TIGR databases to retrieve orthologous genes. The relationships among these sequences were assessed employing phylogenomic analyses to examine molecular evolution and phylogeny. The members of the ILR1-like family analysed were ILL1, ILL2, ILL3, ILL6, ILR1 and IAR3. Present evidence suggests that IAR3 has undergone the least evolution and is most conserved. This conclusion is based on IAR3 having the largest number of total interspecific orthologues, orthologous species and unique orthologues. Although less conserved than IAR3, DNA and protein sequence analyses of ILL1 and ILR1 suggest high conservation. Based on this conservation, IAR3, ILL1 and ILR1 may have had major roles in the physiological evolution of 'higher' plants. ILL3 is least conserved, with the fewest orthologous species and orthologues. The monocotyledonous orthologues for most family-members examined have evolved into two separate molecular clades from dicotyledons, indicating active evolutionary change. The monocotyledon clades are: (a) those possessing a putative endoplasmic reticulum localizing signal; and (b) those that are putative cytoplasmic hydrolases. IAR3, ILL1 and ILL6 are all highly orthologous to a gene in the gymnosperm Pinus taeda, indicating an ancient enzymatic activity. No orthologues could be detected in Chlamydomonas, moss and fern databases.