Fungi are versatile eukaryotes that occupy different ecological niches and are responsible for several important processes, such as organic matter decomposition, symbiotic association and pathogenicity in animals and plants. This group of microorganisms is considered a model for the study of the biology and genetics of eukaryotes. Accordingly, fungi are among those groups of organisms with the largest number of genomes already sequenced or in the process of being sequenced and annotated [23, 24].
The genomes of fungi contain varying numbers and sizes of repeated sequences, usually representing 3% to 10% of the sequenced genome. However, some genomes diverge from this range, such as the genome of Ashbya gossypii, which, surprisingly, contains no detected TEs , and the genome of Tuber melanosporum, which consists of 58% TEs . In Laccaria bicolor, more than 215 genus-specific TEs and a large number of remaining degenerate copies were found . The genome of Mycosphaerella graminicola contains 21.2% of repetitive sequences, and a large percentage of these sequences are in dispensable chromosomes . In the present analysis, the RepeatMasker software, one of the most readily available and widely used bioinformatics tools for the detection, characterization and analysis of repetitive element sequences in the genomes of eukaryotes , along with the LTR-Finder and the Repeat Finder programs, determined that approximately 7% of the M. fijiensis genome consists of complete TEs. Using differences in the dinucleotide profile, Clutterbuck  estimated that approximately 50% of the genome of M. fijiensis is composed of repetitive elements. Compared with analysis based on anomalies in the DFD (dinucleotide frequency distribution), which have little specificity, analysis using RepeatMasker is much more specific because it uses a database (RepBase) of consensus sequences from the principal characterized transposable elements. The anomalies in the DFD may overestimate the number of transposable elements in the genome because they detect any changes in the GC content, including telomeric and centromeric sequences, material from horizontal transfer, satellite regions, supernumerary chromosomes and RIPed sequences, among others. Moreover, RIP appears to be intense in M. fijiensis. RIP is a mechanism that acts on not only transposable elements but also on other duplicated sequences. Thus, Clutterbuck  inferred a large number of repetitive sequences without specifying what percentage of these sequences are actually transposable elements. Moreover, RepeatMasker can fail to detect very degenerate copies of elements and also can miss TEs that are not represented in the database (RepBase). As the evidence suggests that the RIP process operates heavily on the genome of M. fijiensis, it is expected that very degenerate copies are partially identified by the program. However, due to the number of accumulated mutations, very degenerate sequences may have no role in the regulation of genes and, because of decreased homology between the sequences, may not represent targets for ectopic recombination. These considerations drove us to search for intact transposable elements because such elements contain copies less affected by mutations and they can have a real impact on the evolution of this pathogen.
In terms of the types of TEs identified, retrotransposons appear to be largely responsible for the repetitive fraction of the M. fijiensis genome. These elements were found in hundreds of copies and exhibit great family diversity. Gypsy/Ty3 has been the main TE group identified in phytopathogenic fungi  and has also been widely identified in the genome of M. fijiensis. The class II TEs are typically ancient elements found in almost all eukaryotes; however, they are usually found in a small number of copies . The best represented class II elements were those belonging to the Tc1-Mariner superfamily, one of the most diverse and widely distributed in nature. Another superfamily identified that occurs in various species of eukaryotes was the Mutator superfamily. Both superfamilies encode a transposase and are flanked by TIRs; however, they differ in relation to the insertion site. Elements of the Tc1-Mariner superfamily usually insert into TA sequences, while TEs of the Mutator superfamily have insertion sites that vary from 9 to 11 bp . Finally, an element belonging to the Harbinger superfamily exhibited a high accumulation of mutations and did not allow for the detection of conserved domains. Elements belonging to this superfamily generally have two ORFs, one encoding a DNA binding protein and the other encoding a transposase .
There is strong evidence that ectopic recombination events are now or have been very intense in the genome of M. fijiensis. This is because, in addition to finding a large number of degenerate sequences and solo LTRs, 125 identified retrotransposons had different insertion sites flanking the 5’ and 3’ end of the same element. The presence of different insertion sites at the ends of the same TE and the presence of numerous degenerate sequences are indicative of ectopic recombination among retrotransposons. Recombination events can influence the adaptation of this species by promoting rearrangements (deletion, duplication, inversion or translocation) and chromosome breakage . In Magnaporthe grisea, the analysis of the distribution of transposable elements in the genome has highlighted the fact that in the past there was an extensive ectopic recombination. As this organism relies on asexual propagation, recombination events can help improve the adaptation of these microorganisms because many genes that contribute to host specificity are present in regions rich in transposable elements. Thus, recombination events can lead to deletions or alterations in the structure of these genes and therefore altered expression . The involvement of TEs in ectopic recombination has also been inferred in Coprinus cinereus and Verticillium dahliae.
Possible TE activity has been identified in many sequenced fungal genomes. In L. Bicolor, 40 different TE families were observed, but the accumulation of mutations in the nucleotides was less than 5%, indicating that the TEs were recently active. Therefore, the potential activity of these elements could be inferred . In the genome of Fusarium oxysporum, the potential activity of these elements has been identified in several families . The analysis of coding proteins from TEs showed that only three LTR-Copia elements contained uninterrupted ORFs and were potentially active. The high number of stop codons identified in the TEs could be explained by the presence of efficient transposon silencing mechanisms. In fact, our results indicated RIP-like events with preferred mutations in CpG dinucleotides in both class I and II TEs. The RIP index values were highly significant when compared with the set default values and standards set in other TEs analyzed in different fungi , such as PetTra in Penicillium chrysogenum and OPHIO3-1414 in Ophiostoma ulmi, demonstrating that this process must have been or is intense in M. fijiensis. Furthermore, compared to the punt element of Neurospora crassa[30, 34], where RIP is considered a severe event, all of the TEs analyzed in M. fijiensis exhibited higher values. RIP-like events in M. fijiensis have also been identified by Clutterbuck . However, only one transposon with three representatives was analyzed. The present study analyzed a total of 78 transposons. The existence of RIP in certain genomes can carry a high evolutionary cost, as observed in N. crassa, where RIP could be correlated with the absence or paucity of duplicated genes in the genome. Because gene duplication is important for the evolution of any species, the existence of RIP may have a significant impact on the genomes of several fungi . However, there is also the possibility that RIP can be mild, leaving one or more copies of a gene functional, and giving rise to novel alleles .
The hybridization profile found for the Sagui element evidence the recent activity of TEs, given that a large proportion of the hybridization profiles found in different isolates were polymorphic, which can be correlated with the recent activity of the element in M. fijiensis populations. Sagui has been identified and characterized as being potentially active because it possesses complete LTRs and ORFs containing the domains of the key proteins involved in transposition. Only the aspartic proteinase domain was not detected. However, this is an expected result, given that this protein is thought to be difficult to analyze because of its low similarity and different evolution rates [32, 36]. Regarding the Mariner element, although no traces of activity were observed in the analyzed copies, the hybridization profiles of the different isolates showed polymorphisms, consistent with active TEs in the M. fijiensis populations. Another explanation for the few active TEs in the analyzed genome may be the fact that in most sequenced fungi species, the genome is highly stable because it has been maintained under laboratory conditions for long periods of time. However, we must emphasize that defective or non-autonomous elements can be mobilized in trans by related active elements containing proteins with motif sequences recognized by enzymes that are essential to transposition [15, 37]. Moreover, degenerate sequences can still have the ability to modify gene expression of the neighboring genes. Another important aspect is that the hybridization profile detected emphasizes the possibility of the use of such elements as molecular markers to trace the population structure of M. fijiensis in places where this disease has been described.
Genes encoding proteins that may be related to pathogenic mechanisms have been identified around complete TEs. Many genes for ABC and MFS transporters have been identified near TEs-rich regions. Some of these transporters have an important role as drug carriers and, therefore, provide protection to the organism against toxic products and fungicides. In plant pathogens, these transporters may be associated with multidrug resistance, virulence and altered sensitivity to fungicides [38, 39]. Another gene identified near a TE encodes a protein similar to LaeA, a regulator of virulence genes and, possibly, the first antimicrobial target specific for filamentous fungal pathogens of plants and animals . Similarly, TEs have been found near important genes related to the pathogenicity system in two important plant pathogens, M. grisea and F. oxysporum. At first, Khang  studied the gene AVR-Pita in the pertaining to avirulence gene family. These authors discovered that members of this family are associated with different types of transposable elements. The activity of these elements, as well as rearrangements caused by ectopic recombination, can potentially modify the structure or expression of AVR genes, and thus new races of the pathogen may emerge. In F. oxysporum, certain regions of the genome related to pathogenicity have 74% of transposable elements identified in the genome, including 95% of all DNA transposons that may be involved in gene duplication events .
Several genes encoding proteins involved in vital processes were found near TE-related sequences. Genes encoding proteins such as chitin synthase, involved in cell wall biogenesis, were found in regions with a high density of transposon-related sequences. Several sequences encoding serine/threonine kinase proteins have been identified. These protein domains are related to different regulatory pathways in cellular processes, such as growth, sexual/asexual development  and pathogenicity . Our results also identified several genes near TEs encoding proteins with important roles in transcription, translation, replication, cellular respiration, nutrient and ion transport, DNA repair, ubiquitination, apoptosis and cell wall formation and stabilization as well as those involved in important metabolic pathways, such as fatty acid metabolism, pyruvate metabolism and amino acid and vitamin biosynthesis and degradation. Our results show that the insertions of transposable elements in the genome of M. fijiensis are probably harmless. However, the activity of the elements near important genes can potentially modify gene expression, as well as the rearrangements caused by ectopic recombination can modify gene structure.
A final relevant fact regarding the presence and maintenance of transposable elements in the genome of several species is the possible role of TEs in gene regulation. Excluding deleterious insertions, TEs may be linked to the regulation of gene expression. This is a process known as domestication and represents an example of the exaptation of TEs at the molecular level, which would explain their maintenance in the genome of several species . Recently, humans miRNAs derived from TEs have been implicated in the regulation of important pathways, such as cell proliferation, chromosome segregation, mitosis and apoptosis . In addition, miRNAs based on TEs may represent essential components in the maintenance of genomic stability, serving as a safeguard for genome integrity and potentially functioning as an anti-cancer defense mechanism . In fungi, little is known about miRNA regulators. Transposable element domestication through miRNA-based regulation systems may be another important contribution of TEs in fungi. Therefore, further investigations into TE dynamics and their role in regulatory networks via mRNA should be performed in M. fijiensis, especially in light of the strong evidence reported in the present study about the organization and possible impacts of the presence of transposons in the genome of this fungus.