Plant MITEs: useful tools for plant genetics and genomics.

MITEs (Miniature inverted-repeat transposable elements) are reminiscence of non-autonomous DNA (class II) elements, which are distinguished from other transposable elements by their small size, short terminal inverted repeats (TIRs), high copy numbers, genic preference, and DNA sequence identity among family members. Although MITEs were first discovered in plants and still actively reshaping genomes, they have been isolated from a wide range of eukaryotic organisms. MITEs can be divided into Tourist-like, Stowaway-like, and pogo-like groups, according to similarities of their TIRs and TSDs (target site duplications). In despite of several models to explain the origin and amplification of MITEs, their mechanisms of transposition and accumulation in eukaryotic genomes remain poorly understood owing to insufficient experimental data. The unique properties of MITEs have been exploited as useful genetic tools for plant genome analysis. Utilization of MITEs as effective and informative genomic markers and potential application of MITEs in plants systematic, phylogenetic, and genetic studies are discussed.

Transposable elements (TEs) are DNA segments that can insert into new chromosomal locations and often make duplicated copies of themselves in the process (. Barbara McClintock discovered them in maize more than a half century ago as the genetic agents that are responsible for the sectors of altered pigmentation on mutant kernels (. Such elements of various types have been found in many genomes, such as Drosophila melanogaster, yeast (Saccharomyces cerevisiae), Escherichia coli, Caenorhabditis elegans, and humans (reviewed in ref. ).

With the progress of large-scale DNA sequencing, it has become apparent that, far from being rare components of large genomes, TEs are the single largest component of the genetic material of most eukaryotes (. They account for at least 45% of the human genome ( and 50-90% of some grass genomes (.

TEs in general are divided into two major classes according to their modes of transposition 6., 7.. Class I elements (retroelements), or retrotransposons, transpose by means of an RNA intermediate generated by reverse transcription 8., 9.. LTR retrotransposons are characterized by the presence of long terminal repeats. Non-LTR retrotransposons, such as LINEs (long interspersed nuclear elements) and SINEs (short interspersed nuclear elements), lack these characteristic sequences 8., 9.. Class II elements transpose via a DNA intermediate and are characterized by the presence of terminal inverted repeats, or TIRs 10., 11., such as the Ac-Ds family in maize and the P element in Drosophila. Several elements are difficult to classify, mainly because their mechanisms of transposition remain unknown (. It is the case for miniature inverted-repeat transposable elements (MITEs), which were first described in plants ( and later in other organisms (.

Features of MITEs

MITEs were first described for grass genomes 15., 16. and identified in several plant species, including maize 15., 16., 17., rice 15., 16., 18., 19., green pepper (, and Arabidopsis 10., 11., but have also been found in a wide range of other eukaryotic organisms, including Caenorhabditis elegans 21., 22., fungi (, mosquitoes 12., 14., 24., beetles (, and certain vertebrates, like Xenopus (, humans (, and teleost fishes (.

Structurally, MITEs are reminiscence of non-autonomous DNA (class II) elements with their small size (100-600 bp) and short TIRs (10-30 bp, the sequence between TIRs is A+T rich). However, their high copy numbers (1,000 to 15,000 per haploid genome), target-site preference (for TA or TAA, usually 2-3 bp, but up to 9 bp according to the recent reports) 29., 30., and the uniformity of related elements distinguish them from most previously described non-autonomous DNA elements 1., 7., 31., 32., 33..

Although MITEs as an outstanding class of repetitive sequences shares many common features, there are significant sequence similarities within MITE families but not between them (. Members of different subfamilies within a family only resemble each other in structure but not sequence identity. Some MITE families, such as Tourist, Stowaway, Bigfoot, and Micron, are predicted to form distinct secondary structures 15., 28., 30., 34., 35. that are very useful for identifying these elements.

Another important feature of MITEs is their preference for inserting into low copy number sequences or genic regions, especially for the regions of 5′ and 3′ to a gene, where matrix attachment regions (MARs) are found 11., 31., 33., 35., 36., 37., 38., 39., 40.. Tikhonov et al. ( discovered 33 MITEs in a 225-Kb region of the maize genome flanking the gene adh1. None of the MITEs in this region was found within the 166-Kb (74%) of sequence that is occupied by LTR retrotransposons. In rice, of 18 Kiddo members in annotated sequences, 7 (40%) are found in introns, 6 (30%) reside less than 530 bp from a coding sequence (CDS), and 4 (20%) rest between 1 and 5 Kb from a CDS and no MITE seems to have hopped in coding sequences yet. The abundance of MITEs (and of DNA transposons in general) in the gene-rich regions of plant genomes was confirmed after the analysis of the complete genome sequence of Arabidopsis (which contains ~1,200 MITEs and ~1,000 other DNA transposons, together constituting ~6% of the genome) and partial genome sequences of rice (~100,000 MITEs and ~10,000 other DNA transposons, forming ~12% of the genome) 12., 41.. It has been noticed that in plants and mosquitoes, MITEs are frequently associated with wild-type alleles of genes, indicating a potential role of these elements in gene regulation and genome organization 13., 14., 18..

In addition, MITEs were also found, in several cases, to insert into each other. For instance, the first Stowaway element was found as an insertion in a sorghum Tourist element (, whereas in another case a Tourist dimer was found in the same organism (. In maize, some Tourist elements (name Tourist-Zm) were present as adjacent or nested insertion (, and such MITE multimers were also reported in other organisms, including rice 41., 42. and mosquito 12., 14.. In rice, among the 6,600 MITEs analyzed, more than 10% were present as multimers and some MITE families had a high frequency of self-insertions. For the Castaway and Gaijin families, this preference was caused by a high frequency of self-insertions; in contrast, Ditto elements were targeted by many other families of repetitive sequences; but the frequency of MITE insertions into class I or other class II elements was surprisingly low; and nested MITE multimers were arisen from independent insertion events (. It has been believed that MITEs preferentially target other MITEs, rather than self-insertion (.

To date, no MITEs have been found to encode any product required for their movement, and their mechanisms of transposition and accumulation in eukaryotic genomes remain poorly understood (. Investigations of the ability of MITEs to transpose have been hindered by the fact that families with low sequence similarities may contain decayed MITE members that have lost their mobility. In contrast, highly homogeneous MITE families such as Hbr and Kiddo may still well be functional and may, therefore, be useful candidates for solving the mystery of MITE transposition (. A common model for the transposition mechanism of Tc1/mariner elements has emerged from the functional study of a limited number of animal transposases (. The N-terminal region of Tc1/mariner transposases contains DNA-binding domains that bind specifically to the TIRs (. A C-terminal domain is characterized by an amino acid signature called the DDE/D motif that consists of two aspartic acid residues and a glutamic acid residue (or a third aspartic acid residue). This motif is required for catalysis of both the DNA cleavage and the strand transfer steps of the “cut and paste” transposition reaction (.

Classification of MITEs in Plants

With the advantage of many genome sequencing projects, vast amounts of DNA sequence from a variety of plant and animal species, including rice, Arabidopsis, human, mouse, and Drosophila melanogaster, have become available for analysis. MITEs, with their high copy numbers, distinct structural features, and compact stature, are relatively easy to be mined from DNA sequence databases 1., 44., 45.. And there are several new software tools, for instance, Find MITE (ref. ; http://www.biochem.vt.edu/aedes), Miropeats (, REPuter (, GENSOR (http://www.girinst.org/Censor_Server.html), RepeatMasker (http://repeatmasker.genome.washington.edu), and REON (ref. ; http://www.gentics.wust.edu/eddy/recon), to be used for mining the databases and rapidly identifying MITEs on the basis of their structural characteristics or sequence homology.

To date, the identified MITEs can be further classified into many subclasses (Table 1). It is likely that additional MITE families remain to be discovered. In plants, elements of the Stowaway family have been identified in numerous dicots and monocots, whereas elements of other families have only been identified in narrow taxonomic groups (. For instance, the Tourist element is known only from grasses 15., 17., the Emigrant family only from Arabidopsis (, and Bigfoot only from Medicago (ref. ; Table 1).

Table 1

Classification of Plant MITEs

Group type	Subclass	Species	Appprox. copy no.	TSDxa	No. of TIRs	Approx. size (bp)	Reference(s)
Tourist-like	Alien	Bell pepper&Solanaceae	2,400	TWA	25	400	20
	At-mPIF2	A.thaliana	20	TWA	14	400	44
	ATTIR16T3A	A.thaliana	100	TWA	16	500	50
	ATTIRIX1	A.thaliana	70	TWA	16-40	350-400	50
	B2	Z. Mays	1,000	TWA	14	130	15., 41.
	Castaway	O.sativa	1,000	TWA	13	350	18
	Ditto	O.sativa	2,000	TWA	15	300	18
	Explorer	O.sativa	2,000	TWA	13	240	18
	Gaijin	O.sativa	3,000	WWW	17	180	18
	Hbr	Ζ. Mays	4,000	TWW	14	310	31., 34.
	Kiddo	O.sativa	NDb	TWA	14	270	39
	MathE1	A.thaliana	50	TWA	25	400	51
	MPing	O.sativa	14-07	TWA	37-268	430	40., 41.
	Os-mPIF2	O.sativa	150	WWW	14	260	44
	Various	Grasses	> 5, 000	TWA	13-100	100-400	15., 16.
	Tourist
	Various Tourist	A.thaliana	> 300	TWA	> 14	300-500	11
	Tourist
	Wanderer	O.sativa	4,000	TWA	10	300	18
	Zm-mPIF	Ζ. Mays	6,000	TWA	13	350	44
Stowaway-like	Stowaway	Various flowering plants	ND	TA	> 10	70-350	16., 52.
	Various	O.sativa	40,000	TA	20-150	100-350	19., 53.
	Stowaway
	Various	A.thaliana	300	TA	25-100	200-300	11
	Stowaway
pogo-like	AtATE	A.thaliana	1,617	TA	23	742	46
	Emigrant	A.thaliana	500-1,000	TA	24	550	10
Unclassified	Bigfoot	Medicago	> 1, 400	9 bp	9	136-319	30
	Crackle	O.sativa	500	8 bp	SIRsc	385	35
	Hairpin	A.thaliana	10	CTWAR	114	238	54
	Micron	O.sativa	100-200	TA	SIRs	400	36
	Pop	O.sativa	50	8 bp	SIRs	125	35
	Snabo-2	O.sativa	150	4 bp?	107	383	52
	Snap	O.sativa	100	7 bp	SIRs	170	35

N= any nucletide; W = A or T; R= A or C;

ND, not determined;

These elements have no TIRs but have subterminal inverted repeats (SIRs).

Two main obstacles were initially encountered in attempts to classify MITEs with respect to existing transposons. First, none of the available MITE sequences revealed clear-cut relationships with TEs that encode known transposases (autonomous elements). Second, and perhaps more significantly, few MITE family has been shown to be actively transposing 1., 32.. Owing to the fact that MITEs lack coding capacity and little sequence similarity exists between subfamilies, the classification of MITEs becomes complicated. Generally, in plants, the majority of characterized MITE families can be divided into two groups based on similarity of their TIRs and TSDs: Tourist-like and Stowaway-like 33., 36.. Much evidence links Tourist and Stowaway MITEs with two superfamilies of transposases, PIF/Harbinger and Tc1/mariner, respectively 33., 55.. However, a third archetypal element called Emigrant ( is closely related to the pogo-like family of elements ( and has different evolutionary history from the other two (. Table 1 summarizes thorough collection of the previously published plant MITEs as well as those recently generated by the systematic mining of the complete genome sequences.

The discovery of PIFa in maize led to the recognition of a new superfamily of transposases, called PIF/Harbinger (. These transposases are probably of ancient origin as they are distantly related to bacterial IS5 transposases, and have been identified in a wide range of eukaryotes, including various flowering plants, a fungus, and nematodes (. Once PIF-like DNA elements were uncovered in the genomic sequences of rice, Arabidopsis, and nematodes, it became feasible to identify their associated Tourist-like MITEs by searching the respective genomic sequences for non-autonomous members (.

Recently, the first active MITE was identified from rice through systematic computational database search and analysis of the mutability of a rice slender mutation of the glume 33., 40., 57.. The miniature Ping (mPing) element is a sequence classified as a Tourist-like MITE of 430 bp, which is present in about 70 copies in Nipponbare and in about 14 copies in 93-11. These all nearly identical mPing elements transpose actively in an indica cell-culture line ( and are activated in cells derived from anther culture (. An mPing-associated Ping element, which has a putative PIF family transposase, is implicated in the recent proliferation of this MITE family in a subspecies of rice (.

Origin and Amplification of MITEs in Plants

In the absence of coding sequences and an enzymatic activity, it has been difficult to determine how MITEs originate and how they attain their high copy numbers. Although descriptions of MITE families have proliferated in the literature, long-standing mechanistic issues concerning the birth, spread, transposition, and death of MITEs remain enigmatic.

A model suggested that the origin of MITEs involved aberrant DNA replication events when DNA polymerases encountered palindromic sequences as templates (. In this model, the 3′ region of a nascent DNA strand may fold back, allowing DNA synthesis to reinitiate using the nascent DNA strand as templates. A stem loop byproduct (the Angel MITE) may result from this aberrant replication. After its excision from genomic DNA, the stem loop can then integrate into other genomic DNA locations with the help of recombinases, providing new sites for amplification of the MITE (Box 1a). It was illustrated by the distribution of new rice-specific element, Kiddo members with respect to genic region (. In this case, most Kiddo members clustered around the genic region and no MITE was in coding sequences, which indicate that the origin of the groups within the Kiddo family resides in introns. Such an arrangement could yield a very large number of Kiddo copies as a result of transcription and subsequent excision by splicing. Reverse-transcription of a proportion of the excised Kiddo RNA elements into DNA might confer transposition capability, permitting integration into nearby genomic DNA locations. Transposition into introns of other genes could lead to further propagation of Kiddo (. But if the model is correct, excision of MITEs would not occur or remain as rare events, as previously suggested (. Contradicting to this model, a more recent report has concluded that MITE did excise and leave footprints in the process (. This supports the notion that MITEs are DNA transposons and hence should be classified as class II elements.

An alternative model was formulated that Stowaway elements originated by internal deletions from a larger autonomous element (like previously described nonautonomous DNA elements) and were amplified to high copy number by the transposase encoded by MITE (ref.1., 32.; Box 1b). There are three implications in the model. First, a MITE family is composed of subfamilies that have arisen from related autonomous elements in a single genome. A single type of autonomous element can give rise to one or multiple MITE families or can activate nonautonomous elements derived from a related autonomous element 1., 32.. The high sequence identity observed for many MITE families indicates that these families might have spread recently throughout their respective host genomes and the very high copy numbers attributed to many MITE families might be actually resulted from independent amplifications of different subfamilies in the same genome (. This is illustrated best in rice (, where Stowaway MITEs account for over 2% of genomic DNA (. However, upon closer inspection it can be seen that there are more than 30 subfamilies of Stowaway MITEs and most of these has attained a copy number less than 1,000. Only a small number of these subfamilies have reached copy numbers that are significantly greater than 2,000 (. A complex subfamily structure is also observed for Tourist MITEs (which make up ~3% of the rice genome). In contrast, larger genomes harbor very high copy numbers of MITE families. For example, there are over 6,000 copies of mPIF in maize that appear to have arisen from a single ancestral element (. Accordingly, each Stowaway and Tourist MITE subfamily probably arose from the activity of related, but distinct, mariner-like and PIF-like autonomous elements, respectively 1., 33..

Second, MITEs are seen as non-autonomous elements that originated from DNA transposons like previously descried nonautonomous elements in a two-step process. The first step, transposition of an autonomous element, gives rise to various, internally deleted, non-autonomous derivatives. This step is very likely to be dependent on transposases and has been observed for other class II families, such as Ac/Ds or P-elements. In the second step, it is proposed that some derivatives can (for unknown reasons) amplify to high copy numbers. The hypothesis is supported by the recent discovery that rice and other plant genomes contain a tremendous diversity of mariner-like and PIF-like transposases 12., 46., 55., 59.. Zhang et al.( isolated a maize Tourist-like MITE family called miniature PIF (mPIF) that shared several features with PIF elements, and suggested that PIFa and these PIF-like elements belong to a new eukaryotic DNA transposon superfamily that is distantly related to the bacterial IS5 group and are responsible for the origin and spread of Tourist-like MITEs (. Phylogenetic analyses indicated that multiple divergent lineages of mariner-like MITE transposases can coexist within a single plant species and plant transposase sequences are monophyletic and extremely heterogeous (.

The final implication of the model is the possible impact of MITE amplification on the evolution of autonomous elements. The birth and explosive amplification of MITEs could paradoxically be a death sentence for the transposase and consequently for the whole subfamily. However, selection would then lead to the diversification of the transposase favoring variants with altered binding sites, thus ushering in a new cycle of birth and death (. With the identification of an active MITE family, such issues as the birth, spread, transposition, and death of MITEs can now be addressed experimentally. Some issues have been resolved already, for example, the prevalence of MITEs in single-copy regions primarily reflects targeting rather than selection (.

To test the model, a semiautomated computational approach was used to identify and compare mariner-like element (MLEs) and the Stowaway MITEs in two draft genome sequences of rice, because several lines of evidence point to plant MLEs as the autonomous partners of the nonautonomous Stowaway MITEs (. 34 different MLEs were found to group into three major clades and 25 families. More than 22,000 Stowaway MITEs were identified and classified into 36 families. On the basis of detailed sequence comparisons, MLEs were confirmed to be the best candidate autonomous elements for Stowaway MITEs. Surprisingly, however, sequence similarity between MLE and Stowaway families was restricted to TIRs and, in a few cases, to adjacent subterminal sequences. These data suggested that most of the Stowaway MITEs in rice were cross-mobilized by MLE transposases encoded by distantly related elements. The Stowaway MITEs were also gained supports from mPing, which was co-mobilized in cell culture with a closely, but not directly related, autonomous Pong element (. In this model, the origin and amplification of MITEs were considered as two different steps that might be separated by a long period of time (ref. ; Box 1c). The more time elapsed between these two steps, the more difficult it would be to recognize the coordination between a MITE family and an autonomous element. Based on recent research results, we may have to draw a conclusion that there are different mechanisms playing major roles in the origin and amplification of MITEs.

Application of MITEs in Plant Biological Researches

Effective and informative genetic markers

MITEs are useful in systematics and potentially useful as molecular markers because of their features, including high copy number, DNA sequence identity, polymorphism, and genic preference 16., 18., 31., 34., 39.. The insertion sites of MITEs are frequently polymorphic with respect to their presence or absence at a particular locus between individuals of the same species. Because MITE excision seems to be extremely rare, MITE insertion polymorphisms have been successfully exploited as genetic markers 31., 44., 60.. For example, Hbr has been successfully developed as a molecular marker and used for genetic characterization of a set of maize inbred lines 31., 44., 60.. A new powerful marker technology called Inter-MITE polymorphism (IMP) was effectively applied in the fingerprinting of barley cultivars and for genetic similarity analysis (. Many new MITE families, such as Kiddo that exists in a high copy number and share high sequence similarity in subclasses, also appear to have value as new molecular markers (.

There are several merits of the MITE markers. First, compared with SSR, RFLP, and AFLP, MITE-marker possesses higher level of polymorphism (. The level of MITE polymorphism as genetic markers is a reflection of their species specificity (e.g. the extent of restriction site polymorphism) and MITE family specific factors. In the latter case, the extent of polymorphism reflects when each family spread through the population. That is, families that are still active or recently having been active will display higher levels of polymorphism than those active in the more distant past. However, because high sequence identity also correlates with recent amplification, it is anticipated that most families displaying high sequence identity will also be highly polymorphic in mapping population. Second, the ability to map markers with confidence is a function of the reproducibility of the protocol and the ability to unambiguously score segregating bands. In this regard, MITE markers are highly robust (. Casa et al. ( modified the AFLP procedure and developed transposon display (TD) technique to generate and display hundreds of genomic fragments anchored in Hbr elements. TD is a highly reproducible PCR based protocol where multiple fragments are simultaneously detected using only a few primers. Third, compared with AFLP, MITE markers are more randomly distributed in the genome in macroscale (. The distribution of markers in the genome has important implication for the general applicability and utility of the marker class. Randomly distributed markers are desirable as they provide maximum genome coverage. However, the distribution of MITE markers is consistent with the distributions of corresponding MITEs families, which usually prefer to the genic regions. Finally, the segregation distortion and non-parental bands of MITE markers are compatible with the RFLP markers and SSRs (.

An additional source of variation for transposon-anchored markers is transposition. The newly discovered active MITE family mPing in rice 33., 40., 57. will play an important role as a marker. And if one of the parents harbors mobile elements, activity will be easy to discern by the appearance of an unusually high number of new bands or the loss of parental bands in the progeny.

But like the parent AFLP technique, MITE markers are mostly dominant, and their use will be limited in studies where discrimination of multiple alleles at a locus is required (. And the ability to score MITE markers is determined, in part, by the number of amplified fragments displayed in each lane in an electrophoresis system. Adding selective bases to the restriction site-specific primer can reduce this in turn. In addition, the large number of products generated by MITE markers increases the probability of band overlapping among fragments that have the same size, i.e., homoplasy (.

Useful tools for molecular cloning

With the rapid progress of plant genome sequence project, more and more plant genome information becomes available. Insertional mutagenesis is the most suitable method for the systematic functional analysis of a large number of genes in the context of the whole plant. This system allows the production of many mutant lines at one time and the induced mutations can be easily detected by polymerase chain reaction (PCR). In Arabidopsis and rice, whose entire genomic sequences have been completed 61., 62., 63., several insertional mutagens were used to produce a large number of mutant lines. These include T-DNA, the maize transposable elements Ac/Ds and En/Spm, and plant retrotransposon, such as rice Tos17 64., 65.. The mutant populations induced by these mutagens are being used for molecular cloning, sometimes referred as forward and reverse genetics. For forward genetics, traditional transposon tagging is still an important method for cloning important genes for functional analysis.

Recently, the feasibility of tagging using Tos17 has been demonstrated and several important genes have been cloned (. Tos17 becomes active under tissue culture conditions and has been used to develop a large-scale series of rice mutants in the rice genome project. Although this mutant series is regarded as a useful tool for analyzing gene functions, its genetagging efficiency is quite low (5-10%) owing to mutations induced by other factors under tissue-culture conditions. In addition, tissue-culture techniques for the indica subspecies have not been fully developed. Thus, the mobility of the mPing transposon in intact rice plants will provide a useful alternative tool for analyses based on reverse genetics in both the indica and japonica subspecies (, and because mPing seems to be inserted in the genic region like other plant MITEs, it should be a useful molecular tool for gene isolation and gene knockout in these important crop plant species 40., 57..

MITEs are known to be preferentially located in the close vicinity of genes. Active transposes under stress such as anther culture and gamma rays ( will facilitate the tagging and thus isolation of functional genes. Although there has been no report on actual identification and isolation of particular genes, with the discoveries of more active plant MITEs, we are sure to see positive results in the near future.

MITEs and plant evolution

Currently, evolution scenarios of plant species are of great interests to biologists. It has been suggested that MITEs were important tools for evolution studies because their frequent occurrence in the regulatory regions of genes 18., 36.. For example, Gaijin-So1 supplies almost the entire 3′-untranslated region of a sugarcane transporter cDNA; this element most likely supplies both the gene’s polyadenylyation signal and site (. MITEs with a known position in the genome may provide a phylogenetic signal for studying plant species evolution. If a number of taxa have a MITE inserted at exactly the same position on the chromosome, this is a good indication for common origins. The allelic diversity of recently activated or still active element in different species will reflect recent transposition events.

Kanazawa et al. ( examined whether the presence or absence of MITEs may reflect evolutionary events such as speciation, expansion of habitats, and differentiation into ecotypes in wild rice species that share the same AA genome with cultivated rice. They have found that presence or absence of MITEs was highly conserved within each wild rice species except for O. rufipogon. In O. rufipogon, different patterns were detected in different ecotypes and the pattern was conserved within each ecotype. Conserved patterns were observed within each species even when different species showed overlapping distributions, such as O. meridionalis and O. rufipogon in Australia, and O. barthii and O. longistaminata in Africa.

Conclusion

Although MITEs were discovered just ten years ago, numerous classes of MITEs have been found in plants and other organisms. However, the identified active elements are extremely limited, such as the rice mPing element. Why there are so many elements discovered, but so little activity observed? What is the ultimate role of the MITEs to host genome and gene if it does exist? How do we learn about the evolution histories of the elements themselves? Peterson and Seberg ( investigated the mode of Stowaway transposition and tried to trace the evolution of the element by the scrutiny of the phylogenetic tree. Turcotte and Bureau ( suggested that the three main types of MITEs have different evolutionary histories despite their similarity in sequence structure. With the identification of more active elements and experimental studies, great progress is sure to be made on such issues.

Another challenge for the future is to determine whether, and if so how frequently, the sequence diversity created by MITE insertions has altered gene expression or gene products, as well as the fate of elements themselves after insertions. Although some studies have tried to touch these issues, the close proximity of Kiddo members to CDSs suggests that the insertion of these elements could probably modify transcriptional, splicing or translational regulation of the gene (. The IR24 rubq2 promoter that contains the Kiddo insertion has been shown to drive high levels of reporter gene expression in transient assays (. Nevertheless, more systemic researches are definitely needed for the future in revealing the functions and functional consequences of MITEs as biological agents in extant host species.