Solo Long Terminal Repeat Research Articles

Comprehensive Identification and Characterization of HML-9 Group in Chimpanzee Genome.

Endogenous retroviruses (ERVs) are related to long terminal repeat (LTR) retrotransposons, comprising gene sequences of exogenous retroviruses integrated into the host genome and inherited according to Mendelian law. They are considered to have contributed greatly to the evolution of host genome structure and function. We previously characterized HERV-K HML-9 in the human genome. However, the biological function of this type of element in the genome of the chimpanzee, which is the closest living relative of humans, largely remains elusive. Therefore, the current study aims to characterize HML-9 in the chimpanzee genome and to compare the results with those in the human genome. Firstly, we report the distribution and genetic structural characterization of the 26 proviral elements and 38 solo LTR elements of HML-9 in the chimpanzee genome. The results showed that the distribution of these elements displayed a non-random integration pattern, and only six elements maintained a relatively complete structure. Then, we analyze their phylogeny and reveal that the identified elements all cluster together with HML-9 references and with those identified in the human genome. The HML-9 integration time was estimated based on the 2-LTR approach, and the results showed that HML-9 elements were integrated into the chimpanzee genome between 14 and 36 million years ago and into the human genome between 18 and 49 mya. In addition, conserved motifs, cis-regulatory regions, and enriched PBS sequence features in the chimpanzee genome were predicted based on bioinformatics. The results show that pathways significantly enriched for ERV LTR-regulated genes found in the chimpanzee genome are closely associated with disease development, including neurological and neurodevelopmental psychiatric disorders. In summary, the identification, characterization, and genomics of HML-9 presented here not only contribute to our understanding of the role of ERVs in primate evolution but also to our understanding of their biofunctional significance.

Identification and Characterization of the HERV-K (HML-8) Group of Human Endogenous Retroviruses in the Genome.

Human endogenous retroviruses (HERVs) can be vertically transmitted in a Mendelian fashion, are stably maintained in the human genome, and are estimated to constitute ∼8% of the genome. HERVs affect human physiology and pathology through their provirus-encoded protein or long terminal repeat (LTR) element effect. Characterization of the genomic distribution is an essential step to understanding the relationships between endogenous retrovirus expression and diseases. However, the poor characterization of human MMTV-like (HML)-8 prevents a detailed understanding of the regulation of the expression of this family in humans and its impact on the host genome. In light of this, the definition of an accurate and updated HERV-K HML-8 genomic map is urgently needed. In this study, we report the results of a comprehensive analysis of HERV-K HML-8 sequence presence and distribution within the human genome and hominoids, with a detailed description of the different structural and phylogenetic aspects characterizing the group. A total of 40 proviruses and 5 solo LTR elements for human were characterized, which included a detailed description of provirus structure, integration time, potentially regulated genes, transcription factor-binding sites, and primer-binding site features. Besides, 9 chimpanzee sequences, 8 gorilla sequences, and 10 orangutan sequences belonging to the HML-8 subgroup were identified. The integration time results showed that the HML-8 elements were integrated into the primate lineage around 35 and 42 million years ago (mya), during primates evolutionary speciation. Overall, the results clarified the composition of the HML-8 groups, providing an exhaustive background for subsequent functional studies.

Reactivation of transposable elements following hybridization in fission yeast.

Hybridization is thought to reactivate transposable elements (TEs) that were efficiently suppressed in the genomes of the parental hosts. Here, we provide evidence for this “genomic shock hypothesis” in the fission yeast Schizosaccharomyces pombe. In this species, two divergent lineages (Sp and Sk) have experienced recent, likely human-induced, hybridization. We used long-read sequencing data to assemble genomes of 37 samples derived from 31 S. pombe strains spanning a wide range of ancestral admixture proportions. A comprehensive TE inventory revealed exclusive presence of long terminal repeat (LTR) retrotransposons. Sequence analysis of active full-length elements, as well as solo LTRs, revealed a complex history of homologous recombination. Population genetic analyses of syntenic sequences placed insertion of many solo LTRs before the split of the Sp and Sk lineages. Most full-length elements were inserted more recently, after hybridization. With the exception of a single full-length element with signs of positive selection, both solo LTRs and, in particular, full-length elements carry signatures of purifying selection indicating effective removal by the host. Consistent with reactivation upon hybridization, the number of full-length LTR retrotransposons, varying extensively from zero to 87 among strains, significantly increases with the degree of genomic admixture. This study gives a detailed account of global TE diversity in S. pombe, documents complex recombination histories within TE elements, and provides evidence for the “genomic shock hypothesis.”

A spontaneous genetically induced epiallele at a retrotransposon shapes host genome function.

Intracisternal A-particles (IAPs) are endogenous retroviruses (ERVs) responsible for most insertional mutations in the mouse. Full-length IAPs harbour genes flanked by long terminal repeats (LTRs). Here, we identify a solo LTR IAP variant (Iap5-1solo) recently formed in the inbred C57BL/6J mouse strain. In contrast to the C57BL/6J full-length IAP at this locus (Iap5-1full), Iap5-1solo lacks DNA methylation and H3K9 trimethylation. The distinct DNA methylation levels between the two alleles are established during preimplantation development, likely due to loss of KRAB zinc finger protein binding at the Iap5-1solo variant. Iap5-1solo methylation increases and becomes more variable in a hybrid genetic background yet is unresponsive to maternal dietary methyl supplementation. Differential epigenetic modification of the two variants is associated with metabolic differences and tissue-specific changes in adjacent gene expression. Our characterisation of Iap5-1 as a genetically induced epiallele with functional consequences establishes a new model to study transposable element repression and host-element co-evolution.

The Cassandra retrotransposon landscape in sugar beet (Beta vulgaris) and related Amaranthaceae: recombination and re-shuffling lead to a high structural variability.

Plant genomes contain many retrotransposons and their derivatives, which are subject to rapid sequence turnover. As non-autonomous retrotransposons do not encode any proteins, they experience reduced selective constraints leading to their diversification into multiple families, usually limited to a few closely related species. In contrast, the non-coding Cassandra terminal repeat retrotransposons in miniature (TRIMs) are widespread in many plants. Their hallmark is a conserved 5S rDNA-derived promoter in their long terminal repeats (LTRs). As sugar beet (Beta vulgaris) has a well-described LTR retrotransposon landscape, we aim to characterize TRIMs in beet and related genomes. We identified Cassandra retrotransposons in the sugar beet reference genome and characterized their structural relationships. Genomic organization, chromosomal localization, and distribution of Cassandra-TRIMs across the Amaranthaceae were verified by Southern and fluorescent in situ hybridization. All 638 Cassandra sequences in the sugar beet genome contain conserved LTRs and thus constitute a single family. Nevertheless, variable internal regions required a subdivision into two Cassandra subfamilies within B. vulgaris. The related Chenopodium quinoa harbours a third subfamily. These subfamilies vary in their distribution within Amaranthaceae genomes, their insertion times and the degree of silencing by small RNAs. Cassandra retrotransposons gave rise to many structural variants, such as solo LTRs or tandemly arranged Cassandra retrotransposons. These Cassandra derivatives point to an interplay of template switch and recombination processes - mechanisms that likely caused Cassandra's subfamily formation and diversification. We traced the evolution of Cassandra in the Amaranthaceae and detected a considerable variability within the short internal regions, whereas the LTRs are strongly conserved in sequence and length. Presumably these hallmarks make Cassandra a prime target for unequal recombination, resulting in the observed structural diversity, an example of the impact of LTR-mediated evolutionary mechanisms on the host genome.

The unusual dRemp retrotransposon is abundant, highly mutagenic, and mobilized only in the second pollen mitosis of some maize lines

The frequent mutations recovered recently from the pollen of select maize lines resulted from the meiotic mobilization of specific low-copy number long-terminal repeat (LTR) retrotransposons, which differ among lines. Mutations that arise at male meiosis produce kernels with concordant mutant phenotypes in both endosperm and embryo because the two sperms that participate in double fertilization are genetically identical. Those are in a majority. However, a small minority of kernels with a mutant endosperm carry a nonconcordant normal embryo, pointing to a postmeiotic or microgametophytic origin. In this study, we have identified the basis for those nonconcordant mutations. We find that all are produced by transposition of a defective LTR retrotransposon that we have termed dRemp (defective retroelement mobile in pollen). This element has several unique properties. Unlike the mutagenic LTR retrotransposons identified previously, dRemp is present in hundreds of copies in all sequenced lines. It seems to transpose only at the second pollen mitosis because all dRemp insertion mutants are nonconcordant yet recoverable in either the endosperm or the embryo. Although it does not move in most lines, dRemp is highly mobile in the Corn Belt inbred M14, identified earlier by breeders as being highly unstable. Lastly, it can be recovered in an array of structures, ranging from solo LTRs to tandem dRemp repeats containing several internal LTRs, suggestive of extensive recombination during retrotransposition. These results shed further light on the spontaneous mutation process and on the possible basis for inbred instability in maize.

What Can Long Terminal Repeats Tell Us About the Age of LTR Retrotransposons, Gene Conversion and Ectopic Recombination?

LTR retrotransposons constitute a significant part of plant genomes and their evolutionary dynamics play an important role in genome size changes. Current methods of LTR retrotransposon age estimation are based only on LTR (long terminal repeat) divergence. This has prompted us to analyze sequence similarity of LTRs in 25,144 LTR retrotransposons from fifteen plant species as well as formation of solo LTRs. We found that approximately one fourth of nested retrotransposons showed a higher LTR divergence than the pre-existing retrotransposons into which they had been inserted. Moreover, LTR similarity was correlated with LTR length. We propose that gene conversion can contribute to this phenomenon. Gene conversion prediction in LTRs showed potential converted regions in 25% of LTR pairs. Gene conversion was higher in species with smaller genomes while the proportion of solo LTRs did not change with genome size in analyzed species. The negative correlation between the extent of gene conversion and the abundance of solo LTRs suggests interference between gene conversion and ectopic recombination. Since such phenomena limit the traditional methods of LTR retrotransposon age estimation, we recommend an improved approach based on the exclusion of regions affected by gene conversion.

Correlation in Expression between LTR Retrotransposons and Potential Host Cis-Targets during Infection of Antherea pernyi with ApNPV Baculovirus.

The published genome sequence of Antheraea yamamai (Saturnnidae) was used to construct a library of long terminal repeat (LTR)-retrotransposons that is representative of the wild silkmoth (Antherea) genus, and that includes 22,666 solo LTRs and 541 full-length LTRs. The LTR retrotransposons of Antheraea yamamai (AyLTRs) could be classified into the three canonical groups of Gypsy, Copia and Belpao. Eleven AyLTRs contained the env gene element, but the relationship with the env element of baculovirus, particularly A. yamamai and pernyi nucleopolyhedrovirus (AyNPV and ApNPV), was distant. A total of 251 “independent” full-length AyLTRs were identified that were located within 100 kb distance (downstream or upstream) of 406 neighboring genes in A. yamamai. Regulation of these genes might occur in cis by the AyLTRs, and the neighboring genes were found to be enriched in GO terms such as “response to stimulus”, and KEGG terms such as “mTOR signaling pathway” among others. Furthermore, the library of LTR-retrotransposons and the A. yamamai genome were used to identify and analyze the expression of LTR-retrotransposons and genes in ApNPV-infected and non-infected A. pernyi larval midguts, using raw data of a published transcriptome study. Our analysis demonstrates that 93 full-length LTR-retrotransposons are transcribed in the midgut of A. pernyi of which 12 significantly change their expression after ApNPV infection (differentially expressed LTR-retrotransposons or DELs). In addition, the expression of differentially expressed genes (DEGs) and neighboring DELs on the chromosome following ApNPV infection suggests the possibility of regulation of expression of DEGs by DELs through a cis mechanism, which will require experimental verification. When examined in more detail, it was found that genes involved in Notch signaling and stress granule (SG) formation were significantly up-regulated in ApNPV-infected A. pernyi larval midgut. Moreover, several DEGs in the Notch and SG pathways were found to be located in the neighborhood of particular DELs, indicating the possibility of DEG-DEL cross-regulation in cis for these two pathways.

Chromosomal distribution of soybean retrotransposon SORE-1 suggests its recent preferential insertion into euchromatic regions.

Retrotransposons constitute a large portion of plant genomes. The chromosomal distribution of a wide variety of retrotransposons has been analyzed using genome sequencing data in several plants, but the evolutionary profile of transposition has been characterized for a limited number of retrotransposon families. Here, we characterized 96 elements of the SORE-1 family of soybean retrotransposons using genome sequencing data. Insertion time of each SORE-1 element into the genome was estimated on the basis of sequence differences between the 5' and 3' long terminal repeats (LTRs). Combining this estimation with information on the chromosomal location of these elements, we found that the insertion of the existing SORE-1 into gene-rich chromosome arms occurred on average more recently than that into gene-poor pericentromeric regions. In addition, both the number of insertions and the proportion of insertions into chromosome arms profoundly increased after 1 million years ago. Solo LTRs were detected in these regions at a similar frequency, suggesting that elimination of SORE-1 via unequal homologous recombination was unbiased. Taken together, these results suggest the preference of a recent insertion of SORE-1 into chromosome arms comprising euchromatic regions. This notion is contrary to an earlier view deduced from an overall profiling of soybean retrotransposons and suggests that the pattern of chromosomal distribution can be more diverse than previously thought between different families of retrotransposons.

Insertion of a solo LTR retrotransposon associates with spur mutations in 'Red Delicious' apple (Malus×domestica).

Insertion of a solo LTR, which possesses strong bidirectional, stem-specific promoter activities, is associated with the evolution of a dwarfing apple spur mutation. Spur mutations in apple scions revolutionized global apple production. Since long terminal repeat (LTR) retrotransposons are tightly related to natural mutations, inter-retrotransposon-amplified polymorphism technique and genome walking were used to find sequences in the apple genome based on these LTRs. In 'Red Delicious' spur mutants, a novel, 2190-bp insertion was identified as a spur-specific, solo LTR (sLTR) located at the 1038th nucleotide of another sLTR, which was 1536 bp in length. This insertion-within-an-insertion was localized within a preexisting Gypsy-50 retrotransposon at position 3,762,767 on chromosome 4. The analysis of transcriptional activity of the two sLTRs (the 2190- and 1536-bp inserts) indicated that the 2190-bp sLTR is a promoter, capable of bidirectional transcription. GUS expression in the 2190-bp-sense and 2190-bp-antisense transgenic lines was prominent in stems. In contrast, no promoter activity from either the sense or the antisense strand of the 1536-bp sLTR was detected. From ~150kb of DNA on each side of the 2190 bp, sLTR insertion site, corresponding to 300kb of the 'Golden Delicious' genome, 23 genes were predicted. Ten genes had predicted functions that could affect shoot development. This first report, of a sLTR insertion associated with the evolution of apple spur mutation, will facilitate apple breeding, cloning of spur-related genes, and discovery of mechanisms behind dwarf habit.

EnHERV: Enrichment analysis of specific human endogenous retrovirus patterns and their neighboring genes.

Human endogenous retroviruses (HERVs) are flanked by long terminal repeats (LTRs), which contain the regulation part of the retrovirus. Remaining HERVs constitute 7% to 8% of the present day human genome, and most have been identified as solo LTRs. The HERV sequences have been associated with several molecular functions as well as certain diseases in human, but their roles in human diseases are yet to be established. We designed EnHERV to make accessible the identified endogenous retrovirus repetitive sequences from Repbase Update (a database of eukaryotic repetitive elements) that are present in the human genome. Defragmentation process was done to improve the RepeatMasker annotation output. The defragmented elements were used as core database in EnHERV. EnHERV is available at http://sysbio.chula.ac.th/enherv and can be searched using either gene lists of user interest or HERV characteristics. Besides the search function, EnHERV also provides an enrichment analysis function that allows users to perform enrichment analysis between selected HERV characteristics and user-input gene lists, especially genes with the expression profile of a certain disease. EnHERV will facilitate exploratory studies of specific HERV characteristics that control gene expression patterns related to various disease conditions. Here we analyzed 25 selected HERV groups/names from all four HERV superfamilies, using the sense and anti-sense directions of the HERV and gene expression profiles from 49 specific tissue and disease conditions. We found that intragenic HERVs were associated with down-regulated genes in most cancer conditions and in psoriatic skin tissues and associated with up-regulated genes in immune cells particularly from systemic lupus erythematosus (SLE) patients. EnHERV allowed the analysis of how different types of LTRs were differentially associated with specific gene expression profiles in particular disease conditions for further studies into their mechanisms and functions.

Copy-number variation analysis in familial nonsyndromic congenital anomalies of the kidney and urinary tract: Evidence for the causative role of a transposable element-associated genomic rearrangement

Most congenital anomalies of the kidney and urinary tract (CAKUT) are sporadic, but familial occurrence has been described, suggesting a genetic contribution. Copy-number variations (CNVs) were detected in patients with CAKUT to identify possible novel genomic regions associated with CAKUT. CNVs were investigated in 7 children with CAKUT from three unrelated families using array comparative genomic hybridization: female monozygotic twins with bilateral duplex collecting system/vesicoureteral reflux (VUR)/unilateral renal hypodyspasia (URHD); two male siblings with VUR/URHD; 3 male second cousins, one with bilateral VUR/URHD, one with bilateral VUR and one with ureterovesical junction obstruction (UVJO). Five patients had a normal constitution of CNVs, one had a duplication of 0.2 Mb on the 5q-arm (5q23.3), probably unrelated to CAKUT, and one with UVJO had a 1.4 Mb deletion on the 17q-arm (17q12) which includes a known CAKUT gene, HNF1B. The phenotype of HNF1B deletion was extended including renal magnesium wasting. A higher coverage in transposable elements (TEs) was found in the deleted region compared with the expected density in any random genomic region. Notably, the 5′ breakpoint was mapped within a solo long terminal repeat (LTR) sequence. Moreover, highly similar members of solo LTR and mammalian interspersed repetitive (MIR) elements, as well as nucleotide sequence microhomology were detected at the breakpoint regions. In conclusion, the deletion detected in one patient suggests this genomic imbalance as causative for UVJO. A not very well known phenotype of HNF1B deletion resulting in both low urinary tract malformations and renal wasting of magnesium was described. The high load in TEs of the deleted region, the presence of highly similar elements, and the microhomology found at breakpoint regions may have contributed to the generation of the deletion. CNV analysis could reveal novel causative genomic regions in patients with CAKUT, and further studies in larger cohorts are needed.

Genomic Landscape of Long Terminal Repeat Retrotransposons (LTR-RTs) and Solo LTRs as Shaped by Ectopic Recombination in Chicken and Zebra Finch.

Transposable elements (TEs) are nearly ubiquitous among eukaryotic genomes, but TE contents vary dramatically among phylogenetic lineages. Several mechanisms have been proposed as drivers of TE dynamics in genomes, including the fixation/loss of a particular TE insertion by selection or drift as well as structural changes in the genome due to mutation (e.g., recombination). In particular, recombination can have a significant and directional effect on the genomic TE landscape. For example, ectopic recombination removes internal regions of long terminal repeat retrotransposons (LTR-RTs) as well as one long terminal repeat (LTR), resulting in a solo LTR. In this study, we focus on the intra-species dynamics of LTR-RTs and solo LTRs in bird genomes. The distribution of LTR-RTs and solo LTRs in birds is intriguing because avian recombination rates vary widely within a given genome. We used published linkage maps and whole genome assemblies to study the relationship between recombination rates and LTR-removal events in the chicken and zebra finch. We hypothesized that regions with low recombination rates would harbor more full-length LTR-RTs (and fewer solo LTRs) than regions with high recombination rates. We tested this hypothesis by comparing the ratio of full-length LTR-RTs and solo LTRs across chromosomes, across non-overlapping megabase windows, and across physical features (i.e., centromeres and telomeres). The chicken data statistically supported the hypothesis that recombination rates are inversely correlated with the ratio of full-length to solo LTRs at both the chromosome level and in 1-Mb non-overlapping windows. We also found that the ratio of full-length to solo LTRs near chicken telomeres was significantly lower than those ratios near centromeres. Our results suggest a potential role of ectopic recombination in shaping the chicken LTR-RT genomic landscape.

Abstract 2966A: A comprehensive investigation of insertional variations of human endogenous retrovirus elements in tumor tissues

Abstract Human endogenous retrovirus (HERV) is a member of LTR-retrotransposon family. Retroviral invasions are thought to have occurred in the germ cell lines of human or human ancestors, and the fixed retroviral elements are recognized as HERV. A complete HERV sequence contains two long terminal repeats (LTRs) located at both ends. Because intact LTRs possess the highly conserved transcriptional promoter- and enhancer-sequences, to investigate insertional variations of HERV is a key process for understanding the etiology of human genetic diseases that remain unclear with the analyses targeting single nucleotide variations. However, genome-wide investigation of repetitive sequences including HERV is methodologically difficult, and comprehensive understanding of insertional variations of HERV, including a somatic retrotransposition of HERV, remains poor. In the previous study, we have identified two novel insertional polymorphisms of solo LTRs using the inverse PCR and cloning methods, and one of them was involved with the susceptibility to lung adenocarcinoma. Here, we detected multiple novel LTR loci in the human cancer tissues by developing the high-throughput sequencing method combined with inverse PCR. In this system, to preferentially detect insertional variations, primer sequences were designed based on the conserved region of the LTRs which have an identical sequence to the mate within the same HML-2 proviruse, because almost of known HERV insertional polymorphisms are found in HML-2, which is thought to be the most recently endogenized element of HERV, and the presence of an identical LTR sequence at both ends is necessary for the retrotransposition of exogenous retrovirus. The sequence reads generated from the high-throughput sequencer were trimmed, and the fragments consistent with the inverse PCR process were selected. Following the mapping to the reference genome, the sites not corresponding to known LTR loci were sorted out and subjected to the filtration to exclude the artificial fragments. As the result, some candidates of novel LTR loci were detected, and several of them were verified to be actually present both in tumor and non-tumor genomes using the Sanger method, suggesting they are insertional polymorphisms. By using this detection system, we are now challenging to investigate a novel LTR site specifically detected in tumor genome. Citation Format: Tomoaki Kahyo, Hidetaka Yamada, Hong Tao, Yusuke Inoue, Nobuya Kurabe, Haruhiko Sugimura. A comprehensive investigation of insertional variations of human endogenous retrovirus elements in tumor tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2966A. doi:10.1158/1538-7445.AM2015-2966A

Genome-wide amplification of proviral sequences reveals new polymorphic HERV-K(HML-2) proviruses in humans and chimpanzees that are absent from genome assemblies.

BackgroundTo date, the human population census of proviruses of the Betaretrovirus–like human endogenous retroviral (HERV-K) (HML-2) family has been compiled from a limited number of complete genomes, making it certain that rare polymorphic loci are under-represented and are yet to be described.ResultsHere we describe a suppression PCR-based method called genome-wide amplification of proviral sequences (GAPS) that selectively amplifies DNA fragments containing the termini of HERV-K(HML-2) proviral sequences and their flanking genomic sequences. We analysed the HERV-K(HML-2) proviral content of 101 unrelated humans, 4 common chimpanzees and three centre d’etude du polymorphisme humain (CEPH) pedigrees (44 individuals). The technique isolated HERV-K(HML-2) proviruses that had integrated in the genomes of the great apes throughout their divergence and included evolutionarily young elements still unfixed for presence/absence.ConclusionsBy examining the HERV-K(HML-2) proviral content of 145 humans we detected a new insertionally polymorphic Type I HERV-K(HML-2) provirus. We also observed provirus versus solo long terminal repeat (LTR) polymorphism within humans at a previously unreported, but ancient, locus. Finally, we report two novel chimpanzee specific proviruses, one of which is dimorphic for a provirus versus solo LTR. Thus GAPS enables the isolation of uncharacterised HERV-K(HML-2) proviral sequences and provides a direct means to assess inter-individual genetic variation associated with HERV-K(HML-2) proviruses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0162-8) contains supplementary material, which is available to authorized users.

MpSaci is a widespread gypsy-Ty3 retrotransposon highly represented by non-autonomous copies in the Moniliophthora perniciosa genome.

Transposons are an important source of genetic variation. The phytopathogen Moniliophthora perniciosa shows high level of variability but little is known about the role of class I elements in shaping its genome. In this work, we aimed the characterization of a new gypsy/Ty3 retrotransposon species, named MpSaci, in the M. perniciosa genome. These elements are largely variable in size, ranging from 4 to 15kb, and harbor direct long terminal repeats (LTRs) with varying degrees of similarity. Approximately, all of the copies are non-autonomous as shifts in the reading frame and stop codons were detected. Only two elements (MpSaci6 and MpSaci9) code for GAG and POL proteins that possess functional domains. Conserved domains that are typically not found in retrotransposons were detected and could potentially impact the expression of neighbor genes. Solo LTRs and several LARDs (large retrotransposon derivative) were detected. Unusual elements containing small sequences with or without interruptions that are similar to gag or different pol domains and presenting LTRs with different levels of similarities were identified. Methylation was observed in MpSaci reverse transcriptase sequences. Distribution analysis indicates that MpSaci elements are present in high copy number in the genomes of C-, S- and L-biotypes of M. perniciosa. In addition, C-biotype isolates originating from the state of Bahia have fragments in common with isolates from the Amazon region and two hybridization profiles related to two chromosomal groups. RT-PCR analysis reveals that the gag geneis constitutively expressed and that the expression is increased at least three-fold with nutrient depravation even though no new insertion were observed. These findings point out that MpSaci collaborated and, even though is primarily represented by non-autonomous elements, still might contribute to the generation of genetic variability in the most important cacao pathogen in Brazil.

Young but not relatively old retrotransposons are preferentially located in gene-rich euchromatic regions in tomato (Solanum lycopersicum) plants.

Long terminal repeat (LTR) retrotransposons are the major DNA components of flowering plants. They are generally enriched in pericentromeric heterochromatin regions of their host genomes, which could result from the preferential insertion of LTR retrotransposons and the low effectiveness of purifying selection in these regions. To estimate the relative importance of the actions of these two factors on their distribution pattern, the LTR retrotransposons in Solanum lycopersicum (tomato) plants were characterized at the genome level, and then the distribution of young elements was compared with that of relatively old elements. The current data show that old elements are mainly located in recombination-suppressed heterochromatin regions, and that young elements are preferentially located in the gene-rich euchromatic regions. Further analysis showed a negative correlation between the insertion time of LTR retrotransposons and the recombination rate. The data also showed there to be more solo LTRs in genic regions than in intergenic regions or in regions close to genes. These observations indicate that, unlike in many other plant genomes, the current LTR retrotransposons in tomatoes have a tendency to be preferentially located into euchromatic regions, probably caused by their severe suppression of activities in heterochromatic regions. These elements are apt to be maintained in heterochromatin regions, probably as a consequence of the pericentromeric effect in tomatoes. These results also indicate that local recombination rates and intensities of purifying selection in different genomic regions are largely responsible for structural variation and non-random distribution of LTR retrotransposons in tomato plants.

Comparative Genomic Analysis Reveals Multiple Long Terminal Repeats, Lineage-Specific Amplification, and Frequent Interelement Recombination for Cassandra Retrotransposon in Pear (Pyrus bretschneideri Rehd.)

Cassandra transposable elements belong to a specific group of terminal-repeat retrotransposons in miniature (TRIM). Although Cassandra TRIM elements have been found in almost all vascular plants, detailed investigations on the nature, abundance, amplification timeframe, and evolution have not been performed in an individual genome. We therefore conducted a comprehensive analysis of Cassandra retrotransposons using the newly sequenced pear genome along with four other Rosaceae species, including apple, peach, mei, and woodland strawberry. Our data reveal several interesting findings for this particular retrotransposon family: 1) A large number of the intact copies contain three, four, or five long terminal repeats (LTRs) (∼20% in pear); 2) intact copies and solo LTRs with or without target site duplications are both common (∼80% vs. 20%) in each genome; 3) the elements exhibit an overall unbiased distribution among the chromosomes; 4) the elements are most successfully amplified in pear (5,032 copies); and 5) the evolutionary relationships of these elements vary among different lineages, species, and evolutionary time. These results indicate that Cassandra retrotransposons contain more complex structures (elements with multiple LTRs) than what we have known previously, and that frequent interelement unequal recombination followed by transposition may play a critical role in shaping and reshaping host genomes. Thus this study provides insights into the property, propensity, and molecular mechanisms governing the formation and amplification of Cassandra retrotransposons, and enhances our understanding of the structural variation, evolutionary history, and transposition process of LTR retrotransposons in plants.

Quadruplex-forming DNA sequences spread by retrotransposons may serve as genome regulators

Transposable elements (TEs) are ubiquitous genome inhabitants in eukaryotes. Increasing evidence shows that TEs are involved in regulatory networks of eukaryotic cells and contribute to genome evolution. Recently, we reported that many plant long-terminal repeat (LTR) retrotransposons contain DNA quadruplex-forming sequences at precise positions inside their LTRs and that quadruplexes are better preserved in evolutionary younger elements. As quadruplexes can modulate molecular processes, quadruplexes found at specific distances upstream and downstream from the endogenous TE promoter can affect transcription of the element. Moreover, quadruplexes found in solo LTRs, as well as in 3' ends of 5'-truncated copies of LINE-1 elements, can affect expression of neighboring genes. Here, we propose that this way retrotransposons can serve as vehicles for spread of DNA quadruplexes. Quadruplexes can thus fulfill a dual regulatory role-to influence both the retrotransposons carrying them and the neighboring host genes, e.g., by direct effect on transcription or by modifying the local chromatin state. Additionally, four-stranded DNA structures may serve as hotspots for recombination-based genome rearrangements.

Shifts in the evolutionary rate and intensity of purifying selection between two Brassica genomes revealed by analyses of orthologous transposons and relics of a whole genome triplication

Recent sequencing of the Brassica rapa and Brassica oleracea genomes revealed extremely contrasting genomic features such as the abundance and distribution of transposable elements between the two genomes. However, whether and how these structural differentiations may have influenced the evolutionary rates of the two genomes since their split from a common ancestor are unknown. Here, we investigated and compared the rates of nucleotide substitution between two long terminal repeats (LTRs) of individual orthologous LTR-retrotransposons, the rates of synonymous and non-synonymous substitution among triplicated genes retained in both genomes from a shared whole genome triplication event, and the rates of genetic recombination estimated/deduced by the comparison of physical and genetic distances along chromosomes and ratios of solo LTRs to intact elements. Overall, LTR sequences and genic sequences showed more rapid nucleotide substitution in B. rapa than in B. oleracea. Synonymous substitution of triplicated genes retained from a shared whole genome triplication was detected at higher rates in B. rapa than in B. oleracea. Interestingly, non-synonymous substitution was observed at lower rates in the former than in the latter, indicating shifted densities of purifying selection between the two genomes. In addition to evolutionary asymmetry, orthologous genes differentially regulated and/or disrupted by transposable elements between the two genomes were also characterized. Our analyses suggest that local genomic and epigenomic features, such as recombination rates and chromatin dynamics reshaped by independent proliferation of transposable elements and elimination between the two genomes, are perhaps partially the causes and partially the outcomes of the observed inter-specific asymmetric evolution.