Combinatory crossover and recombination at CG-only minimal repeats create versatile genomic constituents across primate and mouse genomes

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A. Maddi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7601515/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Crossover and recombination have significant outcomes in genomic diversity and evolution. We previously reported that minimal repeats (MRs) such as AT-only trinucleotide two-repeat units (T2Us) are primary sites of unequal crossover and recombination across the human genome, the combinatory impact of which result in intricate colonies (distance between consecutive T2Us <500 bp) of biological and evolutionary implications. Methods: We studied the combinatory impact and landscape of all types of CG-only T2Us on crossover and recombination in the human genome. To this end, we mapped CCGCCG, CGGCGG, CGCCGC, GGCGGC, GCGGCG, and CGCCGC. Subsequently, we performed a comparative genomics study of several colonies of diverse sizes in other primates and mouse. Results: Most CG-only T2Us formed colonies that were primarily distributed in genic intervals. These colonies were predominantly larger than the colonies in the intergenic intervals. Dense arrays of combinatory overlapping and non-overlapping recombinants of the T2Us in the same colony indicated unequal crossover and recombination at these units. The colonies formed versatile genomic constituents, including gene parts (promoters, untranslated regions, and exonic/intronic sequences), microsatellites, and minisatellites. Cross-species analysis of several colonies revealed that some of the colonies were dynamically shared as phylogenetically distant as in mouse. These colonies were mainly of maximum complexity and size in human. Conclusions: CG-only T2Us are crossover and recombination hotspots across the genomes of primates and mouse. The combinatory recombination of these MRs creates versatile genomic elements of evolutionary and biological relevance across these genomes. Because of their high CG content, propensity for unequal crossover and recombination, enrichment in genic regions, and cross-species occurrence, CG-only MRs may partly contribute to the formation, architecture, and evolution of euchromatic genomic regions. Human Primate Mouse Minimal repeat Crossover Recombination hotspot Euchromatin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Short tandem repeats (STRs), also known as microsatellites/simple sequence repeats, have significant outcomes in evolutionary, biological, and pathological processes [ 1 – 10 ]. However, only a few studies have focused on the minimal repeats (MRs), for example, two repeats of these genomic elements. It is plausible that these basic repeats correlate with some fundamental phenomena across genomes. We recently reported that several types of MRs are ubiquitous hotspots of crossover, recombination, and consequential colonization (≥ 2 consecutive MRs < 500 bp apart from each other) in the human genome [ 11 – 13 ]. Combinatory crossover of AT-only dinucleotide two-repeat units (D2Us) and trinucleotide two-repeat units (T2Us) form an enormous landscape of recombination and crossover in the human genome that is likely to transform our knowledge of the mechanistic and biological outcomes of these events. Here we extended our study to the combinatory impact of all types of CG-only T2Us in the human genome. We found that combinatory unequal crossover and recombination of CG-only T2Us are the main mechanisms involved in colony development and evolution, which create versatile genomic constituents across the human genome. Subsequently, we studied several colonies of diverse sizes in other species, including primates and mouse. Evidence on the cross-species sharedness of recombination hotspots at the T2U MRs in some of these colonies prompts re-examining the notion that recombination hotspots are rarely (if at all) shared between human and closely related species [ 14 – 20 ]. Methods Whole-genome T2U extraction algorithm The human genome assembly GRCh38. p14 was obtained from the UCSC genome browser ( https://hgdownload.soe.ucsc.edu ) to extract all types of CG-only T2Us from the human genome, including GCGGCG, GCCGCC, GGCGGC, CGCCGG, CCGCCG, and CGGCGG. A Java software package (arabfard/Java_Di_STR_Finder) was developed (available at the following link: https://github.com/arabfard/Java_Di_STR_Finder ) to extract the list of these T2Us and their corresponding locations. The Java program began at the first nucleotide of the genome and repeatedly searched for CG-only T2Us. The program used a window frame of six nucleotides to identify each T2U. Once the program found a unit, it recorded its count and location and continued to search for new T2Us from the next starting nucleotide. To validate the program output, the final list of the identified T2Us was manually evaluated using Ensembl genome browser 114 ( https://asia.ensembl.org/index.html ), and the locations of the T2Us were subsequently determined. The output was classified in an Excel file, in which the start and endpoints of each T2U were determined in the genome. The start and end points of the subsequent T2Us were subtracted to identify colonies (≥ 2 consecutive T2Us of < 500 bp apart). A list of the colonies was obtained, the total number of colonies was determined, and the output was stored in an available format (please see Availability of data and materials). Statistical analysis The Poisson distribution was employed to determine the probability distribution of the colonies, using the following formula: $$\:{\lambda\:}=\frac{Colony\:size\text{*}Whole-genome\:count\:of\:CG-only\:T2Us\:\left(609792\right))}{\text{H}\text{u}\text{m}\text{a}\text{n}\:\text{G}\text{e}\text{n}\text{o}\text{m}\text{e}\:\text{S}\text{i}\text{z}\text{e}\:(\simeq\:3\text{g}\text{b})}$$ For example, the largest CG-only T2U colony in the dataset was C266, and spanned approximately 8 kb of the genomic DNA. On the other hand, the total count of CG-only T2Us was 609,792, resulting in λ = 1.62 for C266, meaning that the expected count of CG-only T2Us in the 8 kb region was 1.62, rather than the observed 266 (Poisson distribution probability = 0). It should be noted that the value “0” is not exactly 0, but extremely small and rounded owing to limited numerical precision. Colony visualization The CG-only T2U recombinants consisted of overlapping and non-overlapping types. For example, GGCGGCG was an overlapping recombinant of GGCGGC and GCGGCG (Fig. 1 A). In the non-overlapping recombinants (Fig. 1 B), the continuity of T2U overlaps was disrupted. For example, CCGCCGCCCGCCG was a non-overlapping recombinant of CCGCCGC and CCGCCG. To facilitate tracking, each type of T2U and recombinant was visualized using specific combinations of highlights and text colors. For simplicity, only alleles that gained repeats are depicted. Cross-species colony comparisons Several colonies of diverse sizes in human were studied in several other species (Table 1 ). Orthologue assignment was based on the Ensembl Genome Browser 114 BLASTN and orthologue/paralogue annotations of this database ( https://asia.ensembl.org/index.html ), in conjunction with gene neighborhood analysis, and literature search. The genome assemblies used were as follows: Chimpanzee: Pan_tro_3.0, Gorilla: gorGor4, Macaque: Mmul_10, Mouse lemur: Mmur_3.0, and Mouse: GRCm39. Results Predominant distribution of CG-only T2U colonies in the genic vs. intergenic intervals In total, 609,792 CG-only T2Us (absolute count) were detected across the human genome, of which 462,792 (75.89%) were arranged in 81,118 colonies (Fig. 2 A). Colony size ranged from 2 to 266 T2Us (Fig. 2 B). Chromosome 1 had a higher colony count (n = 6,887) than other chromosomes (Fig. 2 C). To determine the percentage of each chromosome occupied by the colony intervals, colony intervals in each chromosome were normalized to the length of the chromosome. chromosome 1 had the highest percentage of colonies (8.66% of the chromosome length), followed by chromosomes 19, 17, and 22 (Fig. 2 D). The CG-only T2Us were ubiquitously distributed along all human chromosomes (Fig. 3 A) and primarily in or flanking genic regions (Fig. 3 B). Remarkably, colony sizes were predominantly larger in these regions than the colonies in the intergenic intervals (Fig. 3 C) (Suppl. 1). For example, promoters and 5′ untranslated regions (UTRs) were among the regions of high proportions of colonies of sizes > 10. In an extreme example, although the percentage of CG-only T2Us was minimal in the gene-splicing regions, these regions mainly contained colonies at the extreme large end of the colony size spectrum (colony size ≥ 100). Non-coding RNA (ncRNA) genes were also among the sequences that were enriched by colony sizes > 10. The intergenic regions contained the smallest proportion of colony sizes > 10. Sharedness of T2U crossover/recombination hotspots in primates Among the colonies randomly selected and studied in other species (Table 1 ), several were also detected in other primates. For example, C266 was detected in human, chimpanzee, and gorilla, and not any other species, and C133 was specific to human and chimpanzee. The primate-specific colonies were mainly more complex and larger in size in human. Table 1 Sample CG-only T2U colonies of diverse sizes in human and the orthologous colonies in primates and mouse Colony Formula Colony Interval (Chr:Start-End) Location Colony Size Human Chimpanzee Gorilla Macaque Mouse Lemur Mouse C266 [(GGC)2]148 [(GCG)2]118 [(GGC)2]50 [(GCG)2]37 [(CGG)2]4 ](GGC)2[142 ](GCG2)[138 ](CGG)2[2 22:20354460–20361885 Last exon of FAM230G , Intron 7 of a lncRNA gene ( ENSG0000290950 ) C259 [(GGC)2]36 [(GCC)2]38 [(GCG)2]40 [(CGC)2]49 [(CCG)2]48 [(CGG)2]48 [(GGC)2]3 [(GCC)2]14 [(GCG)2]2 ](CGC)2[12 ](CCG)2[15 ](CGG)2[5 [(GGC)2]3 [(GCC)2]9 [(GCG)2]2 ](CGC)2[5 ](CCG)2[9 ](CGG)2[4 [(GGC)2]2 [(GCC)2]11 [(GCG)2]3 ](CGC)2[6 ](CCG)2[6 ](CGG)2[3 [(GGC)2]4 [(GCC)2]2 [(GCG)2]3 ](CGC)2[3 ](CCG)2[3 ](CGG)2[3 21:8249244–8259593 Novel genes ( ENSG00000277671 , ENSG00000309985 , ENSG00000281383 ) , miR-6724-2 and 5_8S_rRNA C134 [(GGC)2]29 [(GCC)2]21 [(GCG)2]26 [(CGC)2]18 [(CCG)2]17 [(CGG)2]23 [(GGC)2]5 [(GCC)2]8 [(GCG)2]7 [(CGC)2]4 [(CCG)2]4 [(CGG)2]6 [(GGC)2]4 [(GCC)2]6 [(GCG)2]3 [(CGC)2]8 [(CCG)2]6 [(CGG)2]2 [(GGC)2]3 [(GCC)2]2 [(GCG)2]2 [(CGC)2]4 [(CGG)2]2 13:109782140–109787586 Promoter, 5' UTR, Exon 1 of IRS2 ; 5' sequence of ENSG00000275741 RNA gene and ENSG00000300524 novel gene; LINC00676 C133 [(GGC)2]42 [(GCG)2]51 [(CGG)2]40 [(GCC)2]1 [(GCG)2]1 ](CGC)2[3 ](CCG)2[2 8:675038–676522 Intron 3 of ERICH1 C123 [(GGC)2]17 [(GCC)2]13 [(GCG)2]20 [(CGC)2]24 [(CCG)2]24 [(CGG)2]25 [(GGC)2]18 [(GCG)2]26 ](CGG)2[24 [(GGC)2]4 [(GCC)2]1 [(GCG)2]9 ](CGC)2[1 ](CCG)[1 ](CGG)2[7 [(GGC)2]4 [(GCC)2]2 [(GCG)2]4 [(CGC)2]4 [(CCG)2]2 [(CGG)2]4 21:8438626–8442627 5.8S ribosomal RNA N5; intron 2 of ENSG00000305511 RNA gene C122 [(GGC)2]15 [(GCC)2]28 [(GCG)2]11 [(CGC)2]29 [(CCG)2]29 [(CGG)2]10 ](GGC)2]14 [(GCC)2]28 [(GCG)2]11 [(CGC)2]28 [(CCG)2]29 [(CGG)2]10 [(GGC)2]13 [(GCC)2]22 [(GCG)2]10 [(CGC)2]25 [(CCG)2]22 [(CGG)2]8 [(GGC)2]14 [(GCC)2]33 [(GCG)2]8 [(CGC)2]40 [(CCG)2]34 [(CGG)2]7 [(GGC)2]6 [(GCC)2]19 [(GCG)2]7 [(CGC)2]27 [(CCG)2]23 [(CGG)2]5 20:62064415–62066226 Promoter and Exon 1 of TAF4 , miR-3195 ; Exon 1 and 2 of ENSG00000283078 gene C119 [(GGC)2]17 [(GCC)2]11 [(GCG)2]21 [(CGC)2]24 [(CCG)2]22 [(CGG)2]24 ](GGC)2]1 [(GCC)2]1 ](CGC)2[1 ](CCG)2[3 ](CGG)2[1 ](GGC)2]4 [(GCC)2]2 [(GCG)2]3 ](CGC)2[4 ](CCG)2[5 ](CGG)2[4 ](GGC)2]2 [(GCC)2]2 [(GCG)2]1 ](CGC)2[2 ](CCG)2[2 ](CGG)2[3 21:8211381–8215364 Intron 2 of a novel gene ( ENSG00000278996 ); 5.8S ribosomal RNA N2; miR-3648-1; ENSG00000280800 RNA gene C110 [(GGC)2]52 [(CGG)2]58 ](GCC)2[5 ](CCG)2[4 [(GCC)2]3 [(GCG)2]1 [(CGC)2]2 [(CCG)2]2 [(CGG)2]3 6:167421795–167424368 Exon 1 and exon 2 of ENSG00000298440 gene; Intron of ENSG00000298421 C87 [(GGC)2]23 [(GCC)2]16 [(GCG)2]13 [(CGC)2]11 [(CCG)2]12 [(CGG)2]12 [(GGC)2]18 [(GCC)2]14 [(GCG)2]10 [(CGC)2]9 [(CCG)2]10 [(CGG)2]9 [(GGC)2]8 [(GCC)2]1 [(GCG)2]4 [(CCG)2]1 [(CGG)2]3 [(GGC)2]17 [(GCC)2]14 [(GCG)2]8 [(CGC)2]10 [(CCG)2]10 [(CGG)2]7 [(GGC)2]5 [(GCC)2]8 [(GCG)2]1 [(CGC)2]5 [(CCG)2]4 [(CGG)2]2 [(GGC)2]3 [(GCC)2]8 [(CGC)2]1 [(CCG)2]4 [(CGG)2]1 17:2055050–2058974 Promoters and Exon 1 and 2 of HIC1 ; Upstream of ENSG00000304643 RNA gene; Downstream of SMG6 C77 [(GCG)2]25 [(CGG)2]52 ](CCG)2[19 ](CCG)2[7 4:189307407–189309555 Intergenic (~ 16.3 kb upstream of ENSG00000249877 C62 [(CCG)2]62 ](GCC)2[2 ](CGC)2[3 ](CCG)2[7 ](CCG)2[3 ](GCC)2[1 ](CCG)2[1 ](GCC)2[1 ](CCG)2[1 8:1641877–1644115 Intron 8 of DLGAP2 C56 [(GGC)2]1 [(GCG)2]1 [(CGG)2]54 7:158425196–158428347 Intron 2 of PTPRN2 a Colony size, chromosomal location, colony interval, and gene and transcript IDs were based on the human genome as reference. Orthologous colonies in other primates and mouse were identified by BLASTN. Instances in which the colonies were partially or not sequenced for the corresponding loci in a species, or were not detected in a particular species were left blank. b Formulas represent the absolute counts of T2Us. Based on the Poisson distribution, the probability of the occurrence of colonies in this table was 0. Sharedness of CG-only T2U crossover/recombination hotspots beyond primates Several T2U colonies that were studied in other species (Table 1 ) were dynamically shared not only across primates but also in mouse. The absolute count of the T2Us and the composition of the colonies were extensively dynamic across these species, adding multiple layers of complexity in these colonies. For example, C259 (Fig. 4 ) and C122 (Fig. 5) were composed of six types of T2Us and consequently, a versatile repertoire of recombinants. While C259 and the orthologous colonies in chimpanzee and bonobo were composed of overlapping and non-overlapping recombinants, the orthologous colonies of this colony in macaque and mouse lacked non-overlapping recombinants and were composed of overlapping recombinants only (Fig. 4 ). The trend for the increased number and versatility of overlapping and non-overlapping recombinants in this colony reached its maximum in human. In C122, non-overlapping recombinants were also detected in mouse (Fig. 5). In both C259 and C122 and orthologous colonies in other species, we found species-specific compositions of recombinants. Models of the emergence of various overlapping and non-overlapping recombinants in C259 and C122 are provided in the Colony visualization section (Fig. 1 ). CG-only T2U colonies form gene parts and may protect against transposable elements (TEs) Unequal crossover and recombination events at CG-only T2Us form gene parts, such as promoters, UTRs, and exonic/intronic sequences of various genes (Table 1 , Fig. 3 B and C, Fig. 6 ). For example, C266 spans the last exon of FAM230G and intron 7 of a novel gene, ENSG0000290950 . C259 spans several mi-RNAs and novel genes, and C134 is in the 3'-UTR of IRS2 and is expressed in several tissues. C134 also contains binding sites for several TFs such as FOXP1, SMARCA4, and FOS. In another example, C122 spans the promoter, 5'-UTR, and exon 1 of TAF4 . Several examined colonies lacked TEs, while densely surrounded by these elements (Fig. 6 ). Crossover and recombination at CG-only T2Us form STRs/microsatellites and minisatellites The crossover and recombination events at CG-only T2Us lead to the emergence of diverse types of STRs/microsatellites i.e., ≥ 3-repeats, for example, (GGC)7 in C259 (Fig. 4 ) and (GCC)6 in C122 (Fig. 5). These STRs were largely versatile with respect to length and composition across the species studied, and in numerous instances species-specific. For example, the length of (GGC)7 in C259 was detected in human only, and not in any of the orthologous colonies of C259. In some colonies, crossover and recombination led to the emergence of minisatellites. For example, in C266, unequal crossover and recombination events at CG-only T2Us resulted in the formation of a minisatellite composition of extensive dynamicity across human, chimpanzee, and gorilla (Fig. 7 ). The main recombinant across this colony was of the overlapping type, GGCGGCG, which resulted from unequal crossover between GGCGGC and GCGGCG. In human and chimpanzee, all GCGGCG T2Us were consumed in overlapping recombinants, whereas traces of this type of T2U were detectable in gorilla (yellow blocks). In another example, the overlapping recombinant, CGGCGGCG (green blocks), which was a plausible recombination among CGGCGG, GGCGGC, and GCGGCG was detectable in the genomes of chimpanzee and gorilla and depleted in the human genome (Fig. 7 ). A few models for the emergence of the overlapping recombinants in this colony are provided in the Visualization section (Fig. 1 ). C266 is located inside intron 7 of a novel gene ( ENSG00000290950 ), highly transcribed in the testis, and predicted to be a binding site for early growth response 1 (EGR1) (Fig. 6 ). Interestingly, testis cord formation is regulated by EGR1-mediated cell migration [ 21 ]. Based on the ReMap ChiP-seq database, C266 has the densest map of regulatory binding sites in comparison to the flanking sequences (Fig. 6 ). Discussion We recently reported that several types of MRs are ubiquitous hotspots of crossover and recombination across the human genome [ 11 – 13 ]. Here we studied the combinatory impact of CG-only T2Us in the formation of crossover and recombination landscape, which enabled identification of thousands of colonies that spread extensively across all human chromosomes. We studied several colonies of diverse sizes in other primates and mouse, and found that these colonies coincided with highly dynamic and species-specific formulae in these species. Each species studied had a complex formula for these colonies, which resulted from dynamic and species-specific recombination events in these colonies. Not only did the absolute count and the combination of the T2Us differed across the selected species, but the distribution of these units in various types of recombinants also differed, adding additional levels of complexity of the genomic events at these sites. The findings of cross-species sharedness of some of the examined colonies prompts re-examining the hypothesis that recombination hotspots rarely (if at all) occur at the same locus, even between human and closely related species [ 14 – 20 ]. The coincidence of dense arrays of CG-only T2Us and recombinants of these T2Us in the same colony signified T2Us as sites of crossover and recombination. Based on the total count of CG-only T2Us in the human genome size of ~ 3 Gb, the expected occurrence of a CG-only T2U is approximately 1 in every 5 kb of genomic DNA. The exceedingly significant occurrence of colonies based on Poisson distribution further argues against a neutral hypothesis. The complexity of the identified T2Us and the flanking sequences of these units align with previous reports that crossovers are associated with mutations at recombination hotspots in human [ 22 , 23 ]. In contrast with AT-only MR colonies that are mainly distributed in intergenic intervals [ 12 ], most of the CG-only T2U colonies reside in genic regions and form gene parts, such as promoters, UTRs, and exonic/intronic sequences. We found that the colonies in genic regions were predominantly larger in size than the colonies in the intergenic intervals. For an extreme example, the gene-splicing regions contained the largest proportion of colonies of the extreme large size. GC content around splice sites affects splicing through pre-mRNA secondary structures and compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched [ 24 ]. Alternative splicing is proposed to have a significant role in adaptive evolution and plasticity at the cross-species level [ 25 ], highlighting the importance of the colonies residing in splicing intervals, alongside colonies in other genic intervals. The ncRNA genes were also among the sequences that were enriched by larger size colonies. Human ncRNAs are functional RNA molecules that do not code for proteins but are vital for cellular processes like gene expression regulation, genome stability, and cell development [ 26 ]. Overall, it is possible that CG-only T2U colonies are, at least in part, factories of gene synthesis across evolution. In line with this hypothesis, CG-rich sequences are enriched in transcription elements, UTRs, and splice sites [ 27 , 28 ]. Because of their high CG content, propensity for unequal crossover and recombination, enrichment in genic regions, and cross-species occurrence, it is plausible that CG-only MRs partly contribute to the formation, architecture, and evolution of euchromatic genomic regions. This notion is also supported by our previous findings on CG-only D2Us, colonies of which were mainly distributed in genic intervals [ 12 ]. Euchromatin is a lightly packed form of chromatin, characterized by its high CG content, accessibility to transcription factors, and active gene expression [ 29 ]. Chromosomes 1 and 19 were among the top chromosomes enriched by CG-only T2U colonies. Chromosome 1 is the largest human chromosome. Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average [ 30 ]. The unusual pattern of high GC and CpG content in chromosome 19 orthologs, particularly outside gene clusters, is present from human to mouse lemur [ 31 ]. Much CpG variation in chromosome 19 exists both within and between primate species with a portion of this variation occurring in regulatory regions. It is plausible that these unusual characteristics in chromosomes 1 and 19 are partially attributable to the high CG-only T2U crossover/recombination and colonization in these chromosomes. Prior to the discovery of MRs as sites of crossover and recombination, a 13-bp “core” motif “CCTCCCTNNCCAC” and its degenerate versions were reported to strongly correlate with hotspot activity when they occurred in both repeat and nonrepeat DNA, and were binding sites for PRDM9 [ 32 – 34 ]. However, the expression level of PRDM9 explains only a fraction of the hotspots [ 35 , 36 ]. Consistent with this, PRDM9-independent recombination sites are expected to rapidly change the recombination landscape and insert vast evolutionary effects across species [ 11 – 13 , 37 ]. CG T2Us can be relevant in both crossover and non-crossover contexts (the latter referring to the exchange of DNA fragments without exchanging flanking DNA arms). In fact, most recombination interactions in meiosis are of the non-crossover type, as opposed to crossovers, which include flanking chromosomal arms. Evidence indicates that there probably is no non-crossover-specific pathway, and that restoration of intermediate events in a single pairing/recombination pathway promotes synaptonemal complex formation [ 38 ]. Crossovers and recombination at CG-only T2Us form STRs/microsatellites and minisatellites. We found species-specific lengths and compositions of these elements in our cross-species study. STRs and minisatellites are largely linked to evolution, speciation, and disease [ 7 – 10 , 39 – 43 ]. Several CG-only T2U colonies of diverse sizes lacked TEs, but were densely surrounded by these elements. TEs contribute to cell- and species-specific chromatin looping and gene regulation in mammalian genomes [ 44 – 46 ]. Although linked to important biological and evolutionary implications, TEs primarily cause gene disruption and large-scale genomic alterations, including inversions, deletions, and duplications [ 46 ]. Although hypothetical at this time, it is possible that CG-only T2Us, at least in part, protect genic/euchromatic regions of genomes against TEs. Limitations and future research directions Other types of CG-only MRs, such as tetra-, penta- and hexanucleotide two-repeat units, and their combinatory recombination need to be examined in the future studies. Functional studies are also required to shed further light on the mechanistic and biological aspects of this phenomenon. Moreover, comprehensive cross-species studies are required to further investigate the evolutionary extent and impact of this phenomenon. Conclusions In conclusion, CG-only T2Us are recombination hotspots across primate and mouse genomes. Colonies formed because of these recombination events create versatile genomic constituents of biological and evolutionary relevance. In conjunction with other types of MRs, such as D2Us, CG-only T2U-mediated crossover and recombination may be involved in the formation and evolution of euchromatic regions. Abbreviations C Colony D2U Dinucleotide 2-repeat unit STR Short tandem repeat NcRNA Non-coding RNA TE Transposable element TF Transcription factor T2U Trinucleotide 2-repeat unit UTR Untranslated region Glossary T2U Two repeats of CG-only trinucleotides. For example, CGGCGG and CGCCGC are T2Us. Absolute count Count of T2Us, regardless of being isolate or part of a recombinant. Colony Three or more consecutive T2Us of <500 bp apart. Throughout the paper, specific colonies were named by adding "C" prefix. Colony size was based on the human genome. For example, C266 was a colony of 266 T2Us in human. Colony size Absolute count of consecutive T2Us of <500 bp apart from each other. Overlapping recombinant Recombinant of T2Us, as a result of overlapping sequences. For example, TATATA is an overlapping recombinant resulting from unequal crossover between TATA and TATA overlaps. In another example, ATATATA is an overlapping recombinant between ATATAT and TATATA overlaps. In this type of recombinant, T2Us overlap. Non-overlapping recombinant Non-overlapping recombinant of T2Us. For example, CGGCGGCCGCCG is a recombinant resulting from non-overlapping recombination between CGGCGG and CCGCCG. Crossover The exchange of DNA between paired homologous chromosomes (one from each parent) that occurs during the development of egg and sperm cells (meiosis). Unequal crossover Unequal crossing-over, also referred to as illegitimate recombination, refers to crossover events that occur between nonequivalent sequences. Non-crossover Recombination interactions, which include DNA fragments, without exchange of the flanking chromosome arms. Recombination hotspot A genomic region (typically in ~kb ranges) that experience intensely high levels of recombination compared to the genomic background. Short tandem repeat Repeats of ≥3. Declarations Ethics approval and consent to participate Not applicable. Availability of data and materials The dataset obtained and subsequently analyzed in this research is available at the following Figshare link: https://figshare.com/articles/dataset/_strong_Genome_wide_human_list_of_all_possible_two-repeats_units_of_CG-rich_trinucleotides_strong_/23612943?file=57431413 Competing Interests The authors have no competing interests to declare that are relevant to the content of this article. Funding No funding was received for conducting this study. Authorship contribution statement Conceptualization: MO; Methodology: MA, AMAM; Investigation: DA; HB, MA, NT, SKh, SA; Visualization: HB, NT, SA; Project administration: MO, AD, HRKH; Supervision: MO; Writing – original draft: MO, MA; Writing – review & editing: MO, HB. All authors read and approved the final manuscript. Acknowledgment Not applicable. References Horton, C.A., A.M. Alexandari, M.G.B. Hayes, E. Marklund, J.M. Schaepe, A.K. Aditham, et al., Short tandem repeats bind transcription factors to tune eukaryotic gene expression. Science, 2023. 381 (6664): p. eadd1250. https://doi.org/10.1126/science.add1250. Maddi, A.M.A., K. Kavousi, M. Arabfard, H. Ohadi, and M. Ohadi, Tandem repeats ubiquitously flank and contribute to translation initiation sites. BMC Genom Data, 2022. 23 (1): p. 59. https://doi.org/10.1186/s12863-022-01075-5. Ohadi, M., E. Valipour, S. Ghadimi-Haddadan, P. Namdar-Aligoodarzi, A. Bagheri, A. 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08:17:24","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":177623,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7601515/v1/fb2ef2960950268190e5d58d.html"},{"id":94175099,"identity":"5ec2d5a1-05e9-4f2e-b61b-50ee97755d3c","added_by":"auto","created_at":"2025-10-23 08:17:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":173411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColony visualization. \u003c/strong\u003eUnequal crossovers among T2Us led to overlapping \u003cstrong\u003eA) \u003c/strong\u003eand non-overlapping recombinants \u003cstrong\u003eB)\u003c/strong\u003e. Alternative pathways may result in the emergence of the same recombinant, a few models of which are depicted. To facilitate tracking, each type of T2U and recombinants were visualized using specific combinations of text colors and highlights. These models reflect only a sample of the colonies that are visualized (C266, C259, and 122). For simplicity, only alleles gaining repeats are depicted.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7601515/v1/268fdd8cc44ba09320feb52c.png"},{"id":94176297,"identity":"68faf2ea-5270-4211-a7fb-3fcb128c6ca4","added_by":"auto","created_at":"2025-10-23 08:25:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":118572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCounts and chromosome-level distribution of CG-only T2Us and colonies in the human genome. \u003c/strong\u003eThe absolute counts of CG-only T2Us in colonies vs. \u0026nbsp;genome-wide are depicted \u003cstrong\u003eA)\u003c/strong\u003e. Most T2Us were arranged in colonies. The colonies ranged from 2 to 266 T2Us \u003cstrong\u003eB)\u003c/strong\u003e. Chromosome-by-chromosome colony count \u003cstrong\u003eC)\u003c/strong\u003e. Normalized percentage of each chromosome occupied by colonies \u003cstrong\u003eD)\u003c/strong\u003e. Chromosomes 1, 19, 17, and 22 showed the highest percentage of enrichment by colonies compared with other chromosomes. T2U: Trinucleotide 2-repeat Unit; CUG: Count of Units in Genome; CUC: Count of Units in Colony\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7601515/v1/316d930559f8ceffcbcdcac7.png"},{"id":94175101,"identity":"a5ce5d2c-c989-4a4c-bf02-b3a5476d3381","added_by":"auto","created_at":"2025-10-23 08:17:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":175790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of CG-only T2Us and colonies in the human genome. \u003c/strong\u003eWhile CG-only T2Us (outer circle) and colonies (inner circle) were ubiquitously spread along all chromosomes \u003cstrong\u003eA)\u003c/strong\u003e, these elements were primarily enriched in or flanking genic region \u003cstrong\u003eB)\u003c/strong\u003e. The colonies residing in genic regions were predominantly larger in size than the colonies in the intergenic intervals \u003cstrong\u003eC)\u003c/strong\u003e. ncRNA=non-coding RNA.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7601515/v1/c0f96d63b183a7a0daae4596.png"},{"id":94175100,"identity":"a117a9d2-4c0b-41c9-9d10-e9514b415dbd","added_by":"auto","created_at":"2025-10-23 08:17:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1366985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExample of a colony dynamically shared across primates and mouse (C259).\u003c/strong\u003e The absolute count of CG-only T2Us and the composition of the colonies were highly dynamic in the studied species. Overlapping and non-overlapping recombinants reached their maximum count and versatility in human. Mouse and macaque lacked non-overlapping recombinants in this colony and the recombinants were of the overlapping type only.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7601515/v1/5fcff77f9d5b66db55bf1c12.png"},{"id":94175104,"identity":"ce3db1eb-58a3-4fb2-b595-6954ac1655ac","added_by":"auto","created_at":"2025-10-23 08:17:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1142813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComplex recombinants in a colony across primates and mouse (C122).\u003c/strong\u003e Extensive divergence was observed across the studied species in this colony. Overlapping and non-overlapping recombinants were detectable across primates and mouse.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7601515/v1/73a520fb81173cbe6953c805.png"},{"id":94175111,"identity":"84df457d-6dea-496e-95bd-73a9caaabb73","added_by":"auto","created_at":"2025-10-23 08:17:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":408776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCG-only T2U colonies form gene parts across primate and mouse genomes. \u003c/strong\u003eSample colonies form gene parts, such as regulatory and coding sequences. For example, C266 is transcribed in the testis, and ChIP-seq data indicate EGR1 binding sites (orange rectangles) in this colony. C259 is transcribed in several tissues, including whole blood, artery aorta, stomach, pituitary, and brain. This colony spans several non-coding genes such as miR-6724-2. C134 is transcribed in several tissues including the prostate and ovaries. Different transcription factors (orange rectangles) contain binding sites for C134. C123 spans several RNA genes. C122 encompasses the promoter and 5’-UTR of \u003cem\u003eTAF4\u003c/em\u003e. miR-3195 is also transcribed in this colony. ENCODE data indicated the enrichment of acetylated histone 3 lysine 27 (H3K27Ac), which correlates with chromatin open status. C87 spans promoters (HIC1_1 and HIC1_2) and exons 1 and 2 of the tumor suppressor gene \u003cem\u003eHIC1\u003c/em\u003e. The colonies lack TEs, while surrounded by these elements. The colonies are indicated by yellow highlight. Data were obtained from the UCSC ENCODE (\u003ca href=\"https://genome.ucsc.edu/\"\u003ehttps://genome.ucsc.edu/\u003c/a\u003e).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7601515/v1/5cef405de625afb67ec7e20f.png"},{"id":94176598,"identity":"664afa5a-e5c1-499a-9a0d-40abbe2939b7","added_by":"auto","created_at":"2025-10-23 08:33:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":664380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmergence of minisatellites as a result of unequal crossover and recombination at CG-only T2Us. \u003c/strong\u003eUnequal crossover and recombination at CG-only T2Us led to the development of minisatellites in some colonies such as C266. This colony was dynamically shared among human, chimpanzee, and gorilla, and not any other species. Various T2U types and recombinants were detectable because of unequal crossover at these sites (models are provided in Fig. 1). The main recombinant, GGCGGCG (navy blocks), was a result of unequal crossover between GGCGGC (blue blocks) and GCGGCG (yellow blocks), the latter of which was all consumed in the overlapping units in human and chimpanzee, whereas in gorilla, several of this T2U were detected in the colony. Moreover, overlapping blocks were detected in gorilla and chimpanzee, such as CGGCGGCG (green blocks), that were depleted from the human colony.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7601515/v1/9397de31079da1e4b4e51e46.png"},{"id":104690929,"identity":"ee852d59-44bc-4083-9eaf-727794665a97","added_by":"auto","created_at":"2026-03-16 06:11:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5996480,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7601515/v1/939b7a7b-0006-4fd5-85b4-a35998a47f21.pdf"},{"id":94176305,"identity":"4a935454-14ac-44e6-bd40-cc500441270b","added_by":"auto","created_at":"2025-10-23 08:25:24","extension":"txt","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5374126,"visible":true,"origin":"","legend":"","description":"","filename":"T2Usannotated.txt","url":"https://assets-eu.researchsquare.com/files/rs-7601515/v1/a54173d413c1dc8e60d6b6b5.txt"}],"financialInterests":"No competing interests reported.","formattedTitle":"Combinatory crossover and recombination at CG-only minimal repeats create versatile genomic constituents across primate and mouse genomes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eShort tandem repeats (STRs), also known as microsatellites/simple sequence repeats, have significant outcomes in evolutionary, biological, and pathological processes [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, only a few studies have focused on the minimal repeats (MRs), for example, two repeats of these genomic elements. It is plausible that these basic repeats correlate with some fundamental phenomena across genomes. We recently reported that several types of MRs are ubiquitous hotspots of crossover, recombination, and consequential colonization (\u0026ge;\u0026thinsp;2 consecutive MRs\u0026thinsp;\u0026lt;\u0026thinsp;500 bp apart from each other) in the human genome [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Combinatory crossover of AT-only dinucleotide two-repeat units (D2Us) and trinucleotide two-repeat units (T2Us) form an enormous landscape of recombination and crossover in the human genome that is likely to transform our knowledge of the mechanistic and biological outcomes of these events. Here we extended our study to the combinatory impact of all types of CG-only T2Us in the human genome. We found that combinatory unequal crossover and recombination of CG-only T2Us are the main mechanisms involved in colony development and evolution, which create versatile genomic constituents across the human genome. Subsequently, we studied several colonies of diverse sizes in other species, including primates and mouse. Evidence on the cross-species sharedness of recombination hotspots at the T2U MRs in some of these colonies prompts re-examining the notion that recombination hotspots are rarely (if at all) shared between human and closely related species [\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eWhole-genome T2U extraction algorithm\u003c/p\u003e\u003cp\u003eThe human genome assembly GRCh38. p14 was obtained from the UCSC genome browser (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hgdownload.soe.ucsc.edu\u003c/span\u003e\u003cspan address=\"https://hgdownload.soe.ucsc.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to extract all types of CG-only T2Us from the human genome, including GCGGCG, GCCGCC, GGCGGC, CGCCGG, CCGCCG, and CGGCGG. A Java software package (arabfard/Java_Di_STR_Finder) was developed (available at the following link: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/arabfard/Java_Di_STR_Finder\u003c/span\u003e\u003cspan address=\"https://github.com/arabfard/Java_Di_STR_Finder\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to extract the list of these T2Us and their corresponding locations. The Java program began at the first nucleotide of the genome and repeatedly searched for CG-only T2Us. The program used a window frame of six nucleotides to identify each T2U. Once the program found a unit, it recorded its count and location and continued to search for new T2Us from the next starting nucleotide. To validate the program output, the final list of the identified T2Us was manually evaluated using Ensembl genome browser 114 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://asia.ensembl.org/index.html\u003c/span\u003e\u003cspan address=\"https://asia.ensembl.org/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the locations of the T2Us were subsequently determined. The output was classified in an Excel file, in which the start and endpoints of each T2U were determined in the genome. The start and end points of the subsequent T2Us were subtracted to identify colonies (\u0026ge;\u0026thinsp;2 consecutive T2Us of \u0026lt;\u0026thinsp;500 bp apart). A list of the colonies was obtained, the total number of colonies was determined, and the output was stored in an available format (please see Availability of data and materials).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe Poisson distribution was employed to determine the probability distribution of the colonies, using the following formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:{\\lambda\\:}=\\frac{Colony\\:size\\text{*}Whole-genome\\:count\\:of\\:CG-only\\:T2Us\\:\\left(609792\\right))}{\\text{H}\\text{u}\\text{m}\\text{a}\\text{n}\\:\\text{G}\\text{e}\\text{n}\\text{o}\\text{m}\\text{e}\\:\\text{S}\\text{i}\\text{z}\\text{e}\\:(\\simeq\\:3\\text{g}\\text{b})}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFor example, the largest CG-only T2U colony in the dataset was C266, and spanned approximately 8 kb of the genomic DNA. On the other hand, the total count of CG-only T2Us was 609,792, resulting in λ\u0026thinsp;=\u0026thinsp;1.62 for C266, meaning that the expected count of CG-only T2Us in the 8 kb region was 1.62, rather than the observed 266 (Poisson distribution probability\u0026thinsp;=\u0026thinsp;0). It should be noted that the value \u0026ldquo;0\u0026rdquo; is not exactly 0, but extremely small and rounded owing to limited numerical precision.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eColony visualization\u003c/h3\u003e\n\u003cp\u003eThe CG-only T2U recombinants consisted of overlapping and non-overlapping types. For example, GGCGGCG was an overlapping recombinant of GGCGGC and GCGGCG (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In the non-overlapping recombinants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), the continuity of T2U overlaps was disrupted. For example, CCGCCGCCCGCCG was a non-overlapping recombinant of CCGCCGC and CCGCCG. To facilitate tracking, each type of T2U and recombinant was visualized using specific combinations of highlights and text colors. For simplicity, only alleles that gained repeats are depicted.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eCross-species colony comparisons\u003c/h3\u003e\n\u003cp\u003eSeveral colonies of diverse sizes in human were studied in several other species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOrthologue assignment was based on the Ensembl Genome Browser 114 BLASTN and\u003c/p\u003e\u003cp\u003eorthologue/paralogue annotations of this database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://asia.ensembl.org/index.html\u003c/span\u003e\u003cspan address=\"https://asia.ensembl.org/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), in\u003c/p\u003e\u003cp\u003econjunction with gene neighborhood analysis, and literature search. The genome assemblies used\u003c/p\u003e\u003cp\u003ewere as follows: Chimpanzee: Pan_tro_3.0, Gorilla: gorGor4, Macaque: Mmul_10, Mouse lemur:\u003c/p\u003e\u003cp\u003eMmur_3.0, and Mouse: GRCm39.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003ePredominant distribution of CG-only T2U colonies in the genic vs. intergenic intervals\u003c/h2\u003e\u003cp\u003eIn total, 609,792 CG-only T2Us (absolute count) were detected across the human genome, of which 462,792 (75.89%) were arranged in 81,118 colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Colony size ranged from 2 to 266 T2Us (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Chromosome 1 had a higher colony count (n\u0026thinsp;=\u0026thinsp;6,887) than other chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To determine the percentage of each chromosome occupied by the colony intervals, colony intervals in each chromosome were normalized to the length of the chromosome. chromosome 1 had the highest percentage of colonies (8.66% of the chromosome length), followed by chromosomes 19, 17, and 22 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe CG-only T2Us were ubiquitously distributed along all human chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and primarily in or flanking genic regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Remarkably, colony sizes were predominantly larger in these regions than the colonies in the intergenic intervals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) (Suppl. 1). For example, promoters and 5\u0026prime; untranslated regions (UTRs) were among the regions of high proportions of colonies of sizes\u0026thinsp;\u0026gt;\u0026thinsp;10. In an extreme example, although the percentage of CG-only T2Us was minimal in the gene-splicing regions, these regions mainly contained colonies at the extreme large end of the colony size spectrum (colony size\u0026thinsp;\u0026ge;\u0026thinsp;100). Non-coding RNA (ncRNA) genes were also among the sequences that were enriched by colony sizes\u0026thinsp;\u0026gt;\u0026thinsp;10. The intergenic regions contained the smallest proportion of colony sizes\u0026thinsp;\u0026gt;\u0026thinsp;10.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eSharedness of T2U crossover/recombination hotspots in primates\u003c/h2\u003e\u003cp\u003eAmong the colonies randomly selected and studied in other species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), several were also detected in other primates. For example, C266 was detected in human, chimpanzee, and gorilla, and not any other species, and C133 was specific to human and chimpanzee. The primate-specific colonies were mainly more complex and larger in size in human.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSample CG-only T2U colonies of diverse sizes in human and the orthologous colonies in primates and mouse\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003eColony Formula\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eColony Interval (Chr:Start-End)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eLocation\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eColony Size\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHuman\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChimpanzee\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGorilla\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMacaque\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMouse Lemur\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMouse\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC266\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[(GGC)2]148\u003c/p\u003e\u003cp\u003e[(GCG)2]118\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e[(GGC)2]50\u003c/p\u003e\u003cp\u003e[(GCG)2]37 [(CGG)2]4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e](GGC)2[142\u003c/p\u003e\u003cp\u003e](GCG2)[138\u003c/p\u003e\u003cp\u003e](CGG)2[2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e22:20354460\u0026ndash;20361885\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eLast exon of \u003cem\u003eFAM230G\u003c/em\u003e,\u003c/p\u003e\u003cp\u003eIntron 7 of a lncRNA gene (\u003cem\u003eENSG0000290950\u003c/em\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC259\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]36\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]38\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]40\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGC)2]49\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CCG)2]48\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGG)2]48\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]3 [(GCC)2]14 [(GCG)2]2 ](CGC)2[12 ](CCG)2[15 ](CGG)2[5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]3 [(GCC)2]9\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]2 ](CGC)2[5\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e](CCG)2[9\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e](CGG)2[4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]2\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]11 [(GCG)2]3 ](CGC)2[6 ](CCG)2[6 ](CGG)2[3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]4\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]2 [(GCG)2]3 ](CGC)2[3 ](CCG)2[3 ](CGG)2[3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e21:8249244\u0026ndash;8259593\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eNovel genes (\u003c/b\u003e\u003cb\u003eENSG00000277671\u003c/b\u003e, \u003cb\u003eENSG00000309985\u003c/b\u003e, \u003cb\u003eENSG00000281383\u003c/b\u003e\u003cb\u003e)\u003c/b\u003e, \u003cb\u003emiR-6724-2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003e5_8S_rRNA\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC134\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]29\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]21\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]26\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGC)2]18\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CCG)2]17\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGG)2]23\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]5\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]8\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]7\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGC)2]4\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CCG)2]4\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGG)2]6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]4\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]6\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]3\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGC)2]8 [(CCG)2]6\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGG)2]2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]3\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]2\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]2\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGC)2]4\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGG)2]2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e13:109782140\u0026ndash;109787586\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003ePromoter, 5' UTR, Exon 1 of\u003c/b\u003e \u003cb\u003eIRS2\u003c/b\u003e; \u003cb\u003e5' sequence of ENSG00000275741 RNA gene and\u003c/b\u003e \u003cb\u003eENSG00000300524\u003c/b\u003e \u003cb\u003enovel gene;\u003c/b\u003e \u003cb\u003eLINC00676\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC133\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]42\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]51 [(CGG)2]40\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]1 [(GCG)2]1 ](CGC)2[3 ](CCG)2[2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e8:675038\u0026ndash;676522\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eIntron 3 of\u003c/b\u003e \u003cb\u003eERICH1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC123\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]17\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]13 [(GCG)2]20 [(CGC)2]24 [(CCG)2]24 [(CGG)2]25\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]18\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]26\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e](CGG)2[24\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]4\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]1\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]9\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e](CGC)2[1\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e](CCG)[1\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e](CGG)2[7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]4\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]2\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]4\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGC)2]4 [(CCG)2]2 [(CGG)2]4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e21:8438626\u0026ndash;8442627\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e5.8S ribosomal RNA N5; intron 2 of\u003c/b\u003e \u003cb\u003eENSG00000305511\u003c/b\u003e \u003cb\u003eRNA gene\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC122\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]15 [(GCC)2]28\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]11 [(CGC)2]29\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CCG)2]29 [(CGG)2]10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e](GGC)2]14\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]28\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]11\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGC)2]28\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CCG)2]29\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGG)2]10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]13 [(GCC)2]22 [(GCG)2]10 [(CGC)2]25 [(CCG)2]22 [(CGG)2]8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]14\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]33\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]8 [(CGC)2]40\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CCG)2]34\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGG)2]7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]6\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]19\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]7 [(CGC)2]27\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CCG)2]23 [(CGG)2]5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e20:62064415\u0026ndash;62066226\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003ePromoter and Exon 1 of\u003c/b\u003e \u003cb\u003eTAF4\u003c/b\u003e, \u003cb\u003emiR-3195\u003c/b\u003e; \u003cb\u003eExon 1 and 2 of\u003c/b\u003e \u003cb\u003eENSG00000283078\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC119\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]17 [(GCC)2]11 [(GCG)2]21 [(CGC)2]24 [(CCG)2]22 [(CGG)2]24\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e](GGC)2]1 [(GCC)2]1 ](CGC)2[1 ](CCG)2[3 ](CGG)2[1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e](GGC)2]4 [(GCC)2]2 [(GCG)2]3 ](CGC)2[4 ](CCG)2[5 ](CGG)2[4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e](GGC)2]2 [(GCC)2]2 [(GCG)2]1 ](CGC)2[2 ](CCG)2[2 ](CGG)2[3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e21:8211381\u0026ndash;8215364\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eIntron 2 of a novel gene (\u003c/b\u003e\u003cb\u003eENSG00000278996\u003c/b\u003e\u003cb\u003e); 5.8S ribosomal RNA N2; miR-3648-1;\u003c/b\u003e \u003cb\u003eENSG00000280800\u003c/b\u003e\u0026nbsp;\u003cb\u003eRNA gene\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC110\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]52\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGG)2]58\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e](GCC)2[5 ](CCG)2[4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]3\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]1\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGC)2]2 [(CCG)2]2 [(CGG)2]3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e6:167421795\u0026ndash;167424368\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eExon 1 and exon 2 of\u003c/b\u003e \u003cb\u003eENSG00000298440\u003c/b\u003e \u003cb\u003egene;\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIntron of\u003c/b\u003e \u003cb\u003eENSG00000298421\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC87\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]23 [(GCC)2]16 [(GCG)2]13 [(CGC)2]11 [(CCG)2]12 [(CGG)2]12\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]18 [(GCC)2]14 [(GCG)2]10 [(CGC)2]9 [(CCG)2]10 [(CGG)2]9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]8\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCC)2]1\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]4\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CCG)2]1 [(CGG)2]3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]17 [(GCC)2]14\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]8 [(CGC)2]10 [(CCG)2]10 [(CGG)2]7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]5 [(GCC)2]8 [(GCG)2]1 [(CGC)2]5 [(CCG)2]4 [(CGG)2]2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]3 [(GCC)2]8 [(CGC)2]1 [(CCG)2]4 [(CGG)2]1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e17:2055050\u0026ndash;2058974\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003ePromoters and Exon 1 and 2 of\u003c/b\u003e \u003cb\u003eHIC1\u003c/b\u003e; \u003cb\u003eUpstream of\u003c/b\u003e \u003cb\u003eENSG00000304643\u003c/b\u003e \u003cb\u003eRNA gene; Downstream of\u003c/b\u003e \u003cb\u003eSMG6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC77\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]25\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(CGG)2]52\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e](CCG)2[19\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e](CCG)2[7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e4:189307407\u0026ndash;189309555\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eIntergenic\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e(~\u0026thinsp;16.3 kb upstream of\u003c/b\u003e \u003cb\u003eENSG00000249877\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC62\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e[(CCG)2]62\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e](GCC)2[2 ](CGC)2[3 ](CCG)2[7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e](CCG)2[3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e](GCC)2[1 ](CCG)2[1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e](GCC)2[1 ](CCG)2[1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e8:1641877\u0026ndash;1644115\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eIntron 8 of\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDLGAP2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC56\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e[(GGC)2]1\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e[(GCG)2]1 [(CGG)2]54\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e7:158425196\u0026ndash;158428347\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eIntron 2 of\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePTPRN2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Colony size, chromosomal location, colony interval, and gene and transcript IDs were based on the human genome as reference. Orthologous colonies in other primates and mouse were identified by BLASTN. Instances in which the colonies were partially or not sequenced for the corresponding loci in a species, or were not detected in a particular species were left blank.\u003c/p\u003e\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e Formulas represent the absolute counts of T2Us. Based on the Poisson distribution, the probability of the occurrence of colonies in this table was 0.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSharedness of CG-only T2U crossover/recombination hotspots beyond primates\u003c/h3\u003e\n\u003cp\u003eSeveral T2U colonies that were studied in other species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were dynamically shared not only across primates but also in mouse. The absolute count of the T2Us and the composition of the colonies were extensively dynamic across these species, adding multiple layers of complexity in these colonies. For example, C259 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and C122 (Fig.\u0026nbsp;5) were composed of six types of T2Us and consequently, a versatile repertoire of recombinants. While C259 and the orthologous colonies in chimpanzee and bonobo were composed of overlapping and non-overlapping recombinants, the orthologous colonies of this colony in macaque and mouse lacked non-overlapping recombinants and were composed of overlapping recombinants only (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The trend for the increased number and versatility of overlapping and non-overlapping recombinants in this colony reached its maximum in human. In C122, non-overlapping recombinants were also detected in mouse (Fig.\u0026nbsp;5). In both C259 and C122 and orthologous colonies in other species, we found species-specific compositions of recombinants. Models of the emergence of various overlapping and non-overlapping recombinants in C259 and C122 are provided in the Colony visualization section (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eCG-only T2U colonies form gene parts and may protect against transposable elements (TEs)\u003c/h3\u003e\n\u003cp\u003eUnequal crossover and recombination events at CG-only T2Us form gene parts, such as promoters, UTRs, and exonic/intronic sequences of various genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). For example, C266 spans the last exon of \u003cem\u003eFAM230G\u003c/em\u003e and intron 7 of a novel gene, \u003cem\u003eENSG0000290950\u003c/em\u003e. C259 spans several mi-RNAs and novel genes, and C134 is in the 3'-UTR of \u003cem\u003eIRS2\u003c/em\u003e and is expressed in several tissues. C134 also contains binding sites for several TFs such as FOXP1, SMARCA4, and FOS. In another example, C122 spans the promoter, 5'-UTR, and exon 1 of \u003cem\u003eTAF4\u003c/em\u003e. Several examined colonies lacked TEs, while densely surrounded by these elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCrossover and recombination at CG-only T2Us form STRs/microsatellites and minisatellites\u003c/h2\u003e\u003cp\u003eThe crossover and recombination events at CG-only T2Us lead to the emergence of diverse types of STRs/microsatellites i.e., \u0026ge;\u0026thinsp;3-repeats, for example, (GGC)7 in C259 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and (GCC)6 in C122 (Fig.\u0026nbsp;5). These STRs were largely versatile with respect to length and composition across the species studied, and in numerous instances species-specific. For example, the length of (GGC)7 in C259 was detected in human only, and not in any of the orthologous colonies of C259.\u003c/p\u003e\u003cp\u003eIn some colonies, crossover and recombination led to the emergence of minisatellites. For example, in C266, unequal crossover and recombination events at CG-only T2Us resulted in the formation of a minisatellite composition of extensive dynamicity across human, chimpanzee, and gorilla (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The main recombinant across this colony was of the overlapping type, GGCGGCG, which resulted from unequal crossover between GGCGGC and GCGGCG. In human and chimpanzee, all GCGGCG T2Us were consumed in overlapping recombinants, whereas traces of this type of T2U were detectable in gorilla (yellow blocks). In another example, the overlapping recombinant, CGGCGGCG (green blocks), which was a plausible recombination among CGGCGG, GGCGGC, and GCGGCG was detectable in the genomes of chimpanzee and gorilla and depleted in the human genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e). A few models for the emergence of the overlapping recombinants in this colony are provided in the Visualization section (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eC266 is located inside intron 7 of a novel gene (\u003cem\u003eENSG00000290950\u003c/em\u003e), highly transcribed in the testis, and predicted to be a binding site for early growth response 1 (EGR1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Interestingly, testis cord formation is regulated by EGR1-mediated cell migration [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Based on the ReMap ChiP-seq database, C266 has the densest map of regulatory binding sites in comparison to the flanking sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe recently reported that several types of MRs are ubiquitous hotspots of crossover and recombination across the human genome [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Here we studied the combinatory impact of CG-only T2Us in the formation of crossover and recombination landscape, which enabled identification of thousands of colonies that spread extensively across all human chromosomes. We studied several colonies of diverse sizes in other primates and mouse, and found that these colonies coincided with highly dynamic and species-specific formulae in these species. Each species studied had a complex formula for these colonies, which resulted from dynamic and species-specific recombination events in these colonies. Not only did the absolute count and the combination of the T2Us differed across the selected species, but the distribution of these units in various types of recombinants also differed, adding additional levels of complexity of the genomic events at these sites. The findings of cross-species sharedness of some of the examined colonies prompts re-examining the hypothesis that recombination hotspots rarely (if at all) occur at the same locus, even between human and closely related species [\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe coincidence of dense arrays of CG-only T2Us and recombinants of these T2Us in the same colony signified T2Us as sites of crossover and recombination. Based on the total count of CG-only T2Us in the human genome size of ~\u0026thinsp;3 Gb, the expected occurrence of a CG-only T2U is approximately 1 in every 5 kb of genomic DNA. The exceedingly significant occurrence of colonies based on Poisson distribution further argues against a neutral hypothesis. The complexity of the identified T2Us and the flanking sequences of these units align with previous reports that crossovers are associated with mutations at recombination hotspots in human [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn contrast with AT-only MR colonies that are mainly distributed in intergenic intervals [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], most of the CG-only T2U colonies reside in genic regions and form gene parts, such as promoters, UTRs, and exonic/intronic sequences. We found that the colonies in genic regions were predominantly larger in size than the colonies in the intergenic intervals. For an extreme example, the gene-splicing regions contained the largest proportion of colonies of the extreme large size. GC content around splice sites affects splicing through pre-mRNA secondary structures and compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Alternative splicing is proposed to have a significant role in adaptive evolution and plasticity at the cross-species level [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], highlighting the importance of the colonies residing in splicing intervals, alongside colonies in other genic intervals. The ncRNA genes were also among the sequences that were enriched by larger size colonies. Human ncRNAs are functional RNA molecules that do not code for proteins but are vital for cellular processes like gene expression regulation, genome stability, and cell development [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Overall, it is possible that CG-only T2U colonies are, at least in part, factories of gene synthesis across evolution. In line with this hypothesis, CG-rich sequences are enriched in transcription elements, UTRs, and splice sites [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Because of their high CG content, propensity for unequal crossover and recombination, enrichment in genic regions, and cross-species occurrence, it is plausible that CG-only MRs partly contribute to the formation, architecture, and evolution of euchromatic genomic regions. This notion is also supported by our previous findings on CG-only D2Us, colonies of which were mainly distributed in genic intervals [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Euchromatin is a lightly packed form of chromatin, characterized by its high CG content, accessibility to transcription factors, and active gene expression [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eChromosomes 1 and 19 were among the top chromosomes enriched by CG-only T2U colonies. Chromosome 1 is the largest human chromosome. Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The unusual pattern of high GC and CpG content in chromosome 19 orthologs, particularly outside gene clusters, is present from human to mouse lemur [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Much CpG variation in chromosome 19 exists both within and between primate species with a portion of this variation occurring in regulatory regions. It is plausible that these unusual characteristics in chromosomes 1 and 19 are partially attributable to the high CG-only T2U crossover/recombination and colonization in these chromosomes.\u003c/p\u003e\u003cp\u003ePrior to the discovery of MRs as sites of crossover and recombination, a 13-bp \u0026ldquo;core\u0026rdquo; motif \u0026ldquo;CCTCCCTNNCCAC\u0026rdquo; and its degenerate versions were reported to strongly correlate with hotspot activity when they occurred in both repeat and nonrepeat DNA, and were binding sites for PRDM9 [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, the expression level of PRDM9 explains only a fraction of the hotspots [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Consistent with this, PRDM9-independent recombination sites are expected to rapidly change the recombination landscape and insert vast evolutionary effects across species [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCG T2Us can be relevant in both crossover and non-crossover contexts (the latter referring to the exchange of DNA fragments without exchanging flanking DNA arms). In fact, most recombination interactions in meiosis are of the non-crossover type, as opposed to crossovers, which include flanking chromosomal arms. Evidence indicates that there probably is no non-crossover-specific pathway, and that restoration of intermediate events in a single pairing/recombination pathway promotes synaptonemal complex formation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCrossovers and recombination at CG-only T2Us form STRs/microsatellites and minisatellites. We found species-specific lengths and compositions of these elements in our cross-species study. STRs and minisatellites are largely linked to evolution, speciation, and disease [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR40 CR41 CR42\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSeveral CG-only T2U colonies of diverse sizes lacked TEs, but were densely surrounded by these elements. TEs contribute to cell- and species-specific chromatin looping and gene regulation in mammalian genomes [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Although linked to important biological and evolutionary implications, TEs primarily cause gene disruption and large-scale genomic alterations, including inversions, deletions, and duplications [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Although hypothetical at this time, it is possible that CG-only T2Us, at least in part, protect genic/euchromatic regions of genomes against TEs.\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eLimitations and future research directions\u003c/h2\u003e\u003cp\u003eOther types of CG-only MRs, such as tetra-, penta- and hexanucleotide two-repeat units, and their combinatory recombination need to be examined in the future studies. Functional studies are also required to shed further light on the mechanistic and biological aspects of this phenomenon. Moreover, comprehensive cross-species studies are required to further investigate the evolutionary extent and impact of this phenomenon.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, CG-only T2Us are recombination hotspots across primate and mouse genomes. Colonies formed because of these recombination events create versatile genomic constituents of biological and evolutionary relevance. In conjunction with other types of MRs, such as D2Us, CG-only T2U-mediated crossover and recombination may be involved in the formation and evolution of euchromatic regions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e Colony\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD2U\u003c/strong\u003e Dinucleotide 2-repeat unit\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSTR\u003c/strong\u003e Short tandem repeat\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNcRNA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eNon-coding RNA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTE\u003c/strong\u003e Transposable element\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTF\u003c/strong\u003e Transcription factor\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT2U\u003c/strong\u003e Trinucleotide 2-repeat unit\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUTR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eUntranslated region\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlossary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT2U\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eTwo repeats of CG-only trinucleotides. For example, CGGCGG and CGCCGC are T2Us.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbsolute count\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eCount of T2Us, regardless of being isolate or part of a recombinant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony\u003c/strong\u003e Three or more consecutive T2Us of \u0026lt;500 bp apart. Throughout the paper, specific colonies were named by adding \"C\" prefix. Colony size was based on the human genome. For example, C266 was a colony of 266 T2Us in human.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony size\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eAbsolute count of consecutive T2Us of \u0026lt;500 bp apart from each other.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverlapping recombinant\u003c/strong\u003e Recombinant of T2Us, as a result of overlapping sequences. For example, TATATA is an overlapping recombinant resulting from unequal crossover between TATA and TATA overlaps. In another example, ATATATA is an overlapping recombinant between ATATAT and TATATA overlaps. In this type of recombinant, T2Us overlap.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNon-overlapping recombinant\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eNon-overlapping recombinant of T2Us. For example, CGGCGGCCGCCG is a recombinant resulting from non-overlapping recombination between CGGCGG and CCGCCG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCrossover\u0026nbsp;\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; The exchange of DNA between paired homologous chromosomes (one from each parent) that occurs during the development of egg and sperm cells (meiosis).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUnequal crossover \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eUnequal crossing-over, also referred to as illegitimate recombination, refers to\u0026nbsp;crossover events that occur between nonequivalent sequences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNon-crossover \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eRecombination interactions, which include DNA fragments, without\u0026nbsp;exchange of the flanking chromosome arms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecombination hotspot \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eA genomic region (typically in ~kb ranges) that experience intensely high levels of recombination compared to the genomic background.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShort tandem repeat\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eRepeats of ≥3.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset obtained and subsequently analyzed in this research is available\u0026nbsp;at the following Figshare link:\u003c/p\u003e\n\u003cp\u003ehttps://figshare.com/articles/dataset/_strong_Genome_wide_human_list_of_all_possible_two-repeats_units_of_CG-rich_trinucleotides_strong_/23612943?file=57431413\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for conducting this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003econtribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: MO; Methodology: MA, AMAM; Investigation: DA; HB, MA, NT, SKh, SA; Visualization: HB, NT, SA; Project administration: MO, AD, HRKH; Supervision: MO; Writing \u0026ndash; original draft: MO, MA; Writing \u0026ndash; review \u0026amp; editing: MO, HB. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHorton, C.A., A.M. Alexandari, M.G.B. Hayes, E. Marklund, J.M. Schaepe, A.K. Aditham, et al., \u003cem\u003eShort tandem repeats bind transcription factors to tune eukaryotic gene expression.\u003c/em\u003e Science, 2023. \u003cstrong\u003e381\u003c/strong\u003e(6664): p. eadd1250. https://doi.org/10.1126/science.add1250.\u003c/li\u003e\n \u003cli\u003eMaddi, A.M.A., K. Kavousi, M. Arabfard, H. Ohadi, and M. Ohadi, \u003cem\u003eTandem repeats ubiquitously flank and contribute to translation initiation sites.\u003c/em\u003e BMC Genom Data, 2022. \u003cstrong\u003e23\u003c/strong\u003e(1): p. 59. https://doi.org/10.1186/s12863-022-01075-5.\u003c/li\u003e\n \u003cli\u003eOhadi, M., E. Valipour, S. Ghadimi-Haddadan, P. 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PMID: 10526579; PMCID: PMC11146969.\u003c/li\u003e\n \u003cli\u003eDiehl AG, Ouyang N, Boyle AP: Transposable elements contribute to cell and species-specific chromatin looping and gene regulation in mammalian genomes. Nat Commun 2020;11(1):1796. https://doi.org/10.1038/s41467-020-15520-5.\u003c/li\u003e\n \u003cli\u003eSenft AD, Macfarlan TS: Transposable elements shape the evolution of mammalian development. Nat Rev Genet 2021;22(11):691-711. https://doi.org/10.1038/s41576-021-00385-1.\u003c/li\u003e\n \u003cli\u003eBhat A, Ghatage T, Bhan S, Lahane GP, Dhar A, Kumar R, Pandita RK, Bhat KM, Ramos KS, Pandita TK. Role of Transposable Elements in Genome Stability: Implications for Health and Disease. Int J Mol Sci. 2022;23(14):7802. doi: 10.3390/ijms23147802. PMID: 35887150; PMCID: PMC9319628.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Human, Primate, Mouse, Minimal repeat, Crossover, Recombination hotspot, Euchromatin","lastPublishedDoi":"10.21203/rs.3.rs-7601515/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7601515/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Crossover and recombination have significant outcomes in genomic diversity and evolution. We previously reported that minimal repeats (MRs) such as AT-only trinucleotide two-repeat units (T2Us) are primary sites of unequal crossover and recombination across the human genome, the combinatory impact of which result in intricate colonies (distance between consecutive T2Us \u0026lt;500 bp) of biological and evolutionary implications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e We studied the combinatory impact and landscape of all types of CG-only T2Us on crossover and recombination in the human genome. To this end, we mapped CCGCCG, CGGCGG, CGCCGC, GGCGGC, GCGGCG, and CGCCGC. Subsequently, we performed a comparative genomics study of several colonies of diverse sizes in other primates and mouse.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Most CG-only T2Us formed colonies that were primarily distributed in genic intervals. These colonies were predominantly larger than the colonies in the intergenic intervals. Dense arrays of combinatory overlapping and non-overlapping recombinants of the T2Us in the same colony indicated unequal crossover and recombination at these units. The colonies formed versatile genomic constituents, including gene parts (promoters, untranslated regions, and exonic/intronic sequences), microsatellites, and minisatellites. Cross-species analysis of several colonies revealed that some of the colonies were dynamically shared as phylogenetically distant as in mouse. These colonies were mainly of maximum complexity and size in human.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e CG-only T2Us are crossover and recombination hotspots across the genomes of primates and mouse. The combinatory recombination of these MRs creates versatile genomic elements of evolutionary and biological relevance across these genomes. Because of their high CG content, propensity for unequal crossover and recombination, enrichment in genic regions, and cross-species occurrence, CG-only MRs may partly contribute to the formation, architecture, and evolution of euchromatic genomic regions.\u003c/p\u003e","manuscriptTitle":"Combinatory crossover and recombination at CG-only minimal repeats create versatile genomic constituents across primate and mouse genomes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-23 08:17:19","doi":"10.21203/rs.3.rs-7601515/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d0515c5e-2759-41ce-9f73-a099a84c2107","owner":[],"postedDate":"October 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T06:11:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-23 08:17:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7601515","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7601515","identity":"rs-7601515","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}