Association between telomere length and Plasmodium falciparum malaria endemicity in sub-Saharan Africans.

Leukocyte telomere length (LTL) varies significantly across human populations, with individuals of African ancestry having longer LTL than non-Africans. However, the genetic and environmental drivers of LTL variation in Africans remain largely unknown. We report here on the relationship between LTL, genetics, and a variety of environmental and climatic factors in ethnically diverse African adults (n = 1,818) originating from Botswana, Tanzania, Ethiopia, and Cameroon. We observe significant variation in LTL among populations, finding that the San hunter-gatherers from Botswana have the longest leukocyte telomeres and that the Fulani pastoralists from Cameroon have the shortest telomeres. Genetic factors explain ∼50% of LTL variation among individuals. Moreover, we observe a significant negative association between Plasmodium falciparum malaria endemicity and LTL while adjusting for age, sex, and genetics. Within Africa, adults from populations indigenous to areas with high malaria exposure have shorter LTL than those in populations indigenous to areas with low malaria exposure. Finally, we explore to what degree the genetic architecture underlying LTL in Africa covaries with malaria exposure.

Development of a telomere vector-based approach to overcome limitations caused by lethal phenotypes in the study of essential genes in Magnaporthe oryzae.

Reverse genetic approaches are common tools in genomics for elucidating gene functions, involving techniques such as gene deletion followed by screening for aberrant phenotypes. If the generation of gene deletion mutants fails, the question arises whether the failure stems from technical issues or because the gene of interest (GOI) is essential, meaning that the deletion causes lethality. In this report, we introduce a novel method for assessing gene essentiality using the phytopathogenic ascomycete Magnaporthe oryzae. The method is based on the observation that telomere vectors are lost in transformants during cultivation without selection pressure. We tested the hypothesis that essential genes can be identified in deletion mutants co-transformed with a telomere vector. The M. oryzae gene MoPKC, described in literature as essential, was chosen as GOI. Using CRISPR/Cas9 technology transformants with deleted GOI were generated and backed up by a telomere vector carrying a copy of the GOI and conferring fenhexamid resistance. Transformants in which the GOI deletion in the genome was not successful lost the telomere vector on media without fenhexamid. In contrast, transformants with confirmed GOI deletion retained the telomere vector even in absence of fenhexamid selection. In the latter case, the maintenance of the telomere indicates that the GOI is essential for the surveillance of the fungi, as it would have been lost otherwise. The method presented here allows to test for essentiality of genes when no mutants can be obtained from gene deletion approaches, thereby expanding the toolbox for studying gene function in ascomycetes.

Genetic polymorphisms affecting telomere length and their association with cardiovascular disease in the Heinz-Nixdorf-Recall study.

Short telomeres are associated with cardiovascular disease (CVD). We aimed to investigate, if genetically determined telomere-length effects CVD-risk in the Heinz-Nixdorf-Recall study (HNRS) population. We selected 14 single-nucleotide polymorphisms (SNPs) associated with telomere-length (p<10-8) from the literature and after exclusion 9 SNPs were included in the analyses. Additionally, a genetic risk score (GRS) using these 9 SNPs was calculated. Incident CVD was defined as fatal and non-fatal myocardial infarction, stroke, and coronary death. We included 3874 HNRS participants with available genetic data and had no known history of CVD at baseline. Cox proportional-hazards regression was used to test the association between the SNPs/GRS and incident CVD-risk adjusting for common CVD risk-factors. The analyses were further stratified by CVD risk-factors. During follow-up (12.1±4.31 years), 466 participants experienced CVD-events. No association between SNPs/GRS and CVD was observed in the adjusted analyses. However, the GRS, rs10936599, rs2487999 and rs8105767 increase the CVD-risk in current smoker. Few SNPs (rs10936599, rs2487999, and rs7675998) showed an increased CVD-risk, whereas rs10936599, rs677228 and rs4387287 a decreased CVD-risk, in further strata. The results of our study suggest different effects of SNPs/GRS on CVD-risk depending on the CVD risk-factor strata, highlighting the importance of stratified analyses in CVD risk-factors.

Transcription stress at telomeres leads to cytosolic DNA release and paracrine senescence.

Transcription stress has been linked to DNA damage -driven aging, yet the underlying mechanism remains unclear. Here, we demonstrate that Tcea1-/- cells, which harbor a TFIIS defect in transcription elongation, exhibit RNAPII stalling at oxidative DNA damage sites, impaired transcription, accumulation of R-loops, telomere uncapping, chromatin bridges, and genome instability, ultimately resulting in cellular senescence. We found that R-loops at telomeres causally contribute to the release of telomeric DNA fragments in the cytoplasm of Tcea1-/- cells and primary cells derived from naturally aged animals triggering a viral-like immune response. TFIIS-defective cells release extracellular vesicles laden with telomeric DNA fragments that target neighboring cells, which consequently undergo cellular senescence. Thus, transcription stress elicits paracrine signals leading to cellular senescence, promoting aging.

Annotations of four high-quality indigenous chicken genomes identify more than one thousand missing genes in subtelomeric regions and micro-chromosomes with high G/C contents.

BACKGROUND: Although multiple chicken genomes have been assembled and annotated, the numbers of protein-coding genes in chicken genomes and their variation among breeds are still uncertain due to the low quality of these genome assemblies and limited resources used in their gene annotations. To fill these gaps, we recently assembled genomes of four indigenous chicken breeds with distinct traits at chromosome-level. In this study, we annotated genes in each of these assembled genomes using a combination of RNA-seq- and homology-based approaches. RESULTS: We identified varying numbers (17,497-17,718) of protein-coding genes in the four indigenous chicken genomes, while recovering 51 of the 274 "missing" genes in birds in general, and 36 of the 174 "missing" genes in chickens in particular. Intriguingly, based on deeply sequenced RNA-seq data collected in multiple tissues in the four breeds, we found 571 ~ 627 protein-coding genes in each genome, which were missing in the annotations of the reference chicken genomes (GRCg6a and GRCg7b/w). After removing redundancy, we ended up with a total of 1,420 newly annotated genes (NAGs). The NAGs tend to be found in subtelomeric regions of macro-chromosomes (chr1 to chr5, plus chrZ) and middle chromosomes (chr6 to chr13, plus chrW), as well as in micro-chromosomes (chr14 to chr39) and unplaced contigs, where G/C contents are high. Moreover, the NAGs have elevated quadruplexes G frequencies, while both G/C contents and quadruplexes G frequencies in their surrounding regions are also high. The NAGs showed tissue-specific expression, and we were able to verify 39 (92.9%) of 42 randomly selected ones in various tissues of the four chicken breeds using RT-qPCR experiments. Most of the NAGs were also encoded in the reference chicken genomes, thus, these genomes might harbor more genes than previously thought. CONCLUSION: The NAGs are widely distributed in wild, indigenous and commercial chickens, and they might play critical roles in chicken physiology. Counting these new genes, chicken genomes harbor more genes than originally thought.

Exchange of subtelomeric regions between chromosomes 4q and 10q reverts the FSHD genotype and phenotype.

The most common form of facioscapulohumeral dystrophy (FSHD1) is caused by a partial loss of the D4Z4 macrosatellite repeat array in the subtelomeric region of chromosome 4. Patients with FSHD1 typically carry 1 to 10 D4Z4 repeats, whereas nonaffected individuals have 11 to 150 repeats. The ~150-kilobyte subtelomeric region of the chromosome 10q exhibits a ~99% sequence identity to the 4q, including the D4Z4 array. Nevertheless, contractions of the chr10 array do not cause FSHD or any known disease, as in most people D4Z4 array on chr10 is flanked by the nonfunctional polyadenylation signal, not permitting the DUX4 expression. Here, we attempted to correct the FSHD genotype by a CRISPR-Cas9-induced exchange of the chr4 and chr10 subtelomeric regions. We demonstrated that the induced t(4;10) translocation can generate recombinant genotypes translated into improved FSHD phenotype. FSHD myoblasts with the t(4;10) exhibited reduced expression of the DUX4 targets, restored PAX7 target expression, reduced sensitivity to oxidative stress, and improved differentiation capacity.

Extrachromosomal telomere DNA derived from excessive strand displacements.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

Giardia telomeres and telomerase.

Giardia duodenalis, the protozoan responsible for giardiasis, is a significant contributor to millions of diarrheal diseases worldwide. Despite the availability of treatments for this parasitic infection, therapeutic failures are alarmingly frequent. Thus, there is a clear need to identify new therapeutic targets. Giardia telomeres were previously identified, but our understanding of these structures and the critical role played by Giardia telomerase in maintaining genomic stability and its influence on cellular processes remains limited. In this regard, it is known that all Giardia chromosomes are capped by small telomeres, organized and protected by specific proteins that regulate their functions. To counteract natural telomere shortening and maintain high proliferation, Giardia exhibits constant telomerase activity and employs additional mechanisms, such as the formation of G-quadruplex structures and the involvement of transposable elements linked to telomeric repeats. Thus, this study aims to address the existing knowledge gap by compiling the available information (until 2023) about Giardia telomeres and telomerase, focusing on highlighting the distinctive features within this parasite. Furthermore, the potential feasibility of targeting Giardia telomeres and/or telomerase as an innovative therapeutic strategy is discussed.

New insights from bidirectional Mendelian randomization: causal relationships between telomere length and mitochondrial DNA copy number in aging biomarkers.

Mitochondrial DNA (mtDNA) copy number and telomere length (TL) are dynamic factors that have been linked to the aging process in organisms. However, the causal relationship between these variables remains uncertain. In this research, instrumental variables (IVs) related to mtDNA copy number and TL were obtained from publicly available genome-wide association studies (GWAS). Through bidirectional Mendelian randomization (MR) analysis, we examined the potential causal relationship between these factors. The forward analysis, with mtDNA copy number as the exposure and TL as the outcome, did not reveal a significant effect (B=-0.004, P>0.05). On the contrary, upon conducting a reverse analysis, it was found that there exists a positive causal relationship (B=0.054, P<0.05). Sensitivity analyses further confirmed the reliability of these results. The outcomes of this study indicate a one-way positive causal relationship, indicating that telomere shortening in the aging process may lead to a decrease in mtDNA copy number, providing new perspectives on their biological mechanisms.

Monochrome Multiplex Quantitative PCR Telomere Length Measurement.

Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. Telomere length (TL) has been associated with various exposures, biological processes, and health outcomes. This article describes the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay protocol routinely conducted in our laboratory for measuring relative mean TL from human DNA. There are several different PCR-based TL measurement methods, but the specific protocol for the MMqPCR method presented in this publication is repeatable, efficient, cost-effective, and suitable for population-based studies. This detailed protocol outlines all information necessary for investigators to establish this assay in their laboratory. In addition, this protocol provides specific steps to increase the reproducibility of TL measurement by this assay, defined by the intraclass correlation coefficient (ICC) across repeated measurements of the same sample. The ICC is a critical factor in evaluating expected power for a specific study population; as such, reporting cohort-specific ICCs for any TL assay is a necessary step to enhance the overall rigor of population-based studies of TL. Example results utilizing DNA samples extracted from peripheral blood mononuclear cells demonstrate the feasibility of generating highly repeatable TL data using this MMqPCR protocol.

Clinical mutations in the TERT and TERC genes coding for telomerase components induced oxidative stress, DNA damage at telomeres and cell apoptosis besides decreased telomerase activity.

Telomeres are nucleoprotein structures at the end of chromosomes that maintain their integrity. Mutations in genes coding for proteins involved in telomere protection and elongation produce diseases such as dyskeratosis congenita or idiopathic pulmonary fibrosis known as telomeropathies. These diseases are characterized by premature telomere shortening, increased DNA damage and oxidative stress. Genetic diagnosis of telomeropathy patients has identified mutations in the genes TERT and TERC coding for telomerase components but the functional consequences of many of these mutations still have to be experimentally demonstrated. The activity of twelve TERT and five TERC mutants, five of them identified in Spanish patients, has been analyzed. TERT and TERC mutants were expressed in VA-13 human cells that express low telomerase levels and the activity induced was analyzed. The production of reactive oxygen species, DNA oxidation and TRF2 association at telomeres, DNA damage response and cell apoptosis were determined. Most mutations presented decreased telomerase activity, as compared to wild-type TERT and TERC. In addition, the expression of several TERT and TERC mutants induced oxidative stress, DNA oxidation, DNA damage, decreased recruitment of the shelterin component TRF2 to telomeres and increased apoptosis. These observations might indicate that the increase in DNA damage and oxidative stress observed in cells from telomeropathy patients is dependent on their TERT or TERC mutations. Therefore, analysis of the effect of TERT and TERC mutations of unknown function on DNA damage and oxidative stress could be of great utility to determine the possible pathogenicity of these variants.

Study of association of leptin with leukocyte telomere length in a Chinese rural population.

BACKGROUND: Previous studies have demonstrated the relationship between adipocyte factors, insulin resistance, and other indicators with telomere length. However, these studies did not consider the influence of changes in different indicators on telomere length over time. Therefore, the aim of this study is to elucidate the impact of changes in adipocyte factors, HOMA-IR, and other indicators on the dynamic variation of telomere length. METHODS: The data were from a cohort study conducted in Ningxia, China. A total of 1624 subjects were analyzed. Adipokines and relative leukocyte telomere length (RLTL) were measured, and changes in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), Homeostatic Model Assessment for ß-Cell Function (HOMA-ß), and Quantitative Insulin Sensitivity Check Index (QUICKI) were calculated. Generalized linear models evaluated associations between changes in adipokines and RLTL changes. Furthermore, univariate analyses examined the effects of changes in adipokines and insulin resistance indicators on ΔRLTL. RESULTS: The research findings indicate that females generally have shorter telomeres compared to males. In comparison to the low-level group of Δleptin (LEP), the high-level group of ΔLEP shows a negative correlation with ΔRLTL (B=-1.32, 95% CI (-2.38, -0.27)). Even after multivariable adjustments, this relationship persists (B=-1.31, 95% CI (-2.24, -0.23)). Further analysis reveals that after adjusting for ΔHOMA-IR, ΔHOMA-ß, and ΔQUICKI, the high-level group of ΔLEP still exhibits a significant negative correlation with ΔRLTL (B=-1.37, 95% CI (-2.43, -0.31)). However, the interaction effects between ΔHOMA-IR, ΔHOMA-ß, ΔQUICKI, and ΔLEP do not affect ΔRLTL. CONCLUSIONS: Elevated levels of leptin were significantly correlated with shortened telomere length. This suggests that increased leptin levels may impact overall individual health by affecting telomere length, underscoring the importance of measures to reduce leptin levels to mitigate the onset and progression of related diseases.

First telomere-to-telomere gapless assembly of the rice blast fungus Pyricularia oryzae.

Rice blast caused by Pyricularia oryzae (syn., Magnaporthe oryzae) was one of the most destructive diseases of rice throughout the world. Genome assembly was fundamental to genetic variation identification and critically impacted the understanding of its ability to overcome host resistance. Here, we report a gapless genome assembly of rice blast fungus P. oryzae strain P131 using PacBio, Illumina and high throughput chromatin conformation capture (Hi-C) sequencing data. This assembly contained seven complete chromosomes (43,237,743 bp) and a circular mitochondrial genome (34,866 bp). Approximately 14.31% of this assembly carried repeat sequences, significantly greater than its previous assembled version. This assembly had a 99.9% complement in BUSCO evaluation. A total of 14,982 genes protein-coding genes were predicted. In summary, we assembled the first telomere-to-telomere gapless genome of P. oryzae, which would be a valuable genome resource for future research on the genome evolution and host adaptation.

BLM helicase unwinds lagging strand substrates to assemble the ALT telomere damage response.

The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.

Reprogramming Chromosome Ends by Functional Histone Acetylation.

Cancers harness embryonic programs to evade aging and promote survival. Normally, sequences at chromosome ends called telomeres shorten with cell division, serving as a countdown clock to limit cell replication. Therefore, a crucial aspect of cancerous transformation is avoiding replicative aging by activation of telomere repair programs. Mouse embryonic stem cells (mESCs) activate a transient expression of the gene Zscan4, which correlates with chromatin de-condensation and telomere extension. Head and neck squamous cell carcinoma (HNSCC) cancers reactivate ZSCAN4, which in turn regulates the phenotype of cancer stem cells (CSCs). Our study reveals a new role for human ZSCAN4 in facilitating functional histone H3 acetylation at telomere chromatin. Next-generation sequencing indicates ZSCAN4 enrichment at telomere chromatin. These changes correlate with ZSCAN4-induced histone H3 acetylation and telomere elongation, while CRISPR/Cas9 knockout of ZSCAN4 leads to reduced H3 acetylation and telomere shortening. Our study elucidates the intricate involvement of ZSCAN4 and its significant contribution to telomere chromatin remodeling. These findings suggest that ZSCAN4 induction serves as a novel link between 'stemness' and telomere maintenance. Targeting ZSCAN4 may offer new therapeutic approaches to effectively limit or enhance the replicative lifespan of stem cells and cancer cells.

Elimination of subtelomeric repeat sequences exerts little effect on telomere essential functions in Saccharomyces cerevisiae.

Telomeres, which are chromosomal end structures, play a crucial role in maintaining genome stability and integrity in eukaryotes. In the baker's yeast Saccharomyces cerevisiae, the X- and Y'-elements are subtelomeric repetitive sequences found in all 32 and 17 telomeres, respectively. While the Y'-elements serve as a backup for telomere functions in cells lacking telomerase, the function of the X-elements remains unclear. This study utilized the S. cerevisiae strain SY12, which has three chromosomes and six telomeres, to investigate the role of X-elements (as well as Y'-elements) in telomere maintenance. Deletion of Y'-elements (SY12YΔ), X-elements (SY12XYΔ+Y), or both X- and Y'-elements (SY12XYΔ) did not impact the length of the terminal TG1-3 tracks or telomere silencing. However, inactivation of telomerase in SY12YΔ, SY12XYΔ+Y, and SY12XYΔ cells resulted in cellular senescence and the generation of survivors. These survivors either maintained their telomeres through homologous recombination-dependent TG1-3 track elongation or underwent microhomology-mediated intra-chromosomal end-to-end joining. Our findings indicate the non-essential role of subtelomeric X- and Y'-elements in telomere regulation in both telomerase-proficient and telomerase-null cells and suggest that these elements may represent remnants of S. cerevisiae genome evolution. Furthermore, strains with fewer or no subtelomeric elements exhibit more concise telomere structures and offer potential models for future studies in telomere biology.

Caenorhabditis elegans telomere-binding proteins TEBP-1 and TEBP-2 adapt the Myb module to dimerize and bind telomeric DNA.

Protecting chromosome ends from misrecognition as double-stranded (ds) DNA breaks is fundamental to eukaryotic viability. The protein complex shelterin prevents a DNA damage response at mammalian telomeres. Mammalian shelterin proteins TRF1 and TRF2 and their homologs in yeast and protozoa protect telomeric dsDNA. N-terminal homodimerization and C-terminal Myb-domain-mediated dsDNA binding are two structural hallmarks of end protection by TRF homologs. Yet our understanding of how Caenorhabditis elegans protects its telomeric dsDNA is limited. Recently identified C. elegans proteins TEBP-1 (also called DTN-1) and TEBP-2 (also called DTN-2) are functional homologs of TRF proteins, but how they bind DNA and whether or how they dimerize is not known. TEBP-1 and TEBP-2 harbor three Myb-containing domains (MCDs) and no obvious dimerization domain. We demonstrate biochemically that only the third MCD binds DNA. We solve the X-ray crystal structure of TEBP-2 MCD3 with telomeric dsDNA to reveal the structural mechanism of telomeric dsDNA protection in C. elegans. Mutagenesis of the DNA-binding site of TEBP-1 and TEBP-2 compromises DNA binding in vitro, and increases DNA damage signaling, lengthens telomeres, and decreases brood size in vivo. Via an X-ray crystal structure, biochemical validation of the dimerization interface, and SEC-MALS analysis, we demonstrate that MCD1 and MCD2 form a composite dimerization module that facilitates not only TEBP-1 and TEBP-2 homodimerization but also heterodimerization. These findings provide fundamental insights into C. elegans telomeric dsDNA protection and highlight how different eukaryotes have evolved distinct strategies to solve the chromosome end protection problem.

Telomere-to-telomere genome assembly of Oldenlandia diffusa.

We report the complete telomere-to-telomere genome assembly of Oldenlandia diffusa which renowned in traditional Chinese medicine, comprising 16 chromosomes and spanning 499.7 Mb. The assembly showcases 28 telomeres and minimal gaps, with a total of only five. Repeat sequences constitute 46.41% of the genome, and 49,701 potential protein-coding genes have been predicted. Compared with O. corymbosa, O. diffusa exhibits chromosome duplication and fusion events, diverging 20.34 million years ago. Additionally, a total of 11 clusters of terpene synthase have been identified. The comprehensive genome sequence, gene catalog, and terpene synthase clusters of O. diffusa detailed in this study will significantly contribute to advancing research in this species' genetic, genomic, and pharmacological aspects.

Associations between weight-adjusted-waist index and telomere length: Results from NHANES: An observational study.

Previous studies have demonstrated the connection between obesity and telomere length. A recently devised metric for determining obesity, the weight-adjusted-waist index (WWI), offers a distinct advantage in predicting fat and lean mass by depicting weight-independent abdominal adiposity. This article presents the results of the inaugural study on the relationship between WWI and telomere length in adult populations. The cross-sectional investigation analyzed data from 3479 participants from the National Health and Nutrition Examination Survey (NHANES) conducted from 1999 to 2000. To inspect linear and nonlinear correlations, we adopted weighted multiple logistic regression analysis and smooth curve fit, respectively. In addition, threshold effects and subgroup analyses were accomplished. In the fully adapted model, a significant adverse association of WWI with telomere length was detected [ß = -0.02, 95% CI: (-0.03, -0.00), P value = 0.01]. The adverse correlation remained consistent across all subcategories. We also discovered an inverted U-shaped curve linking WWI and telomere length, with a conspicuous inflection point of 10.07 cm/√kg. For the first time, our research demonstrated strong links between WWI and telomere length. The inflection point suggests that controlling WWI within an optimum range might be essential for aging and health.

Near telomere-to-telomere genome assemblies of two Chlorella species unveil the composition and evolution of centromeres in green algae.

BACKGROUND: Centromeres play a crucial and conserved role in cell division, although their composition and evolutionary history in green algae, the evolutionary ancestors of land plants, remains largely unknown. RESULTS: We constructed near telomere-to-telomere (T2T) assemblies for two Trebouxiophyceae species, Chlorella sorokiniana NS4-2 and Chlorella pyrenoidosa DBH, with chromosome numbers of 12 and 13, and genome sizes of 58.11 Mb and 53.41 Mb, respectively. We identified and validated their centromere sequences using CENH3 ChIP-seq and found that, similar to humans and higher plants, the centromeric CENH3 signals of green algae display a pattern of hypomethylation. Interestingly, the centromeres of both species largely comprised transposable elements, although they differed significantly in their composition. Species within the Chlorella genus display a more diverse centromere composition, with major constituents including members of the LTR/Copia, LINE/L1, and LINE/RTEX families. This is in contrast to green algae including Chlamydomonas reinhardtii, Coccomyxa subellipsoidea, and Chromochloris zofingiensis, in which centromere composition instead has a pronounced single-element composition. Moreover, we observed significant differences in the composition and structure of centromeres among chromosomes with strong collinearity within the Chlorella genus, suggesting that centromeric sequence evolves more rapidly than sequence in non-centromeric regions. CONCLUSIONS: This study not only provides high-quality genome data for comparative genomics of green algae but gives insight into the composition and evolutionary history of centromeres in early plants, laying an important foundation for further research on their evolution.