Rapid proliferation and nucleolar organizer targeting centromeric retrotransposons in cotton

Centromeric chromatin in most eukaryotes is composed of highly repetitive centromeric retrotransposons and satellite repeats that are highly variable even among closely related species. The evolutionary mechanisms that underlie the rapid evolution of centromeric repeats remain unknown. To obtain insight into the evolution of centromeric repeats following polyploidy, we studied a model diploid progenitor (Gossypium raimondii, D‐genome) of the allopolyploid (AD‐genome) cottons, G. hirsutum and G. barbadense. Sequence analysis of chromatin‐immunoprecipitated DNA showed that the G. raimondii centromeric repeats originated from retrotransposon‐related sequences. Comparative analysis showed that nine of the 10 analyzed centromeric repeats were absent from the centromeres in the A‐genome and related diploid species (B‐, F‐ and G‐genomes), indicating that they colonized the centromeres of D‐genome lineage after the divergence of the A‐ and D‐ ancestral species or that they were ancestrally retained prior to the origin of Gossypium. Notably, six of the nine repeats were present in both the A‐ and D‐subgenomes in tetraploid G. hirsutum, and increased in abundance in both subgenomes. This finding suggests that centromeric repeats may spread and proliferate between genomes subsequent to polyploidization. Two repeats, Gr334 and Gr359 occurred in both the centromeres and nucleolar organizer regions (NORs) in D‐ and AD‐genome species, yet localized to just the NORs in A‐, B‐, F‐, and G‐genome species. Contained within is a story of an established centromeric repeat that is eliminated and allopolyploidization provides an opportunity for reinvasion and reestablishment, which broadens our evolutionary understanding behind the cycles of centromeric repeat establishment and targeting.     

Two combinatorial patterns of telomere histone marks in plants with canonical and non‐canonical telomere repeats

Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are crucial for the maintenance of genome integrity. In most plants, telomeres consist of conserved tandem repeat units comprising the TTTAGGG motif. Recently, non‐canonical telomeres were described in several plants and plant taxons, including the carnivorous plant Genlisea hispidula (TTCAGG/TTTCAGG), the genus Cestrum (Solanaceae; TTTTTTAGGG), and plants from the Asparagales order with either a vertebrate‐type telomere repeat TTAGGG or Allium genus‐specific CTCGGTTATGGG repeat. We analyzed epigenetic modifications of telomeric histones in plants with canonical and non‐canonical telomeres, and further in telomeric chromatin captured from leaves of Nicotiana benthamiana transiently transformed by telomere CRISPR‐dCas9‐eGFP, and of Arabidopsis thaliana stably transformed with TALE_telo C‐3×GFP. Two combinatorial patterns of telomeric histone modifications were identified: (i) an Arabidopsis‐like pattern (A. thaliana, G. hispidula, Genlisea nigrocaulis, Allium cepa, Narcissus pseudonarcissus, Petunia hybrida, Solanum tuberosum, Solanum lycopersicum) with telomeric histones decorated predominantly by H3K9me2; (ii) a tobacco‐like pattern (Nicotiana tabacum, N. benthamiana, C. elegans) with a strong H3K27me3 signal. Our data suggest that epigenetic modifications of plant telomere‐associated histones are related neither to the sequence of the telomere motif nor to the lengths of the telomeres. Nor the phylogenetic position of the species plays the role; representatives of the Solanaceae family are included in both groups. As both patterns of histone marks are compatible with fully functional telomeres in respective plants, we conclude that the described specific differences in histone marks are not critical for telomere functions.     

Telomeric localization of the Arabidopsis‐type heptamer repeat, (TTTAGGG)n,at the chromosome ends in Saccharina japonica (Phaeophyta)

Telomeres generally consist of short repeats of minisatellite DNA sequences and are useful in chromosome identification and karyotype analysis. To date, telomeres have not been characterized in the economically important brown seaweed Saccharina japonica, thus its full cytogenetic research and genetic breeding potential has not been realized. Herein, the tentative sequence of telomeres in S. japonica was identified by PCR amplification with primers designed based on the Arabidopsis‐type telomere sequence (TTTAGGG)_n, which was chosen out of three possible telomeric repeat DNA sequences typically present in plants and algae. After PCR optimization and cloning, sequence analysis of the amplified products from S. japonica genomic DNA showed that they were composed of repeat units, (TTTAGGG)_n, in which the repeat number ranged from 15 to 63 (n = 46). This type of repeat sequence was verified by a Southern blot assay with the Arabidopsis‐type telomere sequence as a probe. The digestion of S. japonica genomic DNA with the exonuclease Bal31 illustrated that the target sequence corresponding to the Arabidopsis‐type telomere sequence was susceptible to Bal31 digestion, suggesting that the repeat sequence was likely located at the outermost ends of the kelp chromosomes. Fluorescence in situ hybridizations with the aforementioned probe provided the initial cytogenetic evidence that the hybridization signals were principally localized at both ends of S. japonica chromosomes. This study indicates that the telomeric repeat of the kelp chromosomes is (TTTAGGG)_n which differs from the previously reported (TTAGGG)_n sequence in Ectocarpus siliculosus through genome sequencing, thereby suggesting distinct telomeres in brown seaweeds.     

Analysis of transposable elements and organellar DNA in male and female genomes of a species with a huge Y chromosome reveals distinct Y centromeres

Few angiosperms have distinct Y chromosomes. Among those that do are Silene latifolia (Caryophyllaceae), Rumex acetosa (Polygonaceae) and Coccinia grandis (Cucurbitaceae), the latter having a male/female difference of 10% of the total genome (female individuals have a 0.85 pg genome, male individuals 0.94 pg), due to a Y chromosome that arose about 3 million years ago. We compared the sequence composition of male and female C. grandis plants and determined the chromosomal distribution of repetitive and organellar DNA with probes developed from 21 types of repetitive DNA, including 16 mobile elements. The size of the Y chromosome is largely due to the accumulation of certain repeats, such as members of the Ty1/copia and Ty3/gypsy superfamilies, an unclassified element and a satellite, but also plastome‐ and chondriome‐derived sequences. An abundant tandem repeat with a unit size of 144 bp stains the centromeres of the X chromosome and the autosomes, but is absent from the Y centromere. Immunostaining with pericentromere‐specific markers for anti‐histone H3Ser10ph and H2AThr120ph revealed a Y‐specific extension of these histone marks. That the Y centromere has a different make‐up from all the remaining centromeres raises questions about its spindle attachment, and suggests that centromeric or pericentromeric chromatin might be involved in the suppression of recombination.     

Identification and characterization of functional centromeres of the common bean

In higher eukaryotes, centromeres are typically composed of megabase‐sized arrays of satellite repeats that evolve rapidly and homogenize within a species' genome. Despite the importance of centromeres, our knowledge is limited to a few model species. We conducted a comprehensive analysis of common bean (Phaseolus vulgaris) centromeric satellite DNA using genomic data, fluorescence in situ hybridization (FISH), immunofluorescence and chromatin immunoprecipitation (ChIP). Two unrelated centromere‐specific satellite repeats, CentPv1 and CentPv2, and the common bean centromere‐specific histone H3 (PvCENH3) were identified. FISH showed that CentPv1 and CentPv2 are predominantly located at subsets of eight and three centromeres, respectively. Immunofluorescence‐ and ChIP‐based assays demonstrated the functional significance of CentPv1 and CentPv2 at centromeres. Genomic analysis revealed several interesting features of CentPv1 and CentPv2: (i) CentPv1 is organized into an higher‐order repeat structure, named Nazca, of 528 bp, whereas CentPv2 is composed of tandemly organized monomers; (ii) CentPv1 and CentPv2 have undergone chromosome‐specific homogenization; and (iii) CentPv1 and CentPv2 are not likely to be commingled in the genome. These findings suggest that two distinct sets of centromere sequences have evolved independently within the common bean genome, and provide insight into centromere satellite evolution.     

A BAC library of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. for the targeted isolation of centromeric DNA and molecular cytogenetics of <Emphasis Type="Italic">Beta</Emphasis> species

We constructed a sugar beet (Beta vulgaris) bacterial artificial chromosome (BAC) library of the monosomic addition line PAT2. This chromosomal mutant carries a single additional chromosome fragment (minichromosome) derived from the wild beet Beta patellaris. Restriction analysis of the mutant line by pulsed-field gel electrophoresis was used to determine HindIII as a suitable enzyme for partial digestion of genomic DNA to generate large-insert fragments which were cloned into the vector pCC1. The library consists of 36,096 clones with an average insert size of 120 kb, and 2.2% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents 5.7 genome equivalents providing the probability of 99.67% that any sequence of the PAT2 genome can be found in the library. Hybridization to high-density filters was used to isolate 89 BACs containing arrays of the centromere-associated satellite repeats pTS5 and pTS4.1. Using the identified BAC clones in fluorescent in situ hybridization experiments with PAT2 and Beta patellaris chromosome spreads their wild beet origin and centromeric localization was demonstrated. Multi-colour FISH with differently labelled satellite repeats pTS5 and pTS4.1 was used to investigate the large-scale organization of the centromere of the PAT2 minichromosome in detail. FISH studies showed that the centromeric satellite pTS5 is flanked on both sides by pTS4.1 arrays and the arms of the minichromosome are terminated by the Arabidopsis-type telomeric sequences. FISH with a BAC, selected from high-density filters after hybridization with an RFLP marker of the genetic linkage group I, demonstrated that it is feasible to correlate genetic linkage groups with chromosomes. Therefore, the PAT2 BAC library provides a useful tool for the characterization of Beta centromeres and a valuable resource for sugar beet genome analysis.     

Centromeric DNA characterization in the model grass Brachypodium distachyon provides insights on the evolution of the genus

Brachypodium distachyon is a well‐established model monocot plant, and its small and compact genome has been used as an accurate reference for the much larger and often polyploid genomes of cereals such as Avena sativa (oats), Hordeum vulgare (barley) and Triticum aestivum (wheat). Centromeres are indispensable functional units of chromosomes and they play a core role in genome polyploidization events during evolution. As the Brachypodium genus contains about 20 species that differ significantly in terms of their basic chromosome numbers, genome size, ploidy levels and life strategies, studying their centromeres may provide important insight into the structure and evolution of the genome in this interesting and important genus. In this study, we isolated the centromeric DNA of the B. distachyon reference line Bd21 and characterized its composition via the chromatin immunoprecipitation of the nucleosomes that contain the centromere‐specific histone CENH3. We revealed that the centromeres of Bd21 have the features of typical multicellular eukaryotic centromeres. Strikingly, these centromeres contain relatively few centromeric satellite DNAs; in particular, the centromere of chromosome 5 (Bd5) consists of only ~40 kb. Moreover, the centromeric retrotransposons in B. distachyon (CRBds) are evolutionarily young. These transposable elements are located both within and adjacent to the CENH3 binding domains, and have similar compositions. Moreover, based on the presence of CRBds in the centromeres, the species in this study can be grouped into two distinct lineages. This may provide new evidence regarding the phylogenetic relationships within the Brachypodium genus.     

Characterisation of an unusual telomere motif (TTTTTTAGGG)n in the plant Cestrum elegans (Solanaceae), a species with a large genome

The characterization of unusual telomere sequence sheds light on patterns of telomere evolution, maintenance and function. Plant species from the closely related genera Cestrum, Vestia and Sessea (family Solanaceae) lack known plant telomeric sequences. Here we characterize the telomere of Cestrum elegans, work that was a challenge because of its large genome size and few chromosomes (1C 9.76 pg; n = 8). We developed an approach that combines BAL31 digestion, which digests DNA from the ends and chromosome breaks, with next‐generation sequencing (NGS), to generate data analysed in RepeatExplorer, designed for de novo repeats identification and quantification. We identify an unique repeat motif (TTTTTTAGGG)_n in C. elegans, occurring in ca. 30 400 copies per haploid genome, averaging ca. 1900 copies per telomere, and synthesized by telomerase. We demonstrate that the motif is synthesized by telomerase. The occurrence of an unusual eukaryote (TTTTTTAGGG)_n telomeric motif in C. elegans represents a switch in motif from the ‘typical’ angiosperm telomere (TTTAGGG)_n. That switch may have happened with the divergence of Cestrum, Sessea and Vestia. The shift in motif when it arose would have had profound effects on telomere activity. Thus our finding provides a unique handle to study how telomerase and telomeres responded to genetic change, studies that will shed more light on telomere function.     

Centromeric and telomeric repeats are stable in nonagenarians as revealed by the double hybridization fluorescent in situ technique

We report a simple method for simultaneous identification of centromeric and telomeric repeat sequences of human chromosomes. Employing this technique, we investigated the stability of centromeres and telomeres in individuals over 90 years of age and compared them with younger controls (<40 years). Our findings suggest that centromeric and telomeric repeats remain apparently stable in nonagenarians. These findings are enigmatic because it has been suggested that centromeres are lost in older individuals. Furthermore, telomeric shortening has been observed in aged lymphocytes and cellular senescence. However, stability of telomeric repeats noted in nonagenarians may be masked by loss followed by compensation by a process called telomeric elongation.     

Assembly and annotation of a draft genome sequence for Glycine latifolia,a perennial wild relative of soybean

Glycine latifolia (Benth.) Newell & Hymowitz (2n = 40), one of the 27 wild perennial relatives of soybean, possesses genetic diversity and agronomically favorable traits that are lacking in soybean. Here, we report the 939‐Mb draft genome assembly of G. latifolia (PI 559298) using exclusively linked‐reads sequenced from a single Chromium library. We organized scaffolds into 20 chromosome‐scale pseudomolecules utilizing two genetic maps and the Glycine max (L.) Merr. genome sequence. High copy numbers of putative 91‐bp centromere‐specific tandem repeats were observed in consecutive blocks within predicted pericentromeric regions on several pseudomolecules. No 92‐bp putative centromeric repeats, which are abundant in G. max, were detected in G. latifolia or Glycine tomentella. Annotation of the assembled genome and subsequent filtering yielded a high confidence gene set of 54 475 protein‐coding loci. In comparative analysis with five legume species, genes related to defense responses were significantly overrepresented in Glycine‐specific orthologous gene families. A total of 304 putative nucleotide‐binding site (NBS)‐leucine‐rich‐repeat (LRR) genes were identified in this genome assembly. Different from other legume species, we observed a scarcity of TIR‐NBS‐LRR genes in G. latifolia. The G. latifolia genome was also predicted to contain genes encoding 367 LRR‐receptor‐like kinases, a family of proteins involved in basal defense responses and responses to abiotic stress. The genome sequence and annotation of G. latifolia provides a valuable source of alternative alleles and novel genes to facilitate soybean improvement. This study also highlights the efficacy and cost‐effectiveness of the application of Chromium linked‐reads in diploid plant genome de novo assembly.     

Endogenous pararetrovirus sequences associated with 24 nt small RNAs at the centromeres of Fritillaria imperialis L. (Liliaceae), a species with a giant genome

Endogenous pararetroviral sequences are the most commonly found virus sequences integrated into angiosperm genomes. We describe an endogenous pararetrovirus (EPRV) repeat in Fritillaria imperialis, a species that is under study as a result of its exceptionally large genome (1C = 42 096 Mbp, approximately 240 times bigger than Arabidopsis thaliana). The repeat (FriEPRV) was identified from Illumina reads using the RepeatExplorer pipeline, and exists in a complex genomic organization at the centromere of most, or all, chromosomes. The repeat was reconstructed into three consensus sequences that formed three interconnected loops, one of which carries sequence motifs expected of an EPRV (including the gag and pol domains). FriEPRV shows sequence similarity to members of the Caulimoviridae pararetrovirus family, with phylogenetic analysis indicating a close relationship to Petuvirus. It is possible that no complete EPRV sequence exists, although our data suggest an abundance that exceeds the genome size of Arabidopsis. Analysis of single nucleotide polymorphisms revealed elevated levels of C→T and G→A transitions, consistent with deamination of methylated cytosine. Bisulphite sequencing revealed high levels of methylation at CG and CHG motifs (up to 100%), and 15–20% methylation, on average, at CHH motifs. FriEPRV's centromeric location may suggest targeted insertion, perhaps associated with meiotic drive. We observed an abundance of 24 nt small RNAs that specifically target FriEPRV, potentially providing a signature of RNA‐dependent DNA methylation. Such signatures of epigenetic regulation suggest that the huge genome of F. imperialis has not arisen as a consequence of a catastrophic breakdown in the regulation of repeat amplification.     

A tandemly repetitive centromeric DNA sequence of the fish Hoplias malabaricus (Characiformes: Erythrinidae) is derived from 5S rDNA

A substantial fraction of the eukaryotic genome consists of repetitive DNA sequences that include satellites, minisatellites, microsatellites, and transposable elements. Although extensively studied for the past three decades, the molecular forces that generate, propagate and maintain repetitive DNAs in the genomes are still discussed. To further understand the dynamics and the mechanisms of evolution of repetitive DNAs in vertebrate genome, we searched for repetitive sequences in the genome of the fish species Hoplias malabaricus. A satellite sequence, named 5SHindIII-DNA, which has a conspicuous similarity with 5S rRNA genes and spacers was identified. FISH experiments showed that the 5S rRNA bona fide gene repeats were clustered in the interstitial position of two chromosome pairs of H. malabaricus, while the satellite 5SHindIII-DNA sequences were clustered in the centromeric position in nine chromosome pairs of the species. The presence of the 5SHindIII-DNA sequences in the centromeres of several chromosomes indicates that this satellite family probably escaped from the selective pressure that maintains the structure and organization of the 5S rDNA repeats and become disperse into the genome. Although it is not feasible to explain how this sequence has been maintained in the centromeric regions, it is possible to hypothesize that it may be involved in some structural or functional role of the centromere organization.     

Robertsonian metacentrics of the house mouse lose telomeric sequences but retain some minor satellite DNA in the pericentromeric area

A combination of cytogenetic and molecular biology techniques were used to study the molecular composition and organisation of the pericentromeric regions of house mouse metacentric chromosomes, the products of Robertsonian (Rb) translocations between telocentrics. Regardless of whether mitotic or meiotic preparations were used, in situ hybridisation failed to reveal pericentromeric telomeric sequences on any of the Rb chromosomes, while all metacentrics retained detectable, although reduced (average 50 kb), amounts of minor satellite DNA in the vicinity of their centromeres. These results were supported by slot blot hybridisation which indicated that mice with 2n=22 Rb chromosomes have 65% of telomeric sequences (which are allocated to the distal telomeres of both Rb and telocentric chromosomes and to the proximal telomeres of telocentrics) and 15% the amount of minor satellite, compared with mice with 2n=40 all-telocentric chromosomes. Pulsed field gel electrophoresis and Southern analysis of DNA from Rb mice showed that the size of the telomeric arrays is similar to that of mice with all-telocentric chromosomes and that the minor satellite sequences were hybridising to larger fragments incorporating major satellite DNA. Since the telomeric sequences are closer to the physical end of the chromosome than the minor satellite sequences, the absence of telomeric sequences and the reduced amount of minor satellite sequences at the pericentromeric region of the Rb metacentrics suggest that the breakpoints for the Rb translocation occur very close to the minor satellite-major satellite border. Moreover, it is likely that the minor satellite is required for centromeric function, 50–67 kb being enough DNA to organise one centromere with a functionally active kinetochore.     

Rolling‐circle amplification of centromeric Helitrons in plant genomes

The unusual eukaryotic Helitron transposons can readily capture host sequences and are, thus, evolutionarily important. They are presumed to amplify by rolling‐circle replication (RCR) because some elements encode predicted proteins homologous to RCR prokaryotic transposases. In support of this replication mechanism, it was recently shown that transposition of a bat Helitron generates covalently closed circular intermediates. Another strong prediction is that RCR should generate tandem Helitron concatemers, yet almost all Helitrons identified to date occur as solo elements in the genome. To investigate alternative modes of Helitron organization in present‐day genomes, we have applied the novel computational tool HelitronScanner to 27 plant genomes and have uncovered numerous tandem arrays of partially decayed, truncated Helitrons in all of them. Strikingly, most of these Helitron tandem arrays are interspersed with other repeats in centromeres. Many of these arrays have multiple Helitron 5′ ends, but a single 3′ end. The number of repeats in any one array can range from a handful to several hundreds. We propose here an RCR model that conforms to the present Helitron landscape of plant genomes. Our study provides strong evidence that plant Helitrons amplify by RCR and that the tandemly arrayed replication products accumulate mostly in centromeres.     

Cloning and characterization of a centromere-specific repetitive DNA element from Sorghum bicolor

 A 823-bp Sau3AI fragment (pSau3A10) was subcloned from a sorghum bacterial artificial chromosome (BAC) clone, 13I16, that contains DNA sequences specific to the centromeres of grass species. Sequence analysis showed that pSau3A10 consists of six copies of an approximately 137-bp monomer. The six monomers were organized into three dimers. The monomers within the dimers shared 62–72% homology and the dimers were 79–82% homologous with each other. Fluorescence in situ hybridization (FISH) analysis indicated that the Sau3A10 family is present only in the centromeres of sorghum chromosomes. Sequencing, Southern hybridization, and Fiber-FISH analyses indicated that the Sau3A10 family is tandemly arranged and is present in uninterrupted stretches of up to at least 81 kb of DNA. Slot-blot analysis estimated that the Sau3A10 family constitutes 1.6–1.9% of the sorghum genome. The long stretches of Sau3A10 sequences were interrupted by other centromeric DNA elements. Southern analysis indicated that the Sau3A10 sequence is one of the most abundant DNA families located in sorghum centromeres and is conserved only in closely related sorghum species. Methylation experiments indicated that the cytosine of the CG sites in sorghum centromeric regions is generally methylated. The structure and organization of the Sau3A10 family shared similarities with centromeric DNA repeats in other eukaryotic species. It is suggested that the Sau3A10 family is probably an important part of sorghum centromeres. Received: 11 November 1997 / Accepted: 17 November 1997     

Wheat centromeric retrotransposons: the new ones take a major role in centromeric structure

The physical map of the hexaploid wheat chromosome 3B was screened using centromeric DNA probes. A 1.1‐Mb region showing the highest number of positive bacterial artificial chromosome (BAC) clones was fully sequenced and annotated, revealing that 96% of the DNA consisted of transposable elements, mainly long terminal repeat (LTR) retrotransposons (88%). Estimation of the insertion time of the transposable elements revealed that CRW (also called Cereba) and Quinta are the youngest elements at the centromeres of common wheat (Triticum spp.) and its diploid ancestors, with Quinta being younger than CRW in both diploid and hexaploid wheats. Chromatin immunoprecipitation experiments revealed that both CRW and Quinta families are targeted by the centromere‐specific histone H3 variant CENH3. Immuno colocalization of retroelements and CENH3 antibody indicated that a higher proportion of Quinta than CRWs was associated with CENH3, although CRWs were more abundant. Long arrays of satellite repeats were also identified in the wheat centromere regions, but they lost the ability to bind with CENH3. In addition to transposons, two functional genes and one pseudogene were identified. The gene density in the centromere appeared to be between three and four times lower than the average gene density of chromosome 3B. Comparisons with related grasses also indicated a loss of microcollinearity in this region. Finally, comparison of centromeric sequences of Aegilops tauschii (DD), Triticum boeoticum (AA) and hexaploid wheat revealed that the centromeres in both the polyploids and diploids are still undergoing dynamic changes, and that the new CRWs and Quintas may have undertaken the core role in kinetochore formation.     

植物着丝粒结构和功能的研究进展

着丝粒是真核生物有丝分裂和减数分裂染色体正确分离和传递所必需的染色体区域。十多年来, 已对包括拟南芥、水稻、玉米在内的一些植物的着丝粒进行了较深入的分子生物学研究。在不同的植物间, 着丝粒DNA的保守性很低, 呈现快速进化, 但着丝粒的DNA序列类型和组织方式基本相似, 一般是由夹杂排列着的卫星DNA串联重复阵列和着丝粒专一的反转录转座子构成。与着丝粒DNA相反, 着丝粒/着丝点的结构性和瞬时蛋白质在包括植物在内的真核生物中保守。与其他真核生物的情况一样, 拥有含着丝粒组蛋白H3(CENH3)的核小体是植物功能着丝粒染色质最基本的特征, CENH3在着丝粒染色质的识别和保持中起着关键作用。     

Significant association between SNPs in the superoxide dismutase 3, extracellular (SOD3) gene and resistance to Aeromonas hydrophila in the freshwater mussel Hyriopsis cumingii

Extracellular superoxide dismutase (SOD3) is a major antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). In this study, the SOD3 gene was identified and characterized from the freshwater mussel Hyriopsis cumingii (Hc‐SOD3). The cDNA sequence consists of 763 bp, encoding a protein of 208 amino acids. The amino acid sequence possesses two CuZnSOD signature sequences, and amino acids required for binding of Cu (His‐93, ‐95, ‐110 and ‐169) and Zn (His‐110, ‐118, ‐129 and Asp‐132) were conserved in Hc‐SOD3. The Hc‐SOD3 genomic sequence was 9165 bp in length, containing four exons and three introns. Eighteen single nucleotide polymorphisms were detected in the Hc‐SOD3 gene from resistant stock (RS) and susceptible stock (SS) of H. cumingii to Aeromonas hydrophila. The genotype and allele distribution were examined in resistant and susceptible stocks. Among them, a C/G substitution at the g.7994C>G locus and G/C substitution at the g.8087G>C locus were significantly associated with resistance/susceptibility of H. cumingii to A. hydrophila, both in genotype (P = 0.017, P = 0.004 respectively) and allele frequency (P = 0.021, P = 0.006 respectively). Linkage disequilibrium analysis revealed that g.7994C>G, g.8001A>G, g.8035G>A, g.8087G>C and g.8191T>A were in linkage disequilibrium. The results suggest that the two polymorphic loci, g.7994C>G and g.8087G>C, could be potential genetic markers for future molecular selection of strains that are resistant to diseases.     

Telomere repeat binding proteins are functional components of Arabidopsis telomeres and interact with telomerase

Although telomere‐binding proteins constitute an essential part of telomeres, in vivo data indicating the existence of a structure similar to mammalian shelterin complex in plants are limited. Partial characterization of a number of candidate proteins has not identified true components of plant shelterin or elucidated their functional mechanisms. Telomere repeat binding (TRB) proteins from Arabidopsis thaliana bind plant telomeric repeats through a Myb domain of the telobox type in vitro, and have been shown to interact with POT1b (Protection of telomeres 1). Here we demonstrate co‐localization of TRB1 protein with telomeres in situ using fluorescence microscopy, as well as in vivo interaction using chromatin immunoprecipitation. Classification of the TRB1 protein as a component of plant telomeres is further confirmed by the observation of shortening of telomeres in knockout mutants of the trb1 gene. Moreover, TRB proteins physically interact with plant telomerase catalytic subunits. These findings integrate TRB proteins into the telomeric interactome of A. thaliana.