The localization of centromere protein A is conserved among tissues.

Centromeres are epigenetically specified by the histone H3 variant CENP-A. Although mammalian centromeres are typically associated with satellite DNA, we previously demonstrated that the centromere of horse chromosome 11 (ECA11) is completely devoid of satellite DNA. We also showed that the localization of its CENP-A binding domain is not fixed but slides within an about 500 kb region in different individuals, giving rise to positional alleles. These epialleles are inherited as Mendelian traits but their position can move in one generation. It is still unknown whether centromere sliding occurs during meiosis or during development. Here, we first improve the sequence of the ECA11 centromeric region in the EquCab3.0 assembly. Then, to test whether centromere sliding may occur during development, we map the CENP-A binding domains of ECA11 using ChIP-seq in five tissues of different embryonic origin from the four horses of the equine FAANG (Functional Annotation of ANimal Genomes) consortium. Our results demonstrate that the centromere is localized in the same region in all tissues, suggesting that the position of the centromeric domain is maintained during development.

First report of Colletotrichum grossum causing apple bitter rot worldwide (Italy).

Apple bitter rot is a globally widespread disease that is observed on fruits both pre-harvest and post-harvest, contributing to considerable economic losses. While the Colletotrichum acutatum species complex are predominant in Europe (Baroncelli et al. 2014; Amaral Carneiro and Baric 2021), in recent years, the Colletotrichum gloeosporioides species complex are emerging, raising many concerns (Amaral Carneiro et al. 2023). Circular, slightly sunken, brown lesions with acervuli produced in concentric spots were observed on 'Story® Inored' cultivar harvested in September 2022 from an organic orchard in Masi (Padova province, Italy), with a disease incidence close to 30%. From ten diseased apples, tissue samples were excised under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to potato dextrose agar medium and incubated in the dark at 25 °C for 7 days, whereafter five single-spore cultures were obtained. Pure colonies grown at 25 °C for 7 days appeared light gray-white on the upper side with floccose aerial mycelium, while the reverse side was dark gray with a distinct margin. Conidia were hyaline, cylindrical in shape with both ends rounded or one end acute and measured 16.6 ± 1.4 × 6.1 ± 0.5 µm [mean ± SD] (n=50) as described by Diao et al. 2017. To identify the species, genomic DNA of a representative isolate (C38) was extracted, beta-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), Apn2-Mat1-2 intergenic spacer (ApnMat) genes and the internal transcribed spacer (ITS) region, were amplified by PCR and Sanger sequenced (Rojas et al. 2010; Weir et al. 2012). The obtained DNA sequences of, TUB2, CAL, GAPDH, GS, ApnMat and ITS were submitted to GenBank under the accession numbers OR025589, OR025586, OR025587, OR025588, OR025585 and OR004800, respectively. A MegaBLAST analysis resulted 100 % identity to the epitype CAUG7 of Colletotrichum grossum (Diao et al. 2017) for GAPDH (KP890159), for TUB2 (KP890171), 99.85% for CAL (KP890147) and 99,5 % for ITS (KP890165). The phylogenetic tree constructed by concatenation with the obtained sequences, as well as references, revealed that the C38 isolate clustered within C. grossum, confirming the BLAST approach. Pathogenicity tests were performed on 40 'Story® Inored' apples cleaned and wounded with a sterilized needle and exposed to two different conditions: 20 apples (10 inoculated with 20 µl of spore suspension (105 ml-1) and 10 with sterile water as control) were incubated at 20°C with a 12-hour photoperiod for 14 days, while the remaining 20 apples, prepared with same approach, were placed at 1°C for 3 months, then at room temperature for 14 days. Symptoms appeared after 6 days on apples incubated at 20°C, whereas those stored at 1°C displayed symptoms at 11 days after being placed at room temperature. In both conditions, lesions were similar to those observed on the original fruits; while the controls remained asymptomatic. Identity of reisolated fungal colonies was confirmed by CAL, GAPDH and GS region sequence analysis. C. grossum has been reported rarely: in 2017 on Capsicum annuum var. grossum in China, in 2018 on Mangifera indica leaves in Cuba, and in 2021 on Rhyncospermum jasminoides in Italy (Diao et al. 2017; Manzano León et al. 2018; Guarnaccia et al. 2021). To the best of our knowledge, this is the first report of apple bitter rot caused by Colletotrichum grossum worldwide.

Different diagnostic approaches for the characterization of the fungal community and Fusarium species complex composition of Italian durum wheat grain and correlation with secondary metabolite accumulation.

BACKGROUND: The evolution of the fungal communities associated with durum wheat was assessed using different diagnostic approaches. Durum wheat grain samples were collected in three different Italian cultivation macro-areas (north, center and south). Fungal isolation was realized by potato dextrose agar (PDA) and by deep-freezing blotter (DFB). Identification of Fusarium isolates obtained from PDA was achieved by partial tef1α sequencing (PDA + tef1α), while those obtained from DFB were identified from their morphological characteristics (DFB + mc). The fungal biomass of eight Fusarium species was quantified in grains by quantitative polymerase chain reaction (qPCR). Fungal secondary metabolites were analyzed in grains by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Correlations between Fusarium detection techniques (PDA + tef1α; DFB + mc and qPCR) and mycotoxins in grains were assessed. RESULTS: Alternaria and Fusarium showed the highest incidence among the fungal genera developed from grains. Within the Fusarium community, PDA + tef1α highlighted that F. avenaceum and F. graminearum were the most represented members, while, DFB + mc detected a high presence of F. proliferatum. Alternaria and Fusarium mycotoxins, principally enniatins, were particularly present in the grain harvested in central Italy. Deoxynivalenol was mainly detected in northern-central Italy. CONCLUSIONS: The adoption of the different diagnostic techniques of Fusarium detection highlighted that, for some species, qPCR was the best method of predicting their mycotoxin contamination in grains. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A Satellite-Free Centromere in Equus przewalskii Chromosome 10.

In mammals, centromeres are epigenetically specified by the histone H3 variant CENP-A and are typically associated with satellite DNA. We previously described the first example of a natural satellite-free centromere on Equus caballus chromosome 11 (ECA11) and, subsequently, on several chromosomes in other species of the genus Equus. We discovered that these satellite-free neocentromeres arose recently during evolution through centromere repositioning and/or chromosomal fusion, after inactivation of the ancestral centromere, where, in many cases, blocks of satellite sequences were maintained. Here, we investigated by FISH the chromosomal distribution of satellite DNA families in Equus przewalskii (EPR), demonstrating a good degree of conservation of the localization of the major horse satellite families 37cen and 2PI with the domestic horse. Moreover, we demonstrated, by ChIP-seq, that 37cen is the satellite bound by CENP-A and that the centromere of EPR10, the ortholog of ECA11, is devoid of satellite sequences. Our results confirm that these two species are closely related and that the event of centromere repositioning which gave rise to EPR10/ECA11 centromeres occurred in the common ancestor, before the separation of the two horse lineages.

Robertsonian Fusion and Centromere Repositioning Contributed to the Formation of Satellite-free Centromeres During the Evolution of Zebras.

Centromeres are epigenetically specified by the histone H3 variant CENP-A and typically associated with highly repetitive satellite DNA. We previously discovered natural satellite-free neocentromeres in Equus caballus and Equus asinus. Here, through ChIP-seq with an anti-CENP-A antibody, we found an extraordinarily high number of centromeres lacking satellite DNA in the zebras Equus burchelli (15 of 22) and Equus grevyi (13 of 23), demonstrating that the absence of satellite DNA at the majority of centromeres is compatible with genome stability and species survival and challenging the role of satellite DNA in centromere function. Nine satellite-free centromeres are shared between the two species in agreement with their recent separation. We assembled all centromeric regions and improved the reference genome of E. burchelli. Sequence analysis of the CENP-A binding domains revealed that they are LINE-1 and AT-rich with four of them showing DNA amplification. In the two zebras, satellite-free centromeres emerged from centromere repositioning or following Robertsonian fusion. In five chromosomes, the centromeric function arose near the fusion points, which are located within regions marked by traces of ancestral pericentromeric sequences. Therefore, besides centromere repositioning, Robertsonian fusions are an important source of satellite-free centromeres during evolution. Finally, in one case, a satellite-free centromere was seeded on an inversion breakpoint. At 11 chromosomes, whose primary constrictions seemed to be associated with satellite repeats by cytogenetic analysis, satellite-free neocentromeres were instead located near the ancestral inactivated satellite-based centromeres; therefore, the centromeric function has shifted away from a satellite repeat containing locus to a satellite-free new position.

Molecular Dynamics and Evolution of Centromeres in the Genus Equus.

The centromere is the chromosomal locus essential for proper chromosome segregation. While the centromeric function is well conserved and epigenetically specified, centromeric DNA sequences are typically composed of satellite DNA and represent the most rapidly evolving sequences in eukaryotic genomes. The presence of satellite sequences at centromeres hampered the comprehensive molecular analysis of these enigmatic loci. The discovery of functional centromeres completely devoid of satellite repetitions and fixed in some animal and plant species represented a turning point in centromere biology, definitively proving the epigenetic nature of the centromere. The first satellite-free centromere, fixed in a vertebrate species, was discovered in the horse. Later, an extraordinary number of satellite-free neocentromeres had been discovered in other species of the genus Equus, which remains the only mammalian genus with numerous satellite-free centromeres described thus far. These neocentromeres arose recently during evolution and are caught in a stage of incomplete maturation. Their presence made the equids a unique model for investigating, at molecular level, the minimal requirements for centromere seeding and evolution. This model system provided new insights on how centromeres are established and transmitted to the progeny and on the role of satellite DNA in different aspects of centromere biology.

Decoding the Equine Genome: Lessons from ENCODE.

The horse reference genome assemblies, EquCab2.0 and EquCab3.0, have enabled great advancements in the equine genomics field, from tools to novel discoveries. However, significant gaps of knowledge regarding genome function remain, hindering the study of complex traits in horses. In an effort to address these gaps and with inspiration from the Encyclopedia of DNA Elements (ENCODE) project, the equine Functional Annotation of Animal Genome (FAANG) initiative was proposed to bridge the gap between genome and gene expression, providing further insights into functional regulation within the horse genome. Three years after launching the initiative, the equine FAANG group has generated data from more than 400 experiments using over 50 tissues, targeting a variety of regulatory features of the equine genome. In this review, we examine how valuable lessons learned from the ENCODE project informed our decisions in the equine FAANG project. We report the current state of the equine FAANG project and discuss how FAANG can serve as a template for future expansion of functional annotation in the equine genome and be used as a reference for studies of complex traits in horse. A well-annotated reference functional atlas will also help advance equine genetics in the pan-genome and precision medicine era.

Telomeric-Like Repeats Flanked by Sequences Retrotranscribed from the Telomerase RNA Inserted at DNA Double-Strand Break Sites during Vertebrate Genome Evolution.

Interstitial telomeric sequences (ITSs) are stretches of telomeric-like repeats located at internal chromosomal sites. We previously demonstrated that ITSs have been inserted during the repair of DNA double-strand breaks in the course of evolution and that some rodent ITSs, called TERC-ITSs, are flanked by fragments retrotranscribed from the telomerase RNA component (TERC). In this work, we carried out an extensive search of TERC-ITSs in 30 vertebrate genomes and identified 41 such loci in 22 species, including in humans and other primates. The fragment retrotranscribed from the TERC RNA varies in different lineages and its sequence seems to be related to the organization of TERC. Through comparative analysis of TERC-ITSs with orthologous empty loci, we demonstrated that, at each locus, the TERC-like sequence and the ITS have been inserted in one step in the course of evolution. Our findings suggest that telomerase participated in a peculiar pathway of DNA double-strand break repair involving retrotranscription of its RNA component and that this mechanism may be active in all vertebrate species. These results add new evidence to the hypothesis that RNA-templated DNA repair mechanisms are active in vertebrate cells.

Insertion of Telomeric Repeats in the Human and Horse Genomes: An Evolutionary Perspective.

Interstitial telomeric sequences (ITSs) are short stretches of telomeric-like repeats (TTAGGG)n at nonterminal chromosomal sites. We previously demonstrated that, in the genomes of primates and rodents, ITSs were inserted during the repair of DNA double-strand breaks. These conclusions were derived from sequence comparisons of ITS-containing loci and ITS-less orthologous loci in different species. To our knowledge, insertion polymorphism of ITSs, i.e., the presence of an ITS-containing allele and an ITS-less allele in the same species, has not been described. In this work, we carried out a genome-wide analysis of 2504 human genomic sequences retrieved from the 1000 Genomes Project and a PCR-based analysis of 209 human DNA samples. In spite of the large number of individual genomes analyzed we did not find any evidence of insertion polymorphism in the human population. On the contrary, the analysis of ITS loci in the genome of a single horse individual, the reference genome, allowed us to identify five heterozygous ITS loci, suggesting that insertion polymorphism of ITSs is an important source of genetic variability in this species. Finally, following a comparative sequence analysis of horse ITSs and of their orthologous empty loci in other Perissodactyla, we propose models for the mechanism of ITS insertion during the evolution of this order.

Occurrence of Albifimbria verrucaria in the Blood of a Female Child With Neuroblastoma.

We report for the first time the occurrence of a filamentous fungus, Albifimbria verrucaria, in the blood of a pediatric neuroblastoma patient. The Albifimbria genus comprises common soil-inhabiting and saprophytic fungi and has been isolated as a plant pathogen in Northern and Southern Italy. As a human pathogen, A. verrucaria has been implicated in keratitis and can produce trichothecene toxins, which are weakly cytotoxic for mammalian cell lines. A. verrucaria was isolated from blood during the follow-up of a previous coagulase-negative Staphylococcus catheter-related infection. Lung nodules, compatible with fungal infection, had been observed on a CT scan 6 months earlier; they still persist. Possible routes of transmission were considered to be airborne, catheter related, or transfusion dependent, as the patient had undergone platelet and red blood cell transfusions during rescue chemotherapy. No filamentous fungi were isolated from sputum or CVCs. In conclusion, we describe an unprecedented fungemia caused by A. verrucaria and show how an unexpected pathogen may be acquired from the environment by patients at high risk due to immunosuppression. The route of transmission remains unknown.

CENP-A binding domains and recombination patterns in horse spermatocytes.

Centromeres exert an inhibitory effect on meiotic recombination, but the possible contribution of satellite DNA to this "centromere effect" is under debate. In the horse, satellite DNA is present at all centromeres with the exception of the one from chromosome 11. This organization of centromeres allowed us to investigate the role of satellite DNA on recombination suppression in horse spermatocytes at the stage of pachytene. To this aim we analysed the distribution of the MLH1 protein, marker of recombination foci, relative to CENP-A, marker of centromeric function. We demonstrated that the satellite-less centromere of chromosome 11 causes crossover suppression, similarly to satellite-based centromeres. These results suggest that the centromere effect does not depend on satellite DNA. During this analysis, we observed a peculiar phenomenon: while, as expected, the centromere of the majority of meiotic bivalent chromosomes was labelled with a single immunofluorescence centromeric signal, double-spotted or extended signals were also detected. Their number varied from 0 to 7 in different cells. This observation can be explained by positional variation of the centromeric domain on the two homologs and/or misalignment of pericentromeric satellite DNA arrays during homolog pairing confirming the great plasticity of equine centromeres.

Birth, evolution, and transmission of satellite-free mammalian centromeric domains.

Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Equus caballus Chromosome 11. Here, we investigated the satellite-free centromeres of Equus asinus by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regions. A subset of these centromeres is associated with DNA amplification. The location of CENPA binding domains can vary in different individuals, giving rise to epialleles. The analysis of epiallele transmission in hybrids (three mules and one hinny) showed that centromeric domains are inherited as Mendelian traits, but their position can slide in one generation. Conversely, centromere location is stable during mitotic propagation of cultured cells. Our results demonstrate that the presence of more than half of centromeres void of satellite DNA is compatible with genome stability and species survival. The presence of amplified DNA at some centromeres suggests that these arrays may represent an intermediate stage toward satellite DNA formation during evolution. The fact that CENPA binding domains can move within relatively restricted regions (a few hundred kilobases) suggests that the centromeric function is physically limited by epigenetic boundaries.

The major horse satellite DNA family is associated with centromere competence.

BACKGROUND: The centromere is the specialized locus required for correct chromosome segregation during cell division. The DNA of most eukaryotic centromeres is composed of extended arrays of tandem repeats (satellite DNA). In the horse, we previously showed that, although the centromere of chromosome 11 is completely devoid of tandem repeat arrays, all other centromeres are characterized by the presence of satellite DNA. We isolated three horse satellite DNA sequences (37cen, 2P1 and EC137) and described their chromosomal localization in four species of the genus Equus. RESULTS: In the work presented here, using the ChIP-seq methodology, we showed that, in the horse, the 37cen satellite binds CENP-A, the centromere-specific histone-H3 variant. The 37cen sequence bound by CENP-A is GC-rich with 221 bp units organized in a head-to-tail fashion. The physical interaction of CENP-A with 37cen was confirmed through slot blot experiments. Immuno-FISH on stretched chromosomes and chromatin fibres demonstrated that the extension of satellite DNA stretches is variable and is not related to the organization of CENP-A binding domains. Finally, we proved that the centromeric satellite 37cen is transcriptionally active. CONCLUSIONS: Our data offer new insights into the organization of horse centromeres. Although three different satellite DNA families are cytogenetically located at centromeres, only the 37cen family is associated to the centromeric function. Moreover, similarly to other species, CENP-A binding domains are variable in size. The transcriptional competence of the 37cen satellite that we observed adds new evidence to the hypothesis that centromeric transcripts may be required for centromere function.

Evaluation of aging influence on renal toxicity caused by segment-specific nephrotoxicants of the proximal tubule in rat.

Little is known concerning the sensitivity of aged rats to xenobiotics inducing kidney damage. To increase this knowledge, the age-dependent response of the kidney to hexachloro-1 : 3-butadiene (HCBD) or potassium dichromate (chromate) was investigated. Rats were treated at different ages with a single dose of segment-specific nephrotoxicants of the proximal tubule, chosen on the basis of their specificity for S(3) and for S(1)-S(2) segments, respectively. The toxicological impact of these xenobiotics has been evaluated through biochemical and genomic markers, and histopathological investigation of kidney samples. HCBD treatment induced tubular necrosis of the S(3) segment of the proximal tubule associated with changes of toxicological markers unrelated to the age. In contrast, chromate treatment induced an increased kidney damage related to the rat age. In fact, histopathological investigation revealed that at 1 month of age tubular vacuolar degeneration was seen affecting S(1)-S(2) segments of the proximal tubule, whereas at 3 months of age tubular necrosis occurred in the same segments associated with tubular dilation of the distal portions. Consistently, biochemical analysis confirmed a direct correlation among genomic and biochemical marker variability and animal age. Altogether, the results show that during aging there is an increased sensitivity of kidney to chromate but not to HCBD-induced damage and evidence differential age-related selectivity of rats for nephrotoxic compounds. Significance for human risk assessment is discussed.