Genome-wide epigenetic modifications in sports horses during training as an adaptation phenomenon.

With his bicentennial breeding history based on athletic performance, the Thoroughbred horse can be considered the equine sport breed. Although genomic and transcriptomic tools and knowledge are at the state of the art in equine species, the epigenome and its modifications in response to environmental stimuli, such as training, are less studied. One of the major epigenetic modifications is cytosine methylation at 5' of DNA molecules. This crucial biochemical modification directly mediates biological processes and, to some extent, determines the organisms' phenotypic plasticity. Exercise indeed affects the epigenomic state, both in humans and in horses. In this study, we highlight, with a genome-wide analysis of methylation, how the adaptation to training in the Thoroughbred can modify the methylation pattern throughout the genome. Twenty untrained horses, kept under the same environmental conditions and sprint training regimen, were recruited, collecting peripheral blood at the start of the training and after 30 and 90 days. Extracted leukocyte DNA was analyzed with the methylation content sensitive enzyme ddRAD (MCSeEd) technique for the first time applied to animal cells. Approximately one thousand differently methylated genomic regions (DMRs) and nearby genes were called, revealing that methylation changes can be found in a large part of the genome and, therefore, referable to the physiological adaptation to training. Functional analysis via GO enrichment was also performed. We observed significant differences in methylation patterns throughout the training stages: we hypothesize that the methylation profile of some genes can be affected early by training, while others require a more persistent stimulus.

Development and Application of a Cleaved Amplified Polymorphic Sequence Marker (Phyto) Linked to the Pc5.1 Locus Conferring Resistance to Phytophthora capsici in Pepper (Capsicum annuum L.).

Phytophthora capsici causes destructive disease in several crop species, including pepper (Capsicum annuum L.). Resistance in this species is physiologically and genetically complex due to many P. capsici virulence phenotypes and different QTLs and R genes among the identified resistance sources. Several primer pairs were designed to follow an SNP (G/A) within the CA_011264 locus linked to the Pc5.1 locus. All primer pairs were designed on DNA sequences derived from CaDMR1, a homoserine kinase (HSK), which is a gene candidate responsible for the major QTL on chromosome P5 for resistance to P. capsici. A panel of 69 pepper genotypes from the Southern Seed germplasm collection was used to screen the primer pairs designed. Of these, two primers (Phyto_for_2 and Phyto_rev_2) surrounding the SNP proved successful in discriminating susceptible and resistant genotypes when combined with a restriction enzyme (BtgI). This new marker (called Phyto) worked as expected in all genotypes tested, proving to be an excellent candidate for marker-assisted selection in breeding programs aimed at introgressing the resistant locus into pure lines.

Evidence of chromatin and transcriptional dynamics for cold development in peach flower bud.

In temperate zones, fruit trees regulate their annual growth cycle to seasonal environmental changes. During the cold season, growth is limited by both environmental and genetic factors. After the exposure to low temperature and fulfillment of chilling requirements, mild temperatures promote the growth and flowering. However, an insufficient chilling exposure may lead to nonuniform blooming, with a negative impact on fruit set. To gain insights into flower development in the fruit tree buds, peach is an interesting model, the flower and vegetative bud being distinct organs. To understand how flower bud development is regulated, we integrated cytological observations and epigenetic and chromatin genome-wide data with transcriptional changes to identify the main regulatory factors involved in flower development during chilling accumulation. We demonstrated that growth cessation does not occur in peach flower buds during chilling accumulation, but that there are changes in transcript abundance of key genes of hormone metabolism and flower bud development, distribution of histone modifications (H3K4me3 and H3K27me3) and DNA methylation. Altogether, our findings indicate that during the cold season the flower bud is in a nondormant state and that the chilling experience allows flower differentiation to be completed.

Identification of Putative Virulence Genes by DNA Methylation Studies in the Cereal Pathogen Fusarium graminearum.

DNA methylation mediates organisms' adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

Differential Methylation Patterns in Apomictic vs. Sexual Genotypes of the Diplosporous Grass Eragrostis curvula.

DNA methylation is an epigenetic mechanism by which a methyl group is added to a cytosine or an adenine. When located in a gene/regulatory sequence it may repress or de-repress genes, depending on the context and species. Eragrostis curvula is an apomictic grass in which facultative genotypes increases the frequency of sexual pistils triggered by epigenetic mechanisms. The aim of the present study was to look for correlations between the reproductive mode and specific methylated genes or genomic regions. To do so, plants with contrasting reproductive modes were investigated through MCSeEd (Methylation Context Sensitive Enzyme ddRad) showing higher levels of DNA methylation in apomictic genotypes. Moreover, an increased proportion of differentially methylated positions over the regulatory regions were observed, suggesting its possible role in regulation of gene expression. Interestingly, the methylation pathway was also found to be self-regulated since two of the main genes (ROS1 and ROS4), involved in de-methylation, were found differentially methylated between genotypes with different reproductive behavior. Moreover, this work allowed us to detect several genes regulated by methylation that were previously found as differentially expressed in the comparisons between apomictic and sexual genotypes, linking DNA methylation to differences in reproductive mode.

Differential Epigenetic Marks Are Associated with Apospory Expressivity in Diploid Hybrids of Paspalum rufum.

Apomixis seems to emerge from the deregulation of preexisting genes involved in sexuality by genetic and/or epigenetic mechanisms. The trait is associated with polyploidy, but diploid individuals of Paspalum rufum can form aposporous embryo sacs and develop clonal seeds. Moreover, diploid hybrid families presented a wide apospory expressivity variation. To locate methylation changes associated with apomixis expressivity, we compare relative DNA methylation levels, at CG, CHG, and CHH contexts, between full-sib P. rufum diploid genotypes presenting differential apospory expressivity. The survey was performed using a methylation content-sensitive enzyme ddRAD (MCSeEd) strategy on samples at premeiosis/meiosis and postmeiosis stages. Based on the relative methylation level, principal component analysis and heatmaps, clearly discriminate samples with contrasting apospory expressivity. Differential methylated contigs (DMCs) showed 14% of homology to known transcripts of Paspalum notatum reproductive transcriptome, and almost half of them were also differentially expressed between apomictic and sexual samples. DMCs showed homologies to genes involved in flower growth, development, and apomixis. Moreover, a high proportion of DMCs aligned on genomic regions associated with apomixis in Setaria italica. Several stage-specific differential methylated sequences were identified as associated with apospory expressivity, which could guide future functional gene characterization in relation to apomixis success at diploid and tetraploid levels.

The Role of APOSTART in Switching between Sexuality and Apomixis in Poa pratensis.

The production of seeds without sex is considered the holy grail of plant biology. The transfer of apomixis to various crop species has the potential to transform plant breeding, since it will allow new varieties to retain valuable traits thorough asexual reproduction. Therefore, a greater molecular understanding of apomixis is fundamental. In a previous work we identified a gene, namely APOSTART, that seemed to be involved in this asexual mode of reproduction, which is very common in Poa pratensis L., and here we present a detailed work aimed at clarifying its role in apomixis. In situ hybridization showed that PpAPOSTART is expressed in reproductive tissues from pre-meiosis to embryo development. Interestingly, it is expressed early in few nucellar cells of apomictic individuals possibly switching from a somatic to a reproductive cell as in aposporic apomixis. Moreover, out of 13 APOSTART members, we identified one, APOSTART_6, as specifically expressed in flower tissue. APOSTART_6 also exhibited delayed expression in apomictic genotypes when compared with sexual types. Most importantly, the SCAR (Sequence Characterized Amplified Region) derived from the APOSTART_6 sequence completely co-segregated with apomixis.

MCSeEd (Methylation Context Sensitive Enzyme ddRAD): A New Method to Analyze DNA Methylation.

Methylation context sensitive enzyme ddRAD (MCSeEd) is a NGS-based method for genome-wide investigations of DNA methylation at different contexts requiring only low to moderate sequencing depth. It is particularly useful for identifying methylation changes in experimental systems challenged by biotic or abiotic stresses or at different developmental stages.

DNA Methylation Changes Induced by Cold in Psychrophilic and Psychrotolerant Naganishia Yeast Species.

The involvement of DNA methylation in the response to cold stress of two different yeast species (Naganishia antarctica, psychrophilic, and Naganishia albida, psychrotolerant), exhibiting different temperature aptitudes, has been studied. Consecutive incubations at respective optimum temperatures, at 4 °C (cold stress) and at optimum temperatures again, were performed. After Methylation Sensitive Amplified Polymorphism (MSAP) fingerprints a total of 550 and 423 clear and reproducible fragments were amplified from N. antarctica and N. albida strains, respectively. The two Naganishia strains showed a different response in terms of level of DNA methylation during cold stress and recovery from cold stress. The percentage of total methylated fragments in psychrophilic N. antarctica did not show any significant change. On the contrary, the methylation of psychrotolerant N. albida exhibited a nonsignificant increase during the incubation at 4 °C and continued during the recovery step, showing a significant difference if compared with control condition, resembling an uncontrolled response to cold stress. A total of 12 polymorphic fragments were selected, cloned, and sequenced. Four fragments were associated to genes encoding for elongation factor G and for chitin synthase export chaperon. To the best of our knowledge, this is the first study on DNA methylation in the response to cold stress carried out by comparing a psychrophilic and a psychrotolerant yeast species.

Methylation content sensitive enzyme ddRAD (MCSeEd): a reference-free, whole genome profiling system to address cytosine/adenine methylation changes.

Methods for investigating DNA methylation nowadays either require a reference genome and high coverage, or investigate only CG methylation. Moreover, no large-scale analysis can be performed for N6-methyladenosine (6 mA) at an affordable price. Here we describe the methylation content sensitive enzyme double-digest restriction-site-associated DNA (ddRAD) technique (MCSeEd), a reduced-representation, reference-free, cost-effective approach for characterizing whole genome methylation patterns across different methylation contexts (e.g., CG, CHG, CHH, 6 mA). MCSeEd can also detect genetic variations among hundreds of samples. MCSeEd is based on parallel restrictions carried out by combinations of methylation insensitive and sensitive endonucleases, followed by next-generation sequencing. Moreover, we present a robust bioinformatic pipeline (available at https://bitbucket.org/capemaster/mcseed/src/master/ ) for differential methylation analysis combined with single nucleotide polymorphism calling without or with a reference genome.

Analysis of DNA Methylation Patterns Associated with In Vitro Propagated Globe Artichoke Plants Using an EpiRADseq-Based Approach.

Globe artichoke represents one of the main horticultural species of the Mediterranean basin, and 'Spinoso sardo' is the most widespread and economically relevant varietal type in Sardinia, Italy. In the last decades, in vitro culture of meristematic apices has increased the frequency of aberrant plants in open-field production. These off-type phenotypes showed highly pinnate-parted leaves and late inflorescence budding, and emerged from some branches of the true-to-type 'Spinoso sardo' plants. This phenomenon cannot be foreseen and is reversible through generations, suggesting the occurrence of epigenetic alterations. Here, we report an exploratory study on DNA methylation patterns in off-type/true-to-type globe artichoke plants, using a modified EpiRADseq technology, which allowed the identification of 2,897 differentially methylated loci (DML): 1,998 in CG, 458 in CHH, and 441 in CHG methylation contexts of which 720, 88, and 152, respectively, were in coding regions. Most of them appeared involved in primary metabolic processes, mostly linked to photosynthesis, regulation of flower development, and regulation of reproductive processes, coherently with the observed phenotype. Differences in the methylation status of some candidate genes were integrated with transcriptional analysis to test whether these two regulation levels might interplay in the emergence and spread of the 'Spinoso sardo' non-conventional phenotype.

Genetic Characterization of the Apple Germplasm Collection in Central Italy: The Value of Local Varieties.

In the last 50 years, intensive farming systems have been boosted by modern agricultural techniques and newly bred cultivars. The massive use of few and related cultivars has dramatically reduced the apple genetic diversity of local varieties, confined to marginal areas. In Central Italy a limited spread of intensive fruit orchards has made it possible to preserve much of the local genetic diversity, but at the same time the coexistence of both modern and ancient varieties has generated some confusion. The characterization and clarification of possible synonyms, homonyms, and/or labeling errors in old local genetic resources is an issue in the conservation and management of living collections. 175 accessions provided by 10 apple collections, mainly local varieties, some of unknown origin, and well-known modern and ancient varieties, were studied by using 19 SSRs, analyzed by STRUCTURE, Ward's clustering and parentage analysis. We were able to identify 25 duplicates, 9 synonyms, and 9 homonyms. As many as 37 unknown accession were assigned to well known local or commercial varieties. Polyploids made up 20%. Some markers were found to be significantly correlated with morphological traits and the loci associated with the fruit over color were related to QTLs for resistance to biotic stresses, aroma compounds, stiffness, and acidity. In conclusion the gene pool of Central Italy seems to be rather consistent and highly differentiated compared with other European studies (F ST = 0.147). The importance of safeguarding this diversity and the impact on the management of the germplasm living collection is discussed.

Soil Selenium (Se) Biofortification Changes the Physiological, Biochemical and Epigenetic Responses to Water Stress in Zea mays L. by Inducing a Higher Drought Tolerance.

Requiring water and minerals to grow and to develop its organs, Maize (Zea mays L.) production and distribution is highly rainfall-dependent. Current global climatic changes reveal irregular rainfall patterns and this could represent for maize a stressing condition resulting in yield and productivity loss around the world. It is well known that low water availability leads the plant to adopt a number of metabolic alterations to overcome stress or reduce its effects. In this regard, selenium (Se), a trace element, can help reduce water damage caused by the overproduction of reactive oxygen species (ROS). Here we report the effects of exogenous Se supply on physiological and biochemical processes that may influence yield and quality of maize under drought stress conditions. Plants were grown in soil fertilized by adding 150 mg of Se (sodium selenite). We verified the effects of drought stress and Se treatment. Selenium biofortification proved more beneficial for maize plants when supplied at higher Se concentrations. The increase in proline, K concentrations and nitrogen metabolism in aerial parts of plants grown in Se-rich substrates, seems to prove that Se-biofortification increased plant resistance to water shortage conditions. Moreover, the increase of SeMeSeCys and SeCys2 forms in roots and aerial parts of Se-treated plants suggest resistance strategies to Se similar to those existing in Se-hyperaccumulator species. In addition, epigenetic changes in DNA methylation due to water stress and Se treatment were also investigated using methylation sensitive amplified polymorphism (MSAP). Results suggest that Se may be an activator of particular classes of genes that are involved in tolerance to abiotic stresses. In particular, PSY (phytoene synthase) gene, essential for maintaining leaf carotenoid contents, SDH (sorbitol dehydrogenase), whose activity regulates the level of important osmolytes during drought stress and ADH (alcohol dehydrogenase), whose activity plays a central role in biochemical adaptation to environmental stress. In conclusion, Se-biofortification could help maize plants to cope with drought stress conditions, by inducing a higher drought tolerance.

Characterization and Phylogenetic Analysis of Ancient Italian Landraces of Pear.

Pear is one of the oldest fruit tree crops and the third most important temperate fruit species. Its domestication took place independently in the Far East (China) and in the Caucasus region. While the origin of Eastern Asian cultivars is clear, that of European cultivars is still in doubt. Italy has a wealth of local varieties and genetic resources safeguarded by several public and private collections to face the erosion caused by the introduction of improved varieties in specialized orchards. The objectives of the present study were: (i) to characterize the existing germplasm through nuclear (SSR) and (ii) to clarify the genetic divergence between local and cultivated populations through chloroplast DNA (cpDNA) markers in order to provide insights into phylogenetic relationships of Pyrus spp. For this reason, 95 entries from five different germplasm collections, including nine European, Mediterranean and Eastern Asian species, were analyzed, and the intergenic accD-psaI sequences were compared to the worldwide distributed dataset encompassing a total of 298 sequences from 26 different Pyrus species. The nine nuclear SSRs were able to identify a total of 179 alleles, with a loci polymorphism P = 0.89. Most of the variation (97%) was found within groups. Five accessions from different sources were confirmed to be the same. Eight out of 20 accessions of unknown origin were identified, and six synonyms were detected. Locus NH030a was found to be monomorphic in all the cultivated accessions and in reference species interfertile with P. communis, leading to hypothesize selection pressures for adaptation to cultivation. The cpDNA sequences of the 95 accessions were represented by 14 haplotypes, six of which (derived from P. communis, P. cossonii and P. ussuriensis) are recorded here for the first time and may suggest the ancient origin of some local varieties. The network analysis of the 298 cpDNA sequences allowed two different haplogroups, Eastern and Western Eurasia, to be defined, supporting recent views of a clear division between Occidental and Oriental species. By combining the results from nuclear and uniparental markers, it was possible to better define many unknown accessions.

Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity.

Inducible epigenetic changes in eukaryotes are believed to enable rapid adaptation to environmental fluctuations. We have found distinct regions of the Arabidopsis genome that are susceptible to DNA (de)methylation in response to hyperosmotic stress. The stress-induced epigenetic changes are associated with conditionally heritable adaptive phenotypic stress responses. However, these stress responses are primarily transmitted to the next generation through the female lineage due to widespread DNA glycosylase activity in the male germline, and extensively reset in the absence of stress. Using the CNI1/ATL31 locus as an example, we demonstrate that epigenetically targeted sequences function as distantly-acting control elements of antisense long non-coding RNAs, which in turn regulate targeted gene expression in response to stress. Collectively, our findings reveal that plants use a highly dynamic maternal 'short-term stress memory' with which to respond to adverse external conditions. This transient memory relies on the DNA methylation machinery and associated transcriptional changes to extend the phenotypic plasticity accessible to the immediate offspring.

Understanding Genetic Diversity and Population Structure of a Poa pratensis Worldwide Collection through Morphological, Nuclear and Chloroplast Diversity Analysis.

Poa pratensis L. is a forage and turf grass species well adapted to a wide range of mesic to moist habitats. Due to its genome complexity little is known regarding evolution, genome composition and intraspecific phylogenetic relationships of this species. In the present study we investigated the morphological and genetic diversity of 33 P. pratensis accessions from 23 different countries using both nuclear and chloroplast molecular markers as well as flow cytometry of somatic tissues. This with the aim of shedding light on the genetic diversity and phylogenetic relationships of the collection that includes both cultivated and wild materials. Morphological characterization showed that the most relevant traits able to distinguish cultivated from wild forms were spring growth habit and leaf colour. The genome size analysis revealed high variability both within and between accessions in both wild and cultivated materials. The sequence analysis of the trnL-F chloroplast region revealed a low polymorphism level that could be the result of the complex mode of reproduction of this species. In addition, a strong reduction of chloroplast SSR variability was detected in cultivated materials, where only two alleles were conserved out of the four present in wild accessions. Contrarily, at nuclear level, high variability exist in the collection where the analysis of 11 SSR loci allowed the detection of a total of 91 different alleles. A Bayesian analysis performed on nuclear SSR data revealed that studied materials belong to two main clusters. While wild materials are equally represented in both clusters, the domesticated forms are mostly belonging to cluster P2 which is characterized by lower genetic diversity compared to the cluster P1. In the Neighbour Joining tree no clear distinction was found between accessions with the exception of those from China and Mongolia that were clearly separated from all the others.

Methylation-sensitive amplified polymorphism (MSAP) marker to investigate drought-stress response in Montepulciano and Sangiovese grape cultivars.

Methylation-sensitive amplified polymorphism (MSAP) is a technique developed for assessing the extent and pattern of cytosine methylation and has been applied to genomes of several species (Arabidopsis, grape, maize, tomato, and pepper). The technique relies on the use of isoschizomers that differ in their sensitivity to methylation.

Use of MSAP markers to analyse the effects of salt stress on DNA methylation in rapeseed (Brassica napus var. oleifera).

Excessive soil salinity is a major ecological and agronomical problem, the adverse effects of which are becoming a serious issue in regions where saline water is used for irrigation. Plants can employ regulatory strategies, such as DNA methylation, to enable relatively rapid adaptation to new conditions. In this regard, cytosine methylation might play an integral role in the regulation of gene expression at both the transcriptional and post-transcriptional levels. Rapeseed, which is the most important oilseed crop in Europe, is classified as being tolerant of salinity, although cultivars can vary substantially in their levels of tolerance. In this study, the Methylation Sensitive Amplified Polymorphism (MSAP) approach was used to assess the extent of cytosine methylation under salinity stress in salinity-tolerant (Exagone) and salinity-sensitive (Toccata) rapeseed cultivars. Our data show that salinity affected the level of DNA methylation. In particular methylation decreased in Exagone and increased in Toccata. Nineteen DNA fragments showing polymorphisms related to differences in methylation were sequenced. In particular, two of these were highly similar to genes involved in stress responses (Lacerata and trehalose-6-phosphatase synthase S4) and were chosen to further characterization. Bisulfite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied. In particular, our data show that salinity stress influences the expression of the two stress-related genes. Moreover, we quantified the level of trehalose in Exagone shoots and found that it was correlated to TPS4 expression and, therefore, to DNA methylation. In conclusion, we found that salinity could induce genome-wide changes in DNA methylation status, and that these changes, when averaged across different genotypes and developmental stages, accounted for 16.8% of the total site-specific methylation differences in the rapeseed genome, as detected by MSAP analysis.

Comparative cytogenetic study on Trifolium subterraneum (2n = 16) and Trifolium israeliticum (2n = 12).

Changes in chromosome number have played an important role in the evolution of the genus Trifolium. Along with a few species of polyploid origin there are several cases of dysploidy as evidenced by the presence of four basic chromosome numbers (x = 8, 7, 6, 5). Trifolium subterraneum and Trifolium israeliticum are related species with chromosome complements 2n = 16 and 2n = 12, respectively. Although they represent an interesting case of speciation based on chromosome number reduction, no attempts to demonstrate their cytogenetic affinity have been carried out to date. With this study we performed a comparative cytogenetic study with the purpose of clarifying the evolutionary relationship between these species and to verify whether genomic rearrangements, other than modification of the chromosome number, are associated with the speciation process. Although karyomorphological analysis supports the hypothesis that chromosome rearrangements had a role in the reduction of the chromosome number, the physical mapping of the rDNA sequences revealed a significant remodelling of the 45S and 5S rDNA sites that greatly contributed to the differentiation of the 2n = 16 and 2n = 12 karyotypes. The nucleotide analysis of 5S rDNA repeats confirmed that the two species are related but constitute distinct entities. The observed genomic changes lead to the hypothesis that the 2n = 12 species is the result of an evolutionary pathway that passed through intermediate forms. It cannot be excluded that the most direct ancestor of T. israeliticum is a species with 2n = 14.