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.

Genetic and Phenotypic Characteristics of Belted Pig Breeds: A Review.

Belted pig breeds have unique, distinguishing phenotypic characteristics. This review summarises the current knowledge on pig breeds displaying a belted coat pattern. Belts of different widths and positions around the animal's trunk characterise specific pig breeds from all around the world. All the breeds included in the present paper have been searched through the FAO domestic animal diversity information system (DAD-IS), Every country was checked to identify all breeds described as having black or red piebald coat pattern variations. Advances in genomic technologies have made it possible to identify the specific genes and genetic markers associated with the belted phenotype and explore the genetic relationships between different local breeds. Thus, the origin, history, and production traits of these breeds, together with all the genomic information related to the mechanism of skin pigmentation, are discussed. By increasing our understanding of these breeds, we can appreciate the richness of our biological and cultural heritage and work to preserve the biodiversity of the world's animals.

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.

Evolutionary studies of the bHLH transcription factors belonging to MBW complex: their role in seed development.

BACKGROUND AND AIMS: The MBW complex consist of proteins belonging to three major families (MYB, bHLH and WDR) involved in various processes throughout plant development: epidermal cell development, mucilage secretory cells and flavonoid biosynthesis. Recently, it has been reported that TT8, encoding a bHLH transcription factor, is involved in the biosynthesis of flavonoids in the seed coat and it also plays a role in bypassing the postzygotic barrier resulting from an unbalance in genetic loads of the parental lines. Here, we focus on the functional evolution, in seed development, of the bHLH proteins that are part of the MBW complex, complemented with a literature review. METHODS: Phylogenetic analyses performed across seed plants and expression analyses in the reproductive tissues of four selected angiosperms (Arabidopsis thaliana, Brassica napus, Capsella rubella and Solanum lycopersicum) allow us to hypothesize on the evolution of its functions. KEY RESULTS: TT8 expression in the innermost layer of the seed coat is conserved in the selected angiosperms. However, except for Arabidopsis, TT8 is also expressed in ovules, carpels and fruits. The homologues belonging to the sister clade of TT8, EGL3/GL3, involved in trichome development, are expressed in the outermost layer of the seed coat, suggesting potential roles in mucilage. CONCLUSIONS: The ancestral function of these genes appears to be flavonoid biosynthesis, and the conservation of TT8 expression patterns in the innermost layer of the seed coat in angiosperms suggests that their function in postzygotic barriers might also be conserved. Moreover, the literature review and the results of the present study suggest a sophisticated association, linking the mechanisms of action of these genes to the cross-communication activity between the different tissues of the seed. Thus, it provides avenues to study the mechanisms of action of TT8 in the postzygotic triploid block, which is crucial because it impacts seed development in unbalanced crosses.

Novel CRISPR/Cas technology in the realm of algal bloom biomonitoring: Recent trends and future perspectives.

In conjunction with global climate change, progressive ocean warming, and acclivity in pollution and anthropogenic eutrophication, the incidence of harmful algal blooms (HABs) and cyanobacterial harmful algal blooms (CHABs) continue to expand in distribution, frequency, and magnitude. Algal bloom-related toxins have been implicated in human health disorders and ecological dysfunction and are detrimental to the national and global economy. Biomonitoring programs based on traditional monitoring protocols were characterised by some limitations that can be efficiently overdone using the CRISPR/Cas technology. In the present review, the potential and challenges of exploiting the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology for early detection of HABs and CHABs-associated toxigenic species were analysed. Based on more than 30 scientific papers, the main results indicate the great potential of CRISPR/Cas technology for this issue, even if the high sensitivity detected for the Cas12 and Cas13 platforms represents a possible interference risk.

From tetraploid to diploid, a pangenomic approach to identify genes lost during synthetic diploidization of Eragrostis curvula.

Introduction: In Eragrostis curvula, commonly known as weeping lovegrass, a synthetic diploidization event of the facultative apomictic tetraploid Tanganyika INTA cv. originated from the sexual diploid Victoria cv. Apomixis is an asexual reproduction by seeds in which the progeny is genetically identical to the maternal plant. Methods: To assess the genomic changes related to ploidy and to the reproductive mode occurring during diploidization, a mapping approach was followed to obtain the first E. curvula pangenome assembly. In this way, gDNA of Tanganyika INTA was extracted and sequenced in 2x250 Illumina pair-end reads and mapped against the Victoria genome assembly. The unmapped reads were used for variant calling, while the mapped reads were assembled using Masurca software. Results: The length of the assembly was 28,982,419 bp distributed in 18,032 contigs, and the variable genes annotated in these contigs rendered 3,952 gene models. Functional annotation of the genes showed that the reproductive pathway was differentially enriched. PCR amplification in gDNA and cDNA of Tanganyika INTA and Victoria was conducted to validate the presence/absence variation in five genes related to reproduction and ploidy. The polyploid nature of the Tanganyika INTA genome was also evaluated through the variant calling analysis showing the single nucleotide polymorphism (SNP) coverage and allele frequency distribution with a segmental allotetraploid pairing behavior. Discussion: The results presented here suggest that the genes were lost in Tanganyika INTA during the diploidization process that was conducted to suppress the apomictic pathway, affecting severely the fertility of Victoria cv.

Epigenetic Modifications in Plant Development and Reproduction.

Plants are exposed to highly fluctuating effects of light, temperature, weather conditions, and many other environmental factors throughout their life. As sessile organisms, unlike animals, they are unable to escape, hide, or even change their position. Therefore, the growth and development of plants are largely determined by interaction with the external environment. The success of this interaction depends on the ability of the phenotype plasticity, which is largely determined by epigenetic regulation. In addition to how environmental factors can change the patterns of genes expression, epigenetic regulation determines how genetic expression changes during the differentiation of one cell type into another and how patterns of gene expression are passed from one cell to its descendants. Thus, one genome can generate many 'epigenomes'. Epigenetic modifications acquire special significance during the formation of gametes and plant reproduction when epigenetic marks are eliminated during meiosis and early embryogenesis and later reappear. However, during asexual plant reproduction, when meiosis is absent or suspended, epigenetic modifications that have arisen in the parental sporophyte can be transmitted to the next clonal generation practically unchanged. In plants that reproduce sexually and asexually, epigenetic variability has different adaptive significance. In asexuals, epigenetic regulation is of particular importance for imparting plasticity to the phenotype when, apart from mutations, the genotype remains unchanged for many generations of individuals. Of particular interest is the question of the possibility of transferring acquired epigenetic memory to future generations and its potential role for natural selection and evolution. All these issues will be discussed to some extent in this review.

Environmental and Genetic Factors Affecting Apospory Expressivity in Diploid Paspalum rufum.

In angiosperms, gametophytic apomixis (clonal reproduction through seeds) is strongly associated with polyploidy and hybridization. The trait is facultative and its expressivity is highly variable between genotypes. Here, we used an F1 progeny derived from diploid apomictic (aposporic) genotypes of Paspalum rufum and two F2 families, derived from F1 hybrids with different apospory expressivity (%AES), to analyze the influence of the environment and the transgenerational transmission of the trait. In addition, AFLP markers were developed in the F1 population to identify genomic regions associated with the %AES. Cytoembryological analyses showed that the %AES was significantly influenced by different environments, but remained stable across the years. F1 and F2 progenies showed a wide range of %AES variation, but most hybrids were not significantly different from the parental genotypes. Maternal and paternal genetic linkage maps were built covering the ten expected linkage groups (LG). A single-marker analysis detected at least one region of 5.7 cM on LG3 that was significantly associated with apospory expressivity. Our results underline the importance of environmental influence in modulating apospory expressivity and identified a genomic region associated with apospory expressivity at the diploid level.

A Review of Unreduced Gametes and Neopolyploids in Alfalfa: How to Fill the Gap between Well-Established Meiotic Mutants and Next-Generation Genomic Resources.

The gene flow mediated by unreduced gametes between diploid and tetraploid plants of the Medicagosativa-coerulea-falcata complex is pivotal for alfalfa breeding. Sexually tetraploidized hybrids could represent the best way to exploit progressive heterosis simultaneously derived from gene diversity, heterozygosity, and polyploidy. Moreover, unreduced gametes combined with parthenogenesis (i.e., apomixis) would enable the cloning of plants through seeds, providing a unique opportunity for the selection of superior genotypes with permanently fixed heterosis. This reproductive strategy has never been detected in the genus Medicago, but features of apomixis, such as restitutional apomeiosis and haploid parthenogenesis, have been reported. By means of an original case study, we demonstrated that sexually tetraploidized plants maintain apomeiosis, but this trait is developmentally independent from parthenogenesis. Alfalfa meiotic mutants producing unreduced egg cells revealed a null or very low capacity for parthenogenesis. The overall achievements reached so far are reviewed and discussed along with the efforts and strategies made for exploiting reproductive mutants that express apomictic elements in alfalfa breeding programs. Although several studies have investigated the cytological mechanisms responsible for 2n gamete formation and the inheritance of this trait, only a very small number of molecular markers and candidate genes putatively linked to unreduced gamete formation have been identified. Furthermore, this scenario has remained almost unchanged over the last two decades. Here, we propose a reverse genetics approach, by exploiting the genomic and transcriptomic resources available in alfalfa. Through a comparison with 9 proteins belonging to Arabidopsis thaliana known for their involvement in 2n gamete production, we identified 47 orthologous genes and evaluated their expression in several tissues, paving the way for novel candidate gene characterization studies. An overall view on strategies suitable to fill the gap between well-established meiotic mutants and next-generation genomic resources is presented and discussed.

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.

Characterization of Differentially Expressed Genes under Salt Stress in Olive.

Climate change, currently taking place worldwide and also in the Mediterranean area, is leading to a reduction in water availability and to groundwater salinization. Olive represents one of the most efficient tree crops to face these scenarios, thanks to its natural ability to tolerate moderate salinity and drought. In the present work, four olive cultivars (Koroneiki, Picual, Royal de Cazorla and Fadak86) were exposed to high salt stress conditions (200 mM of NaCl) in greenhouse, in order to evaluate their tolerance level and to identify key genes involved in salt stress response. Molecular and physiological parameters, as well as plant growth and leaves' ions Na+ and K+ content were measured. Results of the physiological measurements showed Royal de Cazorla as the most tolerant cultivar, and Fadak86 and Picual as the most susceptible ones. Ten candidate genes were analyzed and their complete genomic, CDS and protein sequences were identified. The expression analysis of their transcripts through reverse transcriptase quantitative PCR (RT-qPCR) demonstrated that only OeNHX7, OeP5CS, OeRD19A and OePetD were upregulated in tolerant cultivars, thus suggesting their key role in the activation of a salt tolerance mechanism.

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.

A Reappraisal of the Evolutionary and Developmental Pathway of Apomixis and Its Genetic Control in Angiosperms.

Apomixis sensu stricto (agamospermy) is asexual reproduction by seed. In angiosperms it represents an easy byway of life cycle renewal through gamete-like cells that give rise to maternal embryos without ploidy reduction (meiosis) and ploidy restitution (syngamy). The origin of apomixis still represents an unsolved problem, as it may be either evolved from sex or the other way around. This review deals with a reappraisal of the origin of apomixis in order to deepen knowledge on such asexual mode of reproduction which seems mainly lacking in the most basal angiosperm orders (i.e., Amborellales, Nymphaeales and Austrobaileyales, also known as ANA-grade), while it clearly occurs in different forms and variants in many unrelated families of monocots and eudicots. Overall findings strengthen the hypothesis that apomixis as a whole may have evolved multiple times in angiosperm evolution following different developmental pathways deviating to different extents from sexuality. Recent developments on the genetic control of apomixis in model species are also presented and adequately discussed in order to shed additional light on the antagonist theories of gain- and loss-of-function over sexuality.

Molecular Identification of the "Facciuta Della Valnerina" Local Goat Population Reared in the Umbria Region, Italy.

Italy holds important genetic resources of small ruminant breeds. By distinguishing goat breeds at the DNA level, certification of products from specific breeds can be valorized. The aim of this study was to establish the genetic identity of Facciuta della Valnerina, a local goat population of Italy, compared with the cosmopolitan breeds, Saanen and Camosciata delle Alpi, reared in the same geographic area. A total of 116 microsatellite alleles ranging from 4 to 13 were detected at 16 loci in the three goat populations/breeds. A total of 23 private alleles with frequencies lower than 0.3 were detected in the Facciuta della Valnerina population. The mean numbers of alleles were 6.67, 4.58, and 4.92 in Facciuta della Valnerina, Camosciata delle Alpi, and Saanen, respectively. The expected heterozygosity ranged from 0.20 to 0.86. Most loci were highly polymorphic and informative (polymorphic information content ≥0.50). Factorial correspondence analysis and principal components analysis revealed very clear separation between Facciuta della Valnerina and the two reference goat breeds. Reducing the number of markers from 16 to 12 (on the basis of polymorphic information content and the number of alleles) still allowed us to distinguish the local population, indicating that microsatellite markers are capable of discriminating local livestock breeds at a low cost.

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.

Exploring Heterosis in Melon (Cucumis melo L.).

Heterosis is the superiority of an F1 hybrid over its parents. Since this phenomenon is still unclear in melon, a half diallel experiment based on eight genetically distant breeding lines was conducted in six environments of Central Italy, assessing commercially important traits: yield, total soluble solids (TSS), and days to ripening (DTR). To estimate the additive (general combining ability; GCA) and the non-additive gene effects (specific combining ability; SCA), yield was analyzed by Griffing's methods two and four, and the results were compared to the GGE (Genotype plus Genotype by Environment interaction) biplot methodology; TSS and earliness were evaluated only by Griffing's method four. Overall, GCAs were significantly more relevant than SCAs for all examined traits. Least square means (LsM), mid-parent heterosis (MPH), best-parent heterosis (BPH), as well as Euclidean and Mahalanobis' distances were calculated and compared with the genetic distance (GD). As a few correlations were found statistically significant (only for TSS), it was difficult to predict the value of a hybrid combination only by knowing the genetic distance of its parents. Despite this, heterosis was observed, indicating either the presence of epistatic effects (additive × additive interactions) and/or an underestimate of SCAs embedded within Griffing's method. The significant Env × Entries source of variation suggests development of hybrids in specific environments. The results are discussed with a breeding perspective.