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Diet selection by grazing livestock may affect animal performance as well as the biodiversity of grazed areas. Recent DNA barcoding techniques allow to assess dietary plant composition in faecal samples, which may be additionally integrated by the description of gut microbiota. In this high throughput metabarcoding study, we investigated the diversity of plant, fungal and bacterial taxa in faecal samples of lactating cows of two breeds grazing an Alpine semi-natural grassland during summer. The estimated plant composition of the diet comprised 67 genera and 39 species, which varied remarkably during summer, suggesting a decline of the diet forage value with the advancing of the vegetative season. The fungal community included Neocallimastigomycota gut symbionts, but also Ascomycota and Basidiomycota plant parasite and coprophilous taxa, likely ingested during grazing. The proportion of ingested fungi was remarkably higher than in other studies, and varied during summer, although less than that observed for plants. Some variation related to breed was also detected. The gut bacterial taxa remained stable through the summer but displayed a breed-specific composition. The study provided insights in the reciprocal organisms' interactions affecting, and being affected by, the foraging behaviour: plants showed a high temporal variation, fungi a smaller one, while bacteria had practically none; conversely, the same kingdoms showed the opposite gradient of variation as respect to the animal host breed, as bacteria revealed to be the group mostly characterized by host-specificity. [ABSTRACT FROM AUTHOR]

Pet food industry has grown considerably in the last few years and it is expected to continue with this rate. Despite the economic impact of this sector and the consumer concerns for the increasing number of food and feed adulteration cases, few studies have been published on mislabelling in pet foods. We therefore investigated the capability of a next generation sequencing-based mini-barcoding approach to identify animal species in pet food products. In a preliminary analysis, a 127 bp fragment of the COI gene was tested on both individual specimens and ad hoc mixed fresh samples used as testers, to evaluate its discrimination power and primers effectiveness. Eighteen pet food products of different price categories and forms available on the market (i.e. kibbles, bites, pâté and strips) were analysed through an NGS approach in biological replicates. At least one of the species listed in the ingredients was not detected in half of the products, while seven products showed supplementary species in addition to those stated on the label. Due to the accuracy, sensitivity and specificity demonstrated, this method can be proposed as food genetic traceability system to evaluate both the feed and food quality timely along the supply chain. [ABSTRACT FROM AUTHOR]

Olive (Olea europaea L.) is one of the most important tree crops of the Mediterranean regions. In spite of the increasing appreciation of typical extra virgin olive oils at world level, based on the use of local traditional varieties, very few studies have focused on the genetic characterisation of olive cultivars of regional interest, such as those grown in Veneto, a North-Eastern Italy region. A deep knowledge of the varieties cultivated in this territory is a key step to address the product quality, to increase market demand and to certify the origin of local olive oils. Here we have analyzed olive cultivars and cultivar groups within the olive cultivation area in Veneto, from the Garda Lake to the Euganean and Trevisan hills, by using discriminant SSR markers, in order to obtain a systematic genetic survey of the Veneto regional olive germplasm patrimony. A total of 203 previously uncharacterized olive samples were collected from ancient trees still grown by local farmers. The analyzed samples included also 36 olive reference cultivars from Veneto and neighbour Regions. We found 57 unique molecular profiles out of this set of olive accessions that were split into 15 cultivar groups corresponding to genetically distinct STRUCTURE clusters. Based on a common SSR database, our 239 Venetian accessions were compared with 280 olive reference genotypes representative of the Mediterranean cultivation area. From the genetic structure analysis, it has been observed that 80% of Venetian cultivars clustered in the central Mediterranean group, about 9% and 2% with the eastern and western varieties, respectively, and all the others resulted intermixed among two or three populations. We found that regionally the most common variety was “Casaliva”, corresponding to the widely diffused cultivar “Frantoio”, while others showed identity with known varieties grown in close regions, such as “Leccino”, “Miniol”, “Capolga” and “Bianchera”. Besides these genotypes, others were not matching any known reference and therefore they could be classified as true local varieties of indigenous origin, possibly deriving from the hybridization and selection made by farmers and from their adaptation to the local soil and climate conditions. [ABSTRACT FROM AUTHOR]

Key message: ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes. Abstract: Apomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family including 30-60 members, of which at least eight are expressed at moderate levels in florets. The sequences of these genes show no functional ORFs, but include segments of different protein coding genes. Particularly, N13 shows partial identity to maize gene BT068773 ( RESPONSE REGULATOR 6). Secondary structure predictions as well as mature miRNA and target cleavage detection suggested that N13 is not a miRNA precursor. Moreover, N13 family members produce abundant 24-nucleotide small RNAs along extensive parts of their sequences. Surveys in the GREENC and CANTATA databases indicated similarity with plant long non-coding RNAs (lncRNAs) involved in splicing regulation; consequently, N13 was renamed as PN_LNC_N13. The Paspalum BT068773 predicted ortholog ( N13TAR) originates floral transcript variants shorter than the canonical maize isoform and with possible structural differences between the apomictic and sexual types. PN_LNC_N13 is expressed only in apomictic plants and displays quantitative representation variation across reproductive developmental stages. However, PN_LNC_N13-like homologs and/or its derived sRNAs showed overall a higher representation in ovules of sexual plants at late premeiosis. Our results suggest the existence of a whole family of N13-like lncRNAs possibly involved in splicing regulation, with some members characterized by differential activity across reproductive types. [ABSTRACT FROM AUTHOR]

The Turin Shroud is traditionally considered to be the burial cloth in which the body of Jesus Christ was wrapped after his death approximately 2000 years ago. Here, we report the main findings from the analysis of genomic DNA extracted from dust particles vacuumed from parts of the body image and the lateral edge used for radiocarbon dating. Several plant taxa native to the Mediterranean area were identified as well as species with a primary center of origin in Asia, the Middle East or the Americas but introduced in a historical interval later than the Medieval period. Regarding human mitogenome lineages, our analyses detected sequences from multiple subjects of different ethnic origins, which clustered into a number of Western Eurasian haplogroups, including some known to be typical of Western Europe, the Near East, the Arabian Peninsula and the Indian sub-continent. Such diversity does not exclude a Medieval origin in Europe but it would be also compatible with the historic path followed by the Turin Shroud during its presumed journey from the Near East. Furthermore, the results raise the possibility of an Indian manufacture of the linen cloth. [ABSTRACT FROM AUTHOR]

Microalgae provide one of the more promising strategies for the production of renewable biodiesel and liquid fuels. Coupling biodiesel production and wastewater treatment based on the use of microalgae can be an effective approach for achieving sustainable production. Several pilot programs aimed at lipid production are currently working with Acutodesmus obliquus, and companies offering algal inocula are increasing in number. A. obliquus strain RL01 was isolated from a pretreated leachate, and preliminary analyses indicated a high growth rate, a final biomass concentration of 1.9 g∙L and a lipid content of 47% DW. With the aim of strictly differentiating and unequivocally identifying the new strain in comparison with other A. obliquus lipid producers available in culture collections, we employed a DNA barcoding approach, targeting conventional and non-conventional genome regions. Genomic analyses were performed using two internal transcribed spacers (nuclear ITS1 and ITS2 sequences) and three selected chloroplast regions (the trnL genic intron and the rpl20- psbA and psbM- psbZ intergenic spacers). Our findings raise some concerns about the discriminant power of the ITS2 nuclear target and place particular emphasis on the psbM- psbZ plastid DNA barcode, which is characterized by high polymorphism, for use in studies addressing intraspecific genetic diversity. [ABSTRACT FROM AUTHOR]

Seed is one of the key factors of crop productivity. Therefore, a comprehension of the mechanisms underlying seed formation in cultivated plants is crucial for the quantitative and qualitative progress of agricultural production. In angiosperms, two pathways of reproduction through seed exist: sexual or amphimictic, and asexual or apomictic; the former is largely exploited by seed companies for breeding new varieties, whereas the latter is receiving continuously increasing attention from both scientific and industrial sectors in basic research projects. If apomixis is engineered into sexual crops in a controlled manner, its impact on agriculture will be broad and profound. In fact, apomixis will allow clonal seed production and thus enable efficient and consistent yields of high-quality seeds, fruits, and vegetables at lower costs. The development of apomixis technology is expected to have a revolutionary impact on agricultural and food production by reducing cost and breeding time, and avoiding the complications that are typical of sexual reproduction (e.g., incompatibility barriers) and vegetative propagation (e.g., viral transfer). However, the development of apomixis technology in agriculture requires a deeper knowledge of the mechanisms that regulate reproductive development in plants. This knowledge is a necessary prerequisite to understanding the genetic control of the apomictic process and its deviations from the sexual process. Our molecular understanding of apomixis will be greatly advanced when genes that are specifically or differentially expressed during embryo and embryo sac formation are discovered. In our review, we report the main findings on this subject by examining two approaches: i) analysis of the apomictic process in natural apomictic species to search for genes controlling apomixis and ii) analysis of gene mutations resembling apomixis or its components in species that normally reproduce sexually. In fact, our opinion is that a novel perspective on this old dilemma pertaining to the molecular control of apomixis can emerge from a cross-check among candidate genes in natural apomicts and a high-throughput analysis of sexual mutants. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression in plants. Many miRNA families and their targets have been extensively studied in model species and major crops. We have characterized mature miRNAs along with their precursors and potential targets in Hypericum to generate a comprehensive list of conserved miRNA families and to investigate the regulatory role of selected miRNAs in biological processes that occur in the flower. St. John's wort ( Hypericum perforatum L., 2 n = 4 x = 32), a medicinal plant that produces pharmaceutically important metabolites with therapeutic activities, was chosen because it is regarded as an attractive model system for the study of apomixis. A computational in silico prediction of structure, in combination with an in vitro validation, allowed us to identify 7 pre-miRNAs, including miR156, miR166, miR390, miR394, miR396, and miR414. We demonstrated that H. perforatum flowers share highly conserved miRNAs and that these miRNAs potentially target dozens of genes with a wide range of molecular functions, including metabolism, response to stress, flower development, and plant reproduction. Our analysis paves the way toward identifying flower-specific miRNAs that may differentiate the sexual and apomictic reproductive pathways. [ABSTRACT FROM AUTHOR]

The widespread occurrence of 2n gametes (i.e. gametes with the somatic chromosome number) in the Medicago sativa-coerulea-falcata complex supports the concept that gene flow from diploid to tetraploid species occurs continuously in nature and plays a key role in alfalfa evolution. Breeders realized early that gene transfer between ploidy levels via 2n pollen and 2n eggs would have had potential use in cultivated alfalfa improvement. Cytological investigations provided insights into the types of meiotic abnormalities responsible for the production of 2n gametes. Alterations were defined as genetically equivalent to first (FDR) or second division restitution (SDR) mechanisms. For breeding purposes, data have proven that 2n gametes of the FDR type are more advantageous than those obtained by SDR for transferring parental heterozygosity and retaining epistatic interactions. The use of diploid meiotic mutants that produce 2n gametes is now recognized as one of the most effective methods available for exploiting heterosis and introgressing wild germplasm traits into cultivated tetraploid alfalfa via unilateral (USP) and bilateral sexual polyploidization (BSP) schemes. Both 2n egg and 2n pollen producers could be used for direct gene transfer from wild diploid relatives into cultivated alfalfa by means of 2x-4x and 4x-2x crosses. Although data have shown that forage yield improvement can be achieved when plants are sexually tetraploidized, problems related to reduced plant fertility and seed production remain largely unexplained. Apomixis has the potential of cloning plants through seed and thus provides a unique opportunity for developing superior tetraploid cultivars with permanently fixed heterosis and epistatic effects. A main goal in alfalfa breeding could be the introduction of functional apomixis (i.e. apomeiosis and parthenogenesis) in cultivated alfalfa stocks. In the future, the efficiency of alfalfa breeding programs based on the use of reproductive mutants could be improved by direct selection at the genotype level using RFLPs and PCR-based markers. Suitable DNA markers and detailed linkage maps of alfalfa mutants should help to discover apomictic mutants and address basic genetic issues such as the extent of genomic recombination in polyploid hybrids and the effect of sexual polyploidization on heterosis. Molecular markers have recently been used in alfalfa for studying the inheritance of 2n gamete formation and identifying polymorphisms associated to genes involved in meiotic abnormalities. Molecular tagging of 2n egg and 2n pollen formation not only should explain the genetic control and regulation of these traits, but may also be an essential step towards marker-assisted selection of 2n gamete producers and implementation of USP and BSP breeding schemes. Future perspectives include strategies for the map-based cloning of genomic DNA markers, and screening of EST mini-libraries related to flowers at different developmental stages from meiotic mutants and wild-types can lead to the identification of mRNAs and thus of candidate genes that control 2n gamete formation in alfalfa. [ABSTRACT FROM AUTHOR]

Gametophytic apomixis in Kentucky bluegrass ( Poa pratensis L.) involves the parthenogenetic development of unreduced eggs from aposporic embryo sacs. Marker-assisted selection for the mode of reproduction in P. pratensis would avoid costly and time-consuming phenotypic progeny tests. We developed and tested two SCAR primer pairs that are associated with the mode of reproduction in P. pratensis. The SCAR primers identified the apomictic and sexual genotypes among progenies of sexual x apomictic crosses with very low bias. Furthermore, when tested on a wide range of Italian and exotic P. pratensis germplasm, they were able to unequivocally distinguish sexual from apomictic genotypes. This system should, therefore, allow new selection models to be set up in this species. [ABSTRACT FROM AUTHOR]

Despite the potential that apomixis has for agriculture, there is little information regarding the genetic control of its functional components. We carried out a cytohistological investigation on an F1 segregating population of Poa pratensis obtained from a cross between a sexual and an apomictic parent. About half of the F1 progeny plants were parthenogenic, as adjudicated by an auxin test. The degree of parthenogenesis ranged from 1.44% to 92.9%. Apospory was detected in parthenogenetic plants as well as in two non-parthenogenetic individuals. These results indicate that two distinct genetic factors control apospory and parthenogenesis in P. pratensis and that apospory and parthenogenesis may be developmentally uncoupled. [ABSTRACT FROM AUTHOR]

Mutants showing features of apomixis have been documented in alfalfa ( Medicago sativa L.), a natural outcrossing sexual species. A differential display of mRNAs that combines cDNA-AFLP markers and bulked segregant analysis was carried out with the aim of selecting expressed sequence tags (ESTs) and cloning candidate genes for apomeiosis in mutants of alfalfa characterized by 2 n egg formation at high frequencies. The approach enabled us to select either mutant- or wild type-specific transcript derived-fragments and to detect transcriptional changes potentially related to 2 n eggs. Sequence alignments of a subset of 40 polymorphic clones showed significant homologies to genes of known function. An EST with identity to a β-tubulin gene, highly expressed in the wild type and poorly expressed in the apomeiotic mutants, and an EST with identity to a Mob1-like gene, qualitatively polymorphic between pre- and post-meiotic stages, were selected as candidate genes for apomeiosis because of their putative roles in the cell cycle. A number of clone-specific primers were designed for performing both 5′ and 3′ rapid amplification of cDNA ends to obtain the full-length clones. Southern blot hybridization revealed that both clones belong to a multi-gene family with a minimum of three genomic DNA members each. Northern blot hybridization of total RNA samples and in situ hybridization of whole buds enabled the definition of their temporal and spatial expression patterns in reproductive organs. Experimental achievements towards the elucidation of apomeiotic megasporogenesis in alfalfa are presented and discussed. [ABSTRACT FROM AUTHOR]

The objective of the present study was to evaluate the fertility of tetraploid plants obtained from diploid mutants that produced 2n gametes via bilateral (BSP) and unilateral sexual polyploidization (USP) schemes. Controlled matings between selected plants from BSP and USP were carried out according to a complete diallel. The level of male and female fertility of each plant was estimated within full-sib, half-sib and non-inbred crosses. Crosses with unrelated self-fertile and male-sterile testers were also performed. Pollen size and stainability were determined by acetocarmine staining. The number of ovules per ovary was counted and the callose deposition within ovules examined to evaluate embryo sac fecundity. Cross-fertility was generally much higher for USP than BSP plants (on average, 0.3 vs. 0.03 seeds per flower pollinated). Both male and female fertility were inversely related to the inbreeding level of cross combinations. The in vitro pollen germinability was positively correlated to male fertility of BSP plants. The level of in vivo embryo sac sterility was not correlated to female fertility in either BSP or USP plants. Female fertility was restored in both BSP and USP groups when plants were crossed with unrelated tetraploid testers. Male fertility also increased in USP plants but remained rather low in the BSP plants. Scaling up the ploidy level by means of 2n gamete union resulted in tetraploid plants with low fertility. In particular, the BSP process yielded virtually self-sterile and highly cross-sterile plants. The well documented positive effect of sexual tetraploidization on forage yield was accompanied by worsening of fertility traits. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

The introgression of apomixis in major seed crops, would guarantee self-seeding of superior heterotic seeds over generations. In the grass species Paspalum simplex, apomixis is controlled by a single locus in which recombination is blocked. In the perspective of isolating the genetic determinants of apomixis, we report data on sequencing, in silico mapping and expression analysis of some of the genes contained in two cloned genomic regions of the apomixis locus of P. simplex. In silico mapping allowed us to identify a conserved synteny group homoeologous to the apomixis locus, located on a telomeric position of chromosomes 12, 8, 3 and 4 of rice, Sorghum bicolor, Setaria italica and Brachypodium distachyum, respectively, and on a more centromeric position of maize chromosome 1. Selected genes of the apomixis locus expressed sense and antisense transcripts in reproductively committed cells of sexual and apomictic ovules. Some of the genes considered here expressed apomixis-specific allelic variants which showed partial non-overlapping expression patterns with alleles shared by sexual and apomictic reproductive phenotypes. Our findings open new routes for the isolation of the genetic determinants of apomixis and, in perspective, for its introgression in crop grasses. [ABSTRACT FROM AUTHOR]

The development of F1 hybrid varieties benefits from the synergistic effect of conventional and molecular marker-assisted breeding schemes. A sequencing run was carried out in Foeniculum vulgare (2n = 2x = 22) to develop the first genome draft and to identify microsatellites suitable for implementing multilocus SSR marker assays. A preliminary cytometric analysis allowed us to estimate the genome size (2C = 2.64-2.86 pg), equal to about 1.34 Mbp for 1C genome, and to calculate the sequencing coverage (53×). The genome draft assembly into 300,408 scaffolds and its bioinformatic analysis enabled the annotation of coding and non-coding regions across the genome, including 103,306 SSR elements. A total of 100 microsatellites were randomly chosen among those with dinucleotide and trinucleotide repeat motifs and with a repeat motif length ≥ 25 times and were preliminarily tested. Of these, 27 SSR markers, classified as suitable for genetic diversity analyses, were efficiently organized in five PCR multiplex assays and validated using a core collection of 100 fennel individuals potentially useful for the development of inbred lines and F1 hybrids. All SSR loci were found to be polymorphic, scoring an observed number of marker alleles Na = 207 and an average polymorphism information content PIC = 0.69. The SSR data were used to calculate (i) the degree of homozygosity for the individual inbred lines (0.35 < Ho < 0.96), to eventually plan additional selfing or sibling cycles, and (ii) the degree of genetic similarity for all possible pair-wise comparisons between parental inbred lines (GS = 0.55-0.77), to identify the most divergent combinations for the constitution of experimental F1 hybrids. The integration of genotypic and phenotypic data was useful for implementing guidelines for precision hybrid breeding schemes in fennel. [ABSTRACT FROM AUTHOR]