Annotation and Expression of IDN2-like and FDM-like genes in sexual and aposporous hypericum perforatum L. Accessions

The protein IDN2, together with the highly similar interactors FDM1 and FDM2, is required for RNA-directed DNA methylation (RdDM) and siRNA production. Epigenetic regulation of gene expression is required to restrict cell fate determination in A. thaliana ovules. Recently, three transcripts sharing high similarity with the A. thaliana IDN2 and FDM1-2 were found to be differentially expressed in ovules of apomictic Hypericum perforatum L. accessions. To gain further insight into the expression and regulation of these genes in the context of apomixis, we investigated genomic, transcriptional and functional aspects of the gene family in this species. The H. perforatum genome encodes for two IDN2-like and 7 FDM-like genes. Differential and heterochronic expression of FDM4-like genes was found in H. perforatum pistils. The involvement of these genes in reproduction and seed development is consistent with the observed reduction of the seed set and high variability in seed size in A. thaliana IDN2 and FDM-like knockout lines. Differential expression of IDN2-like and FDM-like genes in H. perforatum was predicted to affect the network of potential interactions between these proteins. Furthermore, pistil transcript levels are modulated by cytokinin and auxin but the effect operated by the two hormones depends on the reproductive phenotype

TRANSCRIPTOMIC ANALYSIS OF OVULE-SPECIFIC CELL LINEAGES TO IDENTIFY GENES RELATED TO APOSPOROUS APOMIXIS IN HYPERICUM PERFORATUM L.

The medicinal plant St. John\u2019s wort (Hypericum perforatum L.) is an attractive model system for the study of aposporous apomixis. The key biological features of apomixis in this species are the by-passing of meiosis, the differentiation of aposporous initials into embryo sacs containing unreduced egg cells, their autonomous development in functional embryos without fertilization, and the formation of viable endosperm either via fertilization-independent means or following fertilization with a sperm nucleus. The aim of this research is to define gene expression changes occurring in the nucellar cell types of the ovules primarily involved in the differentiation of the megaspore mother cells and aposporous initials. To this end, a laser-capture microdissection approach was adopted in combination with the RNA-seq technology in order to restrict the frame of our investigations to a specific portion of the ovule, i.e. the nucellus, at developmental stages preceding the differentiation of aposporous initials. Overall, our gene expression analysis identified 270 and 81 unigenes that were found significantly up- and down-regulated between ovules collected from sexual and apomictic accessions. Ontological annotation of differentially expressed genes indicated that genes up-regulated in apomictic ovules were significantly enriched in ontological terms related to the RNA-directed DNA polymerase activity and the RNA binding. Among these genes, several actors of the RdDM pathway were found, suggesting that the phenotypic expression of early events of aposporous apomixis is associated to changes in de novo DNA methylation mediated by small RNAs. Furthermore, as deregulation of single components of the sexual developmental pathway is believed to be a trigger of the apomictic reproductive program, genes involved in sporogenesis, gametogenesis and response to hormonal stimuli were annotated and investigated in great detail. The expression analysis of candidate genes was performed not only by Real-Time qPCR but also by ISH assays in order to verify the temporal and spatial expression patterns of selected transcripts in the ovule. Finally, the activity of specific genes in relation to embryo sac and/or embryo formation was investigated by using A. thaliana knock-out lines. Overall, our data suggest that phenotypic expression of aposporous apomixis is concomitant with the modulation of key genes involved in the sexual reproductive pathway, hormones and other actors likely playing a crucial role in the RNA-directed DNA methylation pathway

ANNOTATION AND EXPRESSION OF FDM-LIKE GENES IN SEXUAL AND APOMICTIC MODEL SPECIES

Aposporous apomixis is a reproductive strategy that leads to seed production by avoiding female sporogenesis and, eventually, fertilization. In this peculiar reproductive strategy, embryo and endosperm are formed from unreduced gametes developing from somatic cells belonging to the ovule nucellus. Recently gained information on ovule gene expression in the apomictic model species Hypericum perforatum L. demonstrated that a few genes involved in the RNA-directed DNA methylation pathway (RdDM) are differentially expressed in ovules collected from apomictic accessions at pre-meiotic stages. In A. thaliana, the protein Involved in De Novo 2 (IDN2), together with members of the gene family Factors of DNA Methylation (FDM1, FDM2), acts downstream of the RdDM. In this pathway, IDN2/FDM complex are recruited to the chromatin by the ra-siRNAPol V transcript duplex, and then bind the un-methylated DNA to promote DNA methylation of both transposons and protein coding genes. Remarkably, the knock-out of genes involved in the RdDM in sexually reproducing species such as A. thaliana and Z. mays results in phenotypes mimicking early events of aposporous apomixis. Taken together, these findings let us to hypothesize that RNA-directed DNA methylation might be involved in correct patterning of cell fate determination in the ovule in sexual and apomictic species. This research focuses on genes belonging to the gene family known as Factors of DNA Methylation (FDM1-5) and their closely related IDN2 (Involved in De Novo 2). Our research aim is a better understanding of roles played by these genes in the frame of ovule cell fate determination and gametes formation. Bioinformatics analyses were performed in order to identify and annotate all gene family members expressed in H. perforatum ovules. Gene expression differences between pistils collected from sexual and apomictic accessions were confirmed by qPCR and ISH. Correlated experiments were performed by taking advantage of mutant lines available for A. thaliana. IDN2 and FDM1-5 knockout lines were analyzed for alterations in total seed set and plant habits. Mutant lines displayed overlapping phenotypes, including the reduction of seed set. Overall, our phenotypic data are in line with a sporophytic effect resulting in the ovule abortion in A. thaliana. GUS reporter lines were adopted to visualize the FDM promoter activity in ovules at different developmental time points. Furthermore, the development of a protocol suitable for whole-mount qPCR assays allowed rapid and reliable quantification of gene expression in micro-dissected ovules. Our results elucidate the role of FDM and IDN2 genes in both sexual and apomicts plants and add new factors affecting the complex events involved in ovule and gametes formation that should be further investigated

De novo sequencing of the Hypericum perforatum L. flower transcriptome to identify potential genes that are related to plant reproduction sensu lato

BACKGROUND: St. John’s wort (Hypericum perforatum L.) is a medicinal plant that produces important metabolites with antidepressant and anticancer activities. Recently gained biological information has shown that this species is also an attractive model system for the study of a naturally occurring form of asexual reproduction called apomixis, which allows cloning plants through seeds. In aposporic gametogenesis, one or multiple somatic cells belonging to the ovule nucellus change their fate by dividing mitotically and developing functionally unreduced embryo sacs by mimicking sexual gametogenesis. Although the introduction of apomixis into agronomically important crops could have revolutionary implications for plant breeding, the genetic control of this mechanism of seed formation is still not well understood for most of the model species investigated so far. We used Roche 454 technology to sequence the entire H. perforatum flower transcriptome of whole flower buds and single flower verticils collected from obligately sexual and unrelated highly or facultatively apomictic genotypes, which enabled us to identify RNAs that are likely exclusive to flower organs (i.e., sepals, petals, stamens and carpels) or reproductive strategies (i.e., sexual vs. apomictic). RESULTS: Here we sequenced and annotated the flower transcriptome of H. perforatum with particular reference to reproductive organs and processes. In particular, in our study we characterized approximately 37,000 transcripts found expressed in male and/or female reproductive organs, including tissues or cells of sexual and apomictic flower buds. Ontological annotation was applied to identify major biological processes and molecular functions involved in flower development and plant reproduction. Starting from this dataset, we were able to recover and annotate a large number of transcripts related to meiosis, gametophyte/gamete formation, and embryogenesis, as well as genes that are exclusively or preferentially expressed in sexual or apomictic libraries. Real-Time RT-qPCR assays on pistils and anthers collected at different developmental stages from accessions showing alternative modes of reproduction were used to identify potential genes that are related to plant reproduction sensu lato in H. perforatum. CONCLUSIONS: Our approach of sequencing flowers from two fully obligate sexual genotypes and two unrelated highly apomictic genotypes, in addition to different flower parts dissected from a facultatively apomictic accession, enabled us to analyze the complexity of the flower transcriptome according to its main reproductive organs as well as for alternative reproductive behaviors. Both annotation and expression data provided original results supporting the hypothesis that apomixis in H. perforatum relies upon spatial or temporal mis-expression of genes acting during female sexual reproduction. The present analyses aim to pave the way toward a better understanding of the molecular basis of flower development and plant reproduction, by identifying genes or RNAs that may differentiate or regulate the sexual and apomictic reproductive pathways in H. perforatum. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1439-y) contains supplementary material, which is available to authorized users

TRANSCRIPTOMIC ANALYSIS OF OVULE-SPECIFIC CELL LINEAGES TO IDENTIFY GENES RELATED TO APOSPOROUS APOMIXIS IN HYPERICUM PERFORATUM L.

Apomixis defines a plant reproductive strategy that, unlike sexual reproduction, permits the inheritance of the maternal genome without genetic recombination and syngamy. Earliest phenotypical symptoms of the aposporous developmental program in ovules are the avoidance of meiosis (i.e., apomeiosis) and the differentiation of functional unreduced embryo sacs from somatic cells of the nucellus (i.e., aposporous initials). Apospory deviates from sexuality as, in this latter case, the commitment to develop an embryo sac is strictly restricted to the reduced functional megaspore (FM) and failure of the meiotic program is typically not accompanied by the initiation of embryo sac development from cell lineages of the ovule other than the FM. Our research goal is a better understanding on gene expression changes accompanying the onset of aposporous apomixis in the ovule of the model species H. perforatum L. To this purpose, gene expression analyses were performed by adopting the RNA-seq technology on Laser-Capture Microdissected (LCM) ovule cells collected from sexual and apomictic genotypes at pre-meiotic developmental stages. We identified 402 differentially expressed genes (DEGs) (Bonferroni p-value 64 0.05) between ovules belonging to sexual and apomictic genotypes. Among these, 97 transcripts were only found in apomictic libraries, suggesting apomictic-specific expression. At the same time, 25 transcripts were only detected in sexual libraries. Differential expression was validated by RealTime qPCR and in-situ hybridization assay. Among identified DEGs, we found several RNAs whose products are related to biological processes modulated in other aposporous apomictic model species. Ontological annotation revealed an enrichment of the following biological processes in apomictic ovules: RNA binding, RNA splicing and RNA-directed DNA polymerase activity, this latter being associated to putative non-LTR retroelements. The massive expression of TEs in apomictic ovules suggested that DNA methylation is compromised in these cells. To address this question, we investigated the promoter and gene body DNA methylation level of a subset of DEGs by chop-PCR assays. Gene body methylation level of DEGs annotated as putative non-LTR retroelements supports the idea that transcriptomic changes for these genes might be epigenetically controlled. Furthermore, several genes involved in auxin and cytokinin (CK) homeostasis and signalling were found differentially expressed, implying that apomictic ovules might be subjected to alternative hormonal interplays. This let us hypothesise that hormonal response and DNA methylation might be connected to the transcriptional changes observed in apomictic ovules. To address this question, gene expression and promoter methylation studies were performed on flowers treated with synthetic CK and its antagonist PI-55. Gene expression and DNA methylation data will be presented and critically discussed in the frame of ovule and gamete development. Overall, our data suggest that phenotypic expression of early events of aposporous apomixis in H. perforatum is concomitant with the modulation of key genes involved in hormonal homeostasis, DNA methylation and cell cycle progression

Genome-Wide Datasets of Chicories (Cichorium intybus L.) for Marker-Assisted Crop Breeding Applications: A Systematic Review and Meta-Analysis

Cichorium intybus L. is the most economically important species of its genus and among the most important of the Asteraceae family. In chicory, many linkage maps have been produced, several sets of mapped and unmapped markers have been developed, and dozens of genes linked to traits of agronomic interest have been investigated. This treasure trove of information, properly cataloged and organized, is of pivotal importance for the development of superior commercial products with valuable agronomic potential in terms of yield and quality, including reduced bitter taste and increased inulin production, as well as resistance or tolerance to pathogens and resilience to environmental stresses. For this reason, a systematic review was conducted based on the scientific literature published in chicory during 1980-2023. Based on the results obtained from the meta-analysis, we created two consensus maps capable of supporting marker-assisted breeding (MAB) and marker-assisted selection (MAS) programs. By taking advantage of the recently released genome of C. intybus, we built a 639 molecular marker-based consensus map collecting all the available mapped and unmapped SNP and SSR loci available for this species. In the following section, after summarizing and discussing all the genes investigated in chicory and related to traits of interest such as reproductive barriers, sesquiterpene lactone biosynthesis, inulin metabolism and stress response, we produced a second map encompassing 64 loci that could be useful for MAS purposes. With the advent of omics technologies, molecular data chaos (namely, the situation where the amount of molecular data is so complex and unmanageable that their use becomes challenging) is becoming far from a negligible issue. In this review, we have therefore tried to contribute by standardizing and organizing the molecular data produced thus far in chicory to facilitate the work of breeders

Genetic structure of cultivated varieties of radicchio (Cichorium intybus l.): A comparison between F1 hybrids and synthetics

Cichorium intybus L., well known in Italy with the common name "Radicchio", is an important leafy vegetable that is prevalently reproduced by allogamy due to very efficient barriers of self-incompatibility. Marker-assisted breeding is widely used by seed firms to develop new hybrid varieties that manifest genetic distinctiveness, uniformity and stability. A total of 29 mapped microsatellite markers were used for genotyping 504 samples of the Red of Chioggia biotype: First, two synthetics, four F1 hybrids and two derived F2 populations were compared to assess the distinctiveness of their gene pool and structure; then, the uniformity and stability of 3 years of production of a commercial F1 variety were also investigated. Genetic similarity and diversity statistics as well as the genetic structure of populations were analysed, including allele and genotype frequencies. The mean estimates and ranges of genetic similarity enabled the molecular discrimination of OP synthetics from F1 varieties and their F2 progenies and the determination of individual plant memberships. Moreover, the genetic structure of F1 hybrids produced in 3 years unexpectedly revealed two main clusters that discriminate the first 2 years from the 3rd, mainly because of the presence of uncommon specific alleles and different allele frequencies. Overall, this molecular information will enable breeders to determine the genetic distinctness, uniformity and stability of commercial and experimental varieties, as well as their genetic relationships and relatedness. Hence, this work provides a useful tool for achieving the molecular characterisation and genetic identification of different radicchio populations

Genetic Variability of Lucerne Landraces from Central Italy Detected by RAPD Markers

With the aim to characterize six lucerne landraces (Medicago sativa L.), representing a sample of a collection from central Italy, sixty individuals per landrace were evaluated by screening for RAPD markers with three lucerne-specific primers. Twenty-one amplification products were scored as present or absent across all plants. The dendrogram from mean genetic similarity estimates displayed Casalina alone and the other landraces clustered into one distinct group, showing a single branch point with more than 73% of genetic similarity. The discriminant analysis grouped the landraces in a similar manner. The first function maximally separated the group Grosseto, Gubbio and C. Pieve from Latina and L’Aquila while the second function maximally separated Casalina from the rest of landraces. Overall 56% of individual plants were correctly reclassified into their own groups. Owing to their rather narrow geographic provenance, more primers are needed to increase precision in the estimate of the genetic variability

genomic dna fingerprinting of indigenous chicken breeds with molecular markers designed on interspersed repeats

In Italy more than fifty different local breeds of chicken (Gallus gallus L.) are known to have been present in the past. The overall situation is now critical since most of these breeds are becoming extinct or threatened and only a few are subject of conservation plans. The use of molecular markers for the analysis of chicken populations could help in characterizing their genetic variation and preserving them from genetic erosion. valuable and irreplaceable sources of chicken germplasm from indigenous populations of the veneto region were analyzed by means of DNA fingerprinting with molecular markers designed on interspersed mini- and micros-atellite repeats. The identification of either among-breed discriminant or breed-specific markers was based on the S-SAP and M-AFLP systems derived from the AFLP technology. Genomic DNA fingerprints were generated in 84 individuals belonging to six local breeds (Ermellinata, Padovana, Pepoi, Polverara, Robusta Lionata and Robusta Maculata) and one commercial line used as reference standard. A number of variation statistics were computed to assess the genetic variability within and relatedness among breeds: the effective number of alleles per locus (ne= 1.570), total and single-breed genetic diversity (HT= 0.366 and HS= 0.209, respectively) and the fixation index (GST= 0.429). The mean genetic similarity coefficients within and between local breeds were 0.769 and 0.628, respectively. Markers useful for the genetic traceability of breeds revealed significant sequence similarities with either genic or intergenic regions of known chromosome position. Sequence tagged site primers were designed for the most discriminant markers in order to develop multiplex non-radioactive genomic PCR assays. Analysis of the population structure along with individual assignment tests successfully identified all breed clusters and subclusters. The vast majority of animals were correctly allocated to their breed of origin, demonstrating the suitability and reliability of the chosen AFLP-derived marker systems for detecting population structure and tracing individual breeds. The local breeds have been preliminarily identified according to sequence-specific SNPs and haplotypes and the polymorphism information content of genomic AFLP-derived markers is reported and critically discussed

TRANSCRIPTOMIC ANALYSIS OF WHOLE PISTILS AND OVULE CELLS TO IDENTIFY GENES RELATED TO APOSPORY IN HYPERICUM PERFORATUM L.

St. John\u2019s wort (Hypericum perforatum L.) is a medicinal plant that produces important metabolites with antidepressant and anticancer activities. Beside the pharmaceutical interest, recently gained information has shown that H. perforatum is also an attractive model system for the study of aposporous apomixis, that is a reproductive strategy, which, unlike sexual reproduction, permits the inheritance of the maternal genome over generations without genetic recombination events. This asexual mode of seed formation is believed to be a trait with enormous economic and social potential in agriculture. Its innovative use in this area relies upon the idea that indefinitely fixing highly complex genotypes, including hybrid cultivars, through apomixis would have tremendous advantages in plant breeding, biomass and seed production. During the last decades, the understanding of the molecular basis of apomixis in this species has been complicated by the lack of biological data, e.g. genomic or even transcriptomic sequences. The aim of our research project was the sequencing, annotation and comparative investigation of the H. perforatum flower transcriptome, as critical steps toward a better understanding of the genetic control of aposporic and sexual reproduction in the facultative apomict H. perforatum. To this end, next generation sequencing technologies have been used to sequence the flower transcriptomes of obligate sexual and unrelated apomictic H. perforatum genotypes. This approach has enabled the assembly and annotation of large cDNA repositories and their exploitation to design a custom array to be used in flower expression studies. Global gene expression analysis of H. perforatum was initially performed on ovaries collected from sexual and aposporic plant accessions for the purpose of identifying genes and processes potentially associated with apomixis in this model species. Overall, across two selected developmental stages, 224 and 973 unigenes were found to be significantly upand down-regulated. Ontological annotation of differentially expressed genes indicated that terms related to cell cycle, single-organism cellular process DNA (cytosine-5-)-methyltransferase activity, among others, were significantly enriched. In a following step, a laser-capture microdissection approach was adopted in combination the RNA-seq technology with the aim of identifying genes differentially expressed in the ovule cell types primarily involved in the differentiation of the megaspore mother cells and aposporous initials. On the whole, our data suggest that phenotypic expression of apospory is concomitant with the modulation of key genes involved in the sexual reproductive pathway and the responsive to hormonal stimuli. Annotation of all identified flower transcripts as well as their qualitative and quantitative expression data will be presented and critically discussed as they prove a far better understanding of molecular bases of pistil development, embyo sac and egg cell formation in sexual and apomictic H. perforatum