secondees

Dr. Juan Pablo A. Ortiz will undertake a new secondment to work on the analysis of apomixis-related sequences, with a particular focus on the discovery of genes associated with parthenogenesis. This work will include the analysis of transcriptome databases from sexual and apomictic genotypes to identify genes related to hormone cascades, RNA processing and cell signaling that may be involved in parthenogenesis. The analysis will also include the available apomixis controlling locus genomic resources. This work will be carried out in collaboration with Dr Giovanni Gabelli of the DAFNAE group. The results obtained will contribute to WP3 and WP4 and may provide new candidates for the induction of parthenogenesis in natural apomictic species.

Picture: Sharing mate… from left to right: Juan Pablo Ortiz, Lorena Siena & Samela Draga

The purpose of this  secondment is to deepen the characterization of sexual and apomictic genomes of Paspalum simplex. The work plan includes DNA extractions, library preparation and ONT sequencing experiments of tetraploid plants. These activities will contribute to WP3, which aims to analyze the structure and evolution of apomictic genomes and elucidate the gene content and function of the apomixis locus. The other members of the team that will participate in this work are Dra Lorena Siena (IICAR), Dr Andrea Rubini (IBBR) and BSc Davide Perrone (IBBR). Juan Pablo will also work on the construction of an RNAi vector designed to down-regulate the expression of ORC3, one candidate gene identified by Dr Pupilli’s group as a regulator of the endosperm development in apomictic seeds. This construction will be used to generate transgenic maize plants in order to validate its function in the control of maternal genomic excess in the endosperm of a sexual crop. This part of the work will be carried out in collaboration with Dra Francesca De Marchis and Dr Michelle Bellucci, from IBBR. 

Picture: Juan Pablo A. Ortiz (left) & Fulvio Pupilli (right)

Arabidopsis thaliana transgenic plants were generated expressing apomixis-related genes identified in Eragrostis curvula. These plants showed several phenotypes at MMC specification and during embryo sac stages linked to apomixis, and some evidences of loss in DNA methylation. The aim of this work was to monitor the most relevant histone marks during ovule development in these Arabidopsis transgenic lines using immunolabelling and confocal microscopy techniques. 

The objective of this secondment was to initiate the characterization of  Arabidopsis thaliana SALK mutant lines previously obtained for the CHC2 (Clathrin heavy chain 2) and NFD3 (Nuclear fusion defective 3) genes, both found to be differentially expressed in apomictic vs sexual genotypes of Eragrostis curvula in previous analyses. Genotyping with specific primers was carried out and several heterozygote lines were found, which are now being studied. Also, we recovered and grew CRISPR-Cas9 mutants for the SPL7 (Squamosa promoter binding like 7) gene. These plants are now under characterization. In addition, in situ hybridizations (ISH) have been carried out to determine the expression profile of the SPL7 gene in the reproductive tissues of diploid and tetraploid individuals in A. thaliana (ecotype Col0).

The main objective of this secondment is to continue the characterization of a mapping population of Eragrostis curvula that segregates for apomixis by creating a linkage map and selecting individuals with similar molecular markers but different reproductive modes (apomictic and sexual) for future transcriptomic analysis. During these two months, in collaboration with the group of UNIPG, it is proposed to continue the analysis of the phenotypic and genetic data of a mapping population of E. curvula to generate a new consensus map and to identify molecular markers linked to apomeiosis and, ultimately, to discover genes, which may play a potential role in the control of apomictic reproduction. Additionally, I will work in a calendar to associate floral morphological parameters with megasporogenesis and megagametogenesis developmental stages in different apomictic and sexual genotypes of E. curvula (Task 3.3).

Samela Draga is visiting Prof. Pessino laboratory at IICAR-CONICET-UNR from November 3, 2023, to January 18, 2024. The general objective of her work is to continue the project already started at March-July 2023, at IICAR-CONIC           ET-UNR, contributing to the elucidation of the role of the splicing controller BUD13 (a TGS1 interactor) in apomictic and sexual Paspalum. BUD13 displays differential expression in florets of sexual and apomictic plants in at least two aposporous species, Paspalum notatum and Hypericum perforatum. Interestingly, Arabidopsis bud13 mutants were associated with defects in early embryo development. During her visit, she will generate digoxigenin-labeled BUD13 probe, include Paspalum ovaries in paraffin, slice the blocks, arrange the slices on glass slides and perform the ISH analysis. Observations will allow to determine spatial expression patterns of BUD13 in reproductive organs of sexual and apomictic genotypes of Paspalum notatum.

During this first secondment in Rosario from November 9^th to December  14^th 2023, Fulvio Pupilli discussed with Juan Pablo Ortiz, his staff and Olivier Leblanc the recent achievements on Nanopore sequencing and on the assembly of apomictic and sexual Paspalum simplex genomes. Furthermore he reported  the insights toward the characterization of the gene PsARF5  that is differentially expressed in LCM dissected nucellus  tissues of apomictic and sexual genotypes of the same species and discussed with Juan Pablo Ortiz his possible interaction with PnIAA30, a gene under characterization at IICAR. A series of in situ hybridisation experiments for this gene was scheduled for the next secondment. Finally, he prepared a talk on the role of the gene PsORC3 on maternal excess endosperm development in P.simplex and a tribute to Prof. Camilo Luis Quarin for the IV Apomixis Congress 3-7^th December 2023 Rosario, Argentina. 

During this secondment, we follow up the work on Paspalum rufum with Luciana Delgado.  To understand the interelations between ovule primordia architecture and cell fate plasticity leading to apospory , we are performing a 3D cellular morphometric analyses of P.rufum genotypes showing differential expressivity of apospory. The first step is to build a reference 3D atlas of the sexual genotype. After the work done in previous secondments of segmentation, labelling, and data export using MorphographX (MGX) software, we set up for quantitative analysis on a pilot group of 11 ovules at successive stages covering the developmental window of interest. In addition to cell number and cell size quantification, different shape descriptors, at both cellular and organ level, were selected and calculated from features extracted by MGX. Since the pilot data already encompass thousands of cells to analyze, custom scripts in R were written to manage data compilation, visualization and statistical analysis. In addition, P.rufum ovules around meiosis stage were dissected, fixed and cleared; to extend the developmental window covered, and maximize the probability of detection of differential cell growth between sexual and aposporous genotypes.

Paspalum rufum, Luciana Delgado & Daphné Autran

Davide Perrone is visiting IICAR-CONICET in order to continue the experiments initiated during a previous secondment, concerning the transformation of apomictic and sexual Paspalum notatum plants with fluorescent auxin and cytokinin markers. The aim of these experiments is to compare the pattern of hormone distribution in the ovule between sexual and apomictic reproductive types. Moreover, in this particular secondment Davide will also start performing in-situ hybridization experiments aimed at comparing the expression of AUXIN RESPONSE FACTOR 10 (ARF10) in ovules of sexual and apomictic Paspalum notatum biotypes. Finally, in collaboration with IICAR scientists, he will perform miR-seq analyses using raw data previously generated in Milan.

Picture (from left to right): Augusto Villalba (IBONE), Silvina Pessino (IICAR), Maricel Podio (IICAR) and Davide Perrone (Univ. Milan)

Francesca De Marchis is a researcher of the National Research Council, CNR- IBBR – Perugia. During her visit, she will work with  Dr. Juan Pablo A. Ortiz (IICAR) and her expertise in plant biotechnology will be exploited to design a vector for Paspalum notatum transformation. Some candidates genes for triggering parthenogenesis, identified by the IICAR’s research group, will be considered for plant transformation by biolistic technology choosing for their over-expression or down-regulation.

The objective of my secondment at NIAB was to continue with the work started with Dr. Caccamo´s group aiming at the identification and the characterization of a genomic region and genes involved in apomixis. Here we discussed strategies and designed new experiments. The ongoing work consists on the sequencing and the assembly of the genome of the apomictic cultivar Don Walter (Eragrostis curvula). This genome was sequenced at NIAB using the Oxford nanopore platform with the super-high accuracy plant model protocol and one of the students from CERZOS is working here doing the assembly. We also are involved in a transcriptomics project using RNA-seq data obtained from pistil tissues of apomictic and sexual plants in three different developmental stages. We are looking for differentially expressed genes between plants with different reproductive modes and different developmental stages.

Picture: Viviana at The Eagle, Cambridge (UK)

In this secondment, Lorena Siena from IICAR, is visiting IRD for three months. The objective of the visit is to advance the functional characterization of the TGS1 and QGJ genes during Arabidopsis thaliana reproductive development. The activities will be carried out in collaboration with the IRD group and, first, include the exploring of the potential for parthenogenetic embryos in tgs1 mutant lines by using embryo-specific fluorescent reporters transmitted maternally and paternally. In addition, the cellular identity of the extra, functional megaspores observed in qgj ovules will be extensively characterized, e.g. using the pWOX2-CENH3-GFP reporter. Moreover, we will initiate experiments aiming at identifying potential interactions between QGJ and TGS1 by determining QGJ expression pattern (pQGJ-GUS) in tgs1 ovules and, reciprocally, that of TGS1 (pTGS1:TGS1-GUS) in qgj ovules.

Picture: Montpellier aqueduct (@ Lorena Siena, IICAR).

The objective of this secondment at CERZOS was to gain deeper knowledge, skills and practical experiencein teh area of in-situ hybridization and DIC microscopy in plants.

The general objective of Samela’s work at IICAR is to contribute to the elucidation of the role of the splicing protein BUD13 in the apomixis-sexuality switch. During her stay she will analyze the representation of BUD13 in transcriptomic and genomic databases of Paspalum notatum and generate suitable RNA hybridization probes. Moreover, she will classify ovules of sexual and apomictic Paspalum notatum plants according to developmental stages by using DIC cytoembryology, dehydrate them in an ethanol/xylol series and include them in paraffin. In a second visit (from October to December) she will prepare ovule sections for hybridization using sense and antisense digoxigenine-labelled RNA probes detected by colorimetric reaction using an anti-DIG conjugated with alkaline phosphase and NBT/BCIP substrates.  We expect to produce information on the spatial differential expression of these candidate genes in apomictic and sexual genotypes.

Samela Draga (right) & Silvina Pessino (left)

During my secondment at The University of Adelaide (Matthew Tucker’s lab) my work will be focused on unravelling the molecular mechanisms determining a correct female germline progression in Arabidopsis thaliana. In particular, I will try to characterize the main players involved in spore degeneration and functional megaspore selection and differentiation at the end of megasporogenesis in the ovule. I will determine the contribution of phytohormone cross-talk by trying to rescue mutants defective in this key processes using specific regulatory regions active in different domain of the ovule to specifically drive the expression of candidate genes.

From left to right: Rosanna Petrella, Skippy the Kangaroo, Nicola Babolin

A new stay at IRD of Dr. Juan Pablo A. Ortiz from IICAR, carried out as part of a MAD secondment for the characterization of apomictic genomes (WP3), started March 7, 2023. The work plan includes DNA extraction of several samples of Paspalum spp, intended for Illumina sequencing and further genome structural analyses. The visit is also dedicated to preparing a new manuscript describing the sequencing, chromosome-level assembly and annotation of a Paspalum notatum diploid genome. This achievement is a key step for WP3 progression because it will serve as a reference for characterizing tetraploid genomes and determining the structure and evolution of the apomixis region. This manuscript is the result of a collaborative effort between MAD partners from Argentina, Italy, and France. Finally, the program of the international workshop on Nanopore sequencing and bioinformatics, organized by the MAD project in Rosario, Argentina next November will be discussed and decided during this visit.

IRD plant greenhouse. Juan Pablo A. Ortiz (right) & Olivier Leblanc (left)

The objective of this detachment was to analyze genes that show differential expression between sexual and apomictic ovules of Eragrostis curvula plants and, which are also present in the model plant Arabidopsis thaliana. For this purpose, we studied the phenotype of A. thaliana mutants publicly available for some of these genes but, also, that of new transgenic mutant lines generated by the CRISPR-Cas9 strategy. In addition, ovule development in apomictic and sexual genotypes of E. curvula was characterized using confocal microscopy to further our knowledge on the structures and differences between sexual and asexual modes of reproduction.

From left to right: Marta Mendes, Lucia Colombo, Alejandra Díaz  and Cielo Pasten

Davide Perrone (University of Milan, Italy) is visiting Prof. Silvina Pessino laboratory at IICAR CONICET, Rosario, Argentina, from 3rd September to 27th November 2022. His work, conducted in collaboration with Dr. Carolina Colono (IICAR-CONICET-UNR), is aimed at using biolistic transformation to produce DR5 (auxin marker) and TCS (cytokinin marker) Paspalum notatum fluorescent lines with different reproductive modes (sexuality and aposporous apomixis). These plant materials will be useful to study the ovular hormone patterns along different developmental stages in a well-characterized apomictic model. Moreover, Davide is contributing to study the role of AUXIN RESPONSE FACTOR 10 (ARF10) in the transport of auxin within the Arabidopsis ovule, by doing cytoembryological characterizations of arf10 x PIN1 and arf10 x DII-Venus crosses previously obtained at IICAR-CONICET-UNR. Finally, he is involved in the analysis of the functional connexion between AUXIN RESPONSE FACTOR 10 (ARF10) and TRIMETHYL GUANOSINE SYNTHASE (TGS1) (a reported apospory controller) in Paspalum.

From left to rigth: Carolina Colono, Silvina Pessino and Davide Perrone

A new MAD secondment to work on the characterization of apomictic genomes (WP3) started on June 7, 2022. On this occasion, Dr. Juan Pablo A. Ortiz from IICAR (CONICET-UNR, Argentina) is visiting the laboratory of Dr. Olivier Leblanc at IRD (France). The workplan includes the annotation of a genome assembly of a diploid Pensacola bahiagrass ecotype (Paspalum notatum, var. saurae Parodi), obtained previously in a collaboration between laboratories of France, Italy, and Argentina. We also intend to identify and to characterize sequences related to the apomixis controlling locus (ACL) in a tetraploid race. In particular, we will use the ONT “adaptive sampling” technology that allows sequencing of regions of interest. Therefore, we expect to enrich our sequencing with reads specific from the tetraploid apomictic plant and then assemble the ACL. Olivier Leblanc, Cédric Mariac and Julie Orjuela (DIADE research Unit), as well as Juan Manuel Vega (MAD seconded from IICAR) will contribute to this work.

Juan Pablo Ortiz (left) & Olivier Leblanc (right)

In this secondment, Dr Luciana Delgado (IICAR) and Dr Daphné Autran work together to study the communication between cell division, expansion and differentiation throughout female reproductive development in P. rufum, an apomictic species. The overall aim is to build a 3D atlas of early ovule development. During the stay we will establish the optimal conditions for clarification of P. rufum ovules and cell wall staining techniques to obtain high-resolution 3D images. The images will be segmented with MorphoGraphX, classified by developmental stage, and finally a quantitative analysis of cellular descriptors will be performed. We believe that understanding the coordination between reproductive developmental processes with cell growth and division, in an apomictic context could be enlightening for understanding the regulation of apomixis.

Daphné Autran (left) & Luciana Delgado (right)

A new MAD secondment involving researchers from Argentina and Italy started May 9, 2022 for one month! Indeed, Dr. Juan Pablo A. Ortiz from IICAR, (CONICET-UNR) is visiting Dr. Fulvio Pupilli’s lab at the Institute of Biosciences and Bioresources (IBBR-CNR) of Perugia. The working plan is focused on the functional analysis of ORC3, a gene that segregates as completely linked to apomixis, in the formation of the endosperm of Paspalum simplex apomicts. Moreover, it is also intented to progress in the assembly and the characterization of the P. simplex genome using long reads generated using the Oxford Nanopore sequencing technology. Dr. Pupilli group’s members involved in these projects are: Dra. María Eugenia Cáceres, Dr. Andrea Rubini, Dra. Francesca De Marchis and Dr. Michele Bellucci. By the end of the secondment, we expect to have completed the redaction of a manuscript on PsORC3 functional analysis and produced a draft assembly of the P. simplex genome.

Fulvio Pupilli (left) & Juan Pablo A. Ortiz (right)

This secondment is part of the effort undertaken by the MAD project (see WP3 objectives) to determine the nature, the evolution and the functional role of the apospory controlling region (ACR) in Paspalum notatum. By taking advantage of a diploid reference genome and long reads data sets available for tetraploid apomicts, I will first use the adaptive sampling method offered by the ONT sequencing platform to enrich sequencing of ACR long reads and then perform a high coverage assembly for both an obligate apomict and a facultative apomict. Complementary approaches by using open source methods aiming at identifying and filtering structural variations will also be applied.  Altogether, this should allow structural and comparative genomics to determine the genetic content and coding/regulatory capacity within the ACR and provide cues for its functional role in the emergence of apomixis in Paspalum.