secondees

Secondment period means the period spent by the staff member in a host organisation for the purposes of the action. A secondee is a staff member hosted.

During this secondment, I contributed to the work of Luciana Delgado and Daphné Autran on Paspalum rufum, exploring the links between ovule primordium architecture and cell fate plasticity leading to apospory. To determine the patterns of cell proliferation during ovule development in sexual and apomictic Paspalum rufum at pre-meiotic stages, we conducted EdU labeling experiments to mark cells in S-phase of the cell cycle using the Click-iT™ EdU kit. Ovaries were dissected at four early stages, processed for EdU incorporation and detection, then imaged using a ZEISS 880 confocal microscope at the Institute of Molecular and Cellular Biology (IBR-CONICET) in Rosario. These images will be segmented with MorphographX (MGX) to quantify the differential proliferation rates between genotypes. I have also collaborated on a project by Olivier Leblanc and Lorena Siena, constructing plasmids for the functional analysis of the TGS1 gene in Arabidopsis thaliana, with completion planned in Montpellier in 2025.

Picture. Top left: my UNR teaching credential card!; Bottom left: the IBR confocal microscope, and; Right: a diagram of the dissected Paspalum rufum stages.

During this secondment, in the frame of WP6 of the MAD project, Daphné Autran (IRD) collaborated with Luciana Delgado (IICAR-CONICET), with the help of Caroline Michaud (IRD), to decipher ovule morphogenesis in the aposporous specie Paspalum rufum. To determine which are the earliest cellular events associated with a facultative aposporous developmental program, we took advantage of the flowering period of the unique population of contrasting P. rufum plants available at IICAR, Zavalla, Rosario, Argentina. Here, the process of cell proliferation was specifically addressed, by monitoring the number and localization of dividing cells, using living ovules, collected at different developmental stages. After fixation, clearing and staining, the samples were imaged using confocal fluorescence microscopy (available at IBR (Instituto de Biologia de Rosario). The 3D images dataset generated will be next analyzed, to complete the information gathered from the 3D cell growth atlas of P. rufum ovules produced in previous secondments.

Picture: Luciana Delgado (left panel) & Luciana Delgado and Daphné Autran (right panels)

During this secondment Dr. Lorena Siena will analyze the expression pattern of the gene BUD13 during the reproductive development of apomicts of Paspalum notatum and Hypericum perforatum. BUD13 was found to be differentially expressed between apomictic and sexual genotypes in both species in a previous MAD secondment. Interestingly, Arabidopsis BUD13 null mutants showed defects during early embryo development. Research activities will include the design of PCR primers to amplify both genomic and mRNA sequences, qRT-PCR assays, and the analysis of images from in situ hybridization experiments performed on P. notatum ovaries at IICAR-CONICET. In addition, the initial steps of publication draft including the main results of the study will be discussed. The work is carried out in collaboration with Samela Draga as part of her PhD thesis at DAFNAE. The information obtained will contribute to WP4 and may provide a new candidate for parthenogenesis.

Picture: in the screenhouse with a collection of Hypericum perforatum plants

During her visit at the laboratory of Dr. Fulvio Pupilli, Lorena will sequence tetraploid sexual and apomictic plants of Paspalum simplex in order to improve current tetraploid genome assemblies. The information produced will contribute to WP3 and will be used to study the genome structure and evolution and to decipher the gene content of the apomixis locus. This work will be carried out in collaboration with Dr. Andrea Rubini and BCs Davide Perrone from IBBR and Dr Juan Pablo A Ortiz from IICAR. Lorena will also set up in situ hybridization assays to characterize the expression pattern of the P. simplex candidate gene ARF5 in apomictic and sexual ovaries. She will be involved in the design and labelling of the probe, and in the paraffin mounting of the ovaries. The sectioning and hybridization will be carried out at IICAR, CONICET-UNR. These experiments will be performed in collaboration with Dra Eugenia Cáceres from IBBR.

Picture: Lorena Siena (right) & Davide Perrone (left)

The aim of this secondment at UNIPG is to continue the work with Dr. Albertini´s group to identify the genomic region involved in apomixis in Eragrostis curvula and characterize its gene content. To achieve this, we will analyze the data previously obtained from the E. curvula pangenome, which consits of Illumina sequencing of 10 genotypes with similar genomic backgrounds but with different reproductive modes (apomictic and sexual) and ploidy levels (2x, 4x, 6x, 7x and 8x). The data will be used to study the genomic regions involved in apomixis and syntenic relationships with sexual counterparts through a mapping approach to obtain the first E. curvula pangenome assembly.

During the three months (from May 11 to June 8, 2024) he spent at CERZOS, Domenico Loperfido worked in the lab of Prof. Echenique under the supervision of José Carballo and Cristian Gallo from CERZOS and that of Prof. Ezquer from the University of Milan. The objective of the secondment was to investigate candidate genes involved in growth and seed development through association studies aiming at understanding the role these genes may have in the inheritance of apomictic processes. Genomes of different Arabidopsis thaliana accessions were used ; using bioinformatics tools, eg bcftools, vcftools, R, GAPIT, and others, significant SNPs in genes phenotypically associated with the growth and the development of seed components (embryo, endosperm, and seed coat) controlling hybrid vigorous in sexual plants were identified

During this secondement at CERZOS, my primary focus was on the diplosporous grass Eragrostis curvula, aiming to construct a comprehensive pan-genome. The utilization of the diploid sexual variety, cv. Victoria, as the reference genome provided a solid foundation for investigating genomic variation, phylogenetic relationships, and the impact of ploidy on genome evolution and reproductive modes. This pan-genomic study significantly contributes to our understanding of E. curvula‘s genomic diversity, serving as a valuable resource for targeted investigations into the molecular mechanisms governing its reproductive modes and relationships with ploidy.

Picture (from left to rigth: Juan Pablo Selva, Diego Zappacosta, Juan Manuel Rodrigo, Nengi Lawson, Viviana Echenique, Alejandra Diaz & Giacomo Bongiorno

This second visit to IICAR is part of a secondment dedicated to the genomics of apomixis (WP3) and the functional characterization of Paspalum candidate genes relevant for apomixis using Arabidopsis mutants (WP4). Regarding the genomic approaches, we expect to complete a first draft on the assembly and the detailed annotation of a P. notatum diploid genome reference and will focus analyses on understanding the variations between the genome of diploid, sexual plants and that of apomicts using comparative genomics and k-mer approaches. Regarding the functional characterization of Arabidopsis mutants, the effets of tgs1 mutations on RNA splicing are being analyzed using comparative transcriptional analyses and cytological tools (fluorescent reporters under construction). In addition to these activities, I’m involved in organizing the second MAD workshop on ONT sequencing and bioinformatics (Rosario, November 20-25) and the Apomixis2023 conference (Rosario, December 3-7).

Picture: visiting old friends in Cayasta (Santa Fe, Argentina), cradle of the Paspalum family. from left to right: O Leblanc, JPA Ortiz, & L Siena

For some time now, our CERZOS research group has been collaborating with Dr. Caccamo´s group at NIAB on the identification and the characterization of a genomic region involved in apomixis in Eragrostis curvula (Schrad.) Nees and its genes. Some of these, identified previously by our research group to be involved in apomictic pathways, are under functional characterization since many of them do not have an annotation in other species. Expression of these candidate under constitutive promoters in the rice plant model species is one of the approaches used for their characterization. The aim of this secondment at NIAB was to advance the characterization of one of our candidate genes, mainly to determine whether its overexpression led to any defect in embryo sac development. To achieve this, I was involved in the screening of reproductive tissues from spikelets of independent transgenic rice lines generated at NIAB and overexpressing the gene of interest (WP4). We used Differential Interference Contrast (DIC) microscopy to observe reproductive tissues cleared with a methyl salicylate solution, as this technique enables large-scale observations on meiosis and embryo sac development in intact ovules. Thus, the collaboration between NIAB and CERZOS will allow us to get faster characterization of the candiate genes for apomixis identified in Eragrostis.

In this secondment María Sol is visiting IRD to continue with the functional characterization of the IAA16 gene in Arabidopsis thaliana, whose orthologue (IAA30) has been associated with apomixis in Paspalum notatum. The activities will be carried out in the laboratory of Dr. Olivier Leblanc and will include the recovery of double homozygous plants derived from crosses between the iaa16 mutant and; a line carrying the pWOX2-CENH3-GFP gametophytic reporter; tgs1 plants (related to alterations in gametophyte development), and; topless plants (involved in the auxin response during embryogenesis). Phenotypic analyses of the reproductive development of the materials obtained will be carried out using fluorescence or DIC microscopy during the secondment and at IICAR. In addition, the study of mutants for other genes from the IAA16 clade (iaa14 and iaa17) as well as for a new IAA16 insertion will be initiated.

The objective of my secondment at NIAB was to continue with the sequencing and assembly of the apomictic genome of Eragrostis curvula. The genome of Don Walter genotype was sequenced at NIAB institute using the Oxford nanopore platform with the super-high accuracy plant model protocol. These reads were assembled using Flye software and scaffolded with Omni-C technology in order to obtain a chromosome scale assembly. This genome was assembled and annotated obtaining the A and B sub-genomes of the allotetraploid Don Walter. Moreover, during this time in Cambridge, the RNA-seq data obtained from pistil tissues of apomictic and sexual plants in three different developmental stages was analysed obtaining differentially expressed genes. This secondment continues with the collaborative efforts between NIAB and CERZOS-CONICET in order to disclose the molecular basis of apomixis through a bioinformatic approach.

This secondment continues the tasks initiated in 2022 with Dr. Daphné Autran to build a 3D atlas of early ovule development in Paspalum rufum. We will segment and classify the cells within ovules primordia using high resolution images obtained previously, and a previously established and partially automated workflow using the open source software MorphoGraphX. In parallel, we will test different staining protocols to allow incorporation and detection of a thymidine analogue (EdU) for quantifying location and duration of cell divisions during ovule development in different grasses, including two sexual model species (rice and maize) and Paspalum apomicts. This protocol was successfully used in Arabidopsis and a few other species, it will be useful optimizing conditions for grass species showing disparities in flower and ovule morphologies. Quantitative information from the 3D Atlas and cell division in sexual and apomictic genotypes should help us understand the switch between the two reproductive pathways.

Picture: Interphase cell in a young ovule of Paspalum rufum showing fluorescent signal from DNA containing EdU (@ Luciana Delgado, IICAR).

During my secondment in the University of Adelaide (Australia) in A/Prof. Matthew Tucker laboratory I’m willing to better characterize the molecular network between the two MADS-box genes STK and ABS during the fertilization process and seed development in Arabidopsis thaliana.

At the same time I will be involved on the projects run by Prof. Tucker team on barley plant concerning ovule and seed development highly interconnected with my project in Arabidopsis.

​From left to right: Nicola Babolin, Mathew Tucker, Rosanna Petrella

The aim of this secondment is to investigate molecular mechanism determining female germline. In particular, I will investigate the contributions of phytohormones, auxin and cytokinines, in MMC differentiation. The species investigated are Arabidopsis thaliana and Oryza sativa var. Kitaake. In this frame, I will perform Immunolocalization assays in different backgrounds to localize phytohormones during ovule development using spl/nzz mutant and wild type  Arabidopsis and wild type for Oryza sativa.

The aim of this secondment was to analyze the tissue-specific expression of two candidate genes involved in the apomictic reproductive mode in the grass Eragrostis curvula (WP4). For this purpose, we used RNA in situ hybridization technic (RNA-ISH) to determine the expression patterns cell-specific of these two genes in the reproductive organs of E. curvula, both in the full apomictic and sexual genotypes. In addition, we also characterized a gene that shows differential expression between sexual and apomictic E. curvula plants and that is also expressed in the model plant A. thaliana. For the latter objective, we also used ISH to test the RNA abundance and localization of this orthologous gene in the reproductive tissues of E. curvula (apomictic and sexual genotypes) and A. thaliana (diploid and tetraploid genotypes).

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

This secondment aims at pursuing (1) the characterization of the chromosomic region associated with apomixis in tropical forages. As we now have completed the assembly and the annotation of the genome  of a diploid, sexual individual, different approaches will be explored to reveal structural genomic variations that underlie the apomictic development in Paspalum species. To achieve this, we produced > 100 Gb of long reads (Nanopore technology) from several Paspalum apomictic races that will be used for de novo assembly and comparative analyses, and; (2) the functional analysis of tgs1 Arabidopsis mutants and, more particularly, the comparative transcriptional analysis in tgs1 and WT Arabidopsis embryos collected at early developmental stages. These activities are part of WP3 and WP4, respectively.

Picture: Metallic clones in the pampa outside of Rosario, Province of Santa Fe, Argentina (@ O Leblanc)

The objective of this secondment is to extend a tetraploid genetic linkage map of Eragrostis curvula using DarT seq markers and additional F1 genotypes in order to map more precisely the region involved in apomixis. The F1 population was obtained by crossing a sexual (OTA) and an apomictic genotypes (Don Walter) as maternal and paternal parents, respectively. This new linkage map will facilitate the localization of loci controlling apomixis (diplospory) and to study the genome evolution in this grass (WP3). In collaboration with the UNIPG group, I am analyzing molecular markers (DarT seq) obtained in new F1 individuals and in others previously obtained in Bahía Blanca by our group to construct a new integrated linkage map, as well as two parent-specific linkage maps. For this, segregation analysis and detection of polymorphic SNP markers in one or both parent is needed to solve this three linkage maps. Additionally, the haplotype reconstruction using this F1 population will be explores and parental SNP profile.

Maria Sol is a PhD fellow at IICAR-CONICET, Argentina, and she is visiting the laboratory of Dr. Olivier Leblanc at IRD (Montpellier, France). The main objective of her stay is to initiate the functional analysis of Arabidopsis mutants defective for the IAA16 gene, whose orthologue in Paspalum notatum (PsIAA30) is down-regulated during apomictic development (Siena et al., Plants 2022, 11, 472). Heterozygous and homozygous iaa16 mutants will be analyzed by cytoembryological observations to search for possible female reproductive phenotypes reminiscent of aposporous apomixis. These activities will then contribute to determine the effects of IAA16 depletion on the reproductive development of Arabidopsis and to plan further functional characterization for this or other genes that belong to the pathway. During her visit, Maria Sol will work in collaboration with Dr. Olivier Leblanc and Caroline Michaud from IRD, and with Lorena Siena, her PhD supervisor at IICAR.

Lorena Siena (left) & Sol Vega (right)

During this 3-month secondment, Lorena Siena (IICAR, CONICET- Argentina) is visiting IRD with the objective of advancing the functional characterization of the TGS1 gene during the reproductive development of the model plant Arabidopsis thaliana. The activities will be carried out in collaboration with Dr. Olivier Leblanc and his IRD group, and include: complementation tests using the pTGS1:TGS1-GUS construct; determining the origin of the extra embryo sacs observed in tgs1 mutants using reporter lines carrying the pWOX2 and CENH3 genes reporting both  female gametophytic identity and chromosome number (reduced vs. unreduced origin) , and; analysis of RNAseq data obtained from wild type and tgs1 embryos at early stages of embryonic development. This study aims to determine the alterations in gene expression patterns caused by TGS1 depletion; to identify their potential target genes, and; to explore whether alternative splicing controls early embryogenesis in plants.

Olivier Leblanc (left) & Lorena Siena (right)

The objective of this secondment was to continue with the functional characterization of a gene differentially expressed in sexual and apomictic plants of Eragrostis curvula using maize mutants carrying a Mutator insertion in the orthologous gene.

During these two months, I collaborated with the group of Professor Gabriella Consonni to evaluate different maize F2 populations that were planted in an experimental field. Plants were genotyped using specific primers for the transposon insertion located in the first exon of the orthologous gene and specific primers for the wild type allele. Activities also included observations of cleared pistils under the microscope searching for abnormalities during female reproductive development.

From April 29 to July 2nd, 2022  Dr. Carolina Colono and Prof. Silvina Pessino will be visiting Prof. Lucia Colombo laboratory at the University of Milan, to explore the role of the hormonal control in the transition from sexuality to apomixis using the model plant Arabidopsis thaliana.

From left to rigth: Carolina Colono, SIlvina Pessino and Lucia Colombo

The objective of this secondment is to continue the assembly of Eragrostis curvula tetraploid genomes to determine the locus controlling apomixis and study genome evolution in this grass (WP 3). During these two months, I will analyze, in collaboration with the group of UNIPG, the genetic relationships between the sexual diploid (cv Victoria), previously sequenced and published by the 2 institutions, and the apomictic tetraploid cv. Tanganyika. We will first assemble a pan-genome and resolve it into the core genome and the dispensable genome. The reads mapping onto the Victoria reference will be assembled while unmapped reads will be assembled de novo to analyze the presence/absence variations. We will next analyze orthologous genes to find contractions and expansions of gene families and compare regions controlling apomixis in Eragrostis and Paspalum spp. Finally, I will apply the MCSeEd protocol developed by UNIPG to compare differentially methylated regions between apomictic and sexual genotypes (Task 3.3).