Eragrostis curvula is a forage grass species in which multiple cytotypes coexist and shifts between sexual and apomictic reproduction are common. This study investigates genetic variation among these cytotypes in relation to ploidy levels and reproductive modes. To this end, a panel of accessions was assembled to capture the species' intraspecific diversity. The panel was used to construct a whole-genome sequencing-based linear pangenome by iteratively mapping Illumina short reads and de novo assembling unmapped reads, thereby expanding the available reference genome and improving the representation of the species' gene pool. The panel and the resulting pangenome were then used for gene content detection, functional annotation, and variant calling. Using this approach, extensive variation in gene content was observed among E. curvula accessions, with polyploid cytotypes containing a higher fraction of newly assembled pangenome sequences than diploids. In addition, candidate genes associated with polyploidization and apomixis were identified. Polyploid-associated genes were mainly associated with cell-cycle regulation and DNA replication, whereas apomixis-associated genes were mainly associated with post-transcriptional regulation. Moreover, phylogenetic relationships among cytotypes were inferred, and allele-frequency distributions revealed ploidy-associated differences among accessions. Thus, this work identified candidate genes associated with polyploidy and apomictic reproduction, highlighting ploidy-associated changes in gene content and post-transcriptional regulatory processes and providing insights into how polyploidization and reproductive shifts have shaped genome evolution in this agamic grass species.

Grafting is the process of joining parts of two plants, allowing the exchange of molecules such as small RNAs (including microRNAs and small interfering RNAs), messenger RNAs, and proteins between the rootstock and the scion. Genome editing by grafting exploits RNAs, such as tRNA-like sequences (TLS motifs), to deliver the components (RNA) of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system from transgenic rootstock to wild-type scion. The complex Cas9 protein and sgRNA-TLS produced in the scion perform the desired modification without the integration of foreign DNA in the plant genome, resulting in heritable transgene-free genome editing. In this review, we examine the current state of the art of this innovation and how it helps address regulatory problems, improves crop recovery and selection, exceeds the usage of viral vectors, and may reduce potential off-target effects. We also discuss the promise of genome editing by grafting for plants recalcitrant to in vitro culture and for agamic-propagated species that must maintain heterozygosity for plant productivity, fruit quality, and adaptation. Furthermore, we explore the limitations of this technique, including variable efficiency, graft incompatibility among genotypes, and challenges in large-scale application, while highlighting its considerable potential for further improvement and future broader applications for crop breeding.

Urochloa P. Beauv. (formerly classified as Brachiaria (Trin.) Griseb.) is a genus of African perennial grasses that is extensively cultivated in tropical countries for cattle nutrition. Three of the most economically relevant species, Urochloa brizantha, Urochloa decumbens, and Urochloa ruziziensis, form the brizantha agamic complex, which includes allopolyploid series with distinct subgenomes. Investigating the composition and organization of repetitive DNA, a major component of grass genomes, can provide insights into their genomic relationships and evolutionary history. This study aimed to characterize the repetitive DNA landscape of selected Urochloa species belonging to the b rizantha agamic complex; identify and compare major repeat classes across species; and evaluate their potential as cytogenetic markers on mitotic chromosomes using fluorescent in situ hybridization (FISH). Clustering analysis revealed that repetitive DNA constitutes 56%-65% of the genomes, with Ty3/Gypsy retrotransposons, particularly the Athila and Retand lineages, representing the most abundant repeat class. Urochloa decumbens exhibited the highest proportion of Ty3/Gypsy retrotransposons, while U. ruziziensis had the highest satellite DNA content. The chromosomal location of representative satellites (UroSat-1a, UroSat-2a, and UroSat-3) was determined in all three species via FISH. UroSat-1a was detected in all centromeres, while UroSat-2a and UroSat-3 signals varied in number and position. Our findings validate the use of satDNA as cytogenetic markers in the brizantha agamic complex of Urochloa and revealed genomic relationships among different species and ploidy levels.

Saffron (Crocus sativus L.) presents an attractive opportunity for diversifying production and adding value, particularly for small-scale growers and family-based agriculture. However, the agamic propagation of the crop through corms raises concerns regarding disease dispersion. During the summers of 2013 and 2015, symptoms of corm rot were observed in saffron crops in La Consulta, Valle de Uco, Argentina. These symptoms manifested in the form of wilting plants and red-coloured areas on the surface of the corms, in some cases affecting deeper regions. This study aimed to isolate and identify the causal agent responsible for saffron corm rot while also comparing the virulence of four strains isolated on saffron plants. Consistent isolation of Fusarium spp. colonies from affected corms confirmed its association with the disease. The obtained isolates were inoculated into healthy corms, and the reproduction of symptoms was confirmed, as well as subsequent pathogen re-isolation. Morphological and molecular characterisation of the strains was performed using rDNA gene sequencing. Furthermore, disease progression was assessed with fitting epidemiological models to empirical data, which served as estimators of fungal strain aggressiveness. The results conclusively identified Fusarium oxysporum Schltdl. as the causal agent of corm rot, and variations in virulence were observed among the strains on the host plant. After basic molecular and pathological studies, it is postulated that the fungal strains possibly belong to the forma specialis gladioli, but further studies are necessary to confirm that. The present study provides findings that highlight the importance of early detection and the preservation of pathogen-free fields to sustain saffron cultivation. These findings may constitute the initial step for future projects aimed at understanding the epidemiology of the disease better, determining the species/races of the pathogen, and developing effective management strategies.

Pseudogamous apomixis in Paspalum simplex generates seeds with embryos genetically identical to the mother plant and endosperms deviating from the canonical 2(maternal):1(paternal) parental genome contribution into a maternal excess 4m:1p genome ratio. In P. simplex, the gene homologous to that coding for subunit 3 of the ORIGIN OF RECOGNITION COMPLEX (PsORC3) exists in three isogenic forms: PsORC3a is apomixis specific and constitutively expressed in developing endosperm whereas PsORCb and PsORCc are up-regulated in sexual endosperms and silenced in apomictic ones. This raises the question of how the different arrangement and expression profiles of these three ORC3 isogenes are linked to seed development in interploidy crosses generating maternal excess endosperms. We demonstrate that down-regulation of PsORC3b in sexual tetraploid plants is sufficient to restore seed fertility in interploidy 4n×2n crosses and, in turn, its expression level at the transition from proliferating to endoreduplication endosperm developmental stages dictates the fate of these seeds. Furthermore, we show that only when being maternally inherited can PsORC3c up-regulate PsORC3b. Our findings lay the basis for an innovative route-based on ORC3 manipulation-to introgress the apomictic trait into sexual crops and overcome the fertilization barriers in interploidy crosses.

Micropropagation is an in vitro propagation technique, established in the nursery field sector for numerous species, which offers several advantages compared to traditional agamic propagation techniques. In the case of the olive tree, however, despite the advances made through research, it is still little used, due to the recalcitrance to in vitro proliferation and/or rooting of many olive cultivars and the high cost of zeatin, the only cytokinin that makes it possible to achieve a satisfactory proliferation rate in this species. In this context, numerous attempts have been made to identify alternative cytokinin compounds able to improve the proliferation rate of olive tree explants and thus reduce the unitary production cost. In particular, there is a growing interest in the use of natural substances (called in some cases "complex mixtures"), which, when added to the in vitro cultivation substrates, seem to be able to improve proliferation rates. In the present study, neem oil was added to the propagation substrates (partially/totally replacing zeatin) and in the rooting phase for the olive cultivar Moraiolo. In particular, in the proliferation phase, the effect of neem oil (0.1 mL L-1) in substrates containing different zeatin concentrations (0, 1, 2, and 4 mg L-1) was evaluated. For the rooting phase, agarized substrate and soil were used with shoots derived from a standard proliferation substrate (4 mg L-1 zeatin) and from the substrate that gave the best results in the proliferation phase (2 mg L-1 zeatin and 0.1 mL L-1 neem oil). In the proliferation phase, the addition of neem oil in the substrates with low zeatin concentration (1 and 2 mg L-1) induced an increase in the number of adventitious shoots and shoots length. On the contrary, the addition of neem oil in the rooting substrates did not positively influence the rooting phase, but positive results especially in terms of root number and length were observed in explants derived from a neem oil-enriched proliferation substrate compared to the control substrate. Therefore, the present study demonstrated for the first time the positive role of neem oil in the proliferation of olive in vitro with low zeatin concentrations.

Based on field research in south-central Poland, supplemented with a review of herbarium materials, we identified a stable bramble biotype with a range large enough (190 km distance between the outermost stands) to be described as a new regional agamic species, Rubuskaznowskii sp.nov. It belongs to the series Subthyrsoidei(sect. Corylifolii). Although R.kaznowskii has a unique combination of features, it can be potentially mistaken for R.gothicus. It differs from the latter species in many aspects, including: pruinose primocanes, denser indumentum of the abaxial leaf surface, and more curved prickles on the petiole. R.kaznowskii has mainly been observed on rusty soils, in habitats of mixed coniferous and mixed broadleaf forests, usually in sunny places, along forest margins and roads, in clearings and roadside thickets.

Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. We aimed to develop an optimized approach to determine ploidy of germplasm collection of this tropical forage grass group using dried leaf material, including approaches to collect, dry and preserve plant samples for flow cytometry analysis. Our methods enable robust identification of ploidy levels (coefficient of variation of G0/G1 peaks, CV, typically <5%). Ploidy of some 348 forage grass accessions (ploidy range from 2x to 9x), from international genetic resource collections, showing variation in basic chromosome numbers and reproduction modes (apomixis and sexual), were determined using our defined standard protocol. Two major Urochloa agamic complexes are used in the current breeding programs at CIAT and EMBRAPA: the 'brizantha' and 'humidicola' agamic complexes are variable, with multiple ploidy levels. Some U. brizantha accessions have odd level of ploidy (5x), and the relative differences in fluorescence values of the peak positions between adjacent cytotypes is reduced, thus more precise examination of this species is required. Ploidy measurement of U. humidicola revealed aneuploidy.

Within the agamic Pilosella complex, apomixis (asexual reproduction through seed) involves apospory, parthenogenesis, and autonomous endosperm development. Observations of reproductive biology in P. brzovecensis throughout four growing seasons in the garden have shown that both tetraploid and pentaploid plants of this species do not produce viable seeds and reproduce exclusively vegetatively by underground stolons. The reasons for the seed development failure were unknown, therefore our research focused on the analysis of reproductive events in the ovules of this taxon. We found that apospory was initiated in the ovules of both cytotypes. Multiple aposporous initial (AI) cells differentiated in close proximity to the megaspore mother cell (MMC) and suppressed megasporogenesis at the stage of early prophase I. However, none of the AI cells was able to further develop into a multi-nucleate aposporous embryo sac (AES) due to the inhibition of mitotic divisions. It was unusual that callose was accumulated in the walls of AI cells and its synthesis was most likely associated with a response to the dysfunction of these cells. Callose is regarded as the isolating factor and its surprising deposition in the ovules of P. brzovecensis may signal disruption of reproductive processes that cause premature termination of the aposporous development pathway and ultimately lead to ovule sterility. The results of our embryological analysis may be the basis for undertaking advanced molecular studies aimed at fully understanding of the causes of female sterility in P. brzovecensis.

Strains belonging to Pantoea agglomerans species are known for their ability to produce metabolites that can act in synergy with auxins to induce the adventitious root (AR) formation. The latter is critically important in the agamic propagation of several woody species, including pear (Pyrus communis L.), playing a considerable role in the commercial nursery farms including those using micropropagation techniques. When grown on a medium amended with tryptophan, the plant-growth-promoting (PGP) strain P. agglomerans C1 produces a cocktail of auxin and auxin-like molecules that can be utilized as biostimulants to improve the rooting of vegetable (Solanum lycopersicum L.) and woody crop species (Prunus rootstock GF/677 and hazelnut). In this study, we evaluated the morphological and molecular responses induced by strain C1 exometabolites in microcuttings of P. communis L. cv Dar Gazi and the potential benefits arising from their application. Results showed that exometabolites by P. agglomerans C1 induced a direct and earlier emergence of roots from stem tissues and determined modifications of root morphological parameters and root architecture compared to plants treated with the synthetic hormone indole-3-butyric acid (IBA). Transcription analysis revealed differences in the temporal expression pattern of ARF17 gene when IBA and C1 exometabolites were used alone, while together they also determined changes in the expression pattern of other key auxin-regulated plant genes. These results suggest that the phenotypic and molecular changes triggered by P. agglomerans C1 are dependent on different stimulatory and inhibitory effects that auxin-like molecules and other metabolites secreted by this strain have on the gene regulatory network of the plant. This evidence supports the hypothesis that the strategies used to harness the metabolic potential of PGP bacteria are key factors in obtaining novel biostimulants for sustainable agriculture. Our results demonstrate that metabolites secreted by strain C1 can be successfully used to increase the efficiency of micropropagation of pear through tissue culture techniques.

Geographic parthenogenesis, range expansion of apomictic plants after climate changes, has been described for Northern Hemisphere gametophytic apomicts. But similar trends have been observed for sporophytic apomicts of Cerrado, the savannas in Brazil. Eriotheca pubescens is a common Cerrado tree, an agamic complex of either hexaploid/polyembryonic apomicts or tetraploid/monoembryonic sexual individuals. Some populations have been described as a new species, Eriotheca estevesiae, all included in the Eriotheca Stellate Trichome Species Complex (ESTSC). Since breeding systems and ploidy are clearly associated with polyembryony and stomatal size, we used these ancillary features to map the reproductive and ploidy level traits of E. pubescens and E. estevesiae. Leaves and seeds were collected from individuals of 19 populations. Seeds were evaluated for the presence of polyembryony and leaves for stomatal measurements. Eight populations were monoembryonic while another eight were polyembryonic and for other three, the embryonic pattern was not readily verified. E. pubescens polyembryonic and hexaploid populations formed a homogeneous group, but monoembryonic plants were more variable. E. estevesiae populations were monoembryonic with smaller stomata. In contrast, some E. pubescens monoembryonic populations further south presented larger stomata. Despite these outliers, possibly mixed populations, stomatal size and embryonic pattern differed from northern to southern populations. Embryonic pattern and stomatal size indicated that northernmost populations of Eriotheca STSC (E. estevesiae) are diploid and sexual. Southernmost populations, mostly polyembryonic and with large stomata, are hexaploid and apomictic. This is in agreement with geographic parthenogenesis and range expansion of apomictic lineages to southern habitats available after the last glacial maximum.

The species Pantoea agglomerans includes strains that are agronomically relevant for their growth-promoting or biocontrol traits. Molecular analysis demonstrated that the IPDC pathway involved in the conversion of tryptophan (Trp) to indole-3-acetic acid (IAA) is highly conserved among P. agglomerans strains at both gene and protein levels. Results also indicated that the promoter region controlling the inducible expression of ipdC gene differs from the model system Enterobacter cloacae, which is in accordance with the observation that P. agglomerans accumulates higher levels of IAA when cells are collected in the exponential phase of growth. To assess the potential applications of these microorganisms for IAA production, P. agglomerans C1, an efficient auxin-producer strain, was cultivated in 5 L fermenter so as to evaluate the effect of the medium formulation, the physiological state of the cells, and the induction timing on the volumetric productivity. Results demonstrated that higher IAA levels were obtained by using a saline medium amended with yeast extract and saccharose and by providing Trp, which acts both as a precursor and an inducer, to a culture in the exponential phase of growth. Untargeted metabolomic analysis revealed a significant effect of the carbon source on the exometabolome profile relative to IAA-related compounds and other plant bioactive signaling molecules. The IAA-enriched metabolites secreted in the culture medium by P. agglomerans C1 were used as plant biostimulants to run a series of trials at a large-scale nursery farm. Tests were carried out with in vitro and ex vitro systems following the regular protocols used for large-scale plant tree agamic propagation. Results obtained with 4,540 microcuttings of Prunus rootstock GF/677 and 1,080 plantlets of Corylus avellana L. showed that metabolites from strain C1 improved percentage of rooted-explant, number of adventitious root formation, plant survival, and quality of plant as vigor, with an increase in the leaf area between 17.5 and 42.7% compared to IBA-K (indole-3-butyric acid potassium salt)-treated plants.

Mitotic slippage (MS), the incomplete mitosis that results in a doubled genome in interphase, is a typical response of TP53-mutant tumors resistant to genotoxic therapy. These polyploidized cells display premature senescence and sort the damaged DNA into the cytoplasm. In this study, we explored MS in the MDA-MB-231 cell line treated with doxorubicin (DOX). We found selective release into the cytoplasm of telomere fragments enriched in telomerase reverse transcriptase (hTERT), telomere capping protein TRF2, and DNA double-strand breaks marked by γH2AX, in association with ubiquitin-binding protein SQSTM1/p62. This occurs along with the alternative lengthening of telomeres (ALT) and DNA repair by homologous recombination (HR) in the nuclear promyelocytic leukemia (PML) bodies. The cells in repeated MS cycles activate meiotic genes and display holocentric chromosomes characteristic for inverted meiosis (IM). These giant cells acquire an amoeboid phenotype and finally bud the depolyploidized progeny, restarting the mitotic cycling. We suggest the reversible conversion of the telomerase-driven telomere maintenance into ALT coupled with IM at the sub-telomere breakage sites introduced by meiotic nuclease SPO11. All three MS mechanisms converging at telomeres recapitulate the amoeba-like agamic life-cycle, decreasing the mutagenic load and enabling the recovery of recombined, reduced progeny for return into the mitotic cycle.

Most of the grasses of the genus Cenchrus (20-25 species) and Pennisetum (80-140 species) are distributed throughout the tropical and subtropical regions of the world and reproduce both by sexual and apomictic modes. However, the relationships among the Cenchrus-Pennisetum species are not very clear yet. Molecular markers like expressed sequence tag-simple sequence repeats (EST-SSRs) have been reported to be a better choice for resolving the phylogenetic relationships and to estimate the genetic diversity. The present study describes the identification of EST-SSR markers based on the transcriptome data of Cenchrus ciliaris inflorescence and illustrates the genetic diversity and phylogenetic relationships among these species. Of the 378 primer pairs used across 33 accessions of 21 Cenchrus, Pennisetum, and related grass (Bothriochloa, Dichanthium and Panicum) species, 116 EST-SSR markers were found to be polymorphic with an average polymorphism information content (PIC) of 0.49. Fifty-one EST-SSR loci and 520 alleles showed that where the PIC value is >0.5 there the GAG repeat motif was highly polymorphic. Two EST-SSR markers, CcSSR_80 and CcSSR_102, are polymorphic among the Cenchrus species, while they are absent in Pennisetum and the allied species. Five SSR markers (CcSSR_75, CcSSR_85, CcSSR_87, CcSSR_88 and CcSSR_114) showed 100% cross-transferability among the 21 Cenchrus-Pennisetum species. Species-specific alleles could also be detected for seven species of Cenchrus, Pennisetum and Panicum across 10 SSR markers. Assay of polymorphism across these agamic complexes showed that the three SSR markers (CcSSR_26, CcSSR_97 and CcSSR_109) were associated with Cenchrus-Pennisetum complex, and one (CcSSR_47) with Bothriochloa-Dichanthium complex. Markers with high discriminating power, namely CcSSR_4, CcSSR_38, CcSSR_48, CcSSR_66, CcSSR_67 and CcSSR_70, can be used to estimate the allelic sequence divergence across the sexual and apomictic lineages. Genetic diversity analysis using neighbour-joining (NJ) and principal co-ordinate analysis (PCoA) based approaches showed six and five clusters for the 33 accessions, respectively, having congruence in the pattern of clustering. These accessions were grouped according to their mode of reproduction. Cenchrus and Pennisetum species were grouped separately within the same clade, implying monophyletic group within a 'bristle clade'. Thus, this study showed high discrimination power of microsatellite (EST-SSR) markers to resolve the phylogenetic relationships.