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Grapevine embodies a fascinating species as regards phenotypic plasticity and genotype-per-environment interactions. The terroir, namely the set of agri-environmental factors to which a variety is subjected, can influence the phenotype at the physiological, molecular, and biochemical level, representing an important phenomenon connected to the typicality of productions. We investigated the determinants of plasticity by conducting a field-experiment where all terroir variables, except soil, were kept as constant as possible. We isolated the effect of soils collected from different areas, on phenology, physiology, and transcriptional responses of skin and flesh of a red and a white variety of great economic value: Corvina and Glera. Molecular results, together with physio-phenological parameters, suggest a specific effect of soil on grapevine plastic response, highlighting a higher transcriptional plasticity of Glera in respect to Corvina and a marked response of skin compared to flesh. Using a novel statistical approach, we identified clusters of plastic genes subjected to the specific influence of soil. These findings could represent an issue of applicative value, posing the basis for targeted agricultural practices to enhance the desired characteristics for any soil/cultivar combination, to improve vineyards management for a better resource usage and to valorize vineyards uniqueness maximizing the terroir-effect.

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.

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 Hypericumto 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 perforatumL., 2n= 4x= 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. perforatumflowers 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.

The comprehension of molecular processes underlying the development and progression of flowering in plants is a hot topic, not only because that often the products of interest for human and animal nutrition are linked to the development of fruits or seeds, but also because the processes of gametes formation occurring in sexual organs are at the basis of recombination and genetic variability which constitutes the matter on which evolution acts, whether understood as natural or human driven. In the present study, we used an NGS approach to produce a grapevine flower transcriptome snapshot in different whorls and tissues including calyx, calyptra, filament, anther, stigma, ovary, and embryo in both pre- and post-anthesis phases. Our investigation aimed at identifying hub genes that unequivocally distinguish the different tissues providing insights into the molecular mechanisms that are at the basis of floral whorls and tissue development. To this end we have used different analytical approaches, some now consolidated in transcriptomic studies on plants, such as pairwise comparison and weighted-gene coexpression network analysis, others used mainly in studies on animals or human’s genomics, such as the tau (τ) analysis aimed at isolating highly and absolutely tissue-specific genes. The intersection of data obtained by these analyses allowed us to gradually narrow the field, providing evidence about the molecular mechanisms occurring in those whorls directly involved in reproductive processes, such as anther and stigma, and giving insights into the role of other whorls not directly related to reproduction, such as calyptra and calyx. We believe this work could represent an important genomic resource for functional analyses of grapevine floral organ growth and fruit development shading light on molecular networks underlying grapevine reproductive organ determination.

Mps-one-binder (Mob) proteins play an important role in chromosome separation and cell plate formation in yeast. We cloned two Mob1-like genes from alfalfa (Medicago sativaL.) and show that one gene is constitutively expressed while the other is expressed only in flower buds during sporogenesis and gametogenesis. For the analysis of gene expression during reproduction in alfalfa wild-types and apomeiotic mutants, a specific antisense riboprobe was designed for MsMob1transcripts and a polyclonal antibody was raised against MsMob1 proteins. In situmRNA localization as well as protein immunolocalization proved that MsMob1-like genes are specifically expressed in degenerating megaspores of normal ovules and in enlarged megaspore mother cells and embryo sacs of apomeiotic ovules. Gene products were also found in microspore tetrads at the beginning of pollen development as well as in tapetum cells of anthers undergoing programmed cell death to allow pollen dispersal at maturity. Overall results suggest that MsMob1-like genes can play a key role during the reproductive pathway in plants.

Mulberries are members of the genus Morus L., a taxonomic group showing a great genetic variability and adaptability to different environmental conditions. This study deals with the use of AFLP-based fingerprints as a tool for estimating genetic variability within as well as among three different mulberry species (i.e., M. alba L., M. latifolia Poir. and M. bombycis Koidz.). A high level of polymorphism (72.2) was found over all the 48 accessions analyzed. Genetic similarity (GS) within single Morus species ranged from 0.845 (M. bombycis) to 0.884 (M. alba) being intermediate in M. latifolia (0.869). The between-species mean genetic similarity estimates based on pair-wise AFLP marker fingerprint comparison were very similar ranging from 0.861 to 0.874. The partition of the genetic variation over the three Morus species was unexpected a proportion of the among-species genetic diversity as low as GST= 0.084 pointed out that about 92% of the total genetic diversity found among Morus accessions is due to DNA polymorphisms within a species, while only 8% of the total variation was highlighted among species. Our data indicate that some of the introduced accessions showing distinctive phenotypes, clearly differentiated from those revealed in the original habitat where they have been selected and adapted, hide an identical genotype.

Abstract The essential feature of apomixis is that an embryo is formed autonomously by parthenogenesis from an unreduced egg of an embryo sac generated through apomeiosis. The genetic constitution of the offspring is, therefore, usually identical to the maternal parent, a trait of great interest to plant breeders. If apomixis were well understood and harnessed, it could be exploited to indefinitely propagate superior hybrids or specific genotypes bearing complex gene sets. A fundamental contribution to the understanding of the genetic control of the apomictic pathway could be provided by a deep knowledge of molecular mechanisms that regulate the reproductive events. In Poa pratensis the cDNA-AFLP method of mRNA profiling allowed us to visualize a total of 2248 transcript-derived fragments and to isolate 179 sequences that differed qualitatively or quantitatively between apomictic and sexual genotypes at the time of flowering when the primary stages of apomixis occur. Three ESTs were chosen for further molecular characterization because of their cDNA-AFLP expression pattern and BLAST information retrieval. The full-lengths of the newly isolated genes were recovered by RACE and their temporal expression patterns were assessed by RT-PCR. Their putative role in cell signaling transduction cascades and trafficking events required during sporogenesis, gametogenesis and embryogenesis in plants is reported and discussed.

Mutants showing features of apomixis have been documented in alfalfa (Medicago sativaL.), 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 2negg 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 2neggs. 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

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 F1segregating population ofPoa pratensisobtained from a cross between a sexual and an apomictic parent. About half of the F1progeny 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 inP. pratensisand that apospory and parthenogenesis may be developmentally uncoupled

Gametophytic apomixis in Kentucky bluegrass (Poa pratensisL.) involves the parthenogenetic development of unreduced eggs from aposporic embryo sacs. Marker-assisted selection for the mode of reproduction in P. pratensiswould 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. pratensisgermplasm, 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.

Apomixis involves the parthenogenetic development of apomeiotic eggs. It has the potential of cloning plants through seed, and thus furnishes a unique opportunity in alfalfa breeding for developing superior cultivars with permanently fixed heterosis. Megasporogenesis of a meiotic mutant of alfalfa Medicago sativasubsp, falcata(L.) Arcang. (2n= 2x= 16), named PG‐F9, that produced 2nsecond division restitution (SDR) and apomeiotic eggs at high frequencies, was cytologically analyzed by both stain‐clearing and sectioning techniques. The restitutional 2negg formation was mainly due to the omission of the second meiotic division. The absence of cytokinesis was also documented after a normal second meiotic division. The concurrent examination of integument growth, cell appearance, and nucleoins size associated with either the absence of degenerating meiotic products or aposporic initials in the ovules allowed the production of apomeiotic 2nmegaspores to be ascribed to a diplosporic pathway. The mutant PG‐F9 was grown in the field and in a growth chamber and the frequencies of normal, restitutional, and diplosporic cells were recorded under both environments. Restitutional 2nmegaspore formation (dyads and triads with 2nchalazal megaspores) and the suppression or modification of meiosis leading to apomeiotic 2nmegaspore production were influenced by environmental conditions. The frequency of normal tetrads was three times higher in the field (23.08%) than in the growth chamber (7.91%). The same trend was found for functional reduced chalazal megaspores. The frequency of restitutional cells was largely preserved in the two environments (51.92% in the field versus 50.31% in the growth chamber). The omission of normal meiosis resulting in diplosporic cells increased from 16.35% in the field to 26.27% in the growth chamber. The fertility of seed set and seeds per pod values of PG‐F9 in controlled matings carried out in the field and in the growth chamber using different diploid and tetraploid pollen sources furnished information on 2negg viability and partially supported the cytological results. The discrepancy observed indicated that some 2nmegaspores were unable to develop into a functional embryo sac.

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.