Francesco Mercati

Nitrate uptake along the maize primary root: an integrated physiological and molecular approach

The spatial variability of the nitrate (NO₃-) uptake along the maize primary root axis was investigated at physiological and molecular levels. Net NO₃- uptake rate (NNUR) and its kinetic parameters, together with the gene expression of a high-affinity NO₃- transporter (NRT2.1), were evaluated. The activity and the expression of plasma membrane H⁺ -ATPase (PM H⁺ -ATPase), key enzyme in plant nutrition, were also analysed. The NNUR showed a heterogeneous spatial pattern along the root, where the regions closer to the root tip early exhibited higher capacity to absorb NO₃- than the basal regions, because of a higher maximum NNUR and faster induction of the inducible high-affinity transport system (iHATS), the presence of the high-affinity transport system (HATS) also at external NO₃- concentrations >100 µm and an improved NO₃- transport because of lower K(m) values. ZmNRT2.1 transcript abundances were not spatially correlated with NNUR, suggesting that post-translational effects or NAR2 protein co-expression could be involved. By contrast, PM H⁺ -ATPase displayed a similar spatial-temporal pattern as that of nitrate uptake, resulting in higher activity in the root tip than in the basal regions. Increased activities of the enzyme after nitrate supply resulted in enhanced expression of MAH3 and MAH4, PM H⁺ -ATPase subfamily II genes, while MAH1 was not expressed.

Sex‐biased gene expression in dioecious garden asparagus (Asparagus officinalis)

Sex chromosomes have evolved independently in phylogenetically diverse flowering plant lineages. The genes governing sex determination in dioecious species remain unknown, but theory predicts that the linkage of genes influencing male and female function will spur the origin and early evolution of sex chromosomes. For example, in an XY system, the origin of an active Y may be spurred by the linkage of female suppressing and male promoting genes. Garden asparagus (Asparagus officinalis) serves as a model for plant sex chromosome evolution, given that it has recently evolved an XX/XY sex chromosome system. In order to elucidate the molecular basis of gender differences and sex determination, we used RNA-sequencing (RNA-Seq) to identify differentially expressed genes between female (XX), male (XY) and supermale (YY) individuals. We identified 570 differentially expressed genes, and showed that significantly more genes exhibited male-biased than female-biased expression in garden asparagus. In the context of anther development, we identified genes involved in pollen microspore and tapetum development that were specifically expressed in males and supermales. Comparative analysis of genes in the Arabidopsis thaliana, Zea mays and Oryza sativa anther development pathways shows that anther sterility in females probably occurs through interruption of tapetum development before microspore meiosis.

Microsatellite analyses for evaluation of genetic diversity among Sicilian grapevine cultivars

A total of 82 grapevine genotypes were sampled from several areas of the Italian region of Sicily where vineyards are widely spread. The grapevines were characterized using six microsatellite markers (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62 and VrZAG79) to evaluate genetic diversity. Thirty-seven of the 82 cultivars sampled had their names quoted in historical and literary sources, while 45 cultivars from old vineyards did not have their names reported in ancient literature. According to their genetic profiles at SSR loci, 70 different cultivars were found, while interesting cases of synonymies (Regina and Moscato bianco, Alicante and Dolcetta or among different clones of Zibibbo and Catarratto) and cases of homonymy (Frappato and Nerello Mascalese) were discovered. Several genetic parameters were calculated to assess the efficacy of the loci chosen in this work. Pairwise genetic distances between all cultivars were calculated. A dendrogram representing the genetic similarities among cultivars was depicted using the UPGMA method to investigate their relationships, explaining them from a historical point of view. The cluster distribution of cultivars clearly does not reflect their current geographical distribution, suggesting successive introductions of cultivars in Sicily from different areas of origin.

Characterization of Pseudoperonospora cubensis isolates from Europe and Asia using ISSR and SRAP molecular markers

Downy mildew caused by Pseudoperonospora cubensis is a major disease of cucurbits worldwide. New genotypes of the pathogen have recently appeared in the USA, EU and Israel causing breakdown of genetic resistance, expansion of host range, and the appearance of a new A2 mating type. Seventy-eight P. cubensis isolates were collected during 1996–2011 from cucurbits fields in different regions of Turkey, Israel and the Czech Republic and genetic diversity was analysed using highly polymorphic ISSR and SRAP molecular markers. The data acquired showed remarkable genetic diversity within and among the isolates. While isolates from Turkey and Czech Republic exhibited uniform genetic background, the isolates from Israel were clearly distinguished from the others. The results may indicate on migration and/or frequent sexual reproduction of the pathogen in Israel. Moreover the selected markers can be suggested for monitoring genetic diversity within P. cubensis isolates in further studies.

Single nucleotide polymorphism isolated from a novel EST dataset in garden asparagus (Asparagus officinalis L.)

Single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSR) are abundant and evenly distributed co-dominant molecular markers in plant genomes. SSRs are valuable for marker assisted breeding and positional cloning of genes associated traits of interest. Although several high throughput platforms have been developed to identify SNP and SSR markers for analysis of segregant plant populations, breeding in garden asparagus (Asparagus officinalis L.) has been limited by a low content of such markers. In this study massively parallel GS-FLX pyro-sequencing technology (454 Life Sciences) has been used to sequence and compare transcriptome from two genotypes: a rust tolerant male (1770) and a susceptible female (G190). A total of 122,963 and 99,368 sequence reads, with an average length of 245.7bp, have been recovered from accessions 1770 and 190 respectively. A computational pipeline has been used to predict and visually inspect putative SNPs and SSR sequences. Analysis of Gene Ontology (GO) slim annotation assignments for all assembled uniscripts indicated that the 24,403 assemblies represent genes from a broad array of functions. Further, over 1800 putative SNPs and 1000 SSRs were detected. One hundred forty-four SNPs together with 60 selected SSRs were validated and used to develop a preliminary genetic map by using a large BC(1) population, derived from 1770 and G190. The abundance of SNPs and SSRs provides a foundation for the development of saturated genetic maps and their utilization in assisted asparagus breeding programs.

The asparagus genome sheds light on the origin and evolution of a young Y chromosome

Sex chromosomes evolved from autosomes many times across the eukaryote phylogeny. Several models have been proposed to explain this transition, some involving male and female sterility mutations linked in a region of suppressed recombination between X and Y (or Z/W, U/V) chromosomes. Comparative and experimental analysis of a reference genome assembly for a double haploid YY male garden asparagus (Asparagus officinalis L.) individual implicates separate but linked genes as responsible for sex determination. Dioecy has evolved recently within Asparagus and sex chromosomes are cytogenetically identical with the Y, harboring a megabase segment that is missing from the X. We show that deletion of this entire region results in a male-to-female conversion, whereas loss of a single suppressor of female development drives male-to-hermaphrodite conversion. A single copy anther-specific gene with a male sterile Arabidopsis knockout phenotype is also in the Y-specific region, supporting a two-gene model for sex chromosome evolution.Several models have been proposed to explain the emergence of sex chromosomes. Here, through comparative genomics and mutant analysis, Harkess et al. show that linked but separate genes on the Y chromosome are responsible for sex determination in Asparagus, supporting a two-gene model for sex chromosome evolution.

Intra-varietal genetic diversity of the grapevine ( Vitis vinifera L.) cultivar ‘Nero d’Avola’ as revealed by microsatellite markers

The Sicilian grape cultivar ‘Nero d’Avola’ is among the oldest and most cultivated in the island, taking part in the production of several red wines exported worldwide, including DOC wines (Etna Rosso and Cerasuolo di Vittoria). Due to the ancient origin and repeated clonally propagation of the cultivar, phenotypic variability has been observed. Clone identification in this important cultivar has so far relied on phenotypic and chemical traits analyses, often affected by environmental conditions. Genetic markers, such as microsatellites, are particularly useful for cultivar identification, parentage testing, pedigree reconstruction and population structure studies. In the present paper, microsatellites were used to analyze the intra-varietal genetic diversity among 118 plants of ‘Nero d’Avola’, collected in 30 vineyards displaced in different areas of Sicily. Out of 22 microsatellites, 11 showed polymorphism among samples and 15 different phylogenetic groups were identified. Results show that ‘Nero d’Avola’ actually comprises different genetic profiles, although most of clones share a common origin.

Retrotransposon Proliferation Coincident with the Evolution of Dioecy in Asparagus

Current phylogenetic sampling reveals that dioecy and an XY sex chromosome pair evolved once, or possibly twice, in the genus Asparagus. Although there appear to be some lineage-specific polyploidization events, the base chromosome number of 2n = 2× = 20 is relatively conserved across the Asparagus genus. Regardless, dioecious species tend to have larger genomes than hermaphroditic species. Here, we test whether this genome size expansion in dioecious species is related to a polyploidization and subsequent chromosome fusion, or to retrotransposon proliferation in dioecious species. We first estimate genome sizes, or use published values, for four hermaphrodites and four dioecious species distributed across the phylogeny, and show that dioecious species typically have larger genomes than hermaphroditic species. Utilizing a phylogenomic approach, we find no evidence for ancient polyploidization contributing to increased genome sizes of sampled dioecious species. We do find support for an ancient whole genome duplication (WGD) event predating the diversification of the Asparagus genus. Repetitive DNA content of the four hermaphroditic and four dioecious species was characterized based on randomly sampled whole genome shotgun sequencing, and common elements were annotated. Across our broad phylogenetic sampling, Ty-1 Copia retroelements, in particular, have undergone a marked proliferation in dioecious species. In the absence of a detectable WGD event, retrotransposon proliferation is the most likely explanation for the precipitous increase in genome size in dioecious Asparagus species.

High-throughput 18K SNP array to assess genetic variability of the main grapevine cultivars from Sicily

The viticulture of Sicily, for its vocation, is one of the most important and ancient forms in Italy. Autochthonous grapevine cultivars, many of which known throughout the world, have always been cultivated in the island from many centuries. With the aim to preserve this large grapevine diversity, previous studies have already started to assess the genetic variability among the Sicilian cultivars by using morphological and microsatellite markers. In this study, simple sequence repeat (SSR) were utilized to verify the true-to-typeness of a large clone collection (101) belonging to 21 biotypes of the most 10 cultivated Sicilian cultivars. Afterwards, 42 Organization Internationale de la Vigne et du Vin (OIV) descriptors and a high-throughput single nucleotide polymorphism (SNP) genotyping array (Vitis18kSNP) were applied to assess genetic variability among cultivars and biotypes of the same cultivar. Ampelographic traits and high-throughput SNP genotyping platforms provided an accuracy estimation of genetic diversity in the Sicilian germplasm, showing the relationships among cultivars by cluster and multivariate analyses. The large SNP panel defined sub-clusters unable to discern among biotypes, previously classified by ampelographic analysis, belonging to each cultivar. These results suggested that a very large number of SNP did not cover the genome regions harboring few morphological traits. Genetic structure of the collection revealed a clear optimum number of groups for K = 3, clustering in the same group a significant portion of family-related genotypes. Parentage analysis highlighted significant relationships among Sicilian grape cultivars and Sangiovese, as already reported, but also the first evidences of the relationships between Nero d’Avola and both Inzolia and Catarratto. Finally, a small panel of highly informative markers (12 SNPs) allowed us to isolate a private profile for each Sicilian cultivar, providing a new tool for cultivar identification.

An integrated proteomic and metabolomic study to evaluate the effect of nucleus-cytoplasm interaction in a diploid citrus cybrid between sweet orange and lemon

Key messageOur results provide a comprehensive overview how the alloplasmic condition might lead to a significant improvement in citrus plant breeding, developing varieties more adaptable to a wide range of conditions.AbstractCitrus cybrids resulting from somatic hybridization hold great potential in plant improvement. They represent effective products resulting from the transfer of organelle-encoded traits into cultivated varieties. In these cases, the plant coordinated array of physiological, biochemical, and molecular functions remains the result of integration among different signals, which derive from the compartmentalized genomes of nucleus, plastids and mitochondria. To dissect the effects of genome rearrangement into cybrids, a multidisciplinary study was conducted on a diploid cybrid (C2N), resulting from a breeding program aimed to improve interesting agronomical traits for lemon, the parental cultivars ‘Valencia’ sweet orange (V) and ‘femminello’ lemon (F), and the corresponding somatic allotetraploid hybrid (V + F). In particular, a differential proteomic analysis, based on 2D-DIGE and MS procedures, was carried out on leaf proteomes of C2N, V, F and V + F, using the C2N proteome as pivotal condition. This investigation revealed differentially represented protein patterns that can be associated with genome rearrangement and cell compartment interplay. Interestingly, most of the up-regulated proteins in the cybrid are involved in crucial biological processes such as photosynthesis, energy production and stress tolerance response. The cybrid differential proteome pattern was concomitant with a general increase of leaf gas exchange and content of volatile organic compounds, highlighting a stimulation of specific pathways that can be related to observed plant performances. Our results contribute to a better understanding how the alloplasmic condition might lead to a substantial improvement in plant breeding, opening new opportunities to develop varieties more adaptable to a wide range of conditions.