Simona Urso

Improvement of marker-based predictability of Apparent Amylose Content in japonica rice through GBSSI allele mining

BackgroundApparent Amylose Content (AAC), regulated by the Waxy gene, represents the key determinant of rice cooking properties. In occidental countries high AAC rice represents the most requested market class but the availability of molecular markers allowing specific selection of high AAC varieties is limited.ResultsIn this study, the effectiveness of available molecular markers in predicting AAC was evaluated in a collection of 127 rice accessions (125 japonica ssp. and 2 indica ssp.) characterized by AAC values from glutinous to 26%. The analyses highlighted the presence of several different allelic patterns identifiable by a few molecular markers, and two of them, i.e., the SNPs at intron1 and exon 6, were able to explain a maximum of 79.5% of AAC variation. However, the available molecular markers haplotypes did not provide tools for predicting accessions with AAC higher than 24.5%. To identify additional polymorphisms, the re-sequencing of the Waxy gene and 1kbp of the putative upstream regulatory region was performed in 21 genotypes representing all the AAC classes identified. Several previously un-characterized SNPs were identified and four of them were used to develop dCAPS markers.ConclusionsThe addition of the SNPs newly identified slightly increased the AAC explained variation and allowed the identification of a haplotype almost unequivocally associated to AAC higher than 24.5%. Haplotypes at the waxy locus were also associated to grain length and length/width (L/W) ratio. In particular, the SNP at the first intron, which identifies the Wxa and Wxb alleles, was associated with differences in the width of the grain, the L/W ratio and the length of the kernel, most likely as a result of human selection.

Comparative transcriptome profiling of the early response to Magnaporthe oryzae in durable resistant vs susceptible rice (Oryza sativa L.) genotypes.

Durable resistance to blast, the most significant fungal disease of rice, represents an agronomically relevant character. Gigante Vercelli (GV) and Vialone Nano (VN) are two old temperate japonica Italian rice cultivars with contrasting response to blast infection: GV displays durable and broad resistance while VN is highly susceptible. RNA-seq was used to dissect the early molecular processes deployed during the resistance response of GV at 24 h after blast inoculation. Differential gene expression analysis identified 1,070 and 1,484 modulated genes, of which 726 and 699 were up regulated in response to infection in GV and VN, respectively. Gene ontology (GO) enrichment analyses revealed a set of GO terms enriched in both varieties but, despite this commonality, the gene sets contributing to common GO enriched terms were dissimilar. The expression patterns of genes grouped in GV-specific enriched GO terms were examined in detail including at the transcript isoform level. GV exhibited a dramatic up-regulation of genes encoding diterpene phytoalexin biosynthetic enzymes, flavin-containing monooxygenase, class I chitinase and glycosyl hydrolase 17. The sensitivity and high dynamic range of RNA-seq allowed the identification of genes critically involved in conferring GV resistance during the early steps of defence perception-signalling. These included chitin oligosaccharides sensing factors, wall associated kinases, MAPK cascades and WRKY transcription factors. Candidate genes with expression patterns consistent with a potential role as GV-specific functional resistance (R) gene(s) were also identified. This first application of RNA-seq to dissect durable blast resistance supports a crucial role of the prompt induction of a battery of responses including defence-related genes as well as members of gene families involved in signalling and pathogen-related gene expression regulation.

Emerging Knowledge from Genome Sequencing of Crop Species

Extensive insights into the genome composition, organization, and evolution have been gained from the plant genome sequencing and annotation ongoing projects. The analysis of crop genomes provided surprising evidences with important implications in plant origin and evolution: genome duplication, ancestral re-arrangements and unexpected polyploidization events opened new doors to address fundamental questions related to species proliferation, adaptation, and functional modulations. Detailed paleogenomic analysis led to many speculation on how chromosomes have been shaped over time in terms of gene content and order. The completion of the genome sequences of several major crops, prompted to a detailed identification and annotation of transposable elements: new hypothesis related to their composition, chromosomal distribution, insertion models, amplification rate, and evolution patterns are coming up. Availability of full genome sequence of several crop species as well as from many accessions within species is providing new keys for biodiversity exploitation and interpretation. Re-sequencing is enabling high-throughput genotyping to identify a wealth of SNP and afterward to produce haplotype maps necessary to accurately associate molecular variation to phenotype. Conservation genomics is emerging as a powerful tool to explain adaptation, genetic drift, natural selection, hybridization and to estimate genetic variation, fitness and population’s viability.

Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine

Erysiphe necator is the causal agent of powdery mildew (PM), one of the most destructive diseases of grapevine. PM is controlled by sulfur-based and synthetic fungicides, which every year are dispersed into the environment. This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production. PM resistance can be achieved in other crops by knocking out susceptibility S-genes, such as those residing at genetic loci known as MLO (Mildew Locus O). All MLO S-genes of dicots belong to the phylogenetic clade V, including grapevine genes VvMLO7, 11 and 13, which are upregulated during PM infection, and VvMLO6, which is not upregulated. Before adopting a gene-editing approach to knockout candidate S-genes, the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary. This paper reports the knockdown through RNA interference of VvMLO6, 7, 11 and 13. The knockdown of VvMLO6, 11 and 13 did not decrease PM severity, whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%. The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance, whereas a role for VvMLO11 does not seem likely. Cell wall appositions (papillae) were present in both resistant and susceptible lines in response to PM attack. Thirteen genes involved in defense were less upregulated in infected mlo plants, highlighting the early mlo-dependent disruption of PM invasion.

An Agrobacterium tumefaciens-mediated gene silencing system for functional analysis in grapevine

An efficient agroinfiltration-based gene silencing assay was established to evaluate candidate genes likely to be involved in resistance to powdery mildew (Uncinula necator) in grapevine (Vitis vinifera L.). Functional assays were performed using two grapevine genotypes, cv. Superior, mildew-susceptible, and cv. Run1 Mtp3294, mildew-resistant. Constructs encoding a self-complementary “hairpin” RNA for phytoene desaturase (PDS) along with the green fluorescent protein gene were introduced into Agrobacterium tumefaciens. These constructs were used for agroinfiltration of leaf tissues, yielding both efficient transformation and gene silencing events. The agroinfiltration procedure did not interfere with the powdery mildew infection steps. Confocal laser-scanning microscopy analysis revealed co-localization of the GFP signal with cells lacking chlorophyll autofluorescence, further supporting PDS gene silencing. The extent of PDS mRNA degradation was evaluated by qRT-PCR in the agroinfiltrated areas and results supported a significant reduction of the gene transcripts in the silenced regions. Findings of the present work suggest that the developed silencing procedures represent a useful tool for functional characterization of grapevine genes involved in powdery mildew resistance.

Genome-Wide Analysis of japonica Rice Performance under Limited Water and Permanent Flooding Conditions

A rice GWAS panel of 281 accessions of japonica rice was phenotypically characterized for 26 traits related to phenology, plant and seed morphology, physiology and yield for 2 years in field conditions under permanent flooding (PF) and limited water (LW). A genome-wide analysis uncovered a total of 160 significant marker-trait associations (MTAs), of which 32 were LW-specific, 59 were PF-specific, and 69 were in common between the two water management systems. LW-specific associations were identified for several agronomic traits including days to maturation, days from flowering to maturation, leaf traits, plant height, panicle and seed traits, hundred grain weight, yield and tillering. Significant MTAs were detected across all the 12 rice chromosomes, while clusters of effects influencing different traits under LW or in both watering conditions were, respectively, observed on chromosomes 4, 8, and 12 and on chromosomes 1, 3, 4, 5, and 8. The analysis of genes annotated in the Nipponbare reference sequence and included in the regions associated to traits related to plant morphology, grain yield, and physiological parameters allowed the identification of genes that were demonstrated to affect the respective traits. Among these, three (OsOFP2, Dlf1, OsMADS56) and seven (SUI1, Sd1, OsCOL4, Nal1, OsphyB, GW5, Ehd1) candidate genes were, respectively, identified to co-localize with LW-specific associations and associations in common between the two water treatments. For several LW-specific MTAs, or in common among the two treatments, positional co-localizations with previously identified QTLs for rice adaptation to water shortages were observed, a result that further supports the role of the loci identified in this work in conferring adaptation to LW. The most robust associations identified here could represent suitable targets for genomic selection approaches to improve yield-related traits under LW.