Morphological and Genetic Characterization of Maize Landraces Adapted to Marginal Hills in North-West Italy.

The growing interest in maize landraces over the past two decades has led to the need to characterize the Italian maize germplasm. In Italy, hundreds of maize landraces have been developed, but only a few of them have been genetically characterized, and even fewer are currently employed in agriculture or for breeding purposes. In the present study, 13 maize landraces of the west Emilia-Romagna region were morphologically and genetically characterized. These accessions were sampled in 1954 from three provinces, Modena, Parma, and Piacenza, during the characterization project of Italian maize landraces. The morphological characterization of these 13 accessions was performed according to the UPOV protocol CPVO/TP2/3, examining 34 phenotypic traits. A total of 820 individuals were genotyped with 10 SSR markers. The genetic characterization revealed 74 different alleles, a FST mean value of 0.13, and a Nm mean of 1.73 over all loci. Moreover, AMOVA analysis disclosed a low degree of differentiation among accessions, with only 13% of genetic variability found between populations, supporting PCoA analysis results, where the first two coordinates explained only 16% of variability. Structure analysis, supported by PCoA, showed that only four accessions were clearly distinguished for both K = 4 and 6. Italian landraces can be useful resources to be employed in maize breeding programs for the development of new varieties, adapted to different environmental conditions, in order to increase crop resilience and expand the maize cultivation area.

Characterization and Valorization of Maize Landraces from Aosta Valley.

While there is a rich collection of maize germplasm from Italy, it lacks genetic resources from the Aosta Valley, an isolated mountain region where landraces have been preserved in the absence of modern germplasm introductions. These local materials, which are still cultivated mainly at household level, can have high importance from a genetic and historical point of view. In the present study, five landraces named, after the collecting sites, Arnad, Arnad-Crest, Châtillon, Entrebin and Perloz, were sampled in Aosta Valley and subjected to historic, morphologic and genetic characterization. This study provided evidence for the landraces' long presence in Aosta Valley, a significant genetic variability and differentiation among the investigated landraces. Globally, 67 different alleles were detected ranging from 4 for markers phi127 and p-bnlg176 to 10 for phi031, with a mean of 6.7 alleles per locus. Observed heterozygosity levels were comprised from 0.16 to 0.51 and are generalkly lower than expected heterozigosity supporting fixation at some loci. STRUCTURE analysis revealed clear separation between accessions revealing the presence of four ancestral populations. This may be explained by the long reproductive isolation experienced by these materials. Finally, morphological observations confirm the high diversity between landraces revealing that they generally have flint kernels, variable color from yellow to dark red (Châtillon) while Perloz showed kernels with an apical beak. The present work confirms the importance of mountain areas in conserving biodiversity and increases the rich Italian maize germplasm with materials well adapted to marginal areas. Such new genetic variability may be used to breed new materials for more resilient agriculture.

Loss of ZmLIPOXYGENASE4 Decreases Fusarium verticillioides Resistance in Maize Seedlings.

Fusarium verticillioides is one of the most relevant fungal species in maize responsible for ear, stalk and seedling rot, as well as the fumonisin contamination of kernels. Plant lipoxygenases (LOX) synthesize oxylipins that play a crucial role in the regulation of defense mechanisms against pathogens and influence the outcome of pathogenesis. To better uncover the role of these signaling molecules in maize resistance against F. verticillioides, the functional characterization of the 9-LOX gene, ZmLOX4, was carried out in this study by employing mutants carrying Mu insertions in this gene (named as UFMulox4). In this regard, the genotyping of five UFMulox4 identified the mutant UFMu10924 as the only one having an insertion in the coding region of the gene. The impact of ZmLOX4 mutagenesis on kernel defense against F. verticillioides and fumonisin accumulation were investigated, resulting in an increased fungal susceptibility compared to the inbred lines W22 and Tzi18. Moreover, the expression of most of the genes involved in the LOX, jasmonic acid (JA) and green leaf volatiles (GLV) pathways, as well as LOX enzymatic activity, decreased or were unaffected by fungal inoculation in the mutant UFMu10924. These results confirm the strategic role of ZmLOX4 in controlling defense against F. verticillioides and its influence on the expression of several LOX, JA and GLV genes.

Epigenetic Variability Among Saffron Crocus (Crocus sativus L.) Accessions Characterized by Different Phenotypes.

This work represents the first epigenomic study carried out on saffron crocus. Five accessions of saffron, showing differences in tepal pigmentation, yield of saffron and flowering time, were analyzed at the epigenetic level by applying a methylation-sensitive restriction enzyme-sequencing (MRE-seq) approach. Five accession-specific hypomethylomes plus a reference hypomethylome, generated by combining the sequence data from the single accessions, were obtained. Assembled sequences were annotated against existing online databases. In the absence of the Crocus genome, the rice genome was mainly used as the reference as it is the best annotated genome among monocot plants. Comparison of the hypomethylomes revealed many differentially methylated regions, confirming the high epigenetic variability present among saffron accessions, including sequences encoding for proteins that could be good candidates to explain the accessions' alternative phenotypes. In particular, transcription factors involved in flowering process (MADS-box and TFL) and for the production of pigments (MYB) were detected. Finally, by comparing the generated sequences of the different accessions, a high number of SNPs, likely having arisen as a consequence of the prolonged vegetative propagation, were detected, demonstrating surprisingly high genetic variability. Gene ontology (GO) was performed to map and visualize sequence polymorphisms located within the GOs and to compare their distributions among different accessions. As well as suggesting the possible existence of alternative phenotypes with a genetic basis, a clear difference in polymorphic GO is present among accessions based on their geographic origin, supporting a possible signature of selection in the Indian accession with respect to the Spanish ones.

A Genome-Wide Association Study To Understand the Effect of Fusarium verticillioides Infection on Seedlings of a Maize Diversity Panel.

Fusarium verticillioides, which causes ear, kernel and stem rots, has been reported as the most prevalent species on maize worldwide. Kernel infection by F. verticillioides results in reduced seed yield and quality as well as fumonisin contamination, and may affect seedling traits like germination rate, entire plant seedling length and weight. Maize resistance to Fusarium is a quantitative and complex trait controlled by numerous genes with small effects. In the present work, a Genome Wide Association Study (GWAS) of traits related to Fusarium seedling rot was carried out in 230 lines of a maize association population using 226,446 SNP markers. Phenotypes were scored on artificially infected kernels applying the rolled towel assay screening method and three traits related to disease response were measured in inoculated and not-inoculated seedlings: plant seedling length (PL), plant seedling weight (PW) and germination rate (GERM). Overall, GWAS resulted in 42 SNPs significantly associated with the examined traits. Two and eleven SNPs were associated with PL in inoculated and not-inoculated samples, respectively. Additionally, six and one SNPs were associated with PW and GERM traits in not-inoculated kernels, and further nine and thirteen SNPs were associated to the same traits in inoculated kernels. Five genes containing the significant SNPs or physically closed to them were proposed for Fusarium resistance, and 18 out of 25 genes containing or adjacent to significant SNPs identified by GWAS in the current research co-localized within QTL regions previously reported for resistance to Fusarium seed rot, Fusarium ear rot and fumonisin accumulation. Furthermore, linkage disequilibrium analysis revealed an additional gene not directly observed by GWAS analysis. These findings could aid to better understand the complex interaction between maize and F. verticillioides.

Unravelling the genetic basis of Fusarium seedling rot resistance in the MAGIC maize population: novel targets for breeding.

Fungal infection by Fusarium verticillioides is cause of prevalent maize disease leading to substantial reductions in yield and grain quality worldwide. Maize resistance to the fungus may occur at different developmental stages, from seedling to maturity. The breeding of resistant maize genotypes may take advantage of the identification of quantitative trait loci (QTL) responsible for disease resistance already commenced at seedling level. The Multi-parent Advance Generation Intercross (MAGIC) population was used to conduct high-definition QTL mapping for Fusarium seedling rot (FSR) resistance using rolled towel assay. Infection severity level, seedling weight and length were measured on 401 MAGIC maize recombinant inbred lines (RILs). QTL mapping was performed on reconstructed RIL haplotypes. One-fifth of the MAGIC RILs were resistant to FSR and 10 QTL were identified. For FSR, two QTL were detected at 2.8 Mb and 241.8 Mb on chromosome 4, and one QTL at 169.6 Mb on chromosome 5. Transcriptomic and sequencing information generated on the MAGIC founder lines was used to guide the identification of eight candidate genes within the identified FSR QTL. We conclude that the rolled towel assay applied to the MAGIC maize population provides a fast and cost-effective method to identify QTL and candidate genes for early resistance to F. verticillioides in maize.

A Genome Wide Association Study Reveals Markers and Genes Associated with Resistance to Fusarium verticillioides Infection of Seedlings in a Maize Diversity Panel.

Fusarium verticillioides infects maize, causing ear rot, yield loss and contamination by fumonisin mycotoxins. The fungus can be transmitted via kernels and cause systemic infection in maize. Maize resistance to the fungus may occur at different developmental stages, from seedling to maturity. Resistance during kernel germination is part of the plant-pathogen interaction and so far this aspect has not been investigated. In the present study, a genome wide association study (GWAS) of resistance to Fusarium during the seedling developmental stage was conducted in a maize diversity panel using 226,446 SNP markers. Seedling germination and disease phenotypes were scored on artificially inoculated kernels using the rolled towel assay. GWAS identified 164 SNPs significantly associated with the traits examined. Four SNPs were associated with disease severity score after inoculation, 153 were associated with severity in asymptomatic kernels and 7 with the difference between the severity ratings in inoculated and non-inoculated seeds. A set of genes containing or physically near the significant SNPs were identified as candidates for Fusarium resistance at the seedling stage. Functional analysis revealed that many of these genes are directly involved in plant defense against pathogens and stress responses, including transcription factors, chitinase, cytochrome P450, and ubiquitination proteins. In addition, 25 genes were found in high linkage disequilibrium with the associated SNPs identified by GWAS and four of them directly involved in disease resistance. These findings contribute to understanding the complex system of maize-F. verticillioides and may improve genomic selection for Fusarium resistance at the seedling stage.

Epigenetic stability in Saffron (Crocus sativus L.) accessions during four consecutive years of cultivation and vegetative propagation under open field conditions.

Saffron (Crocus sativus L.) is a sterile species that is vegetatively propagated in the field, year by year, via the production of new corms. While Saffron's genetic variability is extremely low, phenotypic variation is frequently observed in the field and epigenetics could be a possible origin of these alternative phenotypes. Present day knowledge on Saffron epigenetics is very low or absent. In the present paper, to deepen existing knowledge, we focused on the epigenetic differences and stability among 17 Saffron accessions, of different geographic origin, during four consecutive years of vegetative propagation under open field conditions. Before the analysis, the selected accessions have been cultivated in the same field for at least three consecutive years. Despite the low genetic variability and the prolonged co-cultivation in the same environment, Methylation-Sensitive Amplified Fragment Length Polymorphism (MS-AFLP) analysis revealed a very high epigenetic difference among accessions, making it possible to discriminate them based on the epigenetic profiles. During the four years of the study, a little variation has been observed within accessions following different patterns, slightly modifying the accession epigenotypes but not enough to even them to a more uniform profile. These results confirm that, under natural conditions, Saffron epigenotypes are highly stable, supporting a role for epigenetics in phenotypic variability.

Phenolic Profile and Susceptibility to Fusarium Infection of Pigmented Maize Cultivars.

Maize is a staple food source in the world, whose ancient varieties or landraces are receiving a growing attention. In this work, two Italian maize cultivars with pigmented kernels and one inbred line were investigated for untargeted phenolic profile, in vitro antioxidant capacity and resistance to Fusariumverticillioides infection. "Rostrato Rosso" was the richest in anthocyanins whilst phenolic acids were the second class in abundance, with comparable values detected between cultivars. Tyrosol equivalents were also the highest in "Rostrato Rosso" (822.4 mg kg-1). Coherently, "Rostrato Rosso" was highly resistant to fungal penetration and diffusion. These preliminary findings might help in breeding programs, aiming to develop maize lines more resistant to infections and with improved nutraceutical value.

Molecular Basis of Resistance to Fusarium Ear Rot in Maize.

The impact of climate change has been identified as an emerging issue for food security and safety, and the increased incidence of mycotoxin contamination in maize over the last two decades is considered a potential emerging hazard. Disease control by chemical and agronomic approaches is often ineffective and increases the cost of production; for this reason the exploitation of genetic resistance is the most sustainable method for reducing contamination. The review focuses on the significant advances that have been made in the development of transcriptomic, genetic and genomic information for maize, Fusarium verticillioides molds, and their interactions, over recent years. Findings from transcriptomic studies have been used to outline a specific model for the intracellular signaling cascade occurring in maize cells against F. verticillioides infection. Several recognition receptors, such as receptor-like kinases and R genes, are involved in pathogen perception, and trigger down-stream signaling networks mediated by mitogen-associated protein kinases. These signals could be orchestrated primarily by hormones, including salicylic acid, auxin, abscisic acid, ethylene, and jasmonic acid, in association with calcium signaling, targeting multiple transcription factors that in turn promote the down-stream activation of defensive response genes, such as those related to detoxification processes, phenylpropanoid, and oxylipin metabolic pathways. At the genetic and genomic levels, several quantitative trait loci (QTL) and single-nucleotide polymorphism markers for resistance to Fusarium ear rot deriving from QTL mapping and genome-wide association studies are described, indicating the complexity of this polygenic trait. All these findings will contribute to identifying candidate genes for resistance and to applying genomic technologies for selecting resistant maize genotypes and speeding up a strategy of breeding to contrast disease, through plants resistant to mycotoxin-producing pathogens.

Presence and impact of allelic variations of two alternative s-kdr mutations, M918T and M918L, in the voltage-gated sodium channel of the green peach aphid Myzus persicae.

BACKGROUND: Pyrethroids have been widely employed in order to control several agricultural pests, including Myzus persicae. Target-site resistance is the main mechanism that confers insensitivity to this class of compounds, and the most common amino acid substitutions are kdr (L1014F) and s-kdr (M918T), but recently another mutation in the s-kdr locus (M918L) has been described in French and Korean populations of M. persicae. RESULTS: Molecular analysis of several Italian populations of M. persicae by pyrosequencing revealed the presence of the new s-kdr mutation (M918L) in different forms. It was found in two different nucleotide polymorphisms (a/t or a/c substitution), in heterozygous or homozygous status, and also in combination with the classic kdr and s-kdr. Bioassays on populations carrying the M918L mutation show that it strongly affects pyrethroid efficacy, particularly of type II pyrethroids such as lambda-cyhalothrin, while it has no effect against DDT. CONCLUSION: This work provides more information about the new s-kdr M918L mutation in M. persicae, describing a more complicated situation arising from the possible combination with the classic L1014F and M918T. Our data open new questions concerning the origin of these new genotypes with different combinations of target-site mutations, and also their possible influence on control strategies.