Mario Augusto Pagnotta

Morphological and molecular characterization of Italian emmer wheat accessions

SummaryThe characterization of 39 Italian ecotypes and cultivars of Triticum turgidum L. spp. dicoccum Shrank ex Schübler (emmer wheat) was performed utilizing agro-morphological and molecular tools. Emmer wheat is a hulled species which grows wild in the Near East and is still cultivated in the Mediterranean Basin. Due to its characteristics, in Italy it is cultivated mainly in marginal lands of central and southern Italy, where local varieties, adapted to the natural environment where they originated, are used. Emmer wheat cultivation has been drastically reduced during the last century as a consequence of its low yield. Nevertheless, more recently, its agronomic and nutritive values, together with its use in health food products, made its cultivation economically viable in the marginal lands with a parallel increase of the cultivated area which is now more than 2000 ha.In the present paper the results of morphological evaluation, carried out in an experimental field in central Italy using a randomized block design with three replications, and molecular characterization are reported. The analysed material showed distinctive molecular traits and the existence of a huge amount of diversity not only between varieties, but also within them. When the accessions were clustered utilizing their genetic distance, the clusters were not always in agreement with the accessions origins. The obtained results gave information that can be useful for: (i) future registration of material, (ii) germplasm conservation and (iii) use of this valuable source of emmer germplasm for future breeding programmes.

Identification of SNP Mutations in DREB1, HKT1, and WRKY1 Genes Involved in Drought and Salt Stress Tolerance in Durum Wheat (Triticum turgidum L. var durum)

Tolerance mechanisms to salinity and drought stress are quite complex. Plants have developed a complex and elaborate signaling network that ensures their adaptation to this stress. For example, salinity tolerance is thought to be due to three main factors: Na(+) exclusion, tolerance to Na(+) in the tissues and osmotic tolerance. Recently, many transcription factors for tolerance to salt and drought stresses have been identified. In this study, multialignments of conserved domains in DREB1, WRKY1 transcription factors (TFs), and HKT-1 have been utilized to design specific primers in order to identify functional single nucleotide polymorphisms (SNPs). These primers have been used to probe on several genotypes of durum wheat that are differentially tolerant to salt and drought stress; they were grown in increasing concentrations of NaCl. The selected portions have been analyzed using high-resolution melting curve (HRM) technology that currently represents one of the most recent and powerful tools for detecting SNP and INDEL mutations. Analyzing the amplification profiles, observed in the resulting melting curves, samples corresponding to different treatment conditions were selected, sequenced, and aligned with the homolog sequences present in gene databases to identify and characterize potential SNP and INDEL mutations. The PCR amplicons, containing single and double SNPs, produced distinctive HRM profiles. By sequencing the polymerase chain reaction (PCR) products, several SNPs have been identified and validated. All the discovered mutations were able to generate changes in amino acid sequences of the corresponding proteins. Most of the identified SNPs were found in salt and drought tolerant durum wheat genotypes. These varieties are of great value for durum wheat breeding works.

Characterization of Italian spring globe artichoke germplasm: morphological and molecular profiles

Globe artichoke is native to the Mediterranean basin with Italy being the leading world producer. Italian autochthonous germplasm is at risk of genetic erosion due to the large cultivation of only a few varietal types, such as the micropropagated clone C3, and to the introduction of new seed-propagated genetic materials. The aim of our study is to assess the genetic variability existing in nine Italian globe artichoke landraces of spring type, using morphological descriptors and DNA markers (Amplified Fragment Lenght Polymorphism and Inter-Simple Sequence Repeats). A significant genetic differentiation among genotypes was found for many traits. Although some genetic variability found within each genotype was probably due to their multiclonal origin, most of them were genetically well-defined. Morphological and molecular profiles give important information to complete the genotype characterization and proceed with the selection of clones to be propagated and to develop lines and/or varieties. The present paper underlines the differences existing in the analyzed germplasm both within and among landraces and the genetic relations among different material, which is an important starting point for future plant breeding programs.

Genetic variation of the durum wheat landrace Haurani from different agro-ecological regions

The durum wheat landrace Haurani (Triticum durum Desf.) is grown under contrasting climatic regions of Syria from Deir Ezzor, in the North-East (230 m altitude, 150 mm mean annual rainfall), to Qunaytra, in the South-West (1060 m altitude, 825 mm mean annual rainfall). In order to assess the genetic variation between and within Haurani populations, samples from eight provinces of Syria (Dara’a, Damascus, Qunaytra, Deir Ezzor, Hassakeh, Aleppo, Homs and Hama) were analysed by RFLPs and seed storage proteins of glutenin subunits as markers. The analyses showed the presence of genetic polymorphisms in all populations with the highest values in those from Homs and Hassakeh. Moreover, the results point out differences in genetic distances between populations; some populations were further apart, such as Damascus and Aleppo, whereas others were closer to each other, for instance Homs and Hama. Cluster analysis identified two distinct groups of populations, characterized by geographical proximity, with similar rainfall and altitude. It is suggested that the similarity of landraces at locations close to each other might be the result of more frequent seed exchanges between farmers or of gene flow due to 5% estimated outcross rate of Haurani.

Characterizing the molecular and morphophysiological diversity of Italian red clover

Red clover (Trifolium pratense L.) is the third major forage species in Europe, but there is limited information on the biodiversity and the genetic structure of landraces and natural populations which evolved in this region. The objective of this study was producing such information for Italian germplasm on the ground of molecular and morphophysiological diversity. The study included 16 Italian natural populations from a wide range of environments, four landraces representing the four traditional commercial ecotypes, and two varieties. Eight morphophysiological traits were assessed in a replicated trial under field conditions, whereas two AFLPs primer combinations with 140 polymorphic markers were recorded on a random sample of 13 genotypes per population. Ordination and classification results based on morphophysiological data clearly kept track of the type of germplasm (i.e. landrace or natural population) and its geographic origin, unlike results based on molecular markers. Euclidean distances among populations based on morphophysiological traits were not correlated with Nei’s genetic distances based on molecular markers according to Mantel’s test. Geographical distances among landrace or natural population material was correlated with distances based on morphophysiological traits but not with those based on molecular markers. The average within-population variation estimated via molecular markers was about 2.6-fold higher than that among populations, preventing an acceptable discrimination among most populations. On average, natural populations tended to have within-population variation similar to varieties and somewhat lower than landraces. Our results have implications for collection, conservation, exploitation and registration in a sui generis system of red clover genetic resources.

HRM technology for the identification and characterization of INDEL and SNPs mutations in genes involved in drought and salt tolerance of durum wheat

WRKY transcription factors are one of the largest families of transcriptional regulators and form an integral part of signalling webs which modulate many plant processes, such as abiotic stress tolerance. In the present paper, an innovative method has been applied to identify novel WRKY-1 alleles involved in the responses to salt and drought stresses in Triticum durum . This technique involves scanning for sequencing variations in cDNA-derived PCR amplicons, using high-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM represents a novel advance in detection of single-nucleotide polymorphisms (SNPs) by measuring temperature-induced strand separation of short PCR amplicons. The use of this approach is still limited in the field of plant biology. Here, HRM analysis has been applied to the discovery and genotyping of durum wheat SNPs. Specific primers have been designed, starting at multi-alignment of WRKY-1-conserved portions. The PCR amplicons, containing single SNPs, produce distinctive HRM profiles, and by sequencing the PCR products identified, SNPs have been characterized and validated. The results showed that all the revealed SNPs are located on salt-tolerant varieties, confirming their value in breeding activities.

Genetic diversity of Syrian pistachio (Pistacia vera L.) varieties evaluated by AFLP markers

Pistachio (Pistacia vera L.) is a strategic nut tree species in the Middle East which holds comparative advantage over other fruit trees in view of its hardiness, income generation opportunities and benefits for the ecosystem. Yet pistachio cultivation depends on a very narrow genetic base, in spite of the existence of many varieties still marginally exploited. Syria is an important center of diversity for pistachio. A country wide ecogeographic survey in this country was carried out to determine the extent of pistachio genetic diversity and its use. As a whole, 114 accessions were collected from 37 farms to assess diversity at morphological and molecular level. Molecular evaluation was carried out using Amplified Fragment Length Polymorphism (AFLP) technique and performed using seven primer pair combinations. Results from the studies allowed the identification of 25 pistachio female varieties in Syria, some of which unique and described for the first time. Three groups of pistachio diversity were identified by cluster analysis which provides useful information about the distribution of genetic diversity in Syria for enhanced use and sustainable conservation.

Quantification and organization of WIS2-1A and BARE-1 retrotransposons in different genomes of Triticum and Aegilops species

A real-time PCR approach was adopted and optimized to estimate and compare, through a relative quantification, the copy number of WIS2-1A and BARE-1 retrotransposons. The aim of this approach was to identify and quantify the presence of these retrotransposons in Triticum and Aegilops species, and to understand better the genome organization of these retroelements. The species were selected to assess and compare the evolution of the different types of genomes between the more recent species such as the diploid Triticum monococcum, tetraploid T. dicoccon and hexaploid T. spelta, and the corresponding genome donors of the ancient diploids Aegilops (Ae. speltoides, Ae. tauschii, Ae. sharonensis and Ae. bicornis) and T. urartu. The results of this study indicated the presence of great variation in copy number both within and among species, and the existence of a non-linear relationship between retrotransposon copy number and ploidy level. For WIS2-1A, as expected, T. monococcum showed the lowest copy number which instead was similar in T. dicoccon and T. spelta; also T. urartu (AA), Ae. speltoides (BB) and Ae. tauschii (DD) showed a higher WIS2-1A copy number. Similar results were observed for BARE-1 retroelements except for Ae. tauschii which as in T. monococcum showed lower retroelements content; a similar content for T. dicoccon and T. urartu, whereas a higher number was found in T. spelta and Ae. speltoides. The results presented here are in accord with previous studies and contribute to unravelling the structure and evolution of polyploidy and repetitive genomes.

Genetic diversity, population structure and phylogenetic inference among Italian Orchids of the Serapias genus assessed by AFLP molecular markers

The means by which new species arise remain a fundamental question in the Orchid family evolution. Even if the Orchid-pollinator interactions are highly specific, events of cross-hybridization in Serapias are possible and represent a central issue of the plants evolutionary biology. Italian Orchids have a wide variety of pollination systems and highly diverse floral traits, considered as some of the main tools for activating the speciation processes. The assess of diversity in Serapias taxa and eventual phylogenetic relationships among Serapias lingua, S. parviflora, S. vomeracea, S. apulica, S. nurrica, S. neglecta, S. cordigera, S. politisii, and some putative inter-specific hybrids were investigated at the DNA level by AFLP markers. The present molecular study on Serapias populations (1) confirms some previous finding, (2) opens new hypotheses on the possible evolutionary and taxonomic relationships existing among the Serapias species, and (3) gives indications about the real consistence of hybrid individuals in a genus characterized by a relevant gene flow.

Genetic diversity and accession structure in European Cynara cardunculus collections

Understanding the distribution of genetic variations and accession structures is an important factor for managing genetic resources, but also for using proper germplasm in association map analyses and breeding programs. The globe artichoke is the fourth most important horticultural crop in Europe. Here, we report the results of a molecular analysis of a collection including globe artichoke and leafy cardoon germplasm present in the Italian, French and Spanish gene banks. The aims of this study were to: (i) assess the diversity present in European collections, (ii) determine the population structure, (iii) measure the genetic distance between accessions; (iv) cluster the accessions; (v) properly distinguish accessions present in the different national collections carrying the same name; and (vi) understand the diversity distribution in relation to the gene bank and the geographic origin of the germplasm. A total of 556 individuals grouped into 174 accessions of distinct typologies were analyzed by different types of molecular markers, i.e. dominant (ISSR and AFLP) and co-dominant (SSR). The data of the two crops (globe artichoke and leafy cardoon) were analyzed jointly and separately to compute, among other aims, the gene diversity, heterozygosity (He, Ho), fixation indexes, AMOVA, genetic distance and structure. The findings underline the huge diversity present in the analyzed material, and the existence of alleles that are able to discriminate among accessions. The accessions were clustered not only on the basis of their typology, but also on the basis of the gene bank they come from. Probably, the environmental conditions of the different field gene banks affected germplasm conservation. These outcomes will be useful in plant breeding to select accessions and to fingerprint varieties. Moreover, the results highlight the particular attention that should be paid to the method used to conserve the Cynara cardunculus germplasm and suggest to the preference of using accessions from different gene banks to run an association map.