Linda Mondini

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.

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.

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.

Drought and Salt Stress in Cereals

Abiotic stresses, such as salinity, drought, extreme temperatures, chemical toxicity and oxidative stress represent a grave threat to agriculture dramatically affecting the crop production around the world. Climate changes are projected to have a significant impact on temperature and precipitation profiles increasing the incidence and severity of climate changes-related stresses and reducing in particular the productivity of rain-fed crops. In fact, drought and salinity stresses determine the primary cause of worldwide crop loss. Plant adaptation to environmental stresses is based on the activation of molecular networks involved in stress perception, signal transduction, and expression of specific stress-related genes and metabolites. Plants respond to the stresses in part by modulating gene expression in order to restore cellular homeostasis, detoxifying the toxins present into the cells and through the recovery of growth.

Using Molecular Techniques to Dissect Plant Genetic Diversity

During the past few decades, the employment of molecular markers to discover polymorphisms in DNA has been playing an increasing role in conservation strategies and use of plant genetic resources (PGR). Molecular markers are indispensable tools for determining the genetic variation and biodiversity with high levels of accuracy and reproducibility in short times. Different typologies of molecular markers exist, specific for the different applications in molecular genetic methods. Molecular tools have been successfully applied in the analysis of specific genes and gene pathways, as well as to increase understanding of gene action, to generate genetic maps and assist in the development of gene transfer technologies. Molecular markers have also had a critical role in studies of phylogeny and species evolution, and have been applied to increase our understanding of the distribution and extent of genetic variation within and between species. The main two groups of molecular markers can be classified on the basis of the analysis method used: polymerase chain reaction (PCR) and non-PCR-based. Recently, a new class of advanced techniques has emerged, primarily derived from a combination of earlier, more basic techniques. Advanced marker techniques tend to amalgamate advantageous features of several basic techniques, in order to increase the sensitivity and resolution to detect genetic discontinuity and distinctiveness. The past several years have seen revolutionary advances in DNA sequencing technologies with the advent of next-generation sequencing (NGS) techniques. NGS methods now allow millions of bases to be sequenced in one round, at moderate prices and in very short times. This paper is an overview of the diverse, predominantly molecular techniques, used in assessing plant genetic diversity, discussing about the most important and recent advances made in molecular marker techniques, their applications, advantages, and limitations applied to plant sciences in order to provide base platform information to the researchers working in the area.