Assessment of molecular sequences and enzyme activity of amylase trypsin inhibitors (ATIs) from a selection of ancient and modern grains

Wheat amylase-trypsin inhibitors (ATIs) are a family of wheat proteins, which play an important role in plant defence against pest attacks. ATIs are also of great interest for their impact on human health and recently ATIs have been identified as major stimulators of innate immune cells. In this study, ten selected wheat samples with different ploidy level and year of release were used for the agronomic trial, for in vitro enzymatic assays and for ATIs gene sequencing. Wheat samples were grown under organic farming management during three consecutive cropping years at two growing areas (Italy and USA). The PCA analysis performed on the deduced amino acid sequences of four representative ATIs genes (WMAI, WDAI, WTAI-CM3, CMx) evidenced that the ten wheat varieties can be differentiated on the basis of their ploidy level, but not with respect to ancient or recently developed wheat genotypes. The results from in vitro alpha-amylase and trypsin inhibitory activities showed high variability among the ten wheat genotypes and the contribution of the genotype and the cropping year was significant for both inhibitory activities. The hexaploid wheat genotypes showed the highest inhibitory activities. Einkorn showed a very low or even absent alpha-amylase inhibitory activity and the highest trypsin inhibitory activity. It was not possible to differentiate ancient and recently developed wheat genotypes on the basis of their ATIs activity. The weather conditions differently affected the two inhibitory activities. In both cultivation areas, higher precipitation and lower high mean temperatures correlated with lower alpha-amylase inhibitory activities, while there were different correlations considering trypsin inhibitory activity for the two growing areas. The protein content negatively correlated with both inhibitory activities in USA and Italy. This information can be important in the understanding of plant defence mechanisms in relation to the effect of both genotype and abiotic and biotic stress.

Wheat and low-input agriculture: agronomic, nutritional and nutraceutical implications

Recently, the increasing interest in organic food products and environmental friendly practices has emphasized the importance of selecting crop varieties suitable for the low-input systems. Additionally, in recent years the relationship between diet and human health has gained much attention among consumers, favoring the investigations on food nutraceutical properties. Among cereals, wheat plays an important role in human nutrition around the world and contributes to the daily intake of essential nutrients such as starch and protein. Moreover, whole grain contains several bioactive compounds that confer to wheat-derived products unique nutraceutical properties (dietary fibre, antioxidants). The present research provided interesting insights for the selection of wheat genotypes suitable for low-input systems and the development of specific breeding programs dedicated to organic farming. The investigation involved 5 old not dwarf genotypes (Andriolo, Frassineto, Gentil rosso, Inallettabile, Verna) and 1 modern dwarf variety (Palesio), grown under biodynamic management, over two consecutive growing seasons (2009/2010, 2010/2011). Results evidenced that under low-input farming some investigated old wheat genotypes (Frassineto, Inallettabile) were comparable to the modern cultivar in terms of whole agronomic performance. As regards the nutritional and nutraceutical properties, some old genotypes (Andriolo, Gentil rosso, Verna) emerged for their relevant content of several investigated phytochemicals (such as insoluble dietary fibre, polyphenols, flavonoids, in vitro antioxidant activity) and nutrients (protein, lipid, minerals). Despite of the low technological features, the six wheat varieties grown under low-input management may efficiently provide raw material for the preparation of traditionally processed bread with valuable sensory and nutritional properties. Results highlighted that old wheat varieties have peculiar phytochemical composition and may be a valuable source of nutraceutical compounds. Some of the genetic material involved in the present study may be used in breeding programs aimed at selecting varieties suitable for low-input farming and rich in health-promoting compounds.

Fusarium species responsible for mycotoxin production in wheat crop: involvement in food safety

Fusarium Head Blight (FHB) is a worldwide cereal disease responsible of significant yield reduction, inferior grain quality, and mycotoxin accumulation. Fusarium graminearum and F. culmorum are the prevalent causal agents. FHB has been endemic in Italy since 1995, while there are no records about its presence in Syria. Forty-eight and forty-six wheat kernel samples were collected from different localities and analyzed for fungal presence and mycotoxin contamination. Fusarium strains were identified morphologically but the molecular confirmation was performed only for some species. Further differentiation of the chemotypes for trichothecene synthesis by F. graminearum and F. culmorum strains was conducted by PCR assays. Fusarium spp. were present in 62.5% of Syrian samples. 3Acetyl-Deoxynivalenol and nivalenol chemotypes were found in F. culmorum whilst all F. graminearum strains belonged to NIV chemotype. Italian samples were infected with Fusarium spp for 67.4%. 15Ac-DON was the prevalent chemotype in F. graminearum, while 3Ac-DON chemotype was detected in F. culmorum. The 60 Syrian Fusarium strains tested for mycotoxin production by HPLC-MS/MS have shown the prevalence of zearalenone while the emerging mycotoxins were almost absent. The analysis of the different Syrian and Italian samples of wheat kernels for their mycotoxin content showed that Syrian kernels were mainly contaminated with storage mycotoxins, aflatoxins and ochratoxin whilst Italian grains with mainly Fusarium mycotoxins. The aggressiveness of several Syrian F. culmorum isolates was estimated using three different assays: floret inoculation in growth chamber, ear inoculation in the field and a validated new Petri-dish test. The study of the behaviour of different Syrian wheat cultivars, grown under different conditions, has revealed that Jory is a FHB Syrian tolerant cultivar. This is the first study in Syria on Fusarium spp. associated to FHB, Fusarium mycotoxin producers and grain quality.

Association mapping of stem rust resistance in durum wheat at the seedling and adult plant stages

In wheat, stem rust is known to rapidly evolve new virulence to resistance genes. While more than 50 stem rust resistance (Sr) loci have been identified in wheat, only a few remain effective, particularly against the highly virulent race Ug99 (TTKSK race) and a mixture of durum-specific races. An association mapping (AM) study based on 183 durum wheat accessions was utilized to identify resistance loci for stem rust response in Ethiopia over four seasons and artificial inoculation with Ug99 (TTKSK race) and a mixture of durum-specific races under field conditions as well as in greenhouse test at seedling stage under controlled conditions for resistance to four highly virulent stem rust races: TRTTF, TTTTF, (TTKSK (Ug99) and JRCQC. The panel was profiled with 1,253 SSR and DArT markers. Twelve QTL-tagging markers were significant (P < 0.05) across three to four seasons. The role of Sr13, Sr9, Sr14, Sr17, and Sr28 was confirmed. Thirteen significant markers were in regions with no Sr genes/QTLs. The results under controlled conditions showed that 15, 20, 19 and 19 chromosome regions harbored markers that showed significant effects for races TRTTF, TTTTF, TTKSK and JRCQC, respectively. These genomic regions showed marker R2 values ranging from 1.13 to 8.34, 1.92 to 17.64, 1.75 to 23.12 and 1.51 to 15.33% for races TRTTF, TTTTF, TTKSK and JRCQC, respectively. The study demonstrates that stem rust resistance in durum wheat is governed in part by shared loci and in part by race-specific ones. The QTLs identified in this study through AM will be useful in the marker-assisted development of durum wheat cultivars with durable stem rust resistance.

A new global wheat marketmodel (GLOWMM) for the analysis of wheat export prices

Food commodity prices fluctuations have important impacts on poverty and food insecurity across the world. Conventional models have not provided a complete picture of recent price spikes in agricultural commodity markets, while there is an urgent need for appropriate policy responses. Perhaps new approaches are needed in order to better understand international spill-overs, the feedback between the real and the financial sectors and also the link between food and energy prices. In this paper, we present results from a new worldwide dynamic model that provides short and long-run impulse responses of wheat international prices to various real shocks.

A multiparental cross population for mapping QTL for relevant agronomic traits in durum wheat (Triticum durum Desf.)

Multiparental cross designs for mapping quantitative trait loci (QTL) in crops are efficient alternatives to conventional biparental experimental populations because they exploit a broader genetic basis and higher mapping resolution. We describe the development and deployment of a multiparental recombinant inbred line (RIL) population in durum wheat (Triticum durum Desf.) obtained by crossing four elite cultivars characterized by different traits of agronomic value. A linkage map spanning 2,663 cM and including 7,594 single nucleotide polymorphisms (SNPs) was produced by genotyping 338 RILs with a wheat-dedicated 90k SNP chip. A cluster file was developed for correct allele calling in the framework of the tetraploid durum wheat genome. Based on phenotypic data collected over four field experiments, a multi-trait quantitative trait loci (QTL) analysis was carried out for 18 traits of agronomic relevance (including yield, yield-components, morpho-physiological and seed quality traits). Across environments, a total of 63 QTL were identified and characterized in terms of the four founder haplotypes. We mapped two QTL for grain yield across environments and 23 QTL for grain yield components. A novel major QTL for number of grain per spikelet/ear was mapped on chr 2A and shown to control up to 39% of phenotypic variance in this cross. Functionally different QTL alleles, in terms of direction and size of genetic effect, were distributed among the four parents. Based on the occurrence of QTL-clusters, we characterized the breeding values (in terms of effects on yield) of most of QTL for heading and maturity as well as yield component and quality QTL. This multiparental RIL population provides the wheat community with a highly informative QTL mapping resource enabling the dissection of the genetic architecture of multiple agronomic relevant traits in durum wheat.

Genetic basis of variation for root traits and response to heat stress in durum wheat

Durum wheat is the second most important wheat species worldwide and the most important crop in several Mediterranean countries including Italy. Durum wheat is primarily grown under rainfed conditions where episodes of drought and heat stress are major factors limiting grain yield. The research presented in this thesis aimed at the identification of traits and genes that underlie root system architecture (RSA) and tolerance to heat stress in durum wheat, in order to eventually contribute to the genetic improvement of this species. In the first two experiments we aimed at the identification of QTLs for root trait architecture at the seedling level by studying a bi-parental population of 176 recombinant inbred lines (from the cross Meridiano x Claudio) and a collection of 183 durum elite accessions. Forty-eight novel QTLs for RSA traits were identified in each of the two experiments, by means of linkage- and association mapping-based QTL analysis, respectively. Important QTLs controlling the angle of root growth in the seedling were identified. In a third experiment, we investigated the phenotypic variation of root anatomical traits by means of microscope-based analysis of root cross sections in 10 elite durum cultivars. The results showed the presence of sizeable genetic variation in aerenchyma-related traits, prompting for additional studies aimed at mapping the QTLs governing such variation and to test the role of aerenchyma in the adaptive response to abiotic stresses. In the fourth experiment, an association mapping experiment for cell membrane stability at the seedling stage (as a proxy trait for heat tolerance) was carried out by means of association mapping. A total of 34 QTLs (including five major ones), were detected. Our study provides information on QTLs for root architecture and heat tolerance which could potentially be considered in durum wheat breeding programs.

Dissecting the Heat-Stress Tolerance QTLome in Durum Wheat

Heat stress negatively affects wheat performance during its entire cycle, particularly during the reproductive stage. In view of the climate change and the prediction of a continued increase in temperature in the new future, it is urgent to concentrate efforts to discover novel genetic sources able to improve the resilience of wheat to heat stress. In this direction, this study addressed two different experiments in durum wheat to identify novel QTLs suitable to be applied in marker-assisted selection for heat tolerance. Chlorophyll fluorescence (ChlF) is a valuable indicator of plant response to environmental changes allowing a detailed assessment of PSII activity in view of its non-invasive measurement and high-throughput phenotyping. In the first study (Chapter 2), the Light-Induced Fluorescence Transient (LIFT) method was used to access ChlF data to map QTLs for ChlF-related traits during the vegetative growth stage in durum wheat under heat stress condition. Our results provide evidence that LIFT consistently measures ChlF at the level of high-throughput phenotyping combined with high accuracy which is required for Genome-Wide Association Study (GWAS) aimed at identifying genomic regions affecting PSII activity. The 50 QTLs identified for ChlF-related traits under heat stress mostly clustered into five chromosomes hotspots unrelated to phenology, a feature that makes these QTLs a valuable asset for marker-assisted breeding programs across different latitudes. In the second study (Chapter 3), a set of 183 accessions suitable for GWAS, was exposed to optimal and high temperature during two crop seasons under field conditions. Important agronomic traits were evaluated in order to identify valuable QTLs for GY and its components. The GWAS analysis identified several QTLs in the single years as well as in the joint analysis. From the total QTLs identified, 13 QTL clusters can be highlighted to be affecting heat tolerance across different years and/or different traits.

Wheat proteins evolution and environmental triggers of Wheat-related diseases

Cereals, and in particular wheat, have always been recognized as a fundamental food worldwide. In particular, the success of wheat is linked with unique properties of the gluten protein fraction used in bread making process to obtain products that are widely used in traditional and modern diets. The rapid increase in the world population let to a parallel increases in food production, particularly of wheat. Increasing yield potential and selection of cultivars much more resistant to plant disease and to environmental factors could have negatively affected the quality of the grain. Moreover, the “green revolution” was characterized by a widespread use of agricultural chemicals and by industrialization of food production that led to a huge rise in the consumption of refined products. Modern baking practices have shortened bread leavening, increased the use of chemical/yeast leavening agents and there is well-documented scientific evidence of the negative effects of ultra-processed food in human healthy. All this changes profoundly modified the human diet and, as a result, may have affected Gluten-related disease (GRDs) that has arisen in the whole word populations. Gluten-related diseases (GRDs) are multifactorial pathologies in which environmental factors and genetic background contribute to a low-grade chronic inflammation of the gastrointestinal tract. Here, I investigated the potential pro-inflammatory effect of different wheat varieties and whether bread making processing are involved in the onset or worsening of gut inflammation. In vitro, ex vivo and in vivo studies conducted throughout my Phd period have shown a pro-inflammatory effect of wheat especially marked in modern varieties and a higher inflammatory response linked to the use of common raising agent as Saccharomyces Cerevisiae and to the addiction of chemical bakery improver substances.

Identification of resistance factors to the main wheat diseases in the Mediterranean area

Several diseases challenge bread and durum wheat productions worldwide. The importance of these cereals requires adequate protection to pathogens that can cause strong yield and grain quality losses. The main work of this thesis was related to phenotype GDP (Global Durum Panel) in the Mediterranean region (Italy, Egypt, Lebanon, Morocco and Turkey) and Argentina across three years (2019-2021) for yellow rust resistance (infection type and severity). GWAS shows in particular, loci in chromosome 1B, 2B, 4B, 5A, 6A, 7B showed high significance across nurseries/years, with various patterns of GxE. The second chapter is about Zymoseptoria tritici, agent of STB (Septoria Tritici Blotch), a foliar pathogen that yearly causes high damages if not controlled. In recent years research in durum wheat breeding is focused on the identification of novel, underexploited resistance genes to be subsequently and conveniently moved into the pre-breeding and breeding stream. The plants were phenotyped for disease height characters, infection type at the flag leaf and infection type at the level of the canopy below the flag leaf. This experiment opens up a rich scenario of analysis and opportunities to investigate and discover new loci of resistance to STB. Third chapter is about Fusarium head blight (FHB) is a fungal disease caused by pathogens belonging to the genus Fusarium. In particular, Fusarium culmorum and Fusarium graminearum species cause severe grain yield losses and accumulation of mycotoxins in wheat that compromise food safety. Over 250 QTL/genes for FHB resistance have been identified in bread wheat, such as Fhb 1 and Fhb 5 but only a small number of FHB resistance loci have been mapped in durum wheat. The aim of this work is to find loci of partial resistance to FHB already present in durum and bread wheat germplasm and therefore easily cumulative.

Gwas analysis for the identification of molecular basis responsible for yield related traits and disease resistance in durum wheat

The PhD thesis was developed in the framework of Innovar H2020 project. This project aimed at using genomics, transcriptomics and phenotyping techniques to update varietal registration procedure used in Europe for Value of Cultivation and Use (VCU) and Distinctiness Uniformity and Stability (DUS) protocols. The phenotypic and genotypic diversity of a durum wheat panel were assessed for different agronomic traits, connected with wheat development, disease resistance and spike fertility. A panel of 253 durum wheat varieties was characterized for VCU and DUS traits and genotyped with Illumina 90K SNP Chip array (Wang et al., 2014). GWAS analysis was performed, detecting strong QTLs confirmed also by literature review. Candidate genes were identified for each trait and molecular markers will be developed to be used for marker assisted selection in breeding programs. As for disease resistance, the panel was evaluated for resistance to Soil-Borne-Cereal-Mosaic-Virus (SBCMV). A major QTL, sbm2, was detected on chromosome 2B responsible for durum wheat resistance (Maccaferri et al., 2011). The sbm2 interval was explored by fine mapping on segregant population using KASP markers and by RNASeq analysis, detecting candidate genes involved in plant-pathogen reaction. As regards yield related traits, detailed analysis was performed on the GNI-2A QTL (Milner et al., 2016), responsible for increased number spike fertility. Fine mapping analysis was performed on durum panel identifying hox2 a strong candidate gene, codifying for transcription factor protein. The gene is paralogue of GNI-1 (Sakuma et al., 2019), and it has a 4 kbp deletion responsible for increased number of florets per spikelet. To conclude, the herein reported thesis shows a complete characterization of agronomic and disease resistance traits in modern durum wheat varieties. The results obtained will augment available information for each variety, identifying informative molecular markers for breeding purposes and QTLs/candidate genes responsible for different agronomic traits.

Characterization of an international tetraploid wheat germplasm including landraces and primitive wheat towards improved resilience to abiotic and biotic stresses and quality

This thesis aimed to characterise two large tetraploid germplasm collections. The Global Durum Panel, involving modern cultivars and landrances and the Tetraploid Global Collection which comprises all the tetraploid wheat subgroups. Two distinct parallel studies were carried out. The first is focused on the characterisation of both collection for yield and quality related traits. The panel were phenotyped for two consecutive years each. In this phase the following traits were collected: the number of fertile spikelets per spike, the number of fertile florets of central spikelet for the spike-related traits. The following grain related traits were also phenotyped: the thousand kernel weight, the average grain area, average grain length, average grain width, grain brightness, grain redness, grain yellowness. GWAS analysis were performed for each collected trait and major QTLs were subjected to candidate gene analysis. Major QTLs emerging from GWA study were located on chromosome 2A with a strong bibliographic evidence for grain number-related traits such as the fertile spikelet number, the number of fertile florets per central spikelet. On the other hand two evident peaks were detected on chromosomes 6A and 7B for grain size and weight related traits. The second work was focused on the characterisation of the Global Durum Panel for root system architecture components, namely the root growth angle. GWAS analysis was perfomed and three major QTLs were detected on chromosome 2A, 6A and 7A. These three QTLs all have a bibliographic evidence.

Exploring mediterranean durum wheat wild relatives and elite diversity panel for future breeding

Durum wheat (Triticum durum) is an important crop that has been used for millennia for human consumption, and modern breeding can take advantage of the wide variability useful for the adaptation to new challenges. Novel beneficial alleles can be found in wild relatives and landraces thus enhancing crop adaptation to many biotic and abiotic stresses. This dissertation considers the source of variability from both before and after wheat domestication, that caused a loss of potentially useful alleles. Chapter 1. is the thesis introduction, which outlines the importance of wheat in the world, providing an historical overview of the domestication, the evolution mechanisms that led to the current forms of durum wheat and the use of wild relatives as a source of germplasm for future breeding programs is crucial. Moreover, the emergence of Z. tritici has been considered as the main pathogen of wheat since it contains extremely high levels of genetic variability and is thus difficult to control. Chapter 2. Considers the contribution of the phenotypic diversity of 242 accessions of Aegilops tauschii from the Open Wild Wheat Consortium, involved in wheat domestication, provided with whole-genome resequencing. The accessions were phenotyped both in the field and in controlled conditions and A k-mer-based GWAS was performed to identify genomic regions involved in useful traits. Chapter 3. Describes the genetic basis of resistance to Z. tritici in a durum wheat elite diversity panel representative of the germplasm bred in Mediterranean. Quantitative trait loci (QTL) analysis results revealed several loci involved in the STB response that were found in several chromosome regions with a high infection rate. The genomic regions associated with STB resistance identified in this study could be of interest for marker assisted selection (MAS) in durum wheat breeding programs.

Wheat adaptation to climate change in the Mediterranean Basin: retracing the past to predict the future

Wheat productivity is alarmingly threatened by climate change in the Mediterranean Basin, where it is mainly cultivated as a rainfed crop and where the latest climatic projections foresee a rise in temperatures and a reduction in precipitation, with important yield losses expected, being drought the main abiotic stress hampering wheat productivity. Assessing and quantifying the alterations in wheat life cycle caused by climate change is thus a key goal, as well as understating the underlying mechanisms of drought resistance. The first part of this thesis is focused on these main topics. A precise quantification of climate change effects on wheat in this area was performed through a case study, coupling phenological, meteorological and grain quality data before and after climate change. Then, accurate and detailed literature search was performed, reviewing the main controversies regarding the reliability of various functional traits to be used as breeding tools for improving wheat drought stress resistance. The second part of this thesis is focused in identifying interesting genetic material to improve wheat drought stress resistance in the Mediterranean Basin, analyzing drought response on a panel of tetraploid wheat accessions in vitro and in vivo as well as in open field trials, chosen in the attempt to represent as much as possible the biodiversity of tetraploid wheat. The third part of this thesis highlights differences in technological, nutritional and nutraceutical quality between modern cultivars and landraces, focusing on lipids, primary metabolites and bioactive compounds. In fact, wheat adaptation to climate change does not only mean to guarantee satisfactory yields in adverse conditions. It also means to provide millions of consumers with a diet-base food crop, with an improved nutraceutical and nutritional quality. Therefore, investigation and selection process for abiotic stress resistance and for improved quality has to go hand in hand.