Powdery mildew is one of the devastating wheat diseases and is caused by the biotrophic fungus Blumeria graminis f.sp. tritici. The identification of natural sources of resistance and breeding for resistant varieties is the most effective way to control this disease, as chemical control is expensive. To date, more than 40 Pm resistance genes have been characterized, while only one of these genes, Pm3, has been cloned. Pm3 is localized on the short arm of wheat chromosome 1A and is present in 17 functional allelic forms (Pm3a to Pm3g, Pm3k to Pm3t). The Pm3 alleles confer race-specific resistance to different wheat powdery mildew races. The objectives of this work were to study the behaviour of powdery mildew resistance Pm3 gene in a collection of tetraploid wheats and to isolate the complete genomic sequence of Pm3 gene in susceptible and resistant genotypes in order to identify new functional alleles. The search for new Pm3 alleles was carried out on a collection of 233 tetraploid wheat genotypes (Triticum turgidum L.). The collection comprises 128 cultivars of durum wheat (ssp. durum), as well as 19 accessions of ssp. turgidum, 20 of ssp. turanicum, 20 of ssp. polonicum, 12 of ssp. carthlicum, 20 of ssp. dicoccum, and 14 of ssp. dicoccoides. The entire set of these accessions was phenotypically characterized for resistance against wheat powdery mildew by screening with one powdery mildew isolate (O2). This screening led to the identification of 34 resistant accessions (14,6% of the total set). The tetraploid wheat collection was analysed for the Pm3 haplotype by screening for the presence of a Pm3-like gene with a STS marker. This STS marker amplifies a 946 bp fragment from the 5' non- coding region of Pm3b and is diagnostic for the presence of a Pm3-like gene. A total of 129 accessions, both resistant and susceptible, out of 233 (55.4%) tested were identified with a likely presence of a Pm3 gene. Subsequently a set of 21 resistant and susceptible lines showing the presence of a Pm3-like gene was screened for the presence of Pm3a to Pm3g alleles. A positive PCR reaction was observed for the Pm3b allele. Five of these lines, either resistant and susceptible, were further analysed with 11 PCR primer combination covering all the Pm3b sequence. Pm3b allele from tetraploid wheat had a size of 4,442 bp, comprising two exons of 4,156 and 86 bp respectively, and an intron of 200 bp. Sequences alignment compared with the Pm3b sequence, used as reference, indicated that the Pm3 genes from the five tetraploid wheat lines contain several “indels” and SNPs in the form of highly polymorphic sequence blocks. The present study demonstrates that allelic variation occurs in functional genes and represents the first step for the identification and sequencing of new functional Pm3 alleles in tetraploid wheat genotypes.

In the present study, a detailed deletion map for wheat chromosomes 5A and 5B is reported, as well as an integrated genetic linkage map of chromosome 5A enriched with single nucleotide polymorphism (SNP) markers, useful both for comparison studies with other existing maps and for mapping major genes and quantitative trait loci (QTLs). Physical mapping of 5,011 SNP markers was obtained using Chinese Spring bin deletion lines for the homoeologous chromosomes of group 5; 509 SNPs were also genetically mapped in a recombinant inbred line population segregating for chromosome 5A only, obtained by crossing the cultivar Chinese Spring and the disomic substitution line Chinese Spring-5A dicoccoides. The whole 5A genetic map, containing 572 markers, covered a total length of 248.7 cM distributed among three linkage groups of 83.5, 117.8 and 47.4, respectively. The majority of SNP markers physically mapped on 5A were mapped to a unique bin, while a small percentage was assigned a double location, suggesting the presence of a segment of 5A short arm which may have undergone a duplication followed by an insertion into the long arm of the same chromosome. A QTL analysis for yield components was performed, identifying a major QTL in the subtelomeric region of chromosome 5A, corresponding to the 5AL15-0.67-0.78 bin; the chromosome segment was 23.5 cM long and included 111 markers. Candidate genes for yield components on chromosome 5A were identified through a syntenic genomic approach by comparison with genomes of model species. Putative function analysis revealed genes involved in basic metabolism and in stress condition responses, including heat shock proteins, chaperones, serine/ threonine protein kinases and membrane transporters, located in the region of the QTL. This information represents an important step for map-based and candidate gene-based cloning of yield QTLs.

Powdery mildew caused by the pathogen Blumeria graminis f. sp. tritici, is a destructive foliar disease in many regions of the world. The tetraploid wheat Triticum turgidum ssp. dicoccum (2n = 4x = 28, genome AABB) shows particular promises as a donor of useful genetic variation for several traits including disease resistances to introgress in cultivated wheats. The accession MG5323, resistant to powdery mildew, was crossed to the susceptible durum wheat cultivar Latino and a set of 122 recombinant inbred lines (RILs) was produced. Segregation analysis of F2 plants combined with the resistance of F1 progeny to the isolate O2, tested under controlled greenhouse conditions, indicated that resistance is controlled by a single dominant allele. Among the 122 RILs, tested under controlled greenhouse conditions with the same isolate, 67 lines were susceptible and 55 were resistant. The segregation in the RILs fitted the 1:1 segregation ratio expected for a single resistance locus (χ 2 =1.18, 0.50 > P > 0.10). Molecular markers (gSSRs, EST-SSRs and RFLP-derived STS) were used to locate and map the resistance gene. Bulked segregant analysis (BSA), indicated that chromosome arm 2BS could be involved in the control of the resistance. Twelve genomic SSR (gSSRs), three ESTderived SSR markers (EST-SSRs) and one RFLP-derived STS, polymorphic between Latino and MG5323 and located on 2BS, were tested in the set of 122 RILs. Two gSSRs and one EST-SSR, physically mapped on bin 2BS3- 0.84-1.00, were found to be tightly linked to the resistance gene. Among the molecular markers observed in the linkage map of 2BS, few gSSRs showed Mendelian segregation (1:1), whereas most markers, showed significant deviation from the expected ratio. The marker EST-SSR, closely linked to the resistance gene, has potential use in marker-assisted selection and pyramiding of genes for resistance to powdery mildew in wheat.

High-density genetic linkage maps of crop species are particularly useful in detecting qualitative and quantitative trait loci (QTLs) for agronomically important traits and in improving the power of classical approaches to identify candidate genes. The aim of this study was to develop a high-density genetic linkage map in a durum wheat recombinant inbred lines population derived from two elite wheat cultivars and to identify and characterize QTLs for yellow pigment content (YPC). A dense map was constructed by genotyping with the wheat 90K iSelect array and consisted of 5,670 loci, comprising 5,019 single nucleotide polymorphism (SNP), 467 DArT, 182 SSR markers and eight genes distributed in 35 linkage groups. Data for yellow pigment content were obtained from four replicated trials conducted at two locations in southern Italy for 2 years. A total of seven QTLs on different chromosome regions (1B, 2A, 2B, 5A, 5B, 7A and 7B) were identified, three of which were consistent in three or four environments and across environments. The genome scan for QTLs and the SNP homology prediction against annotated proteins in wheat and Brachypodium genomes identified two candidate genes of the carotenoid biosynthesis pathway (aldehyde oxidase, AO1, and diphosphomevalonate decarboxylase, DMAPD) significantly associated with YPC. This study provides a basis for further genetic QTL dissection and tools for marker-assisted breeding programs, because SNP markers and some carotenoid candidate genes were found to be tightly linked to major QTLs for YPC.

Totipotent cDNA libraries representative of all the potentially expressed sequences in a genome would be of great benefit to gene expression studies. Here, we report on an innovative method for creating such a library for durum wheat (Triticum turgidum L. var. durum) and its application for gene discovery. The use of suitable quantities of 5-azacytidine during the germination phase induced the demethylation of total DNA, and the resulting seedlings potentially express all of the genes present in the genome. A new wheat microarray consisting of 4925 unigenes was developed from the totipotent cDNA library and used to screen for genes that may contribute to differences in the disease resistance of two near-isogenic lines, the durum wheat cultivar Latino and the line 5BIL-42, which are respectively susceptible and resistant to powdery mildew. Fluorescently labeled cDNA was prepared from the RNA of seedlings of the two near-isogenic wheat lines after infection with a single powdery mildew isolate under controlled conditions in the greenhouse. Hybridization to the microarray identified six genes that were differently expressed in the two lines. Four of the sequences could be assigned putative functions based on their similarity to known genes in public databases. Physical mapping of the six genes localized them to two regions of the genome: the centromeric region of chromosome 5B, where the Pm36 resistance gene was previously localized, and chromosome 6B

The beneficial effects of whole meal wheat products are mainly attributed to dietary fibre and secondary metabolites related to it, such as phenolic compounds. To date, no studies have investigated the effect of fertilization with sewage sludge on the levels of wheat phenolic compounds. The aim of this study was to point out the effects of increasing doses of composted sewage sludge, also in combination with mineral fertilization, on phenolic compounds and antioxidant activity of durum wheat. Moreover, the effects on productivity and technological quality were verified. A randomized block experimental system with six replicates was adopted and seven treatments were compared: unfertilized control (absence of any fertilization or composted sewage sludge application); four doses of composted sewage sludge (3, 6, 9, and 12 Mg ha-1); mineral fertilization (MF) consisting of 120, 100 and 100 kg ha-1 of N, P2O5 and K2O, respectively; combined fertilization with 6 Mg ha-1 of composted sewage sludge and 60 kg ha-1 of N. There was a significantly positive effect of sewage sludge application on productivity as well as on phenolic compounds, antioxidant activity, and technological quality of durum wheat whole meal. Regarding the productivity, the use of 12 Mg ha-1 of composted sewage sludge can effectively substitute mineral fertilization. Regarding phenolics and antioxidant activity, a further increase can be achieved by employing a combination of composted sewage sludge (at the level of 6 Mg ha-1) and mineral fertilization. The phenolic compounds of whole meal raised from 1.31 mg ferulic acid equivalents (FAE) g-1 (unfertilized control) to 1.93 mg FAE g-1 (combined application of 6 Mg ha-1 of sewage sludge and 60 kg ha-1 N). The antioxidant activity range was 1.89-2.02 μmol 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) g-1 d.m., corresponding to a level of scavenging capacity of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical ranging from 56.26% (unfertilized control) to 62.29% (6 Mg ha-1 of sewage sludge plus 60 kg ha-1 N).

The growing sensibility toward those foods that are characterized by natural and healthy features has raised the interest toward alternative wheat cereals. This research was carried out to compare the technological characteristics and the bread-making quality of Khorasan wheat, type Kamut and spelt (cv. Forenza), to those of common (cv. Rio) and durum wheat (cv. Norba). The results obtained show that both Forenza and Kamut gave an acceptable bread-making performance. A certain variability affected flour characteristics (protein content, carotenoid pigments and alveograph indices) over the 2 years of experimentation, due to environmental effects. This reflected on the corresponding breads but the statistical analysis indicated that, on the whole, Kamut bread was characterized by a high content of carotenoid pigments. Regarding sensory properties (profiled by means of 11 descriptors of visual appearance, texture, odor and flavor) and loaf volumes, breads from Forenza and Kamut appeared different from each other but similar to those obtained from Rio and Norba grown in the same environment, respectively.

The accession MG5323 of T. turgidun ssp. dicoccum, resistant to powdery mildew, was crossed to the susceptible durum wheat cultivar Latino and a set of 122 Recombinant Inbred Lines (RILs) was produced. Genetic analysis of F1 and F2 progenies and RIL population, tested with a powdery mildew isolate O2 in controlled greenhouse conditions, indicated that a single dominant gene, temporarily designed Ml5323, controls the powdery mildew resistance at the seedling stage. Molecular markers and bulk segregant analysis were used to characterize and map the powdery mildew resistance gene. Twelve microsatellite markers were found to be linked to the resistance gene and, among them, two gSSRs (Xwmc25, Xwmc243) and one EST-SSR (CA695634), were found to be tightly linked to the resistance gene. By means of Chinese Spring nullisomic-tetrasomic and ditelosomics lines the polymorphic markers and the resistance gene were assigned to chromosome arm 2BS and were physically mapped on bin 2BS3-0.84-1.00 by Chinese Spring deletion lines. The physical mapping of the microsatellite markers linked to the Ml5323 gene confirmed the presence of this locus in the short arm of 2B on bin 2BS3-0.84-1.00 where was also located the Pm26 gene. A test of allelism between Ml5323and Pm26 was carried out in order to estabilish the relationship between these two genes located in the same bin on chromosome 2BS. The mildew resistant accessions TTD140 of ssp. dicoccoides, that carries the recessive gene Pm26 and MG5323 of ssp. dicoccum with the dominant gene Ml5323 were crossed to obtain the F1 progeny (TTD140 x MG5323). Seedlings of TTD140, MG5323 and ten F1(TTD140 x MG5323) were highly resistant to the isolate O2. Two hundred and twelve F2 families from the TTD140 x MG5323 population were evaluated for their powdery mildew reaction under greenhouse conditions following the same procedures used for the Latino x MG5323 cross. The F2 population segregated for resistance and susceptibility to isolate O2 of powdery mildew and showed a significant deviation from the theoretical 15 resistant : 1 susceptible ratio expected for two dominant genes segregating independently. The results obtained indicated that the two genes Pm26 and Ml5323 are different and linked on the bin 2BS3-0.84-1.00.

The tetraploid wheat Triticum turgidum ssp. dicoccum shows particular promises as a donor of useful genetic variation for several traits including disease resistances to introgress in cultivated wheats. The accession MG5323 of ssp. dicoccum, resistant to powdery mildew and leaf rust, was crossed to susceptible durum wheat cultivar Latino and a set of 113 recombinant inbred lines (RILs) was produced. The objective of this work was to identify and map leaf rust and powdery mildew resistance loci from dicoccum accession. The parents and RIL population were phenotyped, under controlled greenhouse conditions, with two Puccinia triticina (VMC03 and 12766) and one Blumeria graminis (O2) isolates. Marker analysis of the RILs was performed using a large set of different molecular markers (SSR, EST-SSR,COS and SNP) leading to the construction of a linkage map containing 7,808 polymorphic loci covering 6,228 cM of genetic distance on the fourteen chromosomes of durum wheat, with an average distance of 0.80 cM between adjacent markers. Linkage analysis allowed the identification of different regions significantly associated with leaf rust and powdery mildew resistances. One major gene conferring resistance to leaf rust was detected on the short arm of chromosome 1B, explaining a total phenotypic variation ranging from 41.89 to 52.56% and an additional minor gene was located on chromosome 7B, explaining 12.16-31.24% of total phenotypic variation. About the powdery mildew resistance a single dominant gene was located on the short arm of chromosome 2B explaining 67.90% of total phenotypic variation. These results allowed the identification of new resistance genes to leaf rust and powdery mildew in a tetrapoid wheat genetic background and the closest linked markers identified can be used for marker-assisted selection (MAS) making feasible the development of resistant durum wheat cultivars using the discovered resistance genes.

The Triticum turgidum ssp. dicoccum (2n = 4x = 28) accession MG5323 showed a useful level of resistance to leaf rust disease. A segregating population of 110 recombinant inbred lines (RILs), derived from a cross between cv Latino (T. turgidum spp. durum), susceptible to leaf rust, and MG5323 was evaluated for reactions of seedlings to two different Puccinia triticina isolates. Genotyping of the RILs was performed with different molecular markers (SSR, EST-SSR and SNP), leading to the construction of a linkage map containing 10,840 loci covering 14 chromosomes, with an average marker density of 0.22 cM/marker. Linkage analysis allowed the identification of three different regions significantly associated with leaf rust resistance, with MG5323 contributing the resistant alleles. A major resistance gene was detected on the short arm of chromosome 1B, explaining a total phenotypic variation ranging from 41.37 to 49.51 %. Two additional minor resistance genes located on chromosome 7B explained a phenotypic variation ranging between 17.77 and 25.81 %. No obvious positional relationships were observed when the map position of the genes was compared with those of other previously identified wheat leaf rust resistance genes, suggesting that new resistance sources to leaf rust were identified in the tetraploid background. A significant positive epistatic effect was detected between quantitative trait loci (QTLs) for each trait, indicating that different QTLs contribute different degrees of resistance. Analysis of the leaf rust responses of the RILs demonstrated that only lines bearing resistant alleles at both loci showed effective leaf rust resistance, indicating that the genes identified behave as complementary genes.

Powdery mildew caused by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a destructive foliar disease on wheat in many regions of the world. Triticum turgidum ssp. dicoccum (2n = 4x = 28) shows particular promises as a donor source of useful genetic variation for several traits, including disease resistances that could be introgressed to cultivated wheats. Accession MG5323, resistant to powdery mildew, was crossed to the susceptible durum cultivar Latino and a set of 122 recombinant inbred lines (RILs) was produced. F1 and F2 progenies and the RIL population were tested with one isolate of Blumeria graminis and data obtained indicated that a single dominant gene, temporarily designated Ml5323, controlled resistance at the seedling stage. Molecular markers were used to characterize and map the powdery mildew resistance gene. Twelve microsatellite markers were linked to the resistance gene, and among them, EST-SSR CA695634 was tightly linked to the resistance gene, which was assigned to chromosome arm 2BS and physically mapped to the gene rich region of fragment length (FL) 0.84–1.00. An allelism test showed that the Ml5323 gene and the resistant gene Pm26 of ssp. dicoccoides localized in the same bin, are not allelic and tightly linked.