Domenica Nigro

Comparison of genomic and EST-derived SSR markers in phylogenetic analysis of wheat

Microsatellite markers (simple sequence repeats, SSRs) are used for a wide range of crop genetic and breeding applications, including genetic diversity assessment, phylogenetic analysis, genotypic profiling and marker-assisted selection. Genomic SSR (gSSR) have attracted more attention because of abundance in plant genome, reproducibility, high level of polymorphism and codominant inheritance. Recently, the availability of data for expressed sequence tags (EST), has given more emphasis to EST-derived SSRs, which belong to the transcribed regions of DNA, and are expected to be more conserved and have a higher transferability rate across species than gSSR markers. In the present study, several gSSR and EST-SSR markers were investigated for their transferability and level of DNA polymorphism in different ancestral tetraploid and diploid Triticum and Aegilops species. The same gSSR and EST-SSR markers were also evaluated for their applicability in the phylogenetic analysis of wheat. Both gSSR and EST-SSR markers showed differences for the average transferability rate and the number of alleles/ locus. Phylogenetic trees based on gSSR and EST-SSR markers were in accordance with phylogenetic relations based on cytogenetic and molecular analyses.

Characterization of Ferredoxin-Dependent Glutamine-Oxoglutarate Amidotransferase (Fd-GOGAT) Genes and Their Relationship with Grain Protein Content QTL in Wheat

Background In higher plants, inorganic nitrogen is assimilated via the glutamate synthase cycle or GS-GOGAT pathway. GOGAT enzyme occurs in two distinct forms that use NADH (NADH-GOGAT) or Fd (Fd-GOGAT) as electron carriers. The goal of the present study was to characterize wheat Fd-GOGAT genes and to assess the linkage with grain protein content (GPC), an important quantitative trait controlled by multiple genes. Results We report the complete genomic sequences of the three homoeologous A, B and D Fd-GOGAT genes from hexaploid wheat (Triticum aestivum) and their localization and characterization. The gene is comprised of 33 exons and 32 introns for all the three homoeologues genes. The three genes show the same exon/intron number and size, with the only exception of a series of indels in intronic regions. The partial sequence of the Fd-GOGAT gene located on A genome was determined in two durum wheat (Triticum turgidum ssp. durum) cvs Ciccio and Svevo, characterized by different grain protein content. Genomic differences allowed the gene mapping in the centromeric region of chromosome 2A. QTL analysis was conducted in the Svevo×Ciccio RIL mapping population, previously evaluated in 5 different environments. The study co-localized the Fd-GOGAT-A gene with the marker GWM-339, identifying a significant major QTL for GPC. Conclusions The wheat Fd-GOGAT genes are highly conserved; both among the three homoeologous hexaploid wheat genes and in comparison with other plants. In durum wheat, an association was shown between the Fd-GOGAT allele of cv Svevo with increasing GPC - potentially useful in breeding programs.

The carotenoid biosynthetic and catabolic genes in wheat and their association with yellow pigments

BackgroundIn plants carotenoids play an important role in the photosynthetic process and photo-oxidative protection, and are the substrate for the synthesis of abscisic acid and strigolactones. In addition to their protective role as antioxidants and precursors of vitamin A, in wheat carotenoids are important as they influence the colour (whiteness vs. yellowness) of the grain. Understanding the genetic basis of grain yellow pigments, and identifying associated markers provide the basis for improving wheat quality by molecular breeding.ResultsTwenty-four candidate genes involved in the biosynthesis and catabolism of carotenoid compounds have been identified in wheat by comparative genomics. Single nucleotide polymorphisms (SNPs) found in the coding sequences of 19 candidate genes allowed their chromosomal location and accurate map position on two reference consensus maps to be determined. The genome-wide association study based on genotyping a tetraploid wheat collection with 81,587 gene-associated SNPs validated quantitative trait loci (QTLs) previously detected in biparental populations and discovered new QTLs for grain colour-related traits. Ten carotenoid genes mapped in chromosome regions underlying pigment content QTLs indicating possible functional relationships between candidate genes and the trait.ConclusionsThe availability of linked, candidate gene-based markers can facilitate breeding wheat cultivars with desirable levels of carotenoids. Identifying QTLs linked to carotenoid pigmentation can contribute to understanding genes underlying carotenoid accumulation in the wheat kernels. Together these outputs can be combined to exploit the genetic variability of colour-related traits for the nutritional and commercial improvement of wheat products.

Structural Analysis of the Wheat Genes Encoding NADH-Dependent Glutamine-2-oxoglutarate Amidotransferases and Correlation with Grain Protein Content

Background Nitrogen uptake and the efficient absorption and metabolism of nitrogen are essential elements in attempts to breed improved cereal cultivars for grain or silage production. One of the enzymes related to nitrogen metabolism is glutamine-2-oxoglutarate amidotransferase (GOGAT). Together with glutamine synthetase (GS), GOGAT maintains the flow of nitrogen from NH4 + into glutamine and glutamate, which are then used for several aminotransferase reactions during amino acid synthesis. Results The aim of the present work was to identify and analyse the structure of wheat NADH-GOGAT genomic sequences, and study the expression in two durum wheat cultivars characterized by low and high kernel protein content. The genomic sequences of the three homoeologous A, B and D NADH-GOGAT genes were obtained for hexaploid Triticum aestivum and the tetraploid A and B genes of Triticum turgidum ssp. durum. Analysis of the gene sequences indicates that all wheat NADH-GOGAT genes are composed of 22 exons and 21 introns. The three hexaploid wheat homoeologous genes have high conservation of sequence except intron 13 which shows differences in both length and sequence. A comparative analysis of sequences among di- and mono-cotyledonous plants shows both regions of high conservation and of divergence. qRT-PCR performed with the two durum wheat cvs Svevo and Ciccio (characterized by high and low protein content, respectively) indicates different expression levels of the two NADH-GOGAT-3A and NADH-GOGAT-3B genes. Conclusion The three hexaploid wheat homoeologous NADH-GOGAT gene sequences are highly conserved – consistent with the key metabolic role of this gene. However, the dicot and monocot amino acid sequences show distinctive patterns, particularly in the transit peptide, the exon 16–17 junction, and the C-terminus. The lack of conservation in the transit peptide may indicate subcellular differences between the two plant divisions - while the sequence conservation within enzyme functional domains remains high. Higher expression levels of NADH-GOGAT are associated with higher grain protein content in two durum wheats.

Isolation and characterisation of cytosolic glutamine synthetase (GSe) genes and association with grain protein content in durum wheat

Abstract. Glutamine synthetase (GS) enzyme (EC 6.3.1.2) plays a central role in assimilating ammonia produced in the leaf from metabolic processes, spanning from assimilation to transamination reactions and catabolic processes. GS is located in both cytoplasm (GS1, GSe and GSr) and plastids (GS2) of plant cells. Glutamine and glutamate, produced by the concerted action of GS and glutamate synthase, are then transported from the leaf to the developing sinks or grain in wheat. The goal of the present study was to characterise GSe genes and to assess the linkage with grain protein content, an important quantitative trait controlled by multiple genes. Here, we report the isolation of the complete cytosolic GS gene sequences of the durum wheat cvv. ‘Ciccio’ and ‘Svevo’ (characterised by low and high protein content, respectively). GSe-A4 located on 4A chromosome comprises 12 exons separated by 11 introns, while the GSe-B4 gene on 4B chromosome comprises 11 exons separated by 10 introns. Quantitative real-time PCR indicated different expression levels of GSe-A4 and GSe-B4 genes in the two wheat cvv. ‘Ciccio’ and ‘Svevo’. The two GSe genes were significantly associated to quantitative trait loci for grain protein content.

Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content.

Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes.

Allelic Variation of Wheat Flour Allergens in a Collection of Wheat Genotypes

Wheat is the most widely grown crop in the world and provides 20% of the daily protein and food calories for 4.5 billion people. Together with rice, it is the most important food crop in the developing world. In the last decades, various symptoms have been recorded across the population due to the consumption of wheat products, also summarized as “wheat allergy.” Wheat allergy is usually reported as a food allergy but can also be a contact allergy as a result of exposure to wheat. Several important wheat allergens have been characterized in the last years through biochemical, immunological, and molecular biological techniques. In the present work, the identification of allelic variation of genes involved in wheat allergy was reported. A collection of wheat genotypes was screened in order to identify new alleles. A total of 14 new alleles were identified for profilin, triosephosphate-isomerase, dehydrin, glyceraldehyde-3-phosphate-dehydrogenase, α/β gliadin, GluB3-23, and Glutathione transferase allergen genes (located on chromosomes 1B, 3B, 6A, and homoelogous groups 5 and 7), potentially related to a minor allergenicity and useful in breeding programs.

Identification and Characterization of the Sucrose Synthase 2 Gene (Sus2) in Durum Wheat

Sucrose transport is the central system for the allocation of carbon resources in vascular plants. Sucrose synthase (SUS), which reversibly catalyzes sucrose synthesis and cleavage, represents a key enzyme in the control of the flow of carbon into starch biosynthesis. In the present study the genomic identification and characterization of the Sus2-2A and Sus2-2B genes coding for SUS in durum wheat (cultivars Ciccio and Svevo) is reported. The genes were analyzed for their expression in different tissues and at different seed maturation stages, in four tetraploid wheat genotypes (Svevo, Ciccio, Primadur, and 5-BIL42). The activity of the encoded proteins was evaluated by specific activity assays on endosperm extracts and their structure established by modeling approaches. The combined results of sucrose synthase 2 expression and activity levels were then considered in the light of their possible involvement in starch yield.

Exploring SNP diversity in wheat landraces germplasm and setting of a molecular barcode for fingerprinting

During the last century wheat landraces were replaced by modern wheat cultivars leading to a gradual process of genetic erosion. Landraces genotyping and phenotyping are strategically useful, as they could broaden the genetic base of modern cultivars. In this research, we explored Single Nucleotide Polymorphism (SNP) markers diversity in a collection of common and durum wheats, including both landraces and Italian elite cultivars. A panel of 6,872 SNP markers was used to analyze the genetic variability among the accessions, using both the Principal Components Analysis (PCA) and the Neighbour Joining clustering method. PCA analysis separated common wheat accessions from durum ones, and allowed to group separately durum landraces from durum elite cultivars. The Neighbour joining clustering validated PCA results, and moreover, separated common wheat landraces from common elite cultivars. The clustering results demonstrated that Italian durum landraces were poorly exploited in modern breeding programs. Combining cluster results with heterozygosity levels observed, it was possible to clarify synonymy and homonymy cases identified for Bianchetta, Risciola, Saragolla, Timilia and Dauno III accessions. The SNP panel was also used to detect the minimum number of markers to discriminate the studied accessions. A set of 33 SNPs were found to be highly informative and used for a molecular barcode, which could be useful for cultivar identification and for the traceability of wheat end-products.