Durum Genome Research Articles

Linkage disequilibrium patterns, population structure and diversity analysis in a worldwide durum wheat collection including Argentinian genotypes

BackgroundDurum wheat (Triticum turgidum L. ssp. durum Desf. Husn) is the main staple crop used to make pasta products worldwide. Under the current climate change scenarios, genetic variability within a crop plays a crucial role in the successful release of new varieties with high yields and wide crop adaptation. In this study we evaluated a durum wheat collection consisting of 197 genotypes that mainly comprised a historical set of Argentinian germplasm but also included worldwide accessions.ResultsWe assessed the genetic diversity, population structure and linkage disequilibrium (LD) patterns in this collection using a 35 K SNP array. The level of polymorphism was considered, taking account of the frequent and rare allelic variants. A total of 1547 polymorphic SNPs was located within annotated genes. Genetic diversity in the germplasm collection increased slightly from 1915 to 2010. However, a reduction in genetic diversity using SNPs with rare allelic variants was observed after 1979. However, larger numbers of rare private alleles were observed in the 2000–2009 period, indicating that a high reservoir of rare alleles is still present among the recent germplasm in a very low frequency. The percentage of pairwise loci in LD in the durum genome was low (13.4%) in our collection. Overall LD and the high (r2 > 0.7) or complete (r2 = 1) LD presented different patterns in the chromosomes. The LD increased over three main breeding periods (1915–1979, 1980–1999 and 2000–2020).ConclusionsOur results suggest that breeding and selection have impacted differently on the A and B genomes, particularly on chromosome 6A and 2A. The collection was structured in five sub-populations and modern Argentinian accessions (cluster Q4) which were clearly differentiated. Our study contributes to the understanding of the complexity of Argentinian durum wheat germplasm and to derive future breeding strategies enhancing the use of genetic diversity in a more efficient and targeted way.

Candidate Genes and Quantitative Trait Loci for Grain Yield and Seed Size in Durum Wheat.

Grain yield (YLD) is affected by thousand kernel weight (TKW) which reflects the combination of grain length (GL), grain width (GW) and grain area (AREA). Grain weight is also influenced by heading time (HT) and plant height (PH). To detect candidate genes and quantitative trait loci (QTL) of yield components, a durum wheat recombinant inbred line (RIL) population was evaluated in three field trials. The RIL was genotyped with a 90K single nucleotide polymorphism (SNP) array and a high-density genetic linkage map with 5134 markers was obtained. A total of 30 QTL were detected including 23 QTL grouped in clusters on 1B, 2A, 3A, 4B and 6B chromosomes. A QTL cluster on 2A chromosome included a major QTL for HT co-located with QTL for YLD, TKW, GL, GW and AREA, respectively. The photoperiod sensitivity (Ppd-A1) gene was found in the physical position of this cluster. Serine carboxypeptidase, Big grain 1 and β-fructofuranosidase candidate genes were mapped in clusters containing QTL for seed size. This study showed that yield components and phenological traits had higher inheritances than grain yield, allowing an accurate QTL cluster detection. This was a requisite to physically map QTL on durum genome and to identify candidate genes affecting grain yield.

Prototype Gluten-Free Breads from Processed Durum Wheat: Use of Monovarietal Flours and Implications for Gluten Detoxification Strategies.

In this investigation, we reported the production of prototype breads from the processed flours of three specific Triticum turgidum wheat genotypes that were selected in our previous investigation for their potential low toxic/immunogenic activity for celiac disease (CD) patients. The flours were subjected to sourdough fermentation with a mixture of selected Lactobacillus strains, and in presence of fungal endoproteases. The breads were characterized by R5 competitive enzyme linked immunosorbent assay in order to quantify the residual gluten, and the differential efficacy in gluten degradation was assessed. In particular, two of them were classified as gluten-free (<20 ppm) and very low-gluten content (<100 ppm) breads, respectively, whereas the third monovarietal prototype retained a gluten content that was well above the safety threshold prescribed for direct consumption by CD patients. In order to investigate such a genotype-dependent efficiency of the detoxification method applied, an advanced proteomic characterization by high-resolution tandem mass spectrometry was performed. Notably, to the best of our knowledge, this is the first proteomic investigation which benefitted, for protein identification, from the full sequencing of the Triticum turgidum ssp. durum genome. The differences of the proteins’ primary structures affecting their susceptibility to hydrolysis were investigated. As a confirmation of the previous immunoassay-based results, two out of the three breads made with the processed flours presented an exhaustive degradation of the epitopic sequences that are relevant for CD immune stimulatory activity. The list of the detected epitopes was analyzed and critically discussed in light of their susceptibility to the detoxification strategy applied. Finally, in-vitro experiments of human gastroduodenal digestion were carried out in order to assess, in-silico, the toxicity risk of the prototype breads under investigation for direct consumption by CD patients. This approach allowed us to confirm the total degradation of the epitopic sequences upon gastro-duodenal digestion.

Transgenerational Effects of Water-Deficit and Heat Stress on Germination and Seedling Vigour-New Insights from Durum Wheat microRNAs.

Water deficiency and heat stress can severely limit crop production and quality. Stress imposed on the parents during reproduction could have transgenerational effects on their progeny. Seeds with different origins can vary significantly in their germination and early growth. Here, we investigated how water-deficit and heat stress on parental durum wheat plants affected seedling establishment of the subsequent generation. One stress-tolerant and one stress-sensitive Australian durum genotype were used. Seeds were collected from parents with or without exposure to stress during reproduction. Generally, stress on the previous generation negatively affected seed germination and seedling vigour, but to a lesser extent in the tolerant variety. Small RNA sequencing utilising the new durum genome assembly revealed significant differences in microRNA (miRNA) expression in the two genotypes. A bioinformatics approach was used to identify multiple miRNA targets which have critical molecular functions in stress adaptation and plant development and could therefore contribute to the phenotypic differences observed. Our data provide the first confirmation of the transgenerational effects of reproductive-stage stress on germination and seedling establishment in durum wheat. New insights gained on the epigenetic level indicate that durum miRNAs could be key factors in optimising seed vigour for breeding superior germplasm and/or varieties.

Identification of a Dominant Chlorosis Phenotype Through a Forward Screen of the Triticum turgidum cv. Kronos TILLING Population.

Durum wheat (Triticum turgidum) derives from a hybridization event approximately 400,000 years ago which led to the creation of an allotetraploid genome. The evolutionary recent origin of durum wheat means that its genome has not yet been fully diploidised. As a result, many of the genes present in the durum genome act in a redundant fashion, where loss-of-function mutations must be present in both gene copies to observe a phenotypic effect. Here, we use a novel set of induced variation within the cv. Kronos TILLING population to identify a locus controlling a dominant, environmentally dependent chlorosis phenotype. We carried out a forward screen of the sequenced cv. Kronos TILLING lines for senescence phenotypes and identified a line with a dominant early senescence and chlorosis phenotype. Mutant plants contained less chlorophyll throughout their development and displayed premature flag leaf senescence. A segregating population was classified into discrete phenotypic groups and subjected to bulked-segregant analysis using exome capture followed by next-generation sequencing. This allowed the identification of a single region on chromosome 3A, Yellow Early Senescence 1 (YES-1), which was associated with the mutant phenotype. While this phenotype was consistent across 4 years of field trials in the United Kingdom, the mutant phenotype was not observed when grown in Davis, CA (United States). To obtain further SNPs for fine-mapping, we isolated chromosome 3A using flow sorting and sequenced the entire chromosome. By mapping these reads against both the cv. Chinese Spring reference sequence and the cv. Kronos assembly, we could identify high-quality, novel EMS-induced SNPs in non-coding regions within YES-1 that were previously missed in the exome capture data. This allowed us to fine-map YES-1 to 4.3 Mb, containing 59 genes. Our study shows that populations containing induced variation can be sources of novel dominant variation in polyploid crop species, highlighting their importance in future genetic screens. We also demonstrate the value of using cultivar-specific genome assemblies alongside the gold-standard reference genomes particularly when working with non-coding regions of the genome. Further fine-mapping of the YES-1 locus will be pursued to identify the causal SNP underpinning this dominant, environmentally dependent phenotype.

Functional analysis of Triticum durum type 1 metallothionein gene (dMT) in response to varying levels of cadmium

The effect of varying levels of cadmium and correlated changes on the expression level of a type 1 metallothionein gene (dMT) were investigated in Triticum durum cv. Balcali-85. Increasing the cadmium concentration resulted in a decrease in the dry weights of roots and shoots, and the effect was stronger in roots. Roots also showed a higher capacity to accumulate cadmium. Southern blot analyses revealed that the dMT gene, delineated by two exons and a non-coding intron region, exists at a single locus in the T. durum genome. Changes in dMT gene expression during cadmium exposure were monitored by two approaches. Northern blot analyses showed that the transcript level in roots increased upon treatment with increasing cadmium, which was quantified by qRT-PCR as 4.5 fold of the base level at 10 μM Cd. These results show a positive correlation between cadmium exposure and expression of dMT gene in durum wheat, and will provide a basis for studies on the role of type 1 metallothioneins in cadmium response.

Durum wheat genetic stocks involving chromosome 1E of diploid wheatgrass: resistance to Fusarium head blight

Durum wheat (Triticum turgidum L., 2n = 4x = 28; AABB genomes) is the preferred wheat for preparing pasta products. Current durum cultivars have little resistance to Fusarium head blight (FHB), or scab, a ravaging fungal disease. In our Durum Germplasm Enhancement (DGE) project, we previously showed that chromosome 1E of diploid wheatgrass Lophopyrum elongatum (Host) A. Love (2n = 2x = 14; EE genome) is an excellent source of FHB resistance. Through hybridization of this grass with the durum cultivar Langdon, coupled with manipulation of chromosome pairing and selection, we added the chromosome 1E pair into the durum genome and produced a fertile durum 1E addition line, called DGE-1. Also by substituting 1E for the durum homoeologues 1A or 1B, we produced, two fertile disomic substitution lines, 1E(1A) and 1E(1B), which were designated as DGE-2 and DGE-3, respectively. The level of FHB resistance of the genetic stocks was studied in the greenhouse and in the field at Prosper, North Dakota. In the greenhouse, the 1E addition showed 32.15 % infection and was better than Langdon at 58.73 %, but not significantly different from 1E(1A) disomic substitution at 45.60 % infection. The 1E(1B) disomic substitution (88.29 % infection) had the highest FHB infection. Under natural conditions in the field, the observed infection rates were essentially similar. Among the three genetic stocks, the 1E addition DGE-1 had the lowest level of infection (at 10 %) followed by the substitution 1E(1A) at 20 %, and the substitution E(1B) at 70 %. The parental cultivar Langdon had 15 % FHB infection. The genetic stocks of durum may help produce several more alien addition and substitution lines involving different chromosomes and may help locate sources of FHB resistance.

Chromosome Engineering of Durum Wheat with Alien Chromatin of Diploid Wheatgrass

Durum wheat (Triticum turgidum L., 2n = 4x = 28; AABB genomes) is an important cereal widely used for human consumption. It is the choice wheat for preparing pasta products. Current durum cultivars have little resistance to Fusarium head blight (FHB), or scab, a ravaging fungal disease caused by the fungal pathogen Fusarium graminearum Schwabe. We showed earlier that diploid wheatgrass (Lophopyrum elongatum (Host) Á. Löve (2n = 2x = 14; EE genome) is an excellent source of FHB resistance, which could be transferred to commercial durum cultivars by hybridization coupled with manipulation of chromosome pairing. We obtained homoeologous pairing (i.e., pairing between wheat and alien chromosomes) in intergeneric hybrids because of the wheatgrass-induced suppression of the pairing-regulating gene Ph1 of wheat, leading to alien chromatin integration into the durum genome. Adopting this chromosome engineering technology, we have transferred diploid wheatgrass chromatin into the durum genome. We have produced fertile hybrid derivatives with varying degrees of FHB resistance. Some derivatives showed only 7.6% infection compared with 72.0% in the parental durum cultivar. Fluorescent genomic in situ hybridization (fl-GISH) analysis was used to confirm the integration of alien chromatin into the durum complement.

Association mapping of yellow pigment in an elite collection of durum wheat cultivars and breeding lines

Association mapping (AM) is an alternative or complementary strategy to QTL mapping for describing associations between genotype and phenotype based on linkage disequilibrium (LD). Yellow pigment (YP), an important end-use quality trait in durum wheat (Triticum turgidum L. var. durum), was evaluated to determine the ability of AM to identify previously published QTL and to identify genomic regions for further genetic dissection. The YP concentration was determined for 93 durum wheat accessions sampled from a variety of geographic origins. Analysis of population structure using distance- and model-based estimates indicated the presence of five subpopulations. Using subpopulation assignments as covariates, significant (P < 0.05) marker-trait associations for YP were detected on all chromosomes of the durum genome. Using AM, genomic regions housing known YP QTL were confirmed, most notably the group 7 chromosomes. In addition, several markers on the group 1, 2, and 3 chromosomes were identified where QTL have yet to be reported. A phytoene synthase gene, Psy1-B1, a potential candidate gene for YP, was significantly associated with YP and was in strong LD with microsatellite markers on the distal end of 7BL. Our results demonstrated that AM complemented traditional QTL mapping techniques and identified novel QTL that should be the target of further genetic dissection.

Molecular linkage map for an intraspecific recombinant inbred population of durum wheat (Triticum turgidum L. var. durum)

Durum wheat (Triticum turgidum L. var. durum) is an economically and nutritionally important cereal crop in the Mediterranean region. To further our understanding of durum genome organization we constructed a durum linkage map using restriction fragment length polymorphisms (RFLPs), simple sequence repeats (SSRs) known as Gatersleben wheat microsatellites (GWMs), amplified fragment length polymorphisms (AFLPs), and seed storage proteins (SSPs: gliadins and glutenins). A population of 110 F9 recombinant inbred lines (RILs) was derived from an intraspecific cross between two durum cultivars, Jennah Khetifa and Cham 1. The two parents exhibit contrasting traits for resistance to biotic and abiotic stresses and for grain quality. In total, 306 markers have been placed on the linkage map – 138 RFLPs, 26 SSRs, 134 AFLPs, five SSPs, and three known genes (one pyruvate decarboxylase and two lipoxygenases). The map is 3598 cM long, with an average distance between markers of 11.8 cM, and 12.1% of the markers deviated significantly from the expected Mendelian ratio 1:1. The molecular markers were evenly distributed between the A and B genomes. The chromosome with the most markers is 1B (41 markers), followed by 3B and 7B, with 25 markers each. The chromosomes with the fewest markers are 2A (11 markers), 5A (12 markers), and 4B (15 markers). In general, there is a good agreement between the map obtained and the Triticeae linkage consensus maps. This intraspecific map provides a useful tool for marker-assisted selection and map-based breeding for resistance to biotic and abiotic stresses and for improvement of grain quality.

Cytoplasmic homology between Aegilops mutica Boiss. and Ae. ovata L.

Triticum aestivum L. em Thell. (2n=42; AABBDD), and T. durumDesf. (2n=28; AABB) genomes were substituted into the cytoplasms of Aegilops muticaBoiss. (2n=14; MtMt), Ae. heldreichiiHolzm. (2n=14; MM), Ae. uniaristataVis. (2n=14; MuMu), and Ae. ovata L. (2n=28; CuCuMoMo), to identify the M-genome diploid cytoplasm donor of Ae. ovata. Substitution of the T. durum genome into Ae. uniaristata cytoplasm resulted in a large proportion of shriveled inviable seeds. A few plump viable seeds were obtained all of which produced male-sterile plants having one univalent or telocentric chromosome from Ae. uniaristata. The T. aestivum plants having Ae. uniaristata or Ae. mutica cytoplasms were fertile. However, Ae. mutica was similar to Ae. ovata in the induction of delayed maturity and tall robust growth habit to the T. durum and T. aestivum plants. Cytoplasms of the other C- and M-genome diploids Ae. umbellulataZhuk. (2n=14; CuCu) and, Ae. heldreichii (2n=14; MM) earlier had been shown to differ from that of Ae. ovata. Therefore, Ae. mutica is the most likely cytoplasm and M-genome donor to Ae. ovata.