Uncovering the genetic basis of gluten aggregation parameters by genome-wide association analysis in wheat (Triticum aestivum L.) using GlutoPeak.

BACKGROUND: Numerous studies have shown that gluten aggregation properties directly affect the processing quality of wheat, however, the genetic basis of gluten aggregation properties were rarely reported. RESULTS: To explore the genetic basis of gluten aggregation properties in wheat, an association population consisted with 207 wheat genotypes were constructed for evaluating nine parameters of aggregation properties on GlutoPeak across three-year planting seasons. A total of 940 significant SNPs were detected for 9 GlutoPeak parameters through genome-wide association analysis (GWAS). Finally, these SNPs were integrated to 68 non-redundant QTL distributed on 20 chromosomes and 54 QTL was assigned as pleiotropic loci which accounting for multiple parameters of gluten aggregation property. Furthermore, the peak SNPs representing 54 QTL domonstrated additive effect on all the traits. There was a significant positive correlation between the number of favorable alleles and the phenotypic values of each parameter. Peak SNPs of two novel QTL, q3AL.2 and q4DL, which contributing to both PMT (peak maximum time) and A3 (area from the first minimum to torque 15 s before the maximum torque) parameters, were selected for KASP (Kompetitive Allele Specific PCR) markers development and the KASP markers can be used for effectively evaluating the quality of gluten aggregation properties in the association population. CONCLUSION: The rapid and efficient GlutoPeak method for gluten measurement can be used for early selection of wheat breeding. This study revealed the genetic loci related to GlutoPeak parameters in association population, which would be helpful to develop wheat elite lines with improved gluten aggregation through molecular marker-assisted breeding.

Genetic dissection of grain iron concentration in hexaploid wheat (Triticum aestivum L.) using a genome-wide association analysis method.

Iron (Fe) is an essential micronutrient of the body. Low concentrations of bioavailable Fe in staple food result in micronutrient malnutrition. Wheat (Triticum aestivum L.) is the most important global food crop and thus has become an important source of iron for people. Breeding nutritious wheat with high grain-Fe content has become an effective means of alleviating malnutrition. Understanding the genetic basis of micronutrient concentration in wheat grains may provide useful information for breeding for high Fe varieties through marker-assisted selection (MAS). Hence, in the present study, genome-wide association studies (GWAS) were conducted for grain Fe. An association panel of 207 accessions was genotyped using a 660K SNP array and phenotyped for grain Fe content at three locations. The genotypic and phenotypic data obtained thus were used for GWAS. A total of 911 SNPs were significantly associated with grain Fe concentrations. These SNPs were distributed on all 21 wheat chromosomes, and each SNP explained 5.79-25.31% of the phenotypic variations. Notably, the two significant SNPs (AX-108912427 and AX-94729264) not only have a more significant effect on grain Fe concentration but also have the reliability under the different environments. Furthermore, candidate genes potentially associated with grain Fe concentration were predicted, and 10 candidate genes were identified. These candidate genes were related to transport, translocation, remobilization, and accumulationof ironin wheat plants. These findings will not only help in better understanding the molecular basis of Fe accumulation in grains, but also provide elite wheat germplasms to develop Fe-rich wheat varieties through breeding.

Genome-wide association study reveals the genetic architecture for calcium accumulation in grains of hexaploid wheat (Triticum aestivum L.).

BACKGROUND: Hexaploid wheat (Triticum aestivum L.) is a leading cereal crop worldwide. Understanding the mechanism of calcium (Ca) accumulation in wheat is important to reduce the risk of human micronutrient deficiencies. However, the mechanisms of Ca accumulation in wheat grain are only partly understood. RESULTS: Here, a genome-wide association study (GWAS) was performed to dissect the genetic basis of Ca accumulation in wheat grain using an association population consisting of 207 varieties, with phenotypic data from three locations. In total, 11 non-redundant genetic loci associated with Ca concentration were identified and they explained, on average, 9.61-26.93% of the phenotypic variation. Cultivars containing more superior alleles had increased grain Ca concentrations. Notably, four non-redundant loci were mutually verified by different statistical models in at least two environments, indicating their stability across different environments. Four putative candidate genes linked to Ca accumulation were revealed from the stable genetic loci. Among them, two genes, associated with the stable genetic loci on chromosomes 4A (AX-108912427) and 3B (AX-110922471), encode the subunits of V-type Proton ATPase (TraesCS4A02G428900 and TraesCS3B02G241000), which annotated as the typical generators of a proton gradient that might be involved in Ca homeostasis in wheat grain. CONCLUSION: To identify genetic loci associated with Ca accumulation, we conducted GWAS on Ca concentrations and detected 11 genetic loci; whereas four genetic loci were stable across different environments. A genetic loci hot spot exists at the end of chromosome 4A and associated with the putative candidate gene TraesCS4A02G428900. The candidate gene TraesCS4A02G428900 encodes V-type proton ATPase subunit e and highly expressed in wheat grains, and it possibly involved in Ca accumulation. This study increases our understanding of the genetic architecture of Ca accumulation in wheat grains, which is potentially helpful for wheat Ca biofortification pyramid breeding.

Dissection of the Genetic Architecture for Quantities of Gliadins Fractions in Wheat (Triticum aestivum L.).

Gliadin is a group of grain storage proteins that confers extensibility/viscosity to the dough and are vital to end-use quality in wheat. Moreover, gliadins are one of the important components for nutritional quality because they contain the nutritional unprofitable epitopes that cause chronic immune-mediated intestinal disorder in genetically susceptible individuals designated celiac disease (CD). The main genetic loci encoding the gliadins were revealed by previous studies; however, the genes related to the content of gliadins and their fractions were less elucidated. To illustrate the genetic basis of the content of gliadins and their fractions comprehensively, a recombinant inbred line (RIL) population that consisted of 196 lines was constructed from the two parents, Luozhen No.1 and Zhengyumai 9987. Quantitative trait loci (QTL) controlling the content of total gliadins and their fractions (ω-, α-, and γ-gliadin) were screened genome-widely under four environments across 2 years. Totally, thirty QTL which explained 1.97-12.83% of the phenotypic variation were detected to be distributed on 17 chromosomes and they were gathered into 12 clusters. One hundred and one pairs of epistatic QTL (E-QTL) were revealed, among which five were involved with the total gliadins and its fractions content QTL located on chromosome 1AS, 1DS, 4DS, 1DL, and 6AS. Three Kompetitive Allele-Specific PCR (KASP) markers were developed from three major QTL clusters located on chromosomes 6A, 6D, and 7D, respectively. The present research not only dissects the genetic loci for improving the content of gliadins and their three fractions, but may also contribute to marker-assisted selection of varieties with appropriate gliadin fractions content for end-use quality and health benefit at the early developmental stages and early breeding generations.

Genome-Wide Association Study Reveals Genomic Regions Associated With Molybdenum Accumulation in Wheat Grains.

Molybdenum (Mo) is an essential micronutrient for almost all organisms. Wheat, a major staple crop worldwide, is one of the main dietary sources of Mo. However, the genetic basis for the variation of Mo content in wheat grains remains largely unknown. Here, a genome-wide association study (GWAS) was performed on the Mo concentration in the grains of 207 wheat accessions to dissect the genetic basis of Mo accumulation in wheat grains. As a result, 77 SNPs were found to be significantly associated with Mo concentration in wheat grains, among which 52 were detected in at least two sets of data and distributed on chromosome 2A, 7B, and 7D. Moreover, 48 out of the 52 common SNPs were distributed in the 726,761,412-728,132,521 bp genomic region of chromosome 2A. Three putative candidate genes, including molybdate transporter 1;2 (TraesCS2A02G496200), molybdate transporter 1;1 (TraesCS2A02G496700), and molybdopterin biosynthesis protein CNX1 (TraesCS2A02G497200), were identified in this region. These findings provide new insights into the genetic basis for Mo accumulation in wheat grains and important information for further functional characterization and breeding to improve wheat grain quality.

Promoter DNA hypermethylation of TaGli-γ-2.1 positively regulates gluten strength in bread wheat.

Introduction: Gliadins are the major components of gluten proteins with vital roles on properties of end-use wheat product and health-relate quality of wheat. However, the function and regulation mechanisms of γ-gliadin genes remain unclear. Objectives: Dissect the effect of DNA methylation in the promoter of γ-gliadin gene on its expression level and gluten strength of wheat. Methods: The prokaryotic expression and reduction-oxidation reactions were performed to identify the effect of TaGli-γ-2.1 on dough strength. Bisulfite analysis and 5-Aza-2'-deoxycytidine treatment were used to verify the regulation of TaGli-γ-2.1 expression by pTaGli-γ-2.1 methylation. The content of gluten proteins composition was measured by RP-HPLC, and the gluten strength was measured by Gluten Index and Farinograph. Results: TaGli-γ-2.1 was classified into a subgroup of γ-gliadin multigene family and was preferentially expressed in the later period of grain filling. Addition of TaGli-γ-2.1 protein fragment into strong gluten wheat flour significantly decreased the stability time. Hypermethylation of three CG loci of pTaGli-γ-2.1 conferred to lower TaGli-γ-2.1 expression. Treatment with 5-Aza-2'-deoxycytidine in seeds of strong gluten wheat varieties increased the expression levels of TaGli-γ-2.1. Furthermore, the accumulations of gliadin and γ-gliadin were significantly decreased in hypermethylated wheat varieties, corresponding with the increasing of gluten index and dough stability time. Conclusion: Epigenetic modification of pTaGli-γ-2.1 affected gluten strength by modulating the proportion of gluten proteins. Hypermethylation of pTaGli-γ-2.1 is a novel genetic resource for enhancing gluten strength in wheat quality breeding.

Identification of genomic regions affecting grain peroxidase activity in bread wheat using genome-wide association study.

BACKGROUND: Peroxidase (POD) activity plays an important role in flour-based product quality, which is mainly associated with browning and bleaching effects of flour. Here, we performed a genome-wide association study (GWAS) on POD activity using an association population consisted with 207 wheat world-wide collected varieties. Our study also provide basis for the genetic improvement of flour color-based quality in wheat. RESULTS: Twenty quantitative trait loci (QTLs) were detected associated with POD activity, explaining 5.59-12.67% of phenotypic variation. Superior alleles were positively correlated with POD activity. In addition, two SNPs were successfully developed to KASP (Kompetitive Allele-Specific PCR) markers. Two POD genes, TraesCS2B02G615700 and TraesCS2D02G583000, were aligned near the QTLs flanking genomic regions, but only TraesCS2D02G583000 displayed significant divergent expression levels (P < 0.001) between high and low POD activity varieties in the investigated association population. Therefore, it was deduced to be a candidate gene. The expression level of TraesCS2D02G583000 was assigned as a phenotype for expression GWAS (eGWAS) to screen regulatory elements. In total, 505 significant SNPs on 20 chromosomes (excluding 4D) were detected, and 9 of them located within 1 Mb interval of TraesCS2D02G583000. CONCLUSIONS: To identify genetic loci affecting POD activity in wheat grain, we conducted GWAS on POD activity and the candidate gene TraesCS2D02G583000 expression. Finally, 20 QTLs were detected for POD activity, whereas two QTLs associated SNPs were converted to KASP markers that could be used for marker-assisted breeding. Both cis- and trans-acting elements were revealed by eGWAS of TraesCS2D02G583000 expression. The present study provides genetic loci for improving POD activity across wide genetic backgrounds and largely improved the selection efficiency for breeding in wheat.

Quantitative traits loci mapping and molecular marker development for total glutenin and glutenin fraction contents in wheat.

BACKGROUND: Glutenin contents and compositions are crucial factors influencing the end-use quality of wheat. Although the composition of glutenin fractions is well known, there has been relatively little research on the genetic basis of glutenin fractions in wheat. RESULTS: To elucidate the genetic basis for the contents of glutenin and its fractions, a population comprising 196 recombinant inbred lines (RILs) was constructed from two parents, Luozhen No.1 and Zhengyumai 9987, which differ regarding their total glutenin and its fraction contents (except for the By fraction). Forty-one additive Quantitative Trait Loci (QTL) were detected in four environments over two years. These QTL explained 1.3% - 53.4% of the phenotypic variation in the examined traits. Forty-three pairs of epistatic QTL (E-QTL) were detected in the RIL population across four environments. The QTL controlling the content of total glutenin and its seven fractions were detected in clusters. Seven clusters enriched with QTL for more than three traits were identified, including a QTL cluster 6AS-3, which was revealed as a novel genetic locus for glutenin and related traits. Kompetitive Allele-Specific PCR (KASP) markers developed from the main QTL cluster 1DL-2 and the previously developed KASP marker for the QTL cluster 6AS-3 were validated as significantly associated with the target traits in the RIL population and in natural varieties. CONCLUSIONS: This study identified novel genetic loci related to glutenin and its seven fractions. Additionally, the developed KASP markers may be useful for the marker-assisted selection of varieties with high glutenin fraction content and for identifying individuals in the early developmental stages without the need for phenotyping mature plants. On the basis of the results of this study and the KASP markers described herein, breeders will be able to efficiently select wheat lines with favorable glutenin properties and develop elite lines with high glutenin subunit contents.

In-depth genetic analysis reveals conditioning of polyphenol oxidase activity in wheat grains by cis regulation of TaPPO2A-1 expression level.

Time-dependent darkening and discoloration of wheat product caused by high polyphenol oxidase enzymes (PPO) activity is the most undesirable character in wheat processing industry. We performed GWAS of PPO activity in wheat grains utilizing an association panel and identified 22 significant SNPs. The most significant GWAS peak on chromosome 2A was verified by QTL analysis of PPO activity. The candidate gene for this GWAS peak was identified as TaPPO2A-1, which was the highest expressed PPO gene in wheat grains. The expression level of TaPPO2A-1 was significantly correlated with PPO activity. The most significant association signal for GWAS of the expression values of TaPPO2A-1 pinpointed to the genomic region containing TaPPO2A-1. The results suggested that cis regulation of TaPPO2A-1 expression is the key factor in regulation of PPO activity in wheat grains. The conclusion was further enhanced by haplotype analysis of seven SNPs in the promoter of TaPPO2A-1.

Identification of Novel Genomic Regions and Superior Alleles Associated with Zn Accumulation in Wheat Using a Genome-Wide Association Analysis Method.

Micronutrient deficiencies, and especially zinc (Zn) deficiency, pose serious health problems to people who mainly depend on cereal-based diets. Here, we performed a genome-wide association study (GWAS) to detect the genetic basis of the Zn accumulation in wheat (Triticum aestivum L.) grains with a diversity panel of 207 bread wheat varieties. To uncover authentic quantitative trait loci (QTL) controlling Zn accumulation, the varieties were planted in three locations. In total, 29 unique loci associated with Zn grain accumulation were identified. Notably, seven non-redundant loci located on chromosomes 1B, 3B, 3D, 4A, 5A, 5B, and 7A, were detected at least in two environments. Of these quantitative trait loci (QTL), six coincided with known QTL or genes, whereas the highest effect QTL on chromosome 3D identified in this study was not reported previously. Searches of public databases revealed that the seven identified QTL coincided with seven putative candidate genes linked to Zn accumulation. Among these seven genes, NAC domain-containing protein gene (TraesCS3D02G078500) linked with the most significant single nucleotide polymorphism (SNP) AX-94729264 on chromosome 3D was relevant to metal accumulation in wheat grains. Results of this study provide new insights into the genetic architecture of Zn accumulation in wheat grains.

Genetic Basis of Gluten Aggregation Properties in Wheat (Triticum aestivum L.) Dissected by QTL Mapping of GlutoPeak Parameters.

Bread wheat is one of the most important crops worldwide, supplying approximately one-fifth of the daily protein and the calories for human consumption. Gluten aggregation properties play important roles in determining the processing quality of wheat (Triticum aestivum L.) products. Nevertheless, the genetic basis of gluten aggregation properties has not been reported so far. In this study, a recombinant inbred line (RIL) population derived from the cross between Luozhen No. 1 and Zhengyumai 9987 was used to identify quantitative trait loci (QTL) underlying gluten aggregation properties with GlutoPeak parameters. A linkage map was constructed based on 8,518 SNPs genotyped by specific length amplified fragment sequencing (SLAF-seq). A total of 33 additive QTLs on 14 chromosomes were detected by genome-wide composite interval mapping (GCIM), four of which accounted for more than 10% of the phenotypic variation across three environments. Two major QTL clusters were identified on chromosomes 1DS and 1DL. A premature termination of codon (PTC) mutation in the candidate gene (TraesCS1D02G009900) of the QTL cluster on 1DS was detected between Luozhen No. 1 and Zhengyumai 9987, which might be responsible for the difference in gluten aggregation properties between the two varieties. Subsequently, two KASP markers were designed based on SNPs in stringent linkage with the two major QTL clusters. Results of this study provide new insights into the genetic architecture of gluten aggregation properties in wheat, which are helpful for future improvement of the processing quality in wheat breeding.

Allelic variation of LMW-GS composition in Chinese wheat landraces of the Yangtze-River region detected by MALDI-TOF-MS.

Low molecular weight glutenin subunits are important components of wheat storage proteins, which play an important role in determining end-use quality of common wheat. A newly established matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) procedure was used to analyze 478 landraces of bread wheat collected from the Yangtze-River region in China. Results indicated that 17 alleles at three loci: Glu-A3, Glu-B3 and Glu-D3 were identified, resulting in 87 different allele combinations. Of the 17 alleles detected at all the Glu-3 loci, five belonged to Glu-A3, seven to Glu-B3 and five to Glu-D3 locus. MALDI-TOF-MS indicated Glu-A3a/c was present in 72.8%, Glu-A3b in 8.4%, Glu-A3d in 8.4%, Glu-A3f in 5.2% and Glu-A3e in 3.6% lines. Seven types of alleles were identified at the Glu-B3 locus: Glu-B3d/i (25.5%), Glu-B3b (21.3%), Glu-B3c (16.9%), Glu-B3h (13.8%), Glu-B3f (8.4%), Glu-B3a (8.2%), and Glu-B3g (5.2%). Five types of Glu-D3 alleles were detected: Glu-D3a (58.4%), Glu-D3c (22.6%), Glu-D3d (15.5%), Glu-D3b (3.3%) and Glu-D3f (0.2%). Four new alleles that showed abnormal MALDI-TOF spectrum patterns were identified at the Glu-A3 and Glu-B3 loci. A detailed study is needed to further characterize these alleles and their potential usage for wheat improvement.

Comparative transcriptome profiling of the early infection of wheat roots by Gaeumannomyces graminis var. tritici.

Take-all, which is caused by the fungal pathogen, Gaeumannomyces graminis var. tritici (Ggt), is an important soil-borne root rot disease of wheat occurring worldwide. However, the genetic basis of Ggt pathogenicity remains unclear. In this study, transcriptome sequencing for Ggt in axenic culture and Ggt-infected wheat roots was performed using Illumina paired-end sequencing. Approximately 2.62 and 7.76 Gb of clean reads were obtained, and 87% and 63% of the total reads were mapped to the Ggt genome for RNA extracted from Ggt in culture and infected roots, respectively. A total of 3,258 differentially expressed genes (DEGs) were identified with 2,107 (65%) being 2-fold up-regulated and 1,151 (35%) being 2-fold down-regulated between Ggt in culture and Ggt in infected wheat roots. Annotation of these DEGs revealed that many were associated with possible Ggt pathogenicity factors, such as genes for guanine nucleotide-binding protein alpha-2 subunit, cellulase, pectinase, xylanase, glucosidase, aspartic protease and gentisate 1, 2-dioxygenase. Twelve DEGs were analyzed for expression by qRT-PCR, and could be generally divided into those with high expression only early in infection, only late in infection and those that gradually increasing expression over time as root rot developed. This indicates that these possible pathogenicity factors may play roles during different stages of the interaction, such as signaling, plant cell wall degradation and responses to plant defense compounds. This is the first study to compare the transcriptomes of Ggt growing saprophytically in axenic cultures to it growing parasitically in infected wheat roots. As a result, new candidate pathogenicity factors have been identified, which can be further examined by gene knock-outs and other methods to assess their true role in the ability of Ggt to infect roots.

Characterization of novel LMW-GS genes at Glu-D3 locus on chromosome 1D in Aegilops tauschii.

The objectives of this study were to clarify the relationship between LMW-GS Glu-D3 gene of Ae. tauschii registered in GenBank and the six Glu-D3 genes including 12 allelic variants of common wheat characterized in our previous studies, and identify novel Glu-D3 genes and haplotypes from Ae. tauschii using gene specific PCR amplification. By searching the NCBI database, 13 LMW-GS genes/pseudogenes of Ae. tauschii were retrieved and classified into five gene families based on their nucleotide similarity with the six Glu-D3 genes of common wheat. Of them, four Ae. tauschii genes, AY585350, AY585354, AY585355 and AY585356 matched GluD3-4, GluD3-5, GluD3-1 and GluD3-2 of common wheat, respectively, and one pseudogene AY585351 matched to GluD3-6, but none of them matched to GluD3-3. In order to identify the Glu-D3 genes from Ae. tauschii corresponding to GluD3-3 and GluD3-6 of common wheat, gene specific primers were developed to amplify 8-18 Ae. tauschii entries. As a result, two novel Glu-D3 genes, designated as GluDt3-3 and GluDt3-6, were identified. GluDt3-3 showed seven allelic variants or haplotypes at the DNA level in eight Ae. tauschii entries, designated as GluDt3-31, GluDt3-32, GluDt3-33, GluDt3-34, GluDt3-35, GluDt3-36 and GluDt3-37, respectively. Two to eight SNPs were found among the seven haplotypes and 1-4 amino acid substitutions among the deduced peptides. Multiple sequence alignments showed that the DNA similarity was 99.6-99.9% among the seven GluDt3-3 haplotypes, and 99.4-99.7% between these haplotypes and those of common wheat GluD3-3 gene. GluDt3-6 presented seven haplotypes in 18 Ae. tauschii entries, designated as GluDt3-61, GluDt3-62, GluDt3-63, GluDt3-64, GluDt3-65, GluDt3-66 and GluDt3-67, respectively. GluDt3-61 from Ae. tauschii entry Ae38 was the only one haplotype with complete coding sequence, and the other six were all pseudogenes. Compared with GluD3-6 gene of common wheat, GluDt3-61 exhibited a 3-bp insertion, a 42-bp deletion and 11 base substitutions, leading to a glutamine insertion in position 52, 14 amino acid deletion in position 84-97 and 10 amino acid mutations in its deduced peptide; GluDt3-62 and GluDt3-63 showed a 6-bp insertion, a 24-bp deletion and 15-21 base substitutions in coding region, of which a nonsense mutation from C to T at position 622 resulted in pseudogenes; GluDt3-64 had five base substitution, including a nonsense mutation at the position 742. GluDt3-65, GluDt3-66 and GluDt3-67 all had a base deletion at position 247, as well as 7-8 base substitutions, which resulted in frameshift mutations in the three haplotypes. The results indicated that Ae. tauschii also contains six Glu-D3 genes and their allelic variants are even richer than those in common wheat.