Wheat Alleles Research Articles

Development of STS markers and establishment of multiplex PCR for Glu-A3 alleles in common wheat ( Triticum aestivum L.)

Low-molecular-weight glutenin subunits (LMW-GS) play a key role in determining the processing quality of the end-use products of common wheat. The objectives of this study were to identify genes at Glu-A3 locus, develop the STS markers, and establish multiplex PCR with the STS markers for Glu-A3 alleles. Gene-specific PCR primers were designed to amplify six near-isogenic lines (NILs) and Glenlea with different Glu-A3 alleles ( a, b, c, d, e, f and g) defined by the protein electrophoretic mobility. Three Glu-A3 genes with complete coding sequence were cloned, designated as GluA3-1, GluA3-2 and GluA3-3, respectively. Seven dominant allele-specific STS (sequence tagged sites) markers were designed based on the SNPs (single nucleotide polymorphisms) among different allelic variants for the discrimination of the Glu-A3 protein alleles a, b, c, d, e, f and g. Four multiplex PCRs were established including Glu-A3b + Glu-A3f, Glu-A3d + Glu-A3f, Glu-A3d + Glu-A3g, and Glu-A3b + Glu-A3e. These markers and multiplex-PCR systems were validated on 141 CIMMYT wheat varieties and advanced lines with different Glu-A3 alleles, confirming that they can be efficiently used in marker-assisted breeding.

Low-molecular-weight glutenins in durum wheat: analysis ofGlu-A3alleles using PCR markers

With 1 figure and 1 table Abstract The aim of the present study was to compare the durum and bread wheat alleles encoded at the Glu-A3 locus (represented by Glu-Ad3 and Glu-Aa3, respectively) using PCR markers designed for bread wheat Glu-Aa3 alleles to distinguish different durum alleles. Diversity in low-molecular-weight glutenin subunits (LMW-GS) was analysed in eight cultivars representing all known Glu-Ad3 alleles present in durum wheat. Six of the eight Glu-Ad3 alleles of durum wheat could be distinguished using a combination of seven different primer sets based on LMW-GS sequences from bread wheat. However, allele a (cv. ‘Mexicali’), allele b (cv. ‘Langdon’) and allele c (cv. ‘Cocorit’) of durum wheat could not be distinguished using any of the markers tested. Amplicons of identical size were produced for alleles f (cv. ‘Clarofino’) and allele g (cv. ‘Claro de Balazote’), using all except one primer set (Glu-A3a), indicating genes of these alleles share a very high degree of identity. The markers described in this study are useful for the DNA-based identification of durum wheat cultivars and the identification of the Glu-Ad3 a, b, c, d, e, f, g and h alleles of durum wheat for the purposes of marker-assisted selection.

Analysis of Pina and Pinb alleles in the micro-core collections of Chinese wheat germplasm by Ecotilling and identification of a novel Pinb allele

Kernel hardness is mainly conditioned by allelic variations of Pina-D1 and Pinb-D1 genes located on the short arm of chromosome 5D. In this work, the Ecotilling approach was optimized to investigate Pina and Pinb alleles in the micro-core collections of Chinese wheat germplasm, and three Pina and eight Pinb alleles were found. Generally, more Pinb alleles were detected in the accessions coming from the regions that grow winter or a mixture of spring and winter wheats. This was particularly evident for the Southwestern winter wheat, Xinjiang winter–spring wheat and Yellow and Huai River Valley winter wheat regions. A novel variant (designated as Pinb-D1x) was discovered in one of the accessions from the Xinjiang winter–spring wheat region. Compared to wild type (WT) allele Pinb-D1a, two nucleotide substitutions occurred in the coding region of Pinb-D1x, one (at nucleotide position 257) resulting in the replacement of a WT cysteine residue by tyrosine and the other (at nucleotide position 382) creating a premature stop codon. The implications of our data to understanding the diversity of Pina and Pinb alleles in wheat and to future molecular breeding of wheat kernel hardness are discussed.

A NAC Gene regulating senescence improves grain protein, zinc, and iron content in wheat.

Enhancing the nutritional value of food crops is a means of improving human nutrition and health. We report here the positional cloning of Gpc-B1, a wheat quantitative trait locus associated with increased grain protein, zinc, and iron content. The ancestral wild wheat allele encodes a NAC transcription factor (NAM-B1) that accelerates senescence and increases nutrient remobilization from leaves to developing grains, whereas modern wheat varieties carry a nonfunctional NAM-B1 allele. Reduction in RNA levels of the multiple NAM homologs by RNA interference delayed senescence by more than 3 weeks and reduced wheat grain protein, zinc, and iron content by more than 30%.

The high grain protein content gene Gpc-B1 accelerates senescence and has pleiotropic effects on protein content in wheat

High grain protein content (GPC) is a frequent target of wheat breeding programmes because of its positive effect on bread and pasta quality. A wild wheat allele at the Gpc-B1 locus with a significant impact on this trait was identified previously. The precise mapping of several senescence-related traits in a set of tetraploid recombinant substitution lines (RSLs) segregating for Gpc-B1 is reported here. Flag leaf chlorophyll degradation, change in peduncle colour, and spike water content were completely linked to the Gpc-B1 locus and to the differences in GPC within a 0.3 cM interval corresponding to a physical distance of only 250 kb. The effect of Gpc-B1 was also examined in different environments and genetic backgrounds using a set of tetraploid and hexaploid pairs of isogenic lines. The results were consistent with those observed in the RSLs. The high GPC allele conferred a shorter duration of grain fill due to earlier flag leaf senescence and increased GPC in all four genetic backgrounds. The effect on grain size was more variable, depending on the genotype-environment combinations. These results are consistent with a model in which the wild-type allele of Gpc-B1 accelerates senescence in flag leaves producing pleiotropic effects on nitrogen remobilization, total GPC, and grain size.

Rapid generation of new powdery mildew resistance genes after wheat domestication

Plant defence against pathogens is controlled by disease resistance (R) gene products that directly or indirectly detect specific pathogen effectors. Plant-pathogen interactions have been proposed to follow a co-evolutionary arms-race model where R genes are recent and evolve rapidly in response to structural changes in matching pathogen effectors. However, the longevity and extensive polymorphism of R genes studied were more consistent with balancing selection maintaining ancient and diverse R genes or alleles. In bread wheat (Triticum aestivum), the Pm3 locus confers race-specific resistance to wheat powdery mildew (Blumeria graminis f.sp. triticii). Here we describe recently generated Pm3 resistance alleles that all derive from one susceptible allele, Pm3CS, which is widespread among hexaploid bread-wheat lines. One group of four Pm3 resistance alleles shows few, clearly delimited, polymorphic sequence blocks of ancient origin, embedded in sequences identical to Pm3CS and possibly derived from gene conversion. A second group of three alleles differs from Pm3CS by only two to five mutations, all non-synonymous, and all in the leucine-rich repeat-encoding region. Transient transformation experiments confirmed that Pm3 resistance specificities are based on one or few amino acid changes. The Pm3CS allele was found in wild tetraploid wheat, the ancestor of hexaploid bread wheat, specifically from southern Turkey, a region proposed to be the site of wheat domestication. Based on these data, we propose that the Pm3 resistance alleles were generated in agricultural ecosystems after domestication of wheat 10,000 years ago. The evolution of Pm3 alleles in wheat is best described by the model of evolved recycling, where novel genetic variation is integrated in plant populations together with recycling of old variation.

A Novel Dwarfing Mutation in a Green Revolution Gene from Brassica rapa

Mutations in the biosynthesis or signaling pathways of gibberellin (GA) can cause dwarfing phenotypes in plants, and the use of such mutations in plant breeding was a major factor in the success of the Green Revolution. DELLA proteins are GA signaling repressors whose functions are conserved in different plant species. Recent studies show that GA promotes stem growth by causing degradation of DELLA proteins via the ubiquitin-proteasome pathway. The most widely utilized dwarfing alleles in wheat (Triticum aestivum; e.g. Rht-B1b and Rht-D1b) encode GA-resistant forms of a DELLA protein that function as dominant and constitutively active repressors of stem growth. All of the previously identified dominant DELLA repressors from several plant species contain N-terminal mutations. Here we report on a novel dwarf mutant from Brassica rapa (Brrga1-d) that is caused by substitution of a conserved amino acid in the C-terminal domain of a DELLA protein. Brrga1-d, like N-terminal DELLA mutants, retains its repressor function and accumulates to high levels, even in the presence of GA. However, unlike wild-type and N-terminal DELLA mutants, Brrga1-d does not interact with a protein component required for degradation, suggesting that the mutated amino acid causes dwarfism by preventing an interaction needed for its degradation. This novel mutation confers nondeleterious dwarf phenotypes when transferred to Arabidopsis (Arabidopsis thaliana) and oilseed rape (Brassica napus), indicating its potential usefulness in other crop species.

Marker‐Assessed Retention of Wheat Chromatin in Wheat (Triticum aestivum) by Jointed Goatgrass (Aegilops cylindrica) Backcross Derivatives

With the advent of herbicide‐resistant wheat (Triticum aestivum L.), there is a concern with the potential for gene migration between wheat and jointed goatgrass (Aegilops cylindrica Host.). This is especially true for genes on the D genome, since this genome is shared by wheat and jointed goatgrass. To study the potential for gene migration, BC1 and BC2 plants were produced with jointed goatgrass as the male recurrent parent. To determine if wheat chromatin was retained at expected Mendelian frequencies in these backcrosses, 14 simple sequence repeats (SSR) markers associated with the long and short arms of the D genome chromosomes were used. Chi‐square analysis showed that 12 of the 14 markers fit the expected ratio for retention of wheat alleles in the BC1 generation. In the BC2 generation, 11 of the 14 markers fit the expected frequencies for the retention of wheat alleles. The markers not fitting the expected frequencies in both generations deviated in the direction of more heterozygotes than expected, indicating a higher than expected retention of wheat alleles. Furthermore, recombination between the D genome chromosomes of the two species was observed. On the basis of these results, it appears that it is possible for a gene on the D genome of wheat to move into jointed goatgrass if the BC1 and BC2 generations were produced in the field.

Waxy proteins and amylose content in diploid Triticeae species with genomes A, S and D

AbstractA collection of 89 accessions of diploid species of wheat was analysed for waxy protein in the grain: 39 accessions of Einkorn wheats, 41 accessions of Sitopsis section wheat and nine accessions of Triticum tauschii. The electrophoretic patterns showed low polymorphism. In each group of wheat, a single and different allele was detected. In accessions of Einkorn wheats that allele had a similar electrophoretical mobility to the Wx‐A1a allele of the bread wheat ‘Chinese Spring’, in accessions of the Sitopsis section it had a similar mobility to that of the Wx‐B1f allele of tetraploid wheat, and in the accessions of T. tauschii, it was similar to the Wx‐D1a allele of the bread wheat ‘Chinese Spring’. The accessions were also analysed for apparent amylose content. Results showed that amylose content ranged from 22 to 35% in Einkorn wheats, from 28 to 41% in the Sitopsis section and from 26 to 35% in accessions of T. tauschii.

HMW and LMW glutenin alleles among putative tetraploid and hexaploid European spelt wheat (Triticum spelta L.) progenitors.

The allelic compositions of high- and low-molecular-weight subunits of glutenins (HMW-GS and LMW-GS) among European spelt ( Triticum spelta L.) and related hexaploid and tetraploid Triticum species were investigated by one- and two-dimensional polyacrylamide-gel electrophoresis (PAGE) and capillary electrophoresis (CE). A total of seven novel glutenin alleles (designated A1a*, B1d*, B1g*, B1f*, B1j*, D1a* at Glu-1 and A3h at the Glu-3 loci, respectively) in European spelt wheat were detected by SDS-PAGE, which were confirmed further by employing A-PAGE and CE methods. Particularly, two HMW-GS alleles, Glu-B1d* coding the subunits 6.1 and 22.1, and Glu-B1f* coding the subunits 13 and 22*, were found to occur in European spelt with frequencies of 32.34% and 5.11%, respectively. These two alleles were present in cultivated emmer (Triticum dicoccum), but they were not observed in bread wheat (Triticum aestivum L.). The allele Glu-B1g* coding for 13* and 19* subunits found in spelt wheat was also detected in club wheat (Triticum compactum L.). Additionally, two alleles coding for LMW-GS, Glu-A3h and Glu-B3d, occurred with high frequencies in spelt, club and cultivated emmer wheat, whereas these were not found or present with very low frequencies in bread wheat. Our results strongly support the secondary origin hypothesis, namely European spelt wheat originated from hybridization between cultivated emmer and club wheat. This is also confirmed experimentally by the artificial synthesis of spelt through crossing between old European emmer wheat, T. dicoccum and club wheat, T. compactum.

Multiplex-PCR typing of high molecular weight glutenin alleles in wheat

In Australian commercial cultivars, each high molecular weight glutenin (Glu-1) homoeologous locus consists of one of two predominant alleles: Glu-A1a (subunit Ax1) or Glu-A1b (subunit Ax2*) at the GluA1 locus, Glu-B1b (Bx7 and By8 subunits) or Glu-B1i (Bx17 and By18 subunits) at the Glu-B1 locus, and Glu-D1d (Dx5 and Dy10 subunits) or Glu-D1a (Dx2 and Dy12 subunits) at the Glu-D1 locus. PCR-based assays have been developed in this study to discriminate between these common alleles at each locus. Primers specific for the Glu-A1 Ax2* gene give a single fragment of 1319 bp only in the presence of this gene. Primers targeting the Glu-B1 locus resulted in a co-dominant marker for which the Bx7 genotype produced two fragments (630 bp and 766 bp) and the Bx17 genotype a single fragment (669 bp). The third pair of primers was specific for the Dx5 gene and resulted in a single band of 478 bp. A multiplexed PCR assay was established which permitted the discrimination of the major HMW glutenins in a single PCR reaction and agarose gel assay. As the HMW glutenin composition of a wheat line is extremely important in determining the functional properties of wheat gluten, these markers are useful for the purposes of marker-assisted breeding. These markers may also be useful for the purpose of DNA-based identification of wheat varieties.

Coleoptile Length of Dwarf Wheat Isolines

The dwarfing alleles in wheat (Triticum aestivum L.) are associated with shorter coleoptile length that can produce unacceptable and erratic stands. The combined effect of temperature (2, 10, 18°C) and five gibberellic add (GA) concentrations [0, 5, 50, 100, 500 mg L -1 of potassium gibberellin A 3 (C 19 H 21 O 6 K)] in the germination medium on the coleoptile length of four homozygous isolines [normal or T-0 (Rht-B1a/Rht-D1a), semidwarf-1 or SD-1 (Rht-B1b/Rht-D1a), semidwarf-2 or SD-2 (Rht-B1alRht-D1b), dwarf or D-12 (Rht-B1b/Rht-D1b)] in 'Marfed' (spring) and 'Burt' (winter) was studied. With each decrease in temperature, the coleoptile length in each isoline and cultivar increased, with the increase greater in those isolines and cultivars having the shortest length at 18°C and containing at least one dwarfing allele (Rht-B1b andlor Rht-Dlb). At 2°C, higher GA concentrations increased coleoptile length over 0 GA mg L -1 in all isolines, with the greatest increase at the 500 mg L -1 concentration. The three midconcentrations (5, 50, 100 mg L -1 ) resulted in an intermediate but almost equal increase. Increasing temperatures decreased the response to GA so that at 18°C, only T-0 and SD-1 responded to GA applications. The reported GA insensitivity was found to be highly temperature dependent, with the Rht-B1b allele having a wider temperature response range than Rht-Dlb. The results suggested that differences in genetic background, possibly related to the winter-spring growth habit, could influence the effect of the dwarfing alleles.

Specific Glu‐D1ƒ Allele Frequency of Japanese Common Wheat Compared with Distribution of Glu‐1 Alleles in Chinese Wheat

ABSTRACTThe quality of wheat (Triticum aestivum L.) grain favored in breadmaking is strongly affected by components of seed storage protein, particularly high molecular weight glutenin subunits (HMW‐GS). The HMW‐GS 2.2 controlled by the Glu‐D1ƒ allele is frequently found in Japanese cultivars and landraces. In the investigation into the factors affecting the distribution of the allele, the available data on HMW‐GS of common wheats from Japan were analyzed and compared with the data for intensity of winter habit and wheat flour hardness. We show that the main factors affecting the Glu‐D1ƒ allele frequency in Japanese wheat were the intensity of natural selection for winter habit and artificial selection for flour hardness. According to a study of the worldwide distribution of Glu‐1 alleles, the Glu‐D1ƒ allele is rare. However, Glu‐D1ƒ allele was the most common Japanese wheat seed storage protein allele. It is well known that Chinese wheat contributed to Japanese landraces, and Japanese landraces contributed to modern cultivars from Japan. However, common Japanese and Chinese wheats differ in the frequencies of Glu‐D1ƒ allele. These results may be explained either by the founder effect or by a selective bottleneck in Japanese common wheat genetic resources.

Spelt-specific alleles in HMW glutenin genes from modern and historical European spelt ( Triticum spelta L.).

A partial promoter region of the high-molecular weight (HMW) glutenin genes was studied in two wheat specimens, a 300 year-old spelt ( Triticum spelta L.) and an approximately 250 year-old bread wheat ( Triticum aestivum L.) from Switzerland. Sequences were compared to a recent Swiss landrace T. spelta'Oberkulmer.' The alleles from the historical bread wheat were most similar to those of modern T. aestivumcultivars, whereas in the historical and the recent spelt specific alleles were detected. Pairwise genetic distances up to 0.03 within 200 bp from the HMW Glu-A1-2, Glu-B1-1 and Glu-B1-2 alleles in spelt to the most-similar alleles from bread wheat suggest a polyphyletic origin. The spelt Glu-B1-1 allele, which was unlike the corresponding alleles in bread wheat, was closer related to an allele found in tetraploid wheat cultivars. The results are discussed in context of the origin of European spelt.

Contrasting Ppd alleles in wheat: effects on sensitivity to photoperiod in different phases

Photoperiod sensitivity is an important feature of flowering time regulation, which enables wheat plants to adapt to a wide range of environments. Although some work has been done on how time to heading or flowering respond to photoperiod in relation to particular Ppd alleles, there is little evidence on whether these alleles contribute to responses at different phases and to associated yield component generation. The aims of this paper were: (i) to analyse the effects of photoperiod on substitution lines with contrasting Ppd alleles, in terms of duration of particular phases, (ii) to determine if there is any relationship between these alleles and the parameters of photoperiod response (photoperiod sensitivity, optimum photoperiod and basic length of the phase), and (iii) to analyse the effects of different photoperiods applied before and after the onset of terminal spikelet on yield component generation. The effects of length and timing of photoperiod extensions on these traits were analysed under field conditions in Chinese Spring and two substitution lines differing in photoperiod sensitivity. Although time to anthesis was similar in the three genotypes in photoperiods longer than 14.5 h, they did differ in their response to photoperiod in particular phases. Sensitivity to photoperiod for daylengths shorter than 14.5 h was also markedly different. The number of leaves generated was affected by photoperiod, determining the duration of the phase from emergence to floral initiation (EM-FI). The length of the phase from floral initiation to terminal spikelet (FI-TS) was determined by the number of spikelets generated and their rate of initiation, which was also affected by photoperiod. The terminal spikelet to anthesis phase (TS-ANT) was only affected by photoperiod in the most sensitive genotype, in which direct photoperiod effects, other than the effects on leaf number and phyllochron, were evident. There was no apparent relation between photoperiod response parameters such as basic length of the phase (Lb) and optimum photoperiod (Po) and particular Ppd alleles.

More than one origin of hexaploid wheat is indicated by sequence comparison of low-copy DNA

Allohexaploid bread wheat is grown on more acreage than any other cereal crop, yet variation at the DNA level seems to be less than that observed in many diploid crop species. A common explanation for the small amount of DNA-level variation is that a severe bottleneck event resulted from the polyploidization events that gave rise to hexaploid wheat, whereby wheat was genetically separated from its progenitors. In this report, we test the extent of the bottleneck separating wheat from its D-genome progenitor, Triticum tauschii, by comparative DNA sequence analysis. Restriction site variation of low-copy DNA sequences amplified by PCR showed an average of 2.9 and 2.4 alleles per primer set in T. tauschii and wheat, respectively. Two different restriction patterns were present in T. tauschii for DNA amplified with a primer set for the A1 locus. Both alleles were also present in wheat. Alleles at the A1 locus were cloned and 527 bp of sequence obtained from 12 and 13 diverse accessions of wheat and T. tauschii, respectively. Average genetic distance among the wheat alleles was similar to that among the T. tauschii alleles (0.0127 and 0.0133, respectively). Nucleotide differences indicated that two distinct alleles existed in T. tauschii, both of which were present in wheat. These data suggest that hexaploid wheat formed at least twice, and that the bottleneck separating wheat from T. tauschii may be less constrictive than previously supposed.Key words: wheat, evolution, DNA.

Variation and classification of B low-molecular-weight glutenin subunit alleles in durum wheat

The B low-molecular-weight (LMW) glutenin subunit composition of a collection of 88 durum wheat cultivars was analyzed. Extensive variation has been found and 18 different patterns were detected. Each cultivar exhibited 4–8 subunits, and altogether 20 subunits of different mobility were identified. The genetic control of all these subunits was determined through the analysis of nine F2 populations and one backcross. Five subunits were controlled at the Glu-A3 locus, 14 at Glu-B3 and 1 at Glu-B2. At the Glu-A3 locus each cultivar possessed from zero to three bands and eight alleles were identified. At the Glu-B3 locus each cultivar showed four or five bands and nine alleles were detected. Only one band was encoded by the Glu-B2 locus. A nomenclature for these alleles is proposed and the relationship between them and the commonly used LMW-model nomenclature is discussed.

Identification of Glutenin Alleles in Wheat and Triticale Using PCR‐Generated DNA Markers

Quality in wheat (Triticum aestivum L. em Thell.) is a very complex trait; however, the water insoluble gluten proteins are responsible for the elasticity and cohesiveness (strength) of dough and are important determinants of breadmaking quality. High molecular weight glutenin subunits encoded by genes on the long arms of Group 1 chromosomes have been associated with gluten strength, and a portion of the variability between cultivars can be attributed to glutenin subunit composition. Of the glutenins, Subunits 5 + 10 encoded by the D genome have been found to have the largest positive effect on dough strength, whereas the allelic Subunits 2 + 12 have a negative effect. Therefore, it has been important to incorporate the genes for the 5 + 10 subunits into bread wheats. There has been considerable interest in improving the dough strength and quality of soft wheats and triticale (X Triticosecale Whittm.) to use them in bread‐like products. Glutenin subunit screening is accomplished using electrophoresis (SDS‐PAGE). In this paper, we report the development of an alternative screening method based on the glutenin genes themselves, using the polymerase chain reaction (PCR). Primers designed from computer analyses were synthesized and tested on the cloned subunit 10 gene (Glu‐D1‐2b), 10 wheat cultivars of known subunit composition, and six triticales. Although the Glu‐D1‐2b (Dy10) and Glu‐D1‐2a (Dy12) genes have 98.9% DNA sequence similarity, marker fragments capable of consistently identifying those genotypes were amplified. Fragments correlating with glutenin subunit composition offering possibilities of extending the PCR screening system to other subunits were noted.

The effects of ‘gibberellin-insensitive’ dwarfing alleles in wheat on grain weight and protein content

Three series of near-isogenic wheat lines differing in dwarfing alleles, in the varietal backgrounds of 'Maris Huntsman', 'Maris Widgeon' and 'Bersee', and the F2 grain on intravarietal F1 hybrids, produced with a chemical hybridising agent, were examined for grain size and protein content. Individual F2 grains from Rht1/rht, Rht2/rht and Rht3/rht F1 spikes were classified for Rht genotype by assaying embryo half grains in a gibberellic acid seedling response test, while the remaining half was used for protein determination. Mean grain weight and protein percentage were lower in all homozygous isogenic lines and the Rht/rht F1 hybrids than in the respective tall lines, in an allele dose-dependent manner. In all the hybrids, the Rht genotype of individual F2 grains, which segregated within the spikes of F1 plants, had no significant effects on grain weight or protein. Consequently, the pleiotropic effects of the Rht alleles on these yield and quality components must be attributed to their presence in maternal plant tissues rather than in the endosperm or embryo tissues of individual grains.