Wx gene in diploid wheat: molecular characterization of five novel alleles from einkorn (Triticum monococcum L. ssp. monococcum) and T. urartu

The Wx gene encodes the granule-bound starch synthase I or waxy protein, which is the sole enzyme responsible for amylose synthesis in wheat seeds. Triticum urartu and einkorn (T. monococcum L. ssp. monococcum), which are related to the A genome of bread wheat, could be important sources of variation for this gene. This study evaluated the Wx gene variability in 52 accessions of these species and compared their nucleotide sequences with the Wx-A1a allele of bread wheat. The level of polymorphism found was high, although not distributed equally between the two species. Five different alleles were found in T. urartu, of which four were novel (Wx-A ^u 1b, -A ^u 1c, -A ^u 1d and -A ^u 1e). All einkorn accessions had the same allele, which was also novel and was named Wx-A ^m 1a. A comparison between the proteins deduced from the novel alleles and the Wx-A1a protein showed that there were up to 33 amino acid changes in both the transit peptide and the mature protein. These results showed that these species, especially T. urartu, are a potential source of novel waxy variants.     

Differential regulation of waxy gene expression in rice endosperm

Summary In order to examine the effects of different alleles on the gene expression at the waxy locus, the Wx gene product which controls the synthesis of amylose was isolated from endosperm starch of rice plants and analysed by electrophoretic techniques. The major protein bound to starch granules was absent in most of waxy strains and increased with the number of Wx alleles in triploid endosperms, suggesting that the major protein is the Wx gene product. In addition to wx alleles which result in the absence or drastic reduction of the Wx gene product and amylose, differentiation of Wx alleles seemed to have occurred among nonwaxy rice strains. At least two Wx alleles with different efficiencies in the production of the major protein as well as amylose were detected. These alleles are discussed in relation to regulation of the gene expression.     

Waxy genes from spelt wheat: new alleles for modern wheat breeding and new phylogenetic inferences about the origin of this species

Background and Aims

Waxy proteins are responsible for amylose synthesis in wheat seeds, being encoded by three waxy genes (Wx-A1, Wx-B1 and Wx-D1) in hexaploid wheat. In addition to their role in starch quality, waxy loci have been used to study the phylogeny of wheat. The origin of European spelt (Triticum aestivum ssp. spelta) is not clear. This study compared waxy gene sequences of a Spanish spelt collection with their homologous genes in emmer (T. turgidum ssp. dicoccum), durum (T. turgidum ssp. durum) and common wheat (T. aestivum ssp. aestivum), together with other Asian and European spelt that could be used to determine the origin of European spelt.

Methods

waxy genes were amplified and sequenced. Geneious Pro software, DNAsp and MEGA5 were used for sequence, nucleotide diversity and phylogenetic analysis, respectively.

Key Results

Three, four and three new alleles were described for the Wx-A1, Wx-B1 and Wx-D1 loci, respectively. Spelt accessions were classified into two groups based on the variation in Wx-B1, which suggests that there were two different origins for the emmer wheat that has been found to be part of the spelt genetic make-up. One of these groups was only detected in Iberian material. No differences were found between the rest of the European spelt and the Asiatic spelt, which suggested that the Iberian material had a different origin from the other spelt sources.

Conclusions

The results suggested that the waxy gene variability present in wheat is undervalued. The evaluation of this variability has permitted the detection of ten new waxy alleles that could affect starch quality and thus could be used in modern wheat breeding. In addition, two different classes of Wx-B1 were detected that could be used for evaluating the phylogenetic relationships and the origins of different types of wheat.     

Molecular characterization and phylogenetic analysis of Wx genes from three Taeniatherum diploid species

In wheat seeds, starch synthase I or the Waxy protein is an enzyme involved in amylose synthesis. The gene encoding this enzyme is Wx and in this study, eight novel Wx alleles were identified in three diploid Taeniatherum species. The variability of these alleles was evaluated, and their nucleotide sequences were compared with those of homologous alleles from wheat. Two types of Taeniatherum Wx alleles were detected in three diploid species Ta. caput-medusae, Ta. asperum, and Ta. crinitum. A phylogenetic analysis indicates that the Taeniatherum Wx alleles were more closely related to Wx alleles from Aegilops species with C, D, M, and U genomes than to Wx alleles of other species. These alleles represent a potential genetic resource that may be useful in wheat breeding programs.     

Identification of SNPs in the<Emphasis Type="Italic"> waxy</Emphasis> gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice

Common non-waxy (Wx) rice cultivars contain two different alleles at the waxy locus, designated Wx ^a and Wx ^b, which encode different levels of granule-bound starch synthases and are hence involved in the control of endosperm amylose content. The Wx ^a allele was predominant in non-waxy indica cultivars, whereas the Wx ^b allele was common to the non-waxy japonica variety. Recently, some of the molecular mechanisms underlying the differentiation of Wx ^a from Wx ^b have been characterized. One structural difference between these two alleles was shown to be due to alternative splicing caused by a single-base substitution (AGGT to AGTT) at a donor site of the first intron within the Wx gene. In the case of waxy (wx) rice, it was not possible to distinguish whether the each wx allele was derived from Wx ^a or Wx ^b alleles by phenotypic analysis. However, we succeeded in developing a derived cleaved amplified polymorphic sequence (dCAPS) marker for the detection of the one-base splicing mutation without the need for sequencing. A mismatch primer was used to generate a restriction site in the Wx ^a allele (AGGT) but not in the Wx ^b allele (AGTT). Three hundred fifty-three waxy rice strains that are widely found in Asia were then employed for analysis using this dCAPS marker. Our findings suggested that waxy rice strains have both Wx ^a- and Wx ^b-derived alleles, but that the Wx ^b-derived allele was predominant, and its distribution was independent of indica-japonica differentiation. The wild relatives of cultivated rice all possessed the AGGT allele. It was concluded that the waxy mutations, and the corresponding rice cultivation, originated from japonica during the evolution and domestication process of rice and was preferentially selected by most Asian peoples.Communicated by J. Heslop-Harrison     

Dosage effects of the three Wx genes on amylose synthesis in wheat endosperm

Amylose synthesis in wheat endosperm is mainly controlled by the granule-bound starch synthase of about 60 kDa, the so-called waxy (Wx) protein. The Wx proteins are the product of the Wx genes at a triplicate set of single-copy homoeoloci located on chromosomes 7A (Wx-A1), 4A (Wx-B1) and 7D (Wx-D1). Using Chinese Spring and its aneuploid lines, including nullisomic-tetrasomics, tetrasomics, ditelosomics and deletion stocks, together with single-chromosome substitution lines for these chromosomes, the effects of varying the dosage of whole chromosomes and chromosome arms, as well as the effects of null alleles, upon amylose synthesis were investigated. Nullisomic 4A and the deletion of chromosome segments carrying the Wx-B1 gene reduced the amylose content by more than 3%. A reasonable agreement was found in the substitution lines. This confirms that the absence of the Wx-B1 gene, or else substitution of this gene by its null allele, has the most striking effect on decreasing amylose synthesis. The removal of chromosomes carrying either the Wx-A1 or the Wx-D1 gene reduces the amylose content by less than 2%. A similar reduction was revealed by substitution of these two genes by the null alleles. Double dosages of chromosomes 7A, 4A and 7D did not increase amylose content, while the tetrasomic chromosomes produced more of the respective Wx proteins. This suggests that a certain level of Wx gene activity or of the Wx proteins led to the maximum amount of amylose.     

Domesticated emmer wheat [T. turgidum L. subsp. dicoccon (Schrank) Thell.] as a source for improvement of zinc efficiency in durum wheat

Modern durum wheat (AABB) is more sensitive to zinc (Zn) deficiency than bread wheat (AABBDD). One strategy to increase productivity and expansion of durum wheat industry in Zn-deficient soils is to improve its ability to grow and yield in such soils. This ability is termed Zn efficiency. In a growth room experiment using soil culture, we assessed the potential of Triticum turgidum L. subsp. dicoccon (Shrank) Thell. (domesticated emmer wheat, AABB) as a genetic resource for further improvement of Zn efficiency in modern durum wheat. Twenty four accessions of domesticated emmer wheat, four durum landraces/cultivars, and two bread wheat cultivars/ advanced breeders lines of known Zn efficiency were tested under Zn deficiency and Zn sufficiency. Significant variation was observed among genotypes in Zn deficiency symptoms, dry matter production, shoot Zn concentration, shoot Zn content and Zn utilisation efficiency (physiological efficiency). We identified domesticated emmer wheat accessions with greater Zn efficiency than modern durum wheat and even bread wheat genotypes. These accessions could be used in breeding programs to improve Zn efficiency of durum wheat. The results suggest that Zn efficiency of durum or bread wheat is likely to be determined collectively by its progenitors.     

Analysis of agronomic and domestication traits in a durum × cultivated emmer wheat population using a high-density single nucleotide polymorphism-based linkage map

Key message

Development of a high-density SNP map and evaluation of QTL shed light on domestication events in tetraploid wheat and the potential utility of cultivated emmer wheat for durum wheat improvement.

Abstract

Cultivated emmer wheat (Triticum turgidum ssp. dicoccum) is tetraploid and considered as one of the eight founder crops that spawned the Agricultural Revolution about 10,000 years ago. Cultivated emmer has non-free-threshing seed and a somewhat fragile rachis, but mutations in genes governing these and other agronomic traits occurred that led to the formation of today’s fully domesticated durum wheat (T. turgidum ssp. durum). Here, we evaluated a population of recombinant inbred lines (RILs) derived from a cross between a cultivated emmer accession and a durum wheat variety. A high-density single nucleotide polymorphism (SNP)-based genetic linkage map consisting of 2,593 markers was developed for the identification of quantitative trait loci. The major domestication gene Q had profound effects on spike length and compactness, rachis fragility, and threshability as expected. The cultivated emmer parent contributed increased spikelets per spike, and the durum parent contributed higher kernel weight, which led to the identification of some RILs that had significantly higher grain weight per spike than either parent. Threshability was governed not only by the Q locus, but other loci as well including Tg-B1 on chromosome 2B and a putative Tg-A1 locus on chromosome 2A indicating that mutations in the Tg loci occurred during the transition of cultivated emmer to the fully domesticated tetraploid. These results not only shed light on the events that shaped wheat domestication, but also demonstrate that cultivated emmer is a useful source of genetic variation for the enhancement of durum varieties.     

Sequence diversity, haplotype analysis, association mapping and functional marker development in the waxy and starch synthase IIa genes for grain-yield-related traits in hexaploid wheat (Triticum aestivum L.)

Development of high-yielding cereal crops could meet increasing global demands for food, feed and bio-fuels. Wheat is one of the world??s most important cereal crops. The biosynthesis of starch is the major determinant of yield in wheat. Two starch biosynthesis genes, the waxy (Wx) genes and the starch synthase IIa (SSIIa) genes, were amplified and sequenced in 92 diverse wheat genotypes using genome-specific primers. Nucleotide diversity, haplotype analysis and association mapping were performed. The first exon (5??-UTR) and the first intron of the three homoeologous Wx genes were isolated using expressed sequence tag sequences. The Wx genes contained 12 exons separated by 11 introns. SNP (single nucleotide polymorphism) frequency ranged from 1 SNP/3,648?bp for Wx-D1 to 1 SNP/135?bp for SSIIa-A1, with an average of 1 SNP/230?bp. The average SNP frequencies in exon and intron regions were 1 SNP/322?bp and 1 SNP/228?bp, respectively. Thirty, 23 and 5 SNPs were identified and formed five, six and five haplotypes for SSIIa-A1, SSIIa-B1 and SSIIa-D1, respectively. However, no association was found between these SNPs and seven yield-related traits. Twenty-two, 15 and 1 SNPs were detected and formed nine, five and two haplotypes for Wx-A1, Wx-B1 and Wx-D1, respectively. Three unique nucleotides C+A+T at SNP5, SNP6 and SNP12 formed Wx-B1-H3, which was significantly associated with increased grain weight, thousand kernel weight, and total starch content in three spring wheat genotypes and five winter wheat genotypes. Cost-effective and co-dominant SNP markers were developed using temperature-switch (TS)-PCR and are being used for marker-assisted selection of doubled haploid lines with enhanced grain yield and starch content in winter wheat breeding programs.     

Patterns and impacting factors of gene evolutionary rate between wild and cultivated emmer wheat (Triticum turgidum)

Tetraploid emmer wheat (Triticum turgidum L., BBAA) is the founder progenitor of bread wheat, providing the valuable genetic resource and gene pool for wheat improvement. However, the evolutionary trajectory of tetraploid wheat, especially the evolutionary fate of different types of genes has not been well studied. In this study, the rate of non-synonymous substitution (d_N) and synonymous substitution (d_S) was calculated by comparing the orthologs between the wild emmer and cultivated durum wheat at the whole genome and subgenome levels to obtain the positively selected genes (PSGs) and negatively selected genes (NSGs). Then, mutation rate, gene length, exon number, GC content, codon bias, and expression level were comprehensively investigated and compared between the PSGs and NSGs. Within both wild emmer and cultivated durum wheat, PSGs between A and B subgenome displayed shorter gene and exon lengths as well as fewer exon numbers compared with NSGs, whereas from wild emmer to cultivated durum wheat, PSGs showed longer gene length and more exon numbers. Furthermore, PSGs displayed much higher expression levels and stronger codon usage bias, but lower genetic diversity compared with NSGs. Finally, two PSGs TdER1-6B, and TdLC7-2A, were found to play the crucial roles in regulating grain width and plant height of tetraploid wheat, respectively. This study systematically investigated the evolutionary, structural, and functional difference between PSGs and NSGs in tetraploid wheat, which will contribute to a better understanding of the selective mode and evolutionary trajectory during wheat domestication and evolution.     

Differential effects of the null alleles at the three Wx loci on the starch-pasting properties of wheat

The amylose/amylopectin ratio and the pasting properties of wheat starch are important in producing marketable flour products, especially Japanese noodles. To determine if null mutations at the three Wx loci confer differences in starch-pasting viscosity, we analyzed the variation associated with the null mutations in three separate sets of recombinant substitution lines of chromosomes 7A, 4A and 7D produced from crosses between Chinese Spring and three single-chromosome substitution lines carrying the null Wx alleles. Differential effects of null alleles at the three Wx loci on starch-pasting properties were revealed. With respect to chromosome 4A, the effect of the Wx-B1b allele, giving a higher peak and breakdown viscosity, was unambiguous. In addition, a QTL of minor effect was identified near the centromere on the short arm. The presence or absence of the Wx-A1 protein gave some variation in peak and breakdown viscosity, but the effects of Wx-Alb were much smaller than those of the Wx-Blb allele. Associated effects of the Wx-D1 locus were detected for the breakdown viscosity as the null Wx-D1b allele produced a higher viscosity than the wild-type Wx-D1a. While negative correlations between amylose content and breakdown viscosity were common in the three populations, the null mutations at the Wx loci produced some variation independent of amylose content. The genetic variation detected for breakdown viscosity was more evident than that for peak viscosity in all three recombinant populations. Received: 20 July 1999 / Accepted: 7 October 1999     

Molecular characterization and diversity of the Pina and Pinb genes in cultivated and wild diploid wheat

Grain hardness is one of the most important characteristics of wheat quality. Soft endosperm is associated with the presence of two proteins in the wild form, puroindoline a and b. The puroindoline genes and their derived proteins are present in the putative wheat diploid ancestors which are thought to be the donors of the A, B and D genomes in common and durum wheat. In this study, we investigated the variability of grain hardness in einkorn, along with the nucleotide diversity of Pina and Pinb genes in a collection of einkorn wheat and T. urartu, in addition to studying the neutrality and linkage disequilibrium of these genes. Various alleles were detected for Pina and Pinb genes including three novel alleles for the Pinb locus: Pinb-A ^m 1i, Pinb-A ^m 1j and Pinb-A ^m 1k. Some differences were found in grain hardness between the different genotypes. The neutrality test showed a different pattern of variation between the two Pin genes. The genetic analysis of a diploid wheat collection has demonstrated that these species are a potential source of novel puroindoline variants. Our data suggest that, although further studies must be carried out, these variants could be used to expand the range of grain texture in durum and common wheat, which would permit the development of new materials adapted to novel uses in the baking and pasta industry.     

Evolution and dispersal of emmer wheat (Triticum sp.) from novel haplotypes of Ppd-1 (photoperiod response) genes and their surrounding DNA sequences

The sequence data from 5' UTR, intronic, coding and 3' UTR regions of Ppd-A1 and Ppd-B1 were investigated for a total of 158 accessions of emmer wheat landraces comprising 19 of wild emmer wheat (Triticum dicoccoides), 45 of hulled emmer wheat (T. dicoccum) and 94 of free-threshing (FT) emmer wheat (T. durum etc.). We detected some novel types of deletions in the coding regions from 22 hulled emmer accessions and 20 FT emmer accessions. Emmer wheat accessions with these deletions could produce predicted proteins likely to lack function. We also observed some novel mutations in Ppd-B1. Sixty-seven and forty-one haplotypes were found in Ppd-A1 and Ppd-B1, respectively. Some mutations found in this study have not been known, so they have potential for useful genetic resources for wheat breeding. On the basis of sequence data from the 5' UTR region, both Ppd-A1 and Ppd-B1 haplotypes were divided into two groups (Type AI/AII and Type BI/BII). Types AI and AII of Ppd-A1 suggested gene flow between wild and hulled emmer. On the other hand, Types BI and BII of Ppd-B1 suggested gene flow between wild and FT emmer. More than half of hulled emmer accessions were Type AII/BI but few FT emmer accessions were of this type. Therefore, over half of the hulled emmer did not contribute to evolution of FT emmer.     

Population divergence in the wheat leaf rust fungus Puccinia triticina is correlated with wheat evolution

Co-evolution of fungal pathogens with their host species during the domestication of modern crop varieties has likely affected the current genetic divergence of pathogen populations. The objective of this study was to determine if the evolutionary history of the obligate rust pathogen on wheat, Puccinia triticina, is correlated with adaptation to hosts with different ploidy levels. Sequence data from 15 loci with different levels of polymorphism were generated. Phylogenetic analyses (parsimony, Bayesian, maximum likelihood) showed the clear initial divergence of P. triticina isolates collected from Aegilops speltoides (the likely B genome donor of modern wheat) in Israel from the other isolates that were collected from tetraploid (AB genomes) durum wheat and hexaploid (ABD genomes) common wheat. Coalescence-based genealogy samplers also indicated that P. triticina on A. speltoides, diverged initially, followed by P. triticina isolates from durum wheat in Ethiopia and then by isolates from common wheat. Isolates of P. triticina found worldwide on cultivated durum wheat were the most recently coalesced and formed a clade nested within the isolates from common wheat. By a relative time scale, the divergence of P. triticinia as delimited by host specificity appears very recent. Significant reciprocal gene flow between isolates from common wheat and isolates from durum wheat that are found worldwide was detected, in addition to gene flow from isolates on common wheat to isolates on durum wheat in Ethiopia.     

Polymorphism of omega-gliadins in durum wheat as revealed by the two-step APAGE/SDS-PAGE technique

Polymorphism of omega-gliadins was studied in 243 durum wheats from 27 countries using the two-step one-dimensional APAGE/SDS-PAGE technique. A total of 12 bands of different mobility were observed, and four of them were found to be different from those previously detected by Khelifi et al. (1992) in bread wheat. Fifteen alleles, six coded by the Gli-A1 locus and nine coded by the Gli-B1 locus, were identified, accounting for 19 different electrophoretic patterns. Seven new alleles were detected: two at the Gli-A1 locus and five at the Gli-B1 locus. The polymorphism found at the Gli-A1 and Gli-B1 loci was slightly greater than that found in bread wheat. Allelic differences between both species were higher at the Gli-B1 locus. A comparison of the frequencies of alleles in both species was carried out. The null allele, Gli-A1e, was more common in durum wheat than in bread wheat. The Gli-B1b allele, present in 60% of the bread wheats, was found in only 2% of the durum wheats and Gli-B1e, very common in durum wheat (45%), was rare in bread wheat (4%). The Gli-B1IV allele, common in durum wheat (28%), was not detected in bread wheat.     

Strong presence of the high grain protein content allele of NAM-B1 in Fennoscandian wheat

Grain protein content in wheat has been shown to be affected by the NAM-B1 gene where the wildtype allele confers high levels of protein and micronutrients but can reduce yield. Two known non-functional alleles instead increase yield but lead to lower levels of protein and micronutrients. The wildtype allele in hexaploid bread wheat is so far mainly known from historical specimens and a few lines with an emmer wheat introgression. Here we report a screening for the wildtype allele in wheats of different origin. First, a worldwide core collection of 367 bread wheats with worldwide origin was screened and five accessions carrying the wildtype NAM-B1 allele were found. Several of these could be traced to a Fennoscandian origin and the wildtype allele was more frequent in spring wheat. These findings, together with the late maturation of spring wheat, suggested that the faster maturation caused by the wildtype allele might have preserved it in areas with a short growing season. Thus a second set consisting of 138 spring wheats of a northern origin was screened and as many as 33?% of the accessions had the wildtype allele, all of a Fennoscandian origin. The presence of the wildtype allele in landraces and cultivars is in agreement with the use of landraces in Fennoscandian wheat breeding. Last, 22 spelt wheats, a wheat type previously suggested to carry the wildtype allele, were screened and five wildtype accessions found. The wildtype NAM-B1 accessions found could be a suitable material for plant breeding efforts directed towards increasing the nutrient content of bread wheat.     

Detection of allelic variation at the Wx locus with single-segment substitution lines in rice (Oryza sativa L.)

Apparent amylose content (AAC) is a key determinant of eating and cooking quality in rice and it is mainly controlled by the Wx gene which encodes a granule-bound starch synthase (GBSS). In this study, sixteen single-segment substitution lines harboring the Wx gene from 16 different donors and their recipient HJX74 were used to detect the naturally occurring allelic variation at the Wx locus. The AAC in the materials varied widely and could be grouped into glutinous, low, intermediate, and two high AAC sub-classes, high I (24.36?C25.20%) and high II (25.81?C26.19%), under different experimental environments, which showed a positive correlation with the enzymatic activity of GBSS. One insertion/deletion (InDel) and three single nucleotide polymorphisms in the Wx gene were detected and their combinations resulted in the variation of five classes of AAC. Based on the results of AAC phenotypes, GBSS activities and cDNA sequences, five Wx alleles, wx, Wx ^t, Wx ^g1, Wx ^g2, and Wx ^g3, were identified, two of which, Wx ^g2 and Wx ^g3, are separated for the first time in this study. Under different cropping seasons, the AAC differed significantly for the Wx ^t and Wx ^g1 alleles, with higher AAC in the fall season than in the spring season, but did not differ significantly for the wx, Wx ^g2, and Wx ^g3 alleles. In conclusion, the present results might contribute to our understanding of the naturally occurring allelic variation at the Wx locus and will facilitate the improvement of rice quality by marker-assisted selection.