Characterization of waxy grain sorghum lines in relation to granule-bound starch synthase

The waxy phenotype, associated with endosperm containing little or no amylose, has been recognized in sorghum (Sorghum bicolor L. Moench) since 1933. Although variants of the waxy gene are well characterized in other cereals, the waxy trait has been assumed to be controlled by a single allele, wx, in sorghum. Recent improvements in technologies encourage re-examination of the waxy sorghums. The objectives of this research were therefore to identify and characterize sorghum lines with differing waxy alleles and to describe the actions of those alleles in crosses. Grain of eight waxy sorghum lines (BTxARG1, BTx630, Tx2907, B.9307, 94C274, 94C278, 94C289, 94C369), three wild-type checks (BWheatland, RTx430, BN122), and F₂ families from crosses among a subset of these lines were evaluated for presence or absence of granule-bound starch synthase (GBSS), the gene product of the wx locus, and wild-type vs. waxy endosperm. The F₂ segregation ratios were tested for fit to a 3:1 ratio using Chi-square analyses. Two distinctly different naturally occurring waxy alleles were identified: One with no GBSS (GBSS−), and one with apparently inactive GBSS present (GBSS+). We propose that the waxy allele with no GBSS be designated wx^a, and that waxy allele with apparently inactive GBSS present be designated wx^b. These two alleles are located in close proximity on the waxy locus. The wx^b allele is dominant to the wx^a allele in terms of GBSS production, and both are recessive to the wild-type Wx in terms of amylose content. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Development of near-isogenic lines of wheat carrying different null Wx alleles and their starch properties

The granule-bound starch synthase (GBSS I) encoded by the Wxgenes, is involved in amylose synthesis. For analyses of mechanisms of amylose synthesis and associated starch properties in hexaploid wheat, eight possible genotypes having different combinations of the three null alleles at the Wx loci with a common genetic background are a prerequisite. A near-isogenic population of doubled haploid (DH) lines was produced from Chinese Spring × waxy Chinese Spring F₁ plants using the wheat × maize method. The Wx protein phenotypes of the DH progeny were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and found that the null alleles at each of the three Wx loci segregated in a Mendelian fashion. A field trial demonstrated no differences between the eight types for ear emergence time, plant height and grain yield traits. Amylose content in the endosperm starch was highest in the wild type while lowest in the waxy type having no Wx proteins. Comparison between single null types and double null types indicated that the amylose synthesis capacity of Wx-A1a allele is the lowest. Pasting properties of starch are the highest in the waxy type, followed by the double null types. Consequently, both peak viscosity and breakdown were negatively correlated with amylose content. The chain-length distribution analysis of amylopectin structure revealed no clear difference among the eight types,suggesting that the reduced GBSS I activity due to introgression of the null Wx alleles does not affect either the chain length or the degree of branching of amylopectin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Amylose content and starch properties generated by five variant <Emphasis Type="Italic">Wx</Emphasis> alleles for granule-bound starch synthase in common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In common and durum wheats (Triticum aestivum L. and T. durum Desf.), variant waxy (Wx) alleles have been reported for three Wx proteins (Wx-A1, -B1 and -D1), responsible for amylose synthesis in flour starch. Five variant alleles, Wx-A1c, -A1e, -B1c, -B1d and -D1c, were examined to elucidate their effects on amylose content in flour starch. Common wheat lines carrying a Wx protein produced by one variant (e.g., Wx-A1c) and one control (e.g., Wx-A1a) allele were bred and their starches were compared. Results showed that Wx-A1e did not produce amylose (waxy phenotype), whereas three alleles (Wx-A1c, -B1c and -B1d) reduced amylose, and -D1c might have increased it slightly. Most data on blue value, swelling power and starch paste clarity in water and dimethyl sulphoxide also suggested the variant Wx alleles either reduced or increased amylose content.     

Evaluation of the waxy endosperm trait in proso millet (Panicum miliaceum)*

The entire USDA‐ARS maintained collection of 650 accessions of proso millet (Panicum miliaceum L.) was evaluated for the presence of accessions with waxy (amylose‐free) endosperm starch. Six accessions, five of which derived from mainland China, were identified. Segregation ratios for waxy endosperm were evaluated in F₂ and F₃ populations derived from crosses between two waxy accessions, PI 436625 (Lung Shu 16) and PI 436626 (Lung Shu 18), and several wild‐type accessions. The waxy trait was found to be under the control of duplicate recessive alleles at two loci, herein designated wx‐1 and wx‐2. Wild‐type alleles at these loci were designated Wx‐1 and Wx‐2. Iodine‐binding revealed a mean grain‐starch amylose concentration of 3.5% in waxy lines and 25.3% in wild‐type proso. Expression of the granule‐bound starch synthase (waxy protein) in waxy lines was reduced to approximately one‐tenth that of wild‐type accessions. The waxy accessions identified now are available for the introgression of this trait into breeding lines adapted to the Great Plains of North America.     

Genetic variation for waxy proteins and starch properties in Italian wheat germplasm

Granule-bound starch synthase (GBSS) is the primary enzymeresponsible for the synthesis of amylose in amyloplasts of cereal endospermcells. In bread wheat there are three structural genes (Wx-A1, Wx-B1,and Wx-D1) encoding for isoforms of GBSS. The loss of one or moreGBSS isoforms results in the reduction (partial-waxy) or absence (waxy) of amylose in the starch. Waxy wheats may find application inthe production of modified food starch and their flour may be used toextend the shelf life of baked products. In order to breed high qualitywheats able to produce bread with delayed staling, the genetic variabilityfor the waxy trait in our germplasm has been investigated. Weanalysed 288 cultivars of bread wheat, 139 cultivars of durum wheat andabout 200 accessions from other Triticum species. Gel electrophoresisshowed 63 bread wheats deficient in the Wx-B1, one in the Wx-A1 and one in the Wx-D1 protein isoforms, as well as one Triticum dicoccum lacking the Wx-A1 isoform. None of the analysedTriticum monococcum, Triticum durum, Triticum speltaand Triticum timopheevi accessions showed mutations at the Wxloci. The wheat accessions with Wx mutations were evaluated with aRapid Visco Analyser (RVA) to investigate starch properties. All theanalysed cultivars showed Peak Viscosity and Final Viscosity different fromthe normal wheat. Other analyses to evaluate the rheological characteristicsof the partial-waxy genotypes are under way and a breedingprogramme to select new waxy wheat varieties is in progress     

Granule‐bound starch synthase (GBSS) diversity of ancient wheat and related species

Granule‐bound starch synthase of ancient wheat and related species was examined by sodium dodecyl sulphate polyacrylamide gel. A total of 13 different alleles were revealed in a collection of three accessions of diploid wheat, six accessions of tetraploid wheat, 49 accessions of spelt wheat, nine accessions of Sitopsis and two accessions of Aegilops tauschii. A new allele named Wx‐A1a′ appeared in four spelt wheat accessions. The tetraploid wheat accessions evaluated did not show any polymorphism; nevertheless the tetraploid accessions of Sitopsis section revealed three novel alleles. The novel allele Wx‐D^dn1g was found in two accessions of A. ventricosa and the Wx‐D^dcm1h and Wx‐D^dcm1i in two accessions of A. crassa. A novel allele named Wx‐A^u1g was found in Triticum urartu, which is different from the also new Wx‐A^m1h allele of T. monococcum. The diploid‐related species accessions revealed two novel alleles named Wx‐B^sl1h and Wx‐B^s1g found, respectively, in A. longissima and A. speltoides. The amylose content was measured for the different alleles found in all evaluated species and no significant effects of the allele composition on the amylose content were detected.     

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

A 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.     

An alanine to threonine change in the Wx-D1 protein reduces GBSS I activity in waxy mutant wheat

The Wx-D1 protein (granule-bound starch synthase) of Kanto 107, Tanikei A6099 (low amylose line), and Tanikei A6599-4 (waxy line) has been analyzed by SDS-PAGE, peptide mapping and DNA sequencing. Kanto 107 and Tanikei A6099 have the same amino acid sequences in the mature protein, but amino acid substitution (alanine to threonine) occurs at position 258 in the mature protein in Tanikei A6599-4. A comparison of deduced amino acid sequences of the mature Wx-D1 protein in these lines indicates that point mutation in the Wx-D1 gene of Tanikei A6599-4 is responsible for its waxy character.This mutant waxy wheat does not show a reduction in amylose content identicalto other waxy wheats, which probably reduces activity of the GBSS I enzyme but does not to a complete loss of activity. We discuss the function of the mutant Wx-D1 protein in starch synthesis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic studies of speciation in cultivated rice. 5. Inter- and intraspecific differentiation in the waxy gene expression of rice

Summary To get an insight in the gene regulation at the waxy locus of rice, the Wx gene product (Wx protein) controlling the synthesis of amylose was examined by electrophoretic techniques. Among nonwaxy rice strains, two different alleles, Wx ^a and Wx ^b, were found at the waxy locus. Wx ^a drastically enhanced the quantitative level of Wx protein as well as the amylose content in endosperm starch as compared with Wx ^b. The alleles acted additively in triploid endosperms. This implies that regulatory elements responsible for the Wx gene expression are on the same chromosome. The distribution patterns of Wx ^a and Wx ^b in five species of Oryza revealed that the regulatory changes are closely related to racial differentiation within a common rice species (O. sativa), suggesting that Wx ^b might have been selected for through the difference in grain quality during domestication.     

Starch characterisation and variability in GBSS loci of synthetic hexaploid wheats and their durum and <Emphasis Type="Italic">Aegilops tauschii</Emphasis> parents

Greater variability in starch properties is found in lower ploidy wheats than in commercial hexaploid wheats. This paper reports on the starch properties and variability in granule bound starch synthase (GBSS) loci of 17 diploid (Aegilops tauschii) and 12 tetraploid (durums) potential progenitors of wheat, compared with 29 synthetic hexaploid wheats produced from such progenitors. Starch properties examined were granule size distribution, swelling power, amylose content, gelatinisation and amylose-lipid dissociation properties. A PCR screening method was able to detect the presence or absence of each of the three GBSS genes. It also detected polymorphisms in eight diploids and nine hexaploids, all displaying the same 25 bases deletion in the D genome allele of GBSS. Two tetraploids and five hexaploids were null 4A for GBSS. There was little difference in the amylose contents and amylose-lipid dissociation peak temperatures of the synthetic hexaploids and the lower ploidy wheats. The synthetic hexaploids showed intermediate swelling power values with the durums giving the highest swelling powers. The durums also had higher B granule contents than the A. tauschii accessions, but not as high as the synthetics. However, the A. tauschii samples gave the highest gelatinisation peak temperatures. The presence of the null 4A mutation was positively correlated with swelling power, amylose content and DSC measurements. The new smaller D genome allele of GBSS was associated with slightly higher swelling power. These results confirm the value of wheat progenitor lines as sources of new starch properties for hexaploid wheat. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Waxy proteins in diploid, tetraploid and hexaploid wheats

Electrophoretic analyses of waxy proteins, encoded by genes present at the Wx‐1 loci, present in several cultivars and accessions of hexaploid wheat, Triticum aestivum, have permitted the detection of null alleles at the Wx‐B1 and Wx‐D1 loci. Polymorphism at the Wx‐A1 and Wx‐B1 loci was also investigated in several accessions of tetraploid wheats, Triticum durum, Triticum dicoccoides and Triticum timopheevi, and in diploid species, Triticum urartu, Triticum boeoticum and Triticum monococcum. One null allele at the Wx‐A1 locus and three polymorphic alleles at Wx‐B1 locus were detected in T. durum; a new allele at one of the two waxy loci was identified in the tetraploid wheat T. timopheevi; no polymorphism was detected in diploid species. Polymerase chain reaction techniques made possible the detection of further polymorphism existing at the Wx‐1 loci and the reason for the lack of expression of the null genotypes to be investigated. The null forms detected at each locus have been used to produce complete sets of partial and total waxy lines in durum and bread wheat.     

SNPs and an insertion sequence in five Wx-A1 alleles as factors for variant Wx-A1 protein in wheat

The waxy (Wx) gene encodes a granule-bound starch synthase (also called Wx protein) that is involved in synthesizing amylose in the starch grains of cereals, including common wheat (Triticum aestivum L.). Because amylose content affects the quality of food products made from wheat flour, Wx alleles affecting amylose content are of interest. Five wheat Wx alleles (Wx-A1c, -A1d, -A1e, -A1i and -A1j) that produce polymorphic Wx proteins on electrophoretic gels were investigated in terms of amylose content in starch and DNA sequences. Measurement and electrophoresis of gelled starch showed that apparent amylose contents of the genotypes were as follows: Wx-A1e, 2.9 % (= waxy phenotype) < -A1i, 8.0 % < -A1c, 16.8 % < -A1j, 22.6 % = level of wild type allele -A1a. DNA sequencing of the five alleles identified single nucleotide polymorphisms (SNPs) and insertion/deletion variations compared to Wx-A1a. A particular SNP causing amino acid changes in Wx-A1e and -A1c was identified as the factor responsible for decreased amylose. A SNP in Wx-A1d should cause an amino acid change and be responsible for an altered Wx-A1d protein. A transposable-like element of 376 bp present in the 3′ untranslated region (UTR) of Wx-A1i most likely lowered the levels of Wx protein and amylose through aberrant mRNA. The fifth allele, Wx-A1j, possessed four SNPs, two of which altered amino acids in the Wx-A1j protein and should cause polymorphism in the Wx protein. Based on the DNA sequences, functional markers for Wx-A1c, -A1d, -A1e and -A1i were developed.     

Development of a kompetitive allele‐specific PCR marker for selection of the mutated Wx‐D1d allele in wheat breeding

Waxy (Wx) protein is a key enzyme for synthesis of amylose in endosperm. Amylose content in wheat grain influences the quality of end‐use products. Seven alleles have been described at the Wx‐D1 locus, but only two of them (Wx‐D1b, Wx‐D1e) were genotyped with codominant markers. The waxy wheat line K107Wx1 developed by treating ‘Kanto 107’ seeds with ethyl methanesulphonate carries the Wx‐D1d allele. However, no molecular basis supports this nomenclature. In the present study, DNA sequence analysis confirmed that a single nucleotide polymorphism in the sixth exon of Wx‐D1 changed tryptophan at position 301 into a termination codon. Based on this sequence variation, a PCR‐based KASP marker was developed to detect this point mutation using 68 BC₈F₁ plants and 297 BC₈F₂ lines derived from the cross ‘Ningmai 14’*9/K107Wx1. Combined with codominant markers for the Wx‐A1 and Wx‐B1 alleles, waxy and non‐waxy near‐isogenic lines were distinguished. The KASP marker was efficient in identifying the mutant allele and can be used to transfer waxiness to elite lines.     

Identification of allelic variations of puroindoline genes controlling grain hardness in wheat using a modified denaturing PAGE

The amount of long chains (LC) of amylopectin in high-amylose rice is thought to be one of the important determinants of its quality when cooked. A wide range of differences in LC content have been reported in rice varieties, which can be clearly divided into four classes based on LC and apparent amylose content: namely, amylose and LC-free, low or medium-amylose and low-LC, high-amylose and medium-LC, high-amylose and high-LC. However, genetic factors controlling LC content have not been fully understood. Here, we performed quantitative trait loci (QTL) analysis of LC content using 157 recombinant inbred lines (RILs) derived from a cross of a low-LC cultivar, Hyogokitanishiki, and a high-LC line, Hokuriku 142. By analyzing randomly selected 15 RILs, it was shown that high LC content (≥11%) was associated with high setback viscosity (≥200 RVU), and that low LC (≤ 3%) was associated with low setback viscosity (≤ 130 RVU), as measured by a Rapid Visco Analyzer. With setback viscosity as an indicator for LC content, QTL analysis was conducted using 60 DNA markers including a CAPS marker that distinguished Wx a and Wx b alleles coding for granule-bound starch synthase I (GBSSI or Wx protein), the enzyme working for amylose biosynthesis. Only one QTL with a peak log of likelihood score at the wx locus was detected, and no line showing setback viscosity corresponding to the medium-LC class appeared. The fact that wx mutants of Hokuriku 142 lacked LC in their rice starch supports the view that the functional Wx allele is indispensable for LC synthesis in addition to amylose synthesis in rice endosperm. We suggest three possible reasons why no line with medium-LC content was observed. First, the locus (loci) responsible for generation of medium-LC may be located very close to the wx locus and not able to be dissected by the population and DNA markers we used. Second, there may be special QTLs for medium-LC cultivars that do not exist in low- or high-LC cultivars. Third, medium-LC cultivars may have an as-yet unidentified Wx allele with lower capability in LC synthesis compared to the Wx allele in high-LC cultivars.     

A PCR-based DNA marker for detection of mutant and normal alleles of the Wx-D1 gene of wheat

To assist waxy wheat breeding a DNA marker was developed to discriminate mutant and normal alleles at the Wx‐D1 locus. This polymerase chain reaction‐based marker distinguishes the mutant from the normal allele by targeting the previously reported deletion basis of the mutant. The marker codominantly identifies the normal allele of the Wx‐D1 gene from the mutant allele originated from the Chinese landrace ‘Baihoumai’. However, attempts with a number of primer combinations targeting this deletion failed to amplify the corresponding fragment from an unrelated wheat line (NP150) that has a mutant null allele at the same locus. This indicates that NP150 has a different mutant allele from that of ‘Baihoumai’. This marker is a useful tool to identify wheat cultivars with mutant and normal alleles of the Wx‐D1 gene, and is used in marker‐assisted selection of the Wx‐D1 gene in our waxy wheat breeding programme.     

Single nucleotide polymorphism genotyping of the barley waxy gene by polymerase chain reaction with confronting two-pair primers

A high‐throughput single nucleotide polymorphism (SNP) genotyping procedure was developed to select amylose‐free barley mutants whose waxy genes had a C‐ to T‐base substitution in exon 5, which converted Gln‐89 of the wild‐type gene into a termination codon. An F₂ population carrying an amylose‐free waxy gene was checked for segregation. Polymerase chain reaction with confronting two‐pair primers (PCR‐CTPP) produced allele‐specific PCR products that have different sizes and are inherited in a co‐dominant manner. Two alleles of the barley waxy gene with SNP were correctly identified in parental strains using the PCR‐CTPP procedure. Segregation of the SNP as detected by PCR‐CTPP in an F₂ population fitted the expected 1:2:1 ratio. The PCR‐CTPP procedure can provide a time saving and cost‐effective alternative to derived cleaved amplified polymorphic sequence in marker‐assisted selection.     

Gene dosage effect of the wheat Wx alleles and their interaction on amylose synthesis in the endosperm

To find out gene dose effect of each of the three homoeologous Wx genes and their interaction on the production of granule-bound starch synthase (GBSS I) and amylose biosynthesis in the endosperm, Chinese Spring and its near-isogenic waxy types were crossed reciprocally and, obtained a plant population with varying doses of each Wx gene. The amount of GBSSI was increased linearly with increasing gene dose of either of Wxloci. In each of the three Wx loci, the change in amylose content was linear up to 3 doses, with a more potent capacity ofWx-B1a at any dose. Higher level of amylose production was observed in the reciprocal F₁ grains than the expected effect of dose/s of each gene or additive effect of different allelic combination by artificially blend starches which have amylose produced by equivalent number ofWx alleles to that of relevant F₁ cross. When Wx-B1a and Wx-A1a were combined, increase in amylase content was not in proportion to increase in gene dosage. The enhanced amylase synthesis was shown by 2-gene and 3-geneinteraction, indicating that not only type of the three Wx genes and its dose but the interaction among them have significant roles in determining the amylose content. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of two multiplex PCR assays targeting improvement of bread-making and noodle qualities in common wheat

Wheat quality properties are genetically determined by the compositions of high and low molecular weight glutenin subunits, grain hardness, polyphenol oxidase (PPO) activity and starch viscosity. Two multiplex PCR assays were developed and validated using 70 cultivars and advanced lines from Chinese autumn‐sown wheat regions. Multiplex PCR I includes molecular markers for genes/loci ω‐secalin, Glu‐B1‐2a (By8), Glu‐D1‐1d (Dx5), Glu‐A3d, Glu‐B3 (for non‐1B·1R type) and Pinb‐D1b targeting improved gluten parameters and pan bread quality. Multiplex PCR II comprises markers for genes/loci Ppo‐A1, Ppo‐D1 and Wx‐B1b targeting improved noodle quality. The results were consistent with those achieved by SDS‐PAGE and RP‐HPLC, indicating that the two multiplex assays were highly effective, with good repeatability and low costs enabling their use in wheat breeding programmes. In total, nine alleles (subunits) at locus Glu‐B1, four at Glu‐D1 and five at Glu‐A3 locus were identified, and the alleles (subunits) Glu‐B1b (7 + 8), Glu‐B1c (7 + 9), Glu‐D1a (2 + 12), Glu‐D1d (5 + 10), Glu‐A3a, Glu‐A3c and Glu‐A3d were most frequently present in the cultivars and lines tested. The 1B·1R translocation was present in 28 (40.0%) lines, whereas the Wx‐B1 null allele for better noodle quality was present in only seven (10.0%) cultivars and advanced lines, and 37 (52.9%) lines had Pinb‐D1b associated with hard grains. The allele Ppo‐A1b on chromosome 2AL associated with lower PPO activity was present in 38 (54.3%) genotypes, whereas the less effective allele Ppo‐D1a on chromosome 2DL, also associated with low PPO activity was present in 45 (64.3%) of genotypes. These two multiplex PCR assays should be effective in marker assisted selection targeting improved pan bread‐making and noodle qualities.     

Saponin polymorphism in the Korean wild soybean (Glycine soja Sieb. and Zucc.)

Seed saponin composition of 3025 wild soybean (Glycine soja Sieb. and Zucc.) accessions collected from nine regions of Korea was analysed by thin‐layer chromatography to determine its polymorphic variation and geographical distribution and find mutants in saponin components. The saponin composition of seed hypocotyls was primarily divided into seven phenotypes, designated as Aa, Ab, AaBc, AbBc, Aa+α, AaBc+α and AbBc+α. The predominant phenotypes were AaBc (55%), Aa (33%), AaBc+α (7.5%) and Aa+α (3.3%). The frequencies of Ab, AbBc and AbBc+α were very low (0.3‐0.5%). Codominant alleles Sg‐1^a and Sg‐1^b and dominant allele Sg‐4 occupied 98.6, 1.1 and 63.3%, respectively. Alleles Sg‐3 and Sg‐5 were found to be dominant in all the analysed accessions except the mutants. Three accessions were discovered as mutants via LC‐PDA/MS/MS. The accession CWS0115 did not produce saponin Aa and Ax, CWS2133 did not produce saponin Aa and Ab and CWS5095 did not produce any group A saponins. These newly determined mutants might be utilized in producing a new soybean variety with good taste as well as in biosynthetic studies.     

Waxy proteins and amylose content in tetraploid wheats Triticum dicoccum Schulb, Triticum durum L. and Triticum polonicum L.

This paper reports the waxy proteins and apparent amylose contents of the tetraploid species Triticum dicoccum, Triticum polonicum and Triticum durum. Three waxy proteins were found in the three species; two showed the same electrophoretic mobility as the alleles Wx-A1a and Wx-B1a of the hexaploid variety ‘Chinese Spring’, while the third showed the same mobility as the allele Wx-B1e belonging to the hexaploid wheat variety ‘Bai Huo’. In apparent amylose content no significant differences between the alleles Wx-B1a and the Wx-B1e were found for each species. However, the mean amylose contents of T. durum and T. polonicum were significantly greater than that of T. dicoccum, regardless of which allele was present. This revised version was published online in July 2006 with corrections to the Cover Date.