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.     

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.     

Polymorphism of waxy proteins in Spanish durum wheats

A collection of 547 durum wheats (103 cultivars and 444 landraces) from Spain was analysed for waxy protein composition. The electrophoretic patterns showed low polymorphism. At the Wx‐A1 locus, 99.8% of the wheats had the Wx‐Ala allele and only one had the null Wx‐Alb allele. The Wx‐Bl locus was more polymorphic and four different alleles were detected: Wx‐Bla (41.3%), Wx‐Blc (42.6%), a new allele, not detected before in bread wheat and named Wx‐Blf (16.0%), and the null Wx‐Blb allele, found for the first time in one durum wheat. Eleven durum wheats with different allelic composition at the Wx‐l loci were analysed for amylose content. Wheats with the Wx‐Bla allele had a lower amylose content than those with Wx‐Blc or Wx‐Blf. The lowest amylase content was found in the only durum wheat having the null Wx‐Blb allele.     

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.     

Polymorphism of two waxy proteins in the emmer group of tetraploid wheat, Triticum dicoccoides, T. dicoccum, and T. durum

Gel-electrophoretic analyses detected polymorphism of two waxy (Wx) proteins, Wx-Al and Wx-Bl, in 334 accessions of the emmer group of tetraploid wheat, Triticum dicoccoides, T. dicoccum and T. durum. The null allele for the Wx-Al protein (Wx-Alb) was found in one accession of T. dicoccoides and seven of T. dicoccum, but it was not present in those T. durum accessions analysed. The null allele for the Wx-Bl protein (Wx-Blb) did not occur in three emmer-wheat species. Wx-Al and Wx-Bl proteins showing alteration in mobility in SDS-PAGE gel or in isoelectric points were found in five accessions and considered to be the products of new alleles, Wx-Ald, Wx-Ale and/or Wx-Bld. Densitometric analysis of Wx protein bands revealed that Wx-Al was present in smaller amounts than Wx-B1 in almost all accessions.     

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.     

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.     

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.     

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.     

Genetic diversity of European spelt wheat (Triticum aestivum ssp. spelta L. em. Thell.) revealed by glutenin subunit variations at the Glu-1 and Glu-3 loci

Summary High and low molecular weight glutenin subunit (HMW-GS and LMW-GS) compositions of 270 European spelts, 15 Iranian spelts and 25 bread wheat cultivars were analyzed by one- and two-dimensional gel electrophoresis. The results revealed a total of 22 HMW-GS alleles (4 at Glu-A1, 11 at Glu-B1 and 7 at Glu-D1) and 32 allele combinations among the three Glu-1 loci. Two major genotypes of HMW-GS: 1, 13+16, 2+12 and 1, 6.1+22.1, 2+12 were found to occur in Central European spelt wheat cultivars and landraces at higher frequencies of 35 and 28%, respectively. The Glu-B1 locus displayed the greatest variation and genetic diversity index (H) was 0.69 whereas Glu-A1 and Glu-D1 showed H index values of 0.26 and 0.19, respectively. The dendrogram constructed by HMW and LMW glutenin subunit bands revealed that European spelts form a separated cluster from common wheat suggesting that spelt and common wheat form distinct groups. In addition, all 15 Iranian spelt land variety accessions differed from European spelts and possessed similar HMW-GS alleles to common wheat. Because of a wider polymorphism Central European spelt wheats are an important genetic reserviour for improving common wheat quality. Both authors contributed equally to this work     

A novel waxy allele in sorghum landraces in East Asia

A loss of granule‐bound starch synthase I (GBSS I) activity results in starch granules that contain mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles, wx^a, associated with no detectable GBSS I, and wx^b, associated with apparently inactive GBSS I in the endosperm, were reported in sorghum (Sorghum bicolor (L.) Moench). In this study, the waxy alleles in a sorghum core collection were investigated using DNA markers. Of the 337 sorghum accessions examined, 17 accessions that were confirmed to be waxy by a negative iodine staining result and 16 were found to be wx^a. A novel waxy allele, wx^c, was found in a Taiwanese landrace. This allele consists of a +1G to C mutation in the 5′ splice site at the intron 10–exon 11 boundary, a mutation that most likely resulted in the suppression of GBSS I gene expression. A DNA marker specific for wx^c was produced to distinguish the wx^c allele from other alleles, allowing the identification of heterozygous non‐waxy plants.     

Polymorphism of waxy proteins in Iberian hexaploid wheats

A collection of 130 cultivars of bread wheat, 332 landraces of bread wheat and 144 spelt wheats was analysed for waxy proteins in the grain. The electrophoretic patterns showed very low polymorphism and most of the hexaploid wheats had the Wx-Ala, Wx-D1a and Wx-B1 alleles of ‘Chinese Spring’. Two alleles were detected at Wx-A1 (Wx-A1a, and Wx-A1b (null)), the latter was present in only 5.1% of the bread wheat landraces and 7.6% ofthe spelt wheats. No allelic variation was found at the Wx-D1 locus and all the hexaploid wheats had the Wx-D1a allele. Wx-B1 was the most polymorphic locus, with three alleles detected: Wx-B1a, Wx-B1b (null) and Wx-Blc coding for a Wx-B1 protein with a slightly different mobility from Wx-B1a. The null Wx-B1b allele was found in 10.8% of the bread wheat cultivars, 21.4% of the bread wheat landraces and 12.5% of the spelt wheats. Among the 604 hexaploid wheats analysed, only two bread wheat landraces (0.6%) and two spelt wheats (1.4%) had the null allele at both Wx-A1 and Wx-B1 loci.     

Genetic polymorphisms at Gli-Dt gliadin loci in Aegilops tauschii as revealed by acid polyacrylamide gel and capillary electrophoresis

Gliadin variation at Gli‐D^t1 and Gli‐D^t2 loci in 198 Aegilops tauschii accessions was studied by acid polyacrylamide gel electrophoresis (A‐PAGE) and capillary electrophoresis (CE). High genetic polymorphisms were found at both gliadin coding loci, revealing a total of 184 and 169 gliadin variants at the Gli‐D^t1 and Gli‐D^t2 loci, respectively. In particular, 12 gliadin blocks encoded by different alleles were apparently expressed and readily identified in six synthetic hexaploids produced by hybridization between Triticum durum and Ae. tauschii accessions. Compared with Ae. tauschii ssp. eusquarrosa, the gliadin profile of the D genome in Ae. tauschii ssp. strangulata more resembles that of T. aestivum, supporting the view that the subspecies strangulata is the most likely progenitor of bread wheat. Capillary electrophoresis analysis showed that the method is capable of separating and characterizing gliadins with speed, in high resolution using small sample amounts, and is well‐suited to detect protein alleles and to identify desirable genotypes in wheat quality improvement.     

Resistance in spelt wheat to yellow rust

Summary Seven spelt wheat accessions of different origin were hybridized with the susceptible bread wheat cultivar Taichung 29 in order to study the genetics of their resistance to yellow rust (Puccinia striiformis Westend. f. sp. tritici). One Iranian and five European accessions were found to carry Yr5 of Triticum aestivum ssp. spelta var. album, whereas a factor for resistance in the Iranian accession 415 was confirmed to be genetically distinct from Yr5. The alleles for resistance in each of the accessions studied showed a monogenic dominant mode of inheritance. Twenty-eight spelt wheat accessions, including those studied for their resistance to yellow rust, were subjected to polyacrylamide-gel-electrophoresis to study variation for gliadin storage protein patterns. Thirteen distinct patterns were revealed, implying the presence of duplicates within the studied spelt wheat collection.     

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.     

Spanish spelt: a separate gene pool within the spelt germplasm

The renewed interest in spelt (Triticum spelta L.) for wheat improvement programmes requires the study of the available genetic diversity. The purpose of this study was to assess genetic diversity within a Spanish spelt collection. Sixty‐six Spanish spelt accessions, 19 accessions of T. spelta and T. macha from different origins, three bread wheat cultivars (T. aestivum) and one accession of T. dicoccum were screened using simple sequence repeats (SSRs). The diversity observed within the Spanish group was comparable with that observed in the other wheat varieties, despite their broader geographical diversity. Indeed, the highest polymorphic information content value calculated with SSRs for Spanish material (0.90) is similar to that observed for the other wheat varieties (0.98). Principal component analysis explained 46.5% of the cumulative variation and confirmed the Spanish accessions as a separate group. This study showed the Spanish spelt collection to be a variable and unique genetic resource for wheat and spelt breeding programmes.     

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.     

小麦Wx基因近等基因系的创制及其对直链淀粉含量、面条感官品质的影响

为明确不同Wx基因对小麦直链淀粉含量的影响以及筛选面条品质优异的基因型,以糯小麦品系Caiwx (aabbdd)为3个Wx基因隐性突变供体亲本,以扬麦01-2 (AABBDD)为轮回亲本,利用连续回交结合花粉碘染、STS标记和同工酶标记检测方法,创制了8种Wx基因纯合基因型的近等基因系,其基因型分别为AABBDD、AABBdd、AAbbDD、AAbbdd、aaBBDD、aaBBdd、aabbDD和aabbdd。利用这些基因型探讨了不同Wx基因对直链淀粉含量及面条感官品质的影响。结果表明,各系的直链淀粉含量为0.9%~24.8%,系间差异显著;糯小麦型(aabbdd)直链淀粉含量最低,双缺失型和单缺失型其次,双缺失型中aaBBdd型含量最高,单缺失型中AAbbDD型含量最低,表明Wx-B1对直链淀粉的合成作用最大。糯小麦型面条的色泽、表观、软硬度、黏性、韧性得分以及总分显著低于其他类型及轮回亲本扬麦01-2;单缺失型面条的色泽、表观得分、总分显著高于轮回亲本扬麦01-2,其中aaBBDD型面条品质表现突出,与市售优质面条粉“雪花粉”制作的面条得分相当,而其他7种基因型及轮回亲本扬麦01-2的面条评分均显著低于雪花粉。说明可以通过遗传操作Wx基因培育优质面条小麦品种。     

Development of AS‐PCR marker based on a key mutation confirmed by resequencing of Wx‐mp in Milky Princess and its application in japonica soft rice (Oryza sativa L.) breeding

Soft rice with low amylose content (AC) ranging by 5–15% is a unique type with special eating and appearance quality and has become popular in the rice market. We resequenced the Wx‐mp, a key allele from Milky Princess, a Japanese low AC variety, and found that the +473 mutation in exon 4 is the key mutation in both Wx‐mp and its ancestor allele, Wx‐mq from Milky Queen. Based on this functional mutation, an allele‐specific PCR (AS‐PCR) marker was developed and proven in a breeding population derived from a cross between a Chinese late variety Nan Keng 46 (Wx‐mp/Wx‐mp) and an early line Ning 63121(Wx‐b/Wx‐b). Based on the marker‐aided selection by our newly developed AS‐PCR marker for Wx‐mp and the known ST10 marker for Stvb‐i, a total of 12 Wx‐mp homozygotes were selected from 198 F₂ progenies, and four of them were immune to rice stripe virus (RSV) with averagely 11.3 days earlier heading than Nan Keng 46 without significant change in grain yield.     

Regional patterns of microsatellite diversity in Ethiopian tetraploid wheat accessions

This study was conducted to assess regional patterns of diversity of Ethiopian tetraploid wheat accessions and to identify areas of diversity that can be used as source of new germplasm for developing high yielding and stable varieties. A collection of 133 Ethiopian tetraploid wheat accessions and eight introduced cultivars was analysed using 29 wheat microsatellite markers. A total of 383 alleles were detected with an average value of 13.14 alleles per locus. Relatively more alleles were observed in the B genome than in the A genome. Gene diversity indices ranged from 0.08 to 0.95, with a mean value of 0.72. Accessions collected from the same region were pooled and the number of alleles and gene diversity were calculated over the 29 simple sequence repeats for each region. Higher numbers of alleles were detected in the Shewa region (8.72), followed by Tigray (5.86) and Hararghe (5.76). The highest average gene diversity value was found in Shewa (0.65), followed by Gondar (0.64). No significant correlation was observed between geographic distance and genetic distance. Out of 383 different alleles detected, 93 (24.4%) were observed to be region‐specific. Region‐specific alleles were found across all chromosomes except for Xgwm752, Xgwm155 and Xgwm148. Genetic similarity coefficients were estimated for all the possible 55 pairs of regional comparisons and they ranged from 0.16 to 0.52, with a mean value of 0.50. All provinces were differentiated in the UPGMA cluster diagram.