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.     

小麦Wx基因突变体的建立及其淀粉特性的研究

以小麦品种藁城8901为母本,糯麦1号为父本,用“单粒传”法构建了一个含有8种Wx基因突变类型的重组自交系群体 (RIL) 。应用改良的SDS-PAGE方法对RIL-7的Wx基因突变类型进行检测,发现在228个株系中,正常类型的株系有34个,Wx-A1突变体为26个,Wx-B1突变体为32个,Wx-D1突变体30个,Wx-A1和Wx-B1位点同时突变的有28个,Wx-A1和Wx-D1位点同时突变的有20个,Wx-B1和Wx-D1位点同时突变的为28个,3个Wx基因位点均突变的为16个。卡方测验证明,3个Wx基因位点的突变符合孟德尔遗传规律,属于质量性状遗传。田间试验表明,8种类型之间在初花期、株高和穗粒数上无显著差异,但在穗长、每穗小穗数和千粒重上Wx-A1突变型显著低于其他7种类型,而7种类型间无显著差异。淀粉特性研究表明,不同Wx蛋白缺失显著影响直链淀粉含量、淀粉糊化特性和淀粉凝胶的质构剖面分析 (TPA) 特性。正常类型的直链淀粉含量最高(20.8%),糯麦的最低(1.1%)。糯麦淀粉的峰值黏度和稀澥值较高,但其低谷黏度、最终黏度和反弹值较低,与其他缺失类型间差异达5%显著水平。凝胶的TPA测试表明,随着直链淀粉含量的降低,凝胶的硬度、黏着性、弹性、胶着性和咀嚼性显著降低,而黏聚性和回复性显著升高。直链淀粉含量与糊化特性的低谷黏度、最终黏度、反弹值、峰值时间和糊化温度之间正相关达1%显著水平(r=0.892~0.965),与峰值黏度、稀澥值呈1%水平负相关( r=－0.892, r=－0.945);直链淀粉含量与凝胶TPA参数的黏聚性、回复性呈极显著负相关(r=－0.928, r=－0.829),与凝胶的硬度、黏着性、弹性、胶着性和咀嚼性呈极显著正相关( r=0.869~0.979)。     

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     

Amylose synthesis capacity of the three Wx genes of wheat cv. Chinese Spring

The Wx locus controls amylose synthesis in the cereal endosperm. Hexaploid wheat (Triticum aestivum L.) has the three Wx loci on chromosomes 7A ( Wx-A1), 4A (Wx-B1) and 7D (Wx-D1). To verify the effects of null alleles on reducing amylose content and determine the amylose synthesis capacity of each Wx gene independently and accurately, we produced eight possible types of recombinant lines carrying different null alleles at the Wx loci under the ‘Chinese Spring’ genetic background. Amylose content varied from 0% of the waxy ‘Chinese Spring’ to 25% of the ‘Chinese Spring’ normal type. The reducing effect of the single null alleles was the largest in Wx-B1b, and there was no significant difference between Wx-A1b and Wx-D1b. More than 3% reductions in amylose content were detected in the double null types. The results of the double null lines further demonstrated that for the capacity of amylose synthesis, Wx-B1a predominates and produces 21–22% amylose, followed by Wx-D1a (20–21%) and Wx-A1a (15–18%). These significant differences were partly correlated with variation in the amounts of the Wx proteins produced by different Wx genes. However, comparisons of the double null lines with the single null or normal lines indicated that amylose content was not linearly proportional to the number of the Wx genes, suggesting that the Wx genes act in an epistatic manner. This revised version was published online in July 2006 with corrections to the Cover Date.     

Decrease of Waxy (Wx) Protein in Two Common Wheat Cultivars with Low Amylose Content

Two cultivars with a low amylose content, Kanto107 (K107) and K79, were discovered in Japanese common wheat (Triticum aestivum L.). The amount of Wx protein, identified as a single major starch granule-bound protein of about 61 kD by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), was greatly decreased in those two cultivars. Analysis of their Wx protein with a modified SDS-PAGE and two dimensional-PAGE showed that two of the three Wx proteins, produced by Wx-A1 and Wx-B1 loci, were not detected. It was thus concluded that only one locus, Wx-D1, was active in the two low amylose cultivars. These mutants were termed “partial waxy mutants” and considered to be very useful material for breeding waxy wheat.     

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.     

Altered tissue-specific expression at the Wx gene of the opaque mutants in rice

Amylose content is a major determinant of the eating quality in rice. To elucidate the allelic diversity at the Waxy (Wx) gene which controls the amylose synthesis, two cultivated strains having opaque endosperms were studied. The gene responsible for opaque endosperms was introduced into the genetic background of the Japonica type of rice by successive backcrosses, and the two near-isogenic lines (NILs) were selected from the B5 generation. The genetic experiments revealed that an allele, Wx^op, controls opaque endosperms which show chalky as wx endosperms in spite of the production of amylose. Immunoblotting analysis was carried out to compare the gene expression by using the NILs with 4 different alleles (Wx^a, Wx^b, Wx^op and wx). The level of the gene product bound to starch granules was slightly lower in the NILs with Wx^op than that with Wx^b, showing a positive correlation with amylose content in the endosperm. Extracts from mature anthers indicated that the gene product was markedly reduced in the NILs with Wx^op as well as that with wx, showing an altered expression in the tissue specificity in the Wxop lines. Sequence analysis suggested that the Wx^op had been derived from Wx^a, independently of the origin of Wx^b. The importance of the gene regulation was discussed in relation to diversified phenotypes established during the domestication process.     

Intraspecific differentiation and geographical distribution of Wx alleles for low amylose content in endosperm of foxtail millet, Setaria italica (L.) Beauv.

To clarify the genetic mechanism which controls the variation in amylose content among nonwaxy landraces of foxtail millet, the inheritance of different starch types in endosperm was examined by I₂-KI staining. The level of starch granule bound protein in foxtail millet endosperm was also analyzed using SDS-PAGE. The segregation for starch types in F₂ and F₃ seeds determined by I₂-KI staining showed that there are three different alleles at the waxy (wx) locus. A major protein bound to starch granules was detected in nonwaxy endosperm but it was absent in most of the waxy endosperm, suggesting that the protein is the Wx gene product which is responsible for the synthesis of amylose in endosperm. The level of Wx protein proportionally corresponded to the amylose content among nonwaxy landraces. This implies that two different Wx alleles regulate quantitative levels of Wx protein. Landraces with the allele for low amylose content are from Taiwan, the Philippines, Indonesia and Thailand. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

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.     

Distribution of different Vrn-B1 alleles in hexaploid spring wheat germplasm

In wheat, the transition from the vegetative to reproductive stage is primarily controlled by the series of vernalisation (Vrn-1) genes located on the homoeologous group 5 chromosomes. Up to 2009, only two alleles at the Vrn-B1 locus were known: one dominant, spring, allele (now designated Vrn-B1a) and the other recessive, winter, (vrn-B1) allele. Recently, two additional dominant alleles, Vrn-B1b and Vrn-B1c, were described. In this study, we screened a range of hexaploid spring wheat germplasms for the presence of different Vrn-B1 alleles using new diagnostic molecular markers. Our results show that the Vrn-B1a allele was the most prevalent, being present in 55.3 % of the 2,495 accessions examined, followed by the recessive vrn-B1 allele, which occurred in 31.5 % of the accessions. The novel alleles Vrn-B1b and Vrn-B1c were found in 5.3 and 7.9 % of all accessions, respectively.     

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.     

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.     

水稻Wx复等位基因基于PCR的功能标记开发与利用

直链淀粉含量是影响稻米食味品质的重要因素,主要由蜡质基因(Wx)调控,Wx基因座存在Wxa、Wxb、Wxin、Wxmw等多个复等位基因,通过分子标记辅助选择(marker-assisted selection,MAS)培育中低直链淀粉含量的水稻品种是提高稻米适口性的重要途径。Wx复等位基因的功能由单碱基核苷酸多态性(single nucleotide polymorphisms,SNP)决定,其检测依赖于测序或酶切,存在耗时多、费用昂贵等缺点。为提高选择效率,本文利用Wx复等位基因的SNP差异和四引物扩增受阻突变(tetra-primer ARMS-PCR)原理,进行PCR功能标记的开发。引物设计过程中,针对扩增效率低和非匹配的延伸2个技术难题,提出了调整错配位点的解决方案,成功开发了基于PCR技术的两对功能性标记Waxygt-ARMS2和Waxyac-ARMS2,可以准确区分上述复等位基因型,且具有操作简单、耗费较低的特点。利用新开发标记对辽宁省育成的部分水稻品种进行Wx基因型检测的结果表明,40份辽宁育成品种均携带Wxb基因,遗传基础单一。上述结果为利用Wx基因位点的分子标记培育中低直链淀粉含量的水稻品种,改良辽宁省水稻品种的食味品质提供了技术支撑。     

Influence of allelic variations in glutenin on the quality of pan bread and white salted noodles made from Korean wheat cultivars

Dough rheological properties and end-use quality were evaluated to determine the effects of Glu-1 and Glu-3 alleles on those characteristics in Korean wheat cultivars. SDS-sedimentation volume based on protein weight was positively correlated with mixograph parameters and maximum height of dough and also positively correlated with bread volume, crumb firmness and springiness of cooked noodles. Protein content was negatively correlated with optimum water absorption of noodle dough, lightness of noodle dough sheet and hardness and cohesiveness of cooked noodles. Within Glu-1 loci, 1 or 2* subunit and 5 + 10 subunits showed longer mixing time, higher maximum dough height and larger bread volume than other alleles. Cultivars with 13 + 16 subunits at Glu-B1 locus showed higher protein content and optimum water absorption of mixograph than cultivars with 7 + 8 subunits. At Glu-3 loci, Glu-A3d showed longer mixing time than Glu-A3e, and Glu-B3d and Glu-B3h had stronger mixing properties than Glu-B3i. Glu-B3h had higher bread volume and hardness of cooked noodles than Glu-B3d. Glu-D3a had lower protein content than Glu-D3c, and Glu-D3b showed stronger mixing properties than Glu-D3a. Glu-D3c showed lower hardness of cooked noodles than others.     

Comparison of Starch Levels Reduced by High Temperature During Ripening in Japonica Rice Lines Near‐Isogenic for the Wx Locus

One problem caused by high temperature during ripening in Japonica rice cultivars is a reduction in the amount of starch in the endosperm. To better understand this deleterious effect, we compared the accumulation of the two components of starch, amylose and amylopectin in grains ripened at high (32/28 °C) and low (22/18 °C) day/night temperatures in a set of lines of Japonica cultivar Taichung 65 (T65Wx^b) that are near‐isogenic for the Wx locus, which encodes granule‐bound starch synthase I. In T65Wx^b ripened at high temperature, the amount of starch per grain decreased. However, amylose per grain significantly decreased while amylopectin per grain significantly increased. On the other hand, the amount of amylopectin in T65wx, the amylose‐free line, did not differ significantly at the high and low temperatures. These data indicated that high temperatures during ripening did not directly affect amylopectin accumulation in T65Wx^b and that the reduction in starch in T65Wx^b from the high temperatures was caused by a decrease only of amylose. The results for T65Wx^a and T65Wx^op were also consistent with this conclusion. As a result of the decrease in amylose, the outer region of starch granules from T65Wx^b ripened at the high temperatures also had less I₂KI staining. Because this fact might suggest that a portion of amylose was synthesized inside the developing granules after amylopectin synthesis in rice, the effect of amylose deposition in increasing of the density of starch granules is also discussed.     

Reduced height genes and their importance in winter wheat cultivars grown in southern Russia

Reduced height genes are the genetic basis of the “green revolution”. Two agronomically important gibberellic acid (GA)-insensitive genes, Rht-B1b (Rht1) and Rht-D1b (Rht2), localised on chromosomes 4BS and 4DS, respectively, and the GA-responsive gene Rht8, localised on chromosome 2DS, were introduced into many cultivars worldwide. An alternative GA-insensitive gene Rht-B1e (=Rht11) was introduced into Russian wheat cultivars. In this study, we investigated the importance of Rht-B1b, Rht-B1e, Rht-D1b and Rht8 in south Russian bread wheat cultivars. The cultivars were divided into five groups: (1) Rht8c; (2) Rht-B1b; (3) Rht-B1b, Rht8; (4) Rht-B1e, Rht8; and (5) Rht-D1b, Rht8. In the Krasnodar region of south Russia 3,222,321 ha were evaluated for estimating the commercial value of each of these genes in 2009–2011. The results showed that coupling Rht-B1e with Rht8 or Rht-B1b with Rht8 was more successful compared with the effects of other genes or their combinations. The average yield of cultivars carrying Rht-B1e exceeded the average yield of cultivars from the other groups. Our study demonstrates that Rht-B1e can be recommended for use in breeding programs and the presence of a molecular marker for this allele simplifies its transfer to elite wheat germplasm.     

Glutenin allelic variation and 1AL.1RS effects on dough mixing properties of wheat grown in irrigated and rainfed environments

Wheat (Triticum aestivum L.) glutenin allelic variation and presence of the 1AL.1RS wheat-rye (Secale cereale L.) translocation play important roles in determining end-use quality. This study was conducted to evaluate the effects of high and low molecular weight glutenin alleles and 1AL.1RS on dough mixing properties of 189 recombinant inbred lines (RILs) from the cross TAM 107-R7/‘Arlin’ grown in irrigated and rainfed Colorado (USA) environments. The results indicated that (1) higher values (P < 0.05) of some dough mixing properties were observed for Glu-A1b versus Glu-A1a, Glu-B1b versus Glu-B1c, Glu-D1d versus Glu-D1a, and non-1AL.1RS versus 1AL.1RS; (2) no differences in Mixograph properties were found for Glu-A3c versus Glu-A3e, Glu-B3e versus Glu-B3g, or Glu-D3a versus Glu-D3b; (3) although variation at some glutenin loci had little effect on Mixograph properties, pairwise combinations of glutenin loci or a glutenin locus combined with 1AL.1RS affected most Mixograph traits; and (4) in general, the effects of glutenin alleles and 1AL.1RS on dough mixing properties did not differ greatly between the irrigated and the rainfed environment. These results will be useful for assessing potential wheat quality and directing wheat breeding efforts in Colorado and similar environments.