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.     

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     

A polymorphic microsatellite in the 3'end of 'waxy' genes of wheat, Triticum aestivum

Potential polymorphism of an (AT)_N microsatellite at the 3’end of waxy genes in bread wheat was examined. Primers were designed from a published cDNA sequence of a wheat waxy gene. Polymerase chain reaction (PCR) amplification of genomic DNA from 135 mainly Australian cultivars revealed eight alleles on chromosome 7A. This polymorphic microsatellite is a potential codominant marker for the Wx-A1 locus in breeding programmes. A distinguishable fragment was also amplified from chromosome 7D. This fragment was absent where a plant was null for the waxy gene on chromosome 7D, being a dominant marker for the Wx-D1 locus. The primers were also useful for amplifying genomic DNA from barley, rye and triticale and can be used to detect potential polymorphism in these species.     

山西冬小麦品种Waxy蛋白分析及分子标记研究

利用聚丙烯酰胺凝胶电泳(SDS-PAGE)法分析了山西100份小麦品种(系)Waxy蛋白的缺失类型,筛选出缺失Wx-B1蛋白亚基的材料8份。利用2个STS标记和1个SSR标记对不同Waxy蛋白缺失类型进行鉴定,验证其在分子标记辅助育种中的有效性,结果表明:Wx-7A位点的STS引物在野生型中扩增出1条450 bp的特异带,而在缺失Wx-A1蛋白亚基的突变材料中扩增出1条327 bp的特异带;Wx-4A位点的STS引物在野生型中扩增出1条440 bp的特异带,缺失Wx-B1蛋白亚基的突变材料中没有该扩增片段;Wx-7A,Wx-7D位点的SSR引物在野生型中分别扩增出1条265 bp和1条204 bp的特异带,在缺失Wx-A1和Wx-D1蛋白亚基的突变材料中没有扩增出该特异带。这3个标记可以用于分子标记辅助育种。     

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.     

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     

Distribution of the photoperiod insensitive <Emphasis Type="Italic">Ppd-D1a</Emphasis> allele in Chinese wheat cultivars

Photoperiod response is of great importance for optimal adaptation of bread wheat cultivars to specific environments, and variation is commonly associated with allelic differences at the Ppd-D1 locus on chromosome 2D. A total of 926 Chinese wheat landraces and improved cultivars collected from nine wheat growing zones were tested for their genotypes at the Ppd-D1 locus using allele-specific markers. The average frequency of the photoperiod-insensitive Ppd-D1a allele was 66.0%, with the frequencies of 38.6 and 90.6% in landraces and improved cultivars, respectively. However, the Ppd-D1a allele was present in all improved cultivars released after 1970 except for spring wheats in high latitude northwestern China, and winter wheats in Gansu and Xinjiang. The presence of the Ppd-D1a allele in landraces and improved cultivars increased gradually from north to south, illustrating the relationship between photoperiod response and environment. Ppd-D1a in Chinese wheats is derived from three sources, Japanese landrace Akagomughi and Chinese landraces Mazhamai and Youzimai. The current information is important for understanding the broad adaptation of improved Chinese wheat cultivars. F. P. Yang and X. K. Zhang contributed equally to this work.     

小麦(T. aestivum)Waxy-D1 基因缺失材料的发现及分析

利用SDS-PAGE技术, 从河北省及其它省引进小麦地方品种900份中, 鉴定出了普通小麦Waxy-D1基因缺失材料一份, Waxy-B1基因缺失材料6份。 这些材料的发现, 为我国开展品质育种研究打下了基础, 尤其是Waxy-D1隐性材料尤为稀少, 是至今为止世界上发现的第二份含有该隐性基因的地方品种。 在小麦淀粉遗传控制研究和品质育种     

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.     

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.     

High genetic diversity of Spanish common wheats as judged from gliadin alleles

Gliadin alleles were identified in 100 common wheat cultivars registered and/or grown in Spain during the last 40 years. A very high level of genetic polymorphism was found: in total, 103 allelic variants including one null‐allele were found at the six major Gli loci in the Spanish wheats studied. An average genetic diversity for these six loci was found to be higher (H=0.844) than in any group of wheat cultivars studied previously. Spanish wheats bred in Spain demonstrated even higher genetic diversity (H=0.868), probably because of the occurrence in this group of some landraces (local varieties) assumed to be strongly differentiated to fit local environments. The high level of genetic diversity of wheats grown in Spain was maintained by the introduction of distantly related wheat germplasm from different sources, especially from Italy and CIMMYT. A slight decrease of genetic diversity in recently registered cultivars might be caused by the excessive introduction of French wheats. Thirteen new alleles found in Spanish wheats were catalogued, including Gli‐D2w which encodes the first Gli‐D2‐controlledγ‐gliadin to be found.     

Characterization of CIMMYT bread wheats for high- and low-molecular weight glutenin subunits and other quality-related genes with SDS-PAGE,RP-HPLC and molecular markers

Two hundred and seventy-three CIMMYT bread wheat cultivars and advanced lines grown under irrigated conditions in Mexico during the 2005-06 Yaqui crop cycle were characterized for quality-related genetic traits using gene-specific markers for some high- and low-molecular-weight glutenin subunit (HMW-GS and LMW-GS) genes, polyphenol oxidase (PPO), phytoene synthase (PSY), and waxy genes. Of them, 142 were analyzed for quality parameters including SDS sedimentation volume (SDS-SV), dough mixing time, and Alveograph parameters, and for HMW-GS and LMW-GS compositions using sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and reversed-phase high-performance liquid chromatography (RP-HPLC). For the Ppo-A1 locus tested with the marker PPO18, the frequencies of alleles Ppo-A1a and Ppo-A1b were 79.1 and 20.2%, respectively, and no PCR fragment was amplified in 2 lines (0.73%), whereas 227 lines (83.2%) contained the allele Ppo-D1a and 46 lines (16.8%) had Ppo-D1b detected by markers PPO16 and PPO29. For the marker YP7A, 142 lines (52.0%) were assumed to have the allele Psy-A1a and 131 lines (48.0%) contained the allele Psy-A1b. In the case of the marker YP7B for the gene Psy-B1, the alleles Psy-B1a and Psy-B1b were detected in 155 (56.8%) and 43 (15.8%) lines, respectively, and 75 (27.4%) lines possessed the allele Psy-B1d detected by the marker YP7B-3. All 273 lines contained the alleles Wx-A1a and Wx-D1a as determined by markers MAG264 and MAG269, respectively. Using the marker Wx-B1, 204 lines (74.7%) were presumed to have the Wx-B1a allele and 69 (25.3%) possessed Wx-B1b. The over-expressing allele of Bx7 ^OE and subunit By8*, not clearly seen with SDS-PAGE, were detected by RP-HPLC. The numbers of lines with subunits Ax2*, By8, By9, Bx17, Bx20, Dx5, and Glu-B3j were 90, 16, 57, 5, 46, 118, and 33, respectively, in the 142 lines analyzed with molecular markers, and were consistent with the results obtained by SDS-PAGE, except for one line with the 1A.1R translocation. Subunits Ax1 and Ax2* at the Glu-A1 locus showed significantly better effects on all quality parameters than subunit Null. Subunits 5 + 10 gave significantly better effects for all parameters. Subunit Glu-A3b showed more positive effects than its alternative alleles on SDS-SV and SDS-sedimentation volume/protein content index (SPI). The allele Glu-B3g showed the best effect on SDS-SV and Alveograph W, whereas Glu-B3j, associated with the 1B.1R translocation, exhibited a strongly negative effect on all quality parameters.     

人工合成六倍体小麦后代衍生群体Waxy蛋白亚基的分子标记

四倍体小麦与节节麦杂交培育的人工合成六倍体小麦已广泛应用于国内外小麦品种改良。以引自CIMMYT的Syn768、Syn769和Syn780人工合成六倍体小麦分别与中国四川成都平原主栽普通小麦品种杂交、回交的BC2F2:6后代群体中选育的113份优良高代系为材料,采用SSR特异引物的PAGE凝胶电泳对其Waxy蛋白亚基缺失类型进行了研究。结果表明,在所检测的121份材料中,8份材料缺失Waxy-B1型蛋白亚基,占全部材料的6.6%;没有检测到其他类型的缺失体。从每个人工合成六倍体小麦亲本材料所形成的后代衍生群体来看, Waxy-B1缺失体频率各不相同,说明Waxy蛋白亚基缺失类型在人工合成六倍体小麦后代衍生群体材料中的表现存在着随机性,与亲本的基因型状况关系极大。通过研究人工合成六倍体小麦与普通小麦杂交后代的Waxy蛋白亚基缺失类型,有助于提高分子标记育种效率。     

Prevalence of a novel puroindoline b allele in Yunnan endemic wheats (Triticum aestivum ssp. yunnanense King)

Grain hardness is a major factor influencing the classification and end-use quality of bread wheat. In this study, 40 Yunnan endemic wheats, 21 historical cultivars and 66 current cultivars and advanced lines were investigated for kernel hardness and puroindoline alleles using molecular and biochemical markers. The frequencies of soft, mixed and hard genotypes were 10.0%, 5.0% and 85.0%, respectively, in Yunnan endemic wheats, whereas the corresponding frequencies were 47.6%, 23.8% and 28.6% in historical cultivars, and 36.3%, 6.1% and 57.6% in current cultivars and advanced lines. Four known puroindoline alleles, Pina-D1b, Pinb-D1b, Pinb-D1d and Pinb-D1e, were found in the hard wheat cultivars. Compared with endemic wheats and historical cultivars, current cultivars from Yunnan province have relatively high frequencies of Pina-D1b and Pinb-D1b alleles at 43.5% and 16.1%, respectively. All 32 hard Yunnan endemic wheats (Triticum aestivum ssp. yunnanense King) contained a new puroindoline b allele, designated Pinb-D1u, that was characterized as a single nucleotide (G) deletion at position 127 in the coding sequence of the Pinb gene, leading to a shift of the open reading frame (ORF) from position 14 in the deduced amino acid sequence and a stop codon corresponding to position Leu-18. The average SKCS hardness of genotypes with Pina-D1b (68.2) is significantly higher than those of Pinb-D1b (60.3) and Pinb-D1u (60.5). The study of puroindoline alleles in Yunnan germplasm could provide useful information for improving processing quality and further understanding the molecular basis of kernel hardness in bread wheat.     

Comparative Analysis of High-Molecular-Weight Glutenin Subunit Composition in Various Triticum Species

Variation in high-molecular-weight glutenin subunit composition amongst Triticum durum cvs. of different origins was investigated by SDS-PAGE and compared with that reported for T. dicoccum and T. aestivum. Tetraploid wheat collections (408 cvs.) were found to carry nearly twice as many Glu-A1 and Glu-B1 alleles as a hexaploid wheat sample of comparable size. In each of the taxa considered, allelic variation at the Glu-B1 locus was markedly greater than that observed for Glu-A1. However, because all the Glu-B1 subunits so far discovered exhibit a restricted and distinctive mobility range during. SDS-PAGE, it is suggested that they are derived from a single source, possibly from Aegilops searsii. Most durum cvs. carried a ‘null’Glu-A1 allele and therefore fewer subunits than dicoccums and common wheats. It is argued that differences in the frequency of occurrence of ‘null’Glu-1 alleles between taxa probably resulted from random samplings made by early agriculturalists and breeders, rather than from an inherent tendency of polyploid wheats to suppress the activity of “redundant” genes.     

CIMMYT普通小麦品系Waxy蛋白类型及淀粉糊化特性研究

将来自CIMMYT第35届IBWSN和第20届SAWSN国际鉴定圃的488份品系于2003年种植在内蒙古临河,对其Wx蛋白亚基组成和淀粉糊化特性进行了研究。利用单向SDS-PAGE鉴定出191份Wx-B1蛋白亚基缺失和28份和Wx-D1蛋白亚基缺失品系。除峰值时间外,其余淀粉糊化特性参数在品系间都有较大变异,与中国春小麦相比,CIMMYT小麦表现峰值黏度、低谷黏度、最终黏度高,稀澥值和反弹值大,峰值时间长而稳定。Wx-B1和Wx-D1蛋白亚基缺失品系的最终黏度和反弹值显著高于和大于Wx蛋白亚基正常品系,Wx-D1蛋白亚基缺失品系的峰值黏度和稀澥值显著低于和小于Wx蛋白亚基正常品系,Wx-B1蛋白亚基缺失品系的峰值黏度和稀澥值显著高于和大于Wx-D1蛋白亚基缺失品系。第35届IBWSN品系的淀粉糊化特性优于第20届SAWSN品系,两者低谷黏度、最终黏度和反弹值间差异显著。     

Variation at Glu-1 Loci in Club Wheats

Hexaploid club wheats (Triticum aestivum L.) possess unique end-use quality characteristics and are grouped as a U.S. market subclass of soft white common wheat. Although there have been many reports on associations among high-molecular-weight glutenin storage protein (HMW-Glu) sub-units with end-use quality in hard wheats; there has been very limited work done on surveying the club wheats for these subunits. The HMW-Glu subunits, spike types and grain color were determined for 41 U.S. club wheat cultivars and 79 club accessions obtained from the National Small Grains Collection (NSGC), USDA-ARS. Accession ‘Harlan JR 35’ (PI 420948), which appears morphologically to be a hexaploid club wheat, was determined to be tetraploid. Egyptian line ‘Maya II-Tel's’ (PI 422288) was shown to have previously undescribed HMW-Glu subunits. In the U.S. club wheats the most common HMW-Glu subunits were: null, 49 % (Glu-A1); 6, 37 % (Glu-B1); and 2 + 12, 94 % (Glu-D1). In the NSGC group the most common HMW-Glu sub-units were: 2*, 52 % (Glu-A1); 7 + 8, 31 % (Glu-B1); and 2 + 12, 92 % (Glu-D1). The high frequency of subunits 2 + 12 in the club wheat groups has not been observed previously in numerous surveys of diverse wheat cultivars. The Glu-B1 subunits 6 (without subunit 8) and 20 which have not been reported in US red wheats, were shown to be common in both club wheat groups. A comparison of diversity indices, including previous studies on HMW-Glu subunit frequencies on all market classes of wheat, showed that the club groups are as diverse for the Glu-A1 and Glu-B1 loci and less diverse for the Glu-D1 locus.