Rapid classification of partial waxy wheats using PCR-based markers.

Mutations in the three homeologous waxy loci Wx-A1, Wx-B1, and Wx-D1 of a waxy wheat line have previously been characterized at the molecular level. Using combinations of these mutations, six types of partial waxy wheat plus wild type and waxy wheat (types 1-8) can be produced. Here, we describe primer sets for all three loci that can be used under a single set of conditions, allowing 32 lines to be characterized as types 1-8 in a single PCR run using a 96-well plate. Using multiplex PCR, mutations at the Wx-B1 and Wx-D1 loci can be identified in a single PCR, reducing the number of reactions necessary to identify and select the desired partial waxy wheat line. A single multiplex PCR can be used to detect all three mutations when products are analyzed using capillary electrophoresis on a microchip device. The PCR conditions and primers are effective with a number of cultivars from other countries, indicating that the mutations found at the Wx-A1 and Wx-B1 loci of these cultivars likely have the same origins as the mutations in the corresponding loci of the waxy wheat line used in this study. The PCR selection method described here is an easy and effective alternative to the commonly used SDS-PAGE methods for identification of null alleles.     

Waxy protein deficiency and chromosomal location of coding genes in common wheat

Deficiency of the wheat waxy (Wx) proteins (Wx-A1, Wx-B1 and Wx-D1) was studied in 1,960 cultivars derived from several countries. Gel electrophoretic analyses revealed that the null allele for the Wx-A1 protein occurred frequently in Korean, Japanese and Turkish wheats but was relatively rare in cultivars from other countries and regions. About 48% of the wheats deficient for the Wx-B1 protein were from Australia and India. One Chinese cultivar lacked the WxD1 protein. While 9 Japanese cultivars were deficient in both the Wx-A1 and Wx-B1 proteins, no cultivars lacked both the Wx-A1 and Wx-D1 proteins, both the Wx-B1 and Wx-D1 proteins or all three Wx proteins. Two-dimensional gel electrophoresis revealed polymorphisms of the three Wx proteins that varied according to isoelectric points or molecular weight. The Wx-A1 gene coding the Wx-A1 protein and the Wx-B1 gene coding the Wx-B1 protein were localized in the distal regions of chromosome arms 7AS and 4AL, respectively, by deletion mapping using the deletion lines developed in the common wheat cultivar Chinese Spring.     

小麦糯性基因的多重PCR分子鉴定

采用多重 PCR 的方法, 对其反应条件进行优化, 以获得用于小麦糯性(Wx)基因分析的稳定PCR体系。应用两对引物, 分别扩增小麦 Wx-A1、Wx-B1、Wx-D1 基因, 目的片段大小分别为: 230 bp/265 bp、854 bp和 204 bp。经反复验证, 结果准确可靠, 重复性好, 成本低, 可以在同一PCR反应体系中对 3 个Wx 基因进行同时筛选鉴定。该体系可用于 Wx 蛋白基因的分子标记辅助选择, 可以提高小麦淀粉品质评价和糯麦选育的效率。     

小麦淀粉粒束缚淀粉合成酶基因多态性的分子鉴定

运用6％的SDS-PAGE对14个小麦品种成熟籽粒Wx蛋白的多态性进行了鉴定,结果表明,14个小麦品种根据其Wx蛋白的缺失情况可分为6种组合类型。另外,根据Wx-A1、Wx-D1和Wx-D1这3个位点基因序列和变异情况分别设计了PCR引物,扩增结果表明：Wx-A1位点突变材料扩增产物为327bp,正常材料中扩增不到该特异带;在Wx-B1位点扩增出187bp目标带,突变材料没有该扩增产物;在Wx-D1位点扩增出约700bp目标带,突变材料没有该特异带。与前人的研究结果相比,Wx-B1引物在3个位点的扩增产物长度更短,差异更大,在2％琼脂糖胶上即可清楚分开,缩短了鉴定时间,提高了效率,为大规模筛选优质面条小麦品种提供了可能。     

Identification of three Wx proteins in wheat (Triticum aestivum L.)

Nullisomic analysis of waxy (Wx) protein of hexaploid wheat (Triticum aestivum L.) cv. “Chinese Spring” using two-dimensional polyacrylamide gel electrophoresis revealed that threeWx loci,Wx-A1, Wx-B1, andWx-D1, located on chromosome arms 7AS, 4AL, and 7DS, produce three distinct Wx subunit groups, subunit group-A (SGA), SGB, and SGD, respectively. SGA has a higher molecular weight and a more basic isoelectric point (pI) than the other two. SGB and SGD have the same molecular weight but a slightly different pI range. Owing to the detection of these three subunit groups, we were able to identify the expression of three waxy genes in wheat endosperm and to find two types of mutants among Japanese wheat cultivars, one lacking SGA and the others SGB. These results suggest the possibility of breeding a waxy wheat.     

小麦Wx-B1基因酶切片段长度多态性及其与直链淀粉含量的关系

采用序列标志位点(sequence-tagged sites,STS)引物,对35个小麦品种的Wx-B1基因位点上的近第4个内含子区域的序列进行了扩增,用限制性内切酶BamHI切割.结果表明,扩增片段有的能被酶切,有的不能.酶切片段出现两种长度多态性,其长度与直链淀粉含量(AC)呈负相关.AC在约20%以上的小麦品种扩增的片段能够被BamHI切割,而在约20%以下的不能.以上结果表明,Wx-B1基因在扩增序列区域有多态性,这可以作为一种分子标记在育种中预测不同小麦品种的直链淀粉含量,以改良小麦品质.     

47份外引小麦种质资源Waxy和HMW-GS分子检测与品质性状分析

为有效利用外引小麦种质资源,本研究对收集的47份外引小麦种质材料进行Waxy和HMW-GS等位基因的分子检测,并分析了其直链淀粉、支链淀粉、湿面筋等品质参数。结果表明,在Wx-A1位点存在3种类型:Wx-A1a、Wx-A1g和WxA1b,39份材料(82.98%)为Wx-A1a类型;Wx-B1位点3种类型:Wx-B1a、Wx-B1e和Wx-B1b,37份材料(78.72%)为Wx-B1a类型;Wx-D1位点2种类型:Wx-D1a和Wx-D1b,46份材料具有Wx-D1a类型;共鉴定出8种Wx-1位点等位基因组合,31份材料(65.96%)为Wx-A1a/B1a/D1a。在Glu-A1位点,含有等位基因Ax2^*、Null和Ax1类型的材料分别为18份、18份和11份;在Glu-D1位点,含有等位基因Dx2和Dx5类型的材料分别为23份(48.94%)和21份(44.68%),含有等位基因Dy12和Dy10类型的材料分别为22份(46.81%)和20份(42.55%),具有Dy10+Dy12类型材料2份;共鉴定出19种Glu-A1/D1等位基因组合,7份材料含有Null/Dx5+Dy12。含有Wx-A1a/B1a/D1a材料的直链淀粉含量相对较高,支链淀粉含量相对较低;含有优质等位基因Ax1或Ax2^*兼Dx5+Dy10材料的湿面筋含量相对较高。总体上这些外引种质资源Waxy和HMW-GS等位基因类型丰富,可为种质资源合理利用和现代普通小麦品质改良提供参考依据。     

利用回交结合Wx基因分子标记培育部分糯性小麦

颗粒结合型淀粉合成酶Ⅰ(GBSSI,Waxy蛋白)是小麦胚乳中直链淀粉合成的关键酶。小麦基因组中存在3个Waxy基因(Wx-A1、Wx-B1、Wx-D1)。在白火麦中Wx-D1位点的突变(Wx-D1b)引起Wx-D蛋白的缺失,导致直链淀粉含量下降,其面粉表现出部分糯性。与目前生产上推广品种相比,白火麦的农艺性状较差,产量非常低。为了培育农艺性状优良的部分糯性小麦,我们将白火麦与具有优良农艺性状的小麦品种PH85-16、济南17和烟农15(轮回亲本)分别进行杂交,后代分别与相应的三个轮回亲本回交五代,在每代中均选择农艺性状与轮回亲本相近并含有Wx-D1b的后代。在第六代自交后选择具有Wx-D1b的纯合体,选出的单株连续自交三代。获得了六个农艺性状与轮回亲本相似的品系,它们均携带纯合的Wx-D1b位点。研究表明采用回交的方法并结合基于Wx基因序列的分子标记技术,是培育优良部分糯性小麦的一种非常有效的方法。本研究培育出的部分糯性小麦品系可以直接用于大田生产。     

Differential effects of Wx-A1, -B1 and -D1 protein deficiencies on apparent amylose content and starch pasting properties in common wheat

Waxy (Wx) protein is a granule-bound starch synthase (GBSS) responsible for amylose production in cereal endosperm. Eight isolines of wheat (Triticum aestivum L.) having different combinations of presence and absence of three Wx proteins, Wx-A1, -B1, and -D1, were produced in order to elucidate the effect of Wx protein deficiencies on the apparent amylose content and starch-pasting properties. An improved SDS gel electrophoresis showed that ’Bai Huo’ (a parental wheat) carried a variant Wx-B1 protein from an allele, Wx-B1e. Thus, wheat lines of types 1, 2, 4, and 6 examined in this study contained a variant Wx-B1 allele and not the standard allele, Wx-B1a. The results from 3 years of experiments using 176 lines derived from two cross-combinations showed that apparent amylose content increased the least in type 8 (waxy) having no Wx proteins and, in ascending order, increased in type 5 (only the Wx-A1 protein is present) <type 7 (Wx-D1) <type 6 (Wx-B1) <type 3 (Wx-A1 and -D1) <type 4 (Wx-A1 and -B1) <type 2 (Wx-B1 and -D1) <type 1 (three Wx proteins). However, Tukey’ s studentized range test did not detect significant differences in some cases. Densitometric analysis suggested that the amylose content was related to the amount of the Wx protein in the eight types. Parameters in the Rapid Visco-Analyzer test and swelling power were correlated to amylose content. Consequently, amylose content and pasting properties of starch were determined to be influenced the most by the lack of the Wx-B1 protein, followed by a lack of Wx-D1, and leastly by the Wx-A1 deficiency, which indicated the presence of differential effects of the three null alleles for the Wx protein. Received: 1 February 1999 / Accepted: 10 April 1999     

A novel codominant marker for selection of the null Wx-B1 allele in wheat breeding programs

Waxy protein (granule-bound starch synthase I) is a key enzyme in the synthesis of amylose in endosperm tissue. The amylose content of wheat flour plays a significant role in determining Japanese udon noodle quality. Most wheat cultivars suitable for producing udon noodles have a low amylose level due to a lack of Wx-B1 protein conditioned by null Wx-B1 alleles. It was previously determined that the entire coding region of the wheat Wx-B1 gene is deleted in the most common null allele. However, the extent and breakpoints of the deletion have not been established. In this study, the position of the 3′ deletion breakpoint was refined by mapping with PCR-based markers. Using information from this analysis, a chromosome walk was initiated and the DNA sequence flanking the deletion breakpoints was obtained. The deletion included a 3,872 bp region downstream from the termination codon of Wx-B1 gene. Based on similarity with T. monococcum sequences, it was estimated that approximately 60 kb upstream of the Wx-B1 gene was also deleted. Using this sequence information, a codominant marker for the identification of the Wx-B1 null allele was developed. This marker can unambiguously identify heterozygous plants, which will accelerate the selection of partial waxy mutants carrying the Wx-B1 null allele.     

利用回交法与Wx基因分子标记辅助选择培育糯性小麦

以中国春糯性位点全套近等基因系为研究材料,对小麦Wx基因的6个STS标记和1个CAPS标记进行了筛选。改良PCR扩增条件以及产物检测方式后,从这些标记中筛选出3个标记,包括鉴定Wx-A1、Wx-D1位点的2个共显性STS标记和Wx-B1位点的1个显性STS标记,用于本研究中糯性小麦的分子标记辅助育种。在育种过程中,首先配制全糯材料“98Y1441”与推广品种“川育12”的杂交组合,采用籽粒碘染法从其F2种子中选择全糯基因型个体与回交亲本川育12杂交,如此反复自交、回交,历经数代异地加代繁殖得到BC5F2代回交改良群体。利用上述3个分子标记从该群体中筛选出了8种Wx基因型,经卡方检验,其分离比符合3对基因的分离比例,其中基因型为aabbdd的植株有2株,直链淀粉含量分别为1.81%和0.82%,为全糯小麦;基因型为AAbbdd, aabbDD的部分糯性植株各有1株,直链淀粉含量分别为15.24%和17.57%。研究中获得的BC5 F2代群体的农艺性状接近回交亲本,并明显优于全糯材料“98Y1441”,表明采用回交法与Wx基因分子标记辅助选择相结合,有助于培育高产、优质的全糯和部分糯小麦。     

Molecular-genetic analysis of the breeding bread wheat lines with starch of amylopectin type

PCR-analysis with primers to Wx-loci of T. aestivum L. genome lets us run the controlled selection of genotypes with null-alleles of Wx-genes while creating bread wheat lines with amylopectin type of starch. Microsatellite analysis of selected individual plants which were the carries of three null-alleles of Wx-genes (Wx-A1b, Wx-B1b, Wx-D1b) and the cluster analysis in the complex allowed us to pick out four genotypes, which were very close related genetically to the parent form variety Kuyalnik.     

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

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

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

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

小偃6号及其衍生后代品质相关性状基因的分子检测

小偃6号是我国小麦育种的重要骨干亲本之一。本研究利用UMN19、UMN25、UMN26、T2、T5、T13、S13、S1、T1、Wx-A1、Wx-B1、Wx-D1、YP7A、YP7B-1、PPO18、PPO29等16个功能型分子标记对小偃6号及其衍生后代的品质相关性状的基因组成进行了检测和分析。结果表明:在高分子量谷蛋白亚基两位点(Glu-A1、Glu-D1)和Waxy蛋白基因位点上,分别有78.72%、82.98%的衍生品种与骨干亲本小偃6号等位基因一致,但少数品种具有不同的优良亚基等位基因Ax2*或Dx5+Dy10;在低分子量谷蛋白亚基两位点(Glu-B3、Glu-D3)上,有25.33%的衍生品种与小偃6号等位基因一致;在八氢番茄红素合成酶基因位点Psy-A1、Psy-B1和多酚氧化酶(PPO)活性等位基因位点上,仅有17.02%和38.29%的衍生品种与小偃6号一致;并探讨了不同品质相关性状基因位点在衍生后代传递频率存在差异的原因。     

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

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

Null alleles for gliadin blocks in bread and durum wheat cultivars

Summary Wheat gliadin proteins are coded by clusters of genes (complex loci) located on the short arms of chromosomes of homoeologous groups 1 and 6 in bread (6x) and durum (4x) wheats. The proteins expressed by the various complex loci have been designated gliadin blocks. In a survey of accessions from the Germplasm Institute (C.N.R., Bari, Italy) collection, several different accessions have been found that lack particular blocks of proteins (null alleles). In some bread wheat accessions, seeds do not express gliadins that are coded by chromosomes 1D and 6A in normal cultivars. Similarly, some durum wheat accessions lack -gliadin components coded for by genes on chromosomes 1A and 1B. The missing proteins do not result from the absence of whole chromosomes, but may be the consequence of partial deletion of these genes at a complex locus or result from their silencing.     

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

Background and Aims

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

Methods

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

Key Results

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

Conclusions

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