部分小麦高分子量谷蛋白亚基组成分析

利用十二烷基硫酸钠聚丙烯胺凝胶电泳（SDS－PAGE）分析了85个小麦材料的高分子量谷蛋白亚基的构成,其结果表明：（1）目前生产中应用的优质小麦品种,大部分具有1A上的优质亚基1,1B上的14＋15／17＋18或1D上的5＋10,个别品种还同时聚合有1A,1B,1D上的优质亚基;（2）在所分析的28个八倍体小偃麦中,多数材料含有1,2^*和5＋10等优质亚基;（3）在本实验室创造的材料中,来源于中间偃麦草和普通小麦杂交的后代材料中大部分具有14＋15亚基。此外,个别种质材料还含有Payne亚基命名系统中未命名的一些稀有的高分子量谷蛋白亚基。     

Comparison of bread wheat lines selected by doubled haploid, single-seed descent and pedigree selection methods

 Yield performance of each group of ten spring bread wheat lines selected by doubled haploid (DH), single-seed descent (SSD) and pedigree selection (PS) methods from three F₁ crosses was compared with the aim of evaluating the DH method in breeding programs. Populations of 65–97 DH lines and 110 SSD lines per cross were used for selection. PS lines were developed by repeated selections from 1500 F₂ plants. Yield evaluation was performed at the F₆ generation of SSD and PS lines along with DH lines in a 2-year field experiment. It took only 2 years from the planting of wheat materials for DH production to the planting of selected DH lines for yield evaluation. There was no significant difference in grain yield between DH lines and PS lines selected from an F₁ cross whose parental varieties were closely related in their pedigrees. In two crosses with low coefficients of parentage and a large variation in their progenies, grain yield of selected DH lines was significantly lower than those of selected SSD and PS lines. These results confirm that the DH method can save time in obtaining recombinant inbred lines ready for yield evaluation. However, a larger DH population is required to achieve the same level of genetic advance with the PS method in crosses containing greater genetic variation. Received: 23 December 1997 / Accepted: 12 March 1998     

小麦高分子量谷蛋白亚基对加工品质影响的效应分析

分析了 2 50份小麦材料的高分子量谷蛋白亚基 (HMW- GS)组成以及其中 66份材料的加工品质及面条制作品质。回归分析表明 :HMW- GS与 1 0种加工品质性状均有显著的线性关系。不同亚基对综合品质效应的得分大小依次为 :Glu- Al,1 >2 * >null;Glu- Bl,1 4 +1 5>7+8>1 7+1 8>>7+9;Glu- Dl,5+1 0 >>2 +1 2 >4+1 2。不同基因位点对品质的贡献大小顺序为 :Glu- Dl>Glu- Al>Glu- Bl。首次提出了 HMW- GS综合品质评分系统     

Identification and molecular characterization of a large insertion within the repetitive domain of a high-molecular-weight glutenin subunit gene from hexaploid wheat

High-molecular-weight (HMW) glutenin subunits are a particular class of wheat endosperm proteins containing a large repetitive domain flanked by two short N- and C-terminal non-repetitive regions. Deletions and insertions within the central repetitive domain has been suggested to be mainly responsible for the length variations observed for this class of proteins. Nucleotide sequence comparison of a number of HMW glutenin genes allowed the identification of small insertions or deletions within the repetitive domain. However, only indirect evidence has been produced which suggests the occurrence of substantial insertions or deletions within this region when a large variation in molecular size is present between different HMW glutenin subunits. This paper represents the first report on the molecular characterization of an unusually large insertion within the repetitive domain of a functional HMW glutenin gene. This gene is located at the Glu-D1 locus of a hexaploid wheat genotype and contains an insertion of 561 base pairs that codes for 187 amino acids corresponding to the repetitive domain of a HMW glutenin subunit encoded at the same locus. The precise location of the insertion has been identified and the molecular processes underlying such mutational events are discussed.     

Structural homology of endosperm high molecular weight glutenin subunits of common wheat (Triticum aestivum L.)

Summary Several high molecular weight endosperm glutenin subunits, coded by genes located on chromosomes 1A, 1B and 1D of common wheat, Triticum aestivum L. em. Thell., were isolated from excised gel segments and subjected to amino acid analysis and peptide mapping; the latter was carried out following a limited digestion with trypsin, chymotrypsin or Staphylococcus aureus — V8 protease. Generally, all high molecular weight glutenins had a similar amino acid composition but several significant differences were observed in some of them. Both analyses revealed that the structural similarity among the various subunits was related to the homology of the genes coding them: subunits coded by homoalleles, i.e., different alleles of the same gene, were most similar; those coded by homoeoalleles, i.e., alleles of homoeologous genes, were less similar; whereas subunits coded either by alleles of different genes of the same gene cluster, or by nonhomoeoalleles of homoeologous clusters, were the least similar. Several small peptides derived from protease digestion of various subunits had a higher than expected staining intensity indicating that small peptide repeats may be interspersed within the glutenin subunits. The evolutionary course of the high molecular weight glutenins is discussed.     

Genomic regions for yield and yield parameters in Chinese winter wheat (Triticum aestivum L.) genotypes tested under varying environments correspond to QTL in widely different wheat materials

Field trials with a population of 108 doubled haploid (DH) lines of bread wheat (Triticum aestivum L.) derived from a cross between the Chinese winter wheat cultivars CA9613 and H1488 were carried out at Beijing (China) in 2000/2001 and 2001/2002. In addition, a field trial and a pot experiment were carried out at the experimental field stations of Giessen University (Germany) in the vegetation periods 2004/2005 and 2006/2007. Phenotypic data for major agronomic yield-related traits, i.e. grain weight per ear (GWE), grain number per ear (GNE), plant height and thousand-grain weight (TGW), were recorded in all experiments. In addition, biomass weight per tiller and ear weight were evaluated in the two field trials at Beijing. Based on the phenotypic data and a genetic map comprising 168 SSR markers, an analysis of quantitative trait loci (QTL) was carried out for yield and yield parameters using the composite interval mapping (CIM) approach. A total of 30 QTL were detected for these traits across four environments. Five of these QTL located on chromosomes 1A, 1B, 2B, 2D and 7D exhibited pleiotropic effects. Such pleiotropic gene loci will be very useful for understanding the homologous/homeologous relationships among QTL and designing an appropriate marker-assisted breeding programme including multi-trait selection in order to accumulate (“pyramide”) favorable alleles at different genetic loci.     

Detection and characterization of a glutenin subunit with unusual high Mr at the Glu-A1 locus in hexaploid wheat

A hexaploid wheat landrace collected from the Baluchistan province of Pakistan was found to possess a novel high-molecular-weight glutenin subunit (HMW-GS). The subunit has a very slow electrophoretic mobility as revealed by SDS-PAGE, and its molecular weight is comparable to that of the highest molecular weight glutenin subunit (2.2 encoded in the D-genome) reported so far in hexaploid wheat varieties and landraces of Japanese origin. Evidence obtained from (PCR) gene amplification studies using the primers specific for Glu-1 loci proved that the gene coding for this novel subunit belongs to the Glu-A1 locus located on the long arm of chromosome 1A. Digestion of the amplified gene (PCR product) with restriction enzymes indicated that the novel gene differs from prevailing Glu-A1 alleles (null, 1 and 2^*) by an extra DNA fragment of approximately 600 base pairs. The results also indicated that the novel subunit is most probably a derivative of subunit 2^* that has very likely incorporated the 600-bp fragment following a process of unequal crossing over. The present findings were further substantiated by reserved phase high performance liquid chromatography (RP-HPLC) analysis.     

The agronomic performance of wheat doubled haploid lines derived from wheat x maize crosses

Summary The agronomic performance of 9 doubled haploid (DH) lines of Chinese Spring, 6 DH lines of Hope, 14 DH lines of the single chromosome substitution line Chinese Spring (Hope 5 A) and their respective parents was analyzed under field conditions. Seventeen Chinese Spring DH lines derived from wheat x Hordeum bulbosum crosses were also included for comparison. No significant variation was detected in either population of Chinese Spring DH lines and neither DH population differed from its parent. The Hope DH lines differed significantly for tiller biomass, spikelet number per ear, ear grain weight and 50-grain weight. However, all the variation could be attributed to the poor performance of only one line. Chinese Spring (Hope 5 A) DH lines showed significant variation for ear emergence time, but this was probably due to genetic heterogeneity in the parental stock. Overall, the results suggest that most DH lines produced by the wheat x maize method resemble their wheat parent, and that the variation induced in DH production is likely to be similar to that found in DHs from wheat x Hordeum bulbosum crosses.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Chromosomal control of glutenin subunits in aneuploid lines of wheat: analysis by reversed-phase high-performance liquid chromatography

Summary Glutenin subunits from nullisomic-tetrasomic and ditelocentric lines of the hexaploid wheat variety ‘Chinese Spring’ (CS) and from substitution lines of the durum wheat variety ‘Langdon’ were fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) at 70 °C using a gradient of acetonitrile in the presence of 0.1% trifluoroacetic acid. Nineteen subunits were detected in CS. The presence and amounts of four early-eluted subunits were found, through aneuploid analysis, to be controlled by the long arms of chromosomes 1D (1DL) (peaks 1–2) and 1B (1BL) (peaks 3–4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that these four subunits are the high molecular weight subunits of glutenin, which elute in the order 1Dy, 1Dx, 1By, and 1Bx. Similar amounts of 1DL subunits were present (6.3 and 8.8% of total glutenin), but 1BL subunits differed more in abundance (5.4 and 9.5%, respectively). Results indicate that most late-eluting CS glutenin subunits were coded by structural genes on the short arms of homoeologous group 1 chromosomes: 6 by 1DS, 5 by 1AS, and 4 by 1BS. Glutenin of tetraploid ‘Langdon’ durum wheat separated into nine major subunits: 6 were coded by genes on 1B chromosomes, and 3 on 1A chromosomes. Gene locations for glutenin subunits in the tetraploid durum varieties ‘Edmore’ and ‘Kharkovskaya-5’ are also given. These results should make RP-HPLC a powerful tool for qualitative and quantitative genetic studies of wheat glutenin. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned Stationed at the Northern Regional Research Center, Peoria.     

Effect of gliadins and HMW and LMW subunits of glutenin on dough properties in the F6 recombinant inbred lines from a bread wheat cross

The storage proteins of 64 F₂-derived F₆ recombinant inbred lines (RILs) from the bread wheat cross Prinqual/Marengo were analyzed. Parents differed at four loci: Gli-B1 (coding for gliadins), Glu-B1 (coding for HMW glutenin subunits), Glu-A3/Gli-A1 (coding for LMW glutenin subunits/gliadins) and Glu-D3 (coding for LMW glutenin subunits). The effect of allelic variation at these loci on tenacity, extensibility and dough strength as measured by the Chopin alveograph was determined. Allelic differences at the Glu-B1 locus had a significant effect on only tenacity. None of the allelic differences at either the Glu-A3/Gli-A1 or Glu-D3 loci had a significant effect on quality criteria. Allelic variation at the Gli-B1 locus significantly affected all of the dough properties. Epistatic effects between some of the loci considered contributed significantly to the variation in dough quality. Additive and epistatic effects each accounted for 15% of the variation in tenacity. Epistasis accounted for 15% of the variation in extensibility, whereas additive effects accounted for 4%. Epistasis accounted for 14% of the variation in dough strength, and additivity for 9%. The relative importance of epistatic effects suggest that they should be included in predictive models when breeding for breadmaking quality.     

小麦高分子量麦谷蛋白亚基鉴定及其品质效应研究进展

高分子量谷蛋白亚基(HMW-GS,high molecular weight glutenin subunits)是小麦子粒贮藏蛋白的重要组成成分,其组成、搭配、表达水平及含量决定面团弹性和面包加工品质。本文主要介绍了小麦HMW-GS编码基因的克隆、分子特征、分子标记开发及其在小麦育种中的应用,并综述了不同HMW-GS与面粉加工品质之间的关系,以及HMW-GS基因遗传转化、微量配粉和突变体培育等方面的研究进展,分析了目前研究中存在的主要问题,认为通过分子标记辅助选择和转基因技术聚合优质亚基,培育优质面包小麦品种和明确各个HMW-GS基因的品质效应是今后的研究重点。     

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25°C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25–27°C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.Communicated by W. Harwood     

Detection of quantitative trait loci for heading date based on the doubled haploid progeny of two elite Chinese wheat cultivars

Quantitative trait loci (QTLs) with epistatic and QTL × environment (QE) interaction for heading date were studied using a doubled haploid (DH) population containing 168 progeny lines derived from a cross between two elite Chinese wheat cultivars Huapei 3 × Yumai 57 (Triticum aestivum L.). A genetic map was constructed based on 305 marker loci, consisting of 283 SSR loci and 22 EST-SSR markers, which covered a total length of 2141.7 cM with an average distance of 7.02 cM between adjacent markers in the genome. QTL analyses were performed using a mixed linear model approach. Two main-effect QTLs and two pairs of digenic epistatic effects were detected for heading date on chromosomes 1B, 2B, 5D, 6D, 7A, and 7D at three different environments in 2005 and 2006 cropping seasons. A highly significant QTL with an F-value 148.96, designated as Qhd5D, was observed within the Xbarc320-Xwmc215 interval on chromosome 5DL, accounting for 53.19% of the phenotypic variance and reducing days-to-heading by 2.77 days. The Qhd5D closely links with a PCR marker Xwmc215 with the genetic distance 2.1 cM, which can be used in molecular marker-assisted selection (MAS) in wheat breeding programs. Moreover, the Qhd5D was located on the similar position of well-characterised vernalization sensitivity gene Vrn-D1. We are also spending more efforts to develop near-isogenic lines to finely map the Qhd5D and clone the gene Vrn-D1 through map-based cloning. The Qhd1B with additive effect on heading date has not been reported in previous linkage mapping studies, which might be a photoperiod-sensitive gene homoeologous to the Ppd-H2 gene on chromosome 1B. No main-effect QTLs for heading date were involved in epistatic effects.     

Methods for estimating gene numbers for quantitative characters using doubled haploid lines

Summary Three methods of estimating the numbers of genes segregating for quantitative characters using doubled haploid lines are presented. The first uses estimates of the range and genetical variance of an F₁ or F₂ derived population. The second adapts the genotype assay method of Jinks and Towey (1976) to F₂ derived lines. The third uses the variances of an F₂ derived population. Statistical problems of obtaining meaningful estimates using these methods are discussed and it is concluded that genotype assay is the best method for distinguishing between few and many genes. These methods are illustrated using data from an experiment containing doubled haploid lines of barley developed using the H. bulbosum system.     

Identification of SNPs and development of functional markers for LMW-GS genes at <Emphasis Type="Italic">Glu-D3</Emphasis> and <Emphasis Type="Italic">Glu-B3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Low-molecular-weight glutenin subunits (LMW-GS) have great effect on wheat processing quality, but were numerous and difficult to dissect by SDS-PAGE. The development of functional markers may be the most effective way for a clear discrimination of different LMW-GS genes. In the present study, three different approaches were used to identify SNPs of different genes at Glu-D3 and Glu-B3 loci in bread wheat for the development of six STS markers (3 for Glu-D3 and 3 for Glu-B3 genes) that were validated with distinguished wheat cultivars. Firstly, seven LMW-GS gene sequences ( AY585350, AY585354, AY585355, AY585356, AY585349, AY585351 and AY585353 ) from Aegilops tauschii, the diploid donor of the D-genome of bread wheat, were chosen to design seven pairs of AS-PCR primers for Glu-D3 genes. By amplifying the corresponding genes from five bread wheat cultivars with different Glu-D3 alleles (a, b, c, d and e) and Ae. tauschii, a primer set, S13F2/S13R1, specific to the gene AY585356, was found to be positive to cultivars with alleles Glu-D3c and d. Nevertheless, the other five pairs of primers designed from AY585350, AY585349, AY585353, AY585354 and AY585355, respectively, did not produce specific PCR products to the cultivars tested. Secondly, all the PCR products from the five primer sets without specific characteristics were sequenced and an SNP from the gene AY585350 was detected in the cultivar Hartog, which resulted in the second STS marker S1F1/S1R3 specific to the allelic variant of AY585350. Thirdly, three Glu-D3 sequences (AB062851, AB062865 and AB062872) and three Glu-B3 sequences (AB062852, AB062853 and AB062860) defined by Ikeda et al. (2002) were chosen to query wheat EST and NR databases, and DNA markers were developed based on the putative SNPs among the sequences. Using this approach, four STS markers were developed and validated with 16-19 bread wheat cultivars. The primer set T1F4/T1R1 was also a Glu-D3 gene-specific marker for AB062872, while T2F2/T2R2, T5F3/T5R1 and T13F4/T13R3 were all Glu-B3 gene specific markers for AB062852, BF293671 and AY831800, respectively. The chromosomal locations of the six markers were verified by amplifying the genomic DNA of Ae. tauschii (DD), T. monococcum (AA) and T. turgidum (AABB) entries, as well as Chinese Spring and its group 1 chromosome nulli-tetrasomic lines. The results are useful to discriminate the corresponding Glu-D3 and Glu-B3 genes in wheat breeding programs.     

Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture

The objective of this work was to produce doubled haploid plants from durum wheat through the induction of androgenesis. A microspore culture technique was developed and used to produce fertile doubled haploid plants of agronomic interest. Five cultivars, one selected line, plus a collection of 20 F₁ crosses between different genotypes of high breeding value were used. Studies on several factors such as pre-treatments and media components were carried out in order to develop a protocol to regenerate green haploid plantlets. Anthers were pre-treated in 0.7 M mannitol. Microspores, from anther maceration, were plated on a C₁₇ induction culture medium with ovary co-culture. The optimum regeneration medium J25–8 was used. From 35 microspore isolations, 407 green plantlets were obtained. With this technique mature embryos were obtained. Green plants were regenerated from all genotypes used and approximately 67% of them were spontaneously doubled haploids. Some haploids and a very few polyploids plants were obtained. From the 407 plants, 275 were completely fertile and gave enough seeds to be assayed in the field. This protocol could be used complementary to or instead of the intergeneric crossing with maize as an economically feasible method to obtain doubled haploids from most durum wheat genotypes.     

DNA restriction-fragment variation in the gene family encoding high molecular weight (HMW) glutenin subunits of wheat

Restriction enzyme digests of DNA from nullisomic-tetrasomic and intervarietal chromosome substitution lines of wheat were probed with a high molecular weight (HMW) glutenin cDNA. Three restriction endonucleases were used to investigate restriction-fragment differences among five wheat varieties. The results suggest that the hybridizing fragments contain single gene copies and permit the identification of the subunit encoded by each gene. Restriction-fragment variation associated with previously established allelic differences between varieties was observed. Also, there is a clear relationship between the electrophoretic mobility of a HMW subunit and the length of the central repetitive section of the gene encoding it. These results are discussed with reference to the evolution of the HMW glutenin gene family and the uses of restriction-fragment variation in plant breeding and genetics.N.P.H. was supported by a MRC Training Fellowship in Recombinant DNA Technology and a grant from the Perry Foundation. D.B. is supported by EEC Contract GBI-4-027-UK.     

Wheat storage proteins: diversity of HMW glutenin subunits in wild emmer from Israel

Summary The diversity of HMW glutenin subunits in the tetraploid wild progenitor of wheat, Triticum turgidum var. dicoccoides was studied electrophoretically in 231 individuals representing 11 populations of wild emmer from Israel. The results show that (a) The two HMW glutenin loci, Glu-A1 and Glu-B1, are rich in variation, having 11 and 15 alleles, respectively, (b) Genetic variation in HMW glutenin subunits is often severely restricted in individual populations, supporting an island population genetic model, (c) Significant correlations were found between glutenin diversity and the frequencies of specific glutenin alleles and physical (climate and soil) and biotic (vegetation) variables. Our results suggest that: (a) at least part of the glutenin polymorphisms in wild emmer can be accounted for by environmental factors and (b) the endosperm of wild emmer contains many allelic variants of glutenin storage proteins that are not present in bread wheat and could be utilized in breeding varieties with improved bread-making qualities.