Identification and chromosomal organization of two rye genome-specific RAPD products useful as introgression markers in wheat.

Two rye genome-specific random amplified polymorphic DNA (RAPD) markers were identified for detection of rye introgression in wheat. Both markers were amplified in all of the tested materials that contained rye chromatin such as rye, hexaploid triticale, wheat-rye addition lines, and wheat varieties with 1BL.1RS translocation. Two cloned markers, designated pSc10C and pSc20H, were 1012 bp and 1494 bp, respectively. Sequence analysis showed that both pSc10C and pSc20H fragments were related to retrotransposons, ubiquitously distributed in plant genomes. Using fluorescence in situ hybridization (FISH), probe pSc10C was shown to hybridize predominantly to the pericentromeric regions of all rye chromosomes, whereas probe pSc20H was dispersed throughout the rye genome except at telomeric regions and nucleolar organizing regions. The FISH patterns showed that the two markers should be useful to select or track all wheat-rye translocation lines derived from the whole arms of rye chromosomes, as well as to characterize the positions of the translocation breakpoints generated in the proximal and distal regions of rye arms.     

Inheritance of glutenin protein subunits of wheat

Summary The inheritance of the high-molecular-weight (HMW) glutenin protein subunits in hexaploid wheat has been investigated by using sodium dodecyl sulphate-polyacrylamide gel electrophoresis to examine the segregation of these subunits in 496 test-cross seeds. The parents of the f₁ hybrid were chosen so that the test-cross seeds segregated for all the HMW glutenin bands. Two glutenin subunits from one parent, believed to be controlled by genes on chromosome 1D, segregated as alternatives to two glutenin subunits from the other parent, a result that supports the assumption that these subunits are controlled by allelic genes at each of two loci that are very closely linked. Similar results were obtained for glutenin subunits believed to be controlled by chromosome IB, which suggests that these subunits are controlled also by allelic genes at each of two loci that are very closely linked. A single glutenin subunit band, believed to be controlled by chromosome 1A, segregated as an alternative to a single glutenin band from the other parent, except that one seed did not possess either band. It was concluded that these bands are controlled either by allelic genes or by nonallelic genes that are very closely linked.     

K型小麦细胞质雄性不育保持系T911289中外源遗传物质的初步鉴定

利用荧光基因组原位杂交(GISH)、生化标记和DNA分子标记技术对普通小麦(triticum aestivum L.)K型细胞质雄性不育保持系T911289的染色体组成进行了鉴定与分析。GISH鉴定和黑麦特异散布重复序列的检测结果表明,T911289的外源遗传物质来源于黑麦,黑麦1RS上的微卫星引物SCM9扩增结果和醇溶蛋白酸性聚丙烯酰胺凝胶电泳(A—PAGE)分析、低分子量谷蛋白的sDS_PAGE分析均表明,T911289所含的黑麦遗传物质来源于1RS;A-PAGE和SDS-PAGE分析及小麦1BS上的微卫星引物的扩增结果则表明,‘1911289缺少1BS染色体臂或1BS末端片段。GISH鉴定结果还表明,‘1911289中有罗泊逊易位和小片段易位两种类型的杂交信号,说明T911289是一个异质群体,但其罗泊逊易位又不同于生产上大面积应用的1BL／1RS易位,它可能是一种新的复杂易位形式。虽然T911289的小片段易位未能打破优异农艺性状与劣质蛋白基因的连锁,但这种小片段易位的获得将有利于小麦和黑麦的遗传研究,这种种质材料在育种上的应用价值也应优于罗泊逊易位。     

Production of a monoclonal antibody specific for high molecular weight glutenin subunits (HMW-GS) in wheat and its antigenic determinant

Wheat high molecular weight glutenin subunits (HMW-GS) 1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice. Subcutaneous inoculation of the antigen is performed. The intra-peritoneal injection is completed 3 days before fusion with myeloma cell (SP2/0) via PEG-1500. The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay (ELISA). Positive hybrid cells are further verified three times by limit dilution of the culture cells. A hybridoma cell line is successfully obtained. The monoclonal antibody belongs to lgG1 subclass. In immunoblotting, the antibody binds to all HMW-GS of T. aestivum cultivars, but does not bind to other storage proteins in seeds of wheat. This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat. The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T. durum (durum wheat). Furthermore, it also binds to HMW storage proteins in Secale cereale (rye), Hordeum vulgare (barley). However, it never binds seed storage proteins in other cereals such as maize, oat, rice, foxtail millet, sorghum etc. The antigen determinant recognized by the antibody has been located within hexapeptide [PGQGQQ] or / and nonapeptide [GYYPTSPQQ] in the central repetitive region of HMW-GS.     

Production of a monoclonal antibody specific for high molecular weight glutenin subunits (HMW-GS) in wheat and its antigenic determinant

Wheat high molecular weight glutenin subunits (HMW-GS) 1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice. Subcutaneous inoculation of the antigen is performed. The intra-peritoneal injection is completed 3 days before fusion with myeloma cell (SP2/0) via PEG-1500. The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay (ELISA). Positive hybrid cells are further verified three times by limit dilution of the culture cells. A hybridoma cell line is successfully obtained. The monoclonal antibody belongs to IgG1 subclass. In immunoblotting, the antibody binds to all HMW-GS of T.aestivum cultivars, but does not bind to other storage proteins in seeds of wheat. This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat. The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T. durum (durum wheat). Furthermore, it also binds to HMW storage proteins in Secale cereale (rye),Hordeum vulgare (barley). However, it never binds seed storage proteins in other cereals such as maize, oat, rice, foxtail millet, sorghum etc. The antigen determinant recognized by the antibody has been located within hexapeptide [PGQGQQ] or / and nonapeptide [GYYPTSPQQ] in the central repetitive region of HMW-GS.     

Diversity of Novel Glutenin Subunits in Bread Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Glutenin is a major determinant of baking performance and viscoelasticity, which are responsible for high-quality bread with a light porous crumb structure of a well-leavened loaf. We analyzed the diversity of glutenin genes from six wheat cultivars (Korean cvs. Keumgang and Jinpum, Chinese cvs. China-108 and Yeonnon-78, and Japanese cvs. Norin-61 and Kantou-107). Glutenins contain two types of isoforms such as high molecular weight glutenin subunit (HMW-GS) and low molecular weight glutenin subunit (LMW-GS). Glutenin fractions were extracted from wheat endosperm using Osborne solubility method. A total of 217 protein spots were separated on two-dimensional gel electrophoresis with isoelectric focusing (wide range of pH 3–10). The proteins spots were subjected to tryptic digestion and identified by matrix assisted laser desorption/ionization–time of flight mass spectrometry. HMW-GS (43 isoforms) and LMW-GS (seven isoforms) are directly responsible for producing high-quality bread and noodles. Likewise, all the seed storage proteins are digested to provide nutrients for the embryo during seed germination and seedling growth. We identified the diverse glutenin subunits in wheat cultivars and compared the gluten isoforms among different wheat cultivars according to quality. This work gives an insight on the quality improvement in wheat crop.     

Characterisation and analysis of new HMW-glutenin alleles encoded by the Glu-R1 locus of Secale cereale

This work reports the molecular characterisation of new alleles of the previously reported Glu-R1 locus. Wheat lines carrying the chromosome substitution 1R(1D), rye cultivars and related wild species were analysed. Five new x-type and four y-type Glu-R1 glutenin subunits were isolated and characterised. The coding region of the sequences shows the typical structure of the HMW glutenin genes previously described in wheat, with the N and C-terminal domains flanking the central repetitive region. Tri-, hexa- and nona-peptides found in the central repetitive region of wheat glutenin genes were also present in the rye genes. Duplications and deletions of these motifs are responsible for allelic variation at the Glu-R1 locus. Orthologous genes (from different genomes) were more closely related than paralogous genes (x- and y-type), supporting the hypothesis of gene duplication before Triticeae speciation. Differences in the number and position of cysteine residues identified alleles which in wheat are associated with good dough quality. SDS proteins encoded by some characterised alleles were presumptively identified.     

Role of individual disulfide bonds in the structural maturation of a low molecular weight glutenin subunit.

Gliadins and glutenins are the major storage proteins that accumulate in wheat endosperm cells during seed development. Although gliadins are mainly monomeric, glutenins consist of very large disulfide-linked polymers made up of high molecular weight and low molecular weight subunits. These polymers are among the largest protein molecules known in nature and are the most important determinants of the viscoelastic properties of gluten. As a first step toward the elucidation of the folding and assembly pathways that lead to glutenin polymer formation, we have exploited an in vitro system composed of wheat germ extract and bean microsomes to examine the role of disulfide bonds in the structural maturation of a low molecular weight glutenin subunit. When conditions allowing the formation of disulfide bonds were established, the in vitro synthesized low molecular weight glutenin subunit was recovered in monomeric form containing intrachain disulfide bonds. Conversely, synthesis under conditions that did not favor the formation of disulfide bonds led to the production of large aggregates from which the polypeptides could not be rescued by the post-translational generation of a more oxidizing environment. These results indicate that disulfide bond formation is essential for the conformational maturation of the low molecular weight glutenin subunit and suggest that early folding steps may play an important role in this process, allowing the timely pairing of critical cysteine residues. To determine which cysteines were important to maintain the protein in monomeric form, we prepared a set of mutants containing selected cysteine to serine substitutions. Our results show that two conserved cysteine residues form a critical disulfide bond that is essential in preventing the exposure of adhesive domains and the consequent formation of aberrant aggregates.     

Identification and characterization of high-molecular-weight glutenin genes in Polish triticale cultivars by PCR-based DNA markers

Molecular markers were used to identify the allele/gene composition of complex loci Glu-A1 and Glu-B1 of high-molecular-weight (HMW) glutenin subunits in triticale cultivars. Forty-six Polish cultivars of both winter and spring triticale were analysed with 7 PCR-based markers. Amplified DNA fragments of HMW glutenin Glu-1 genes were separated by agarose slab-gel electrophoresis. Differences between all 3 alleles at the locus Glu-A1 [Glu-A1a (encoding Ax1), 1b (Ax2*), and 1c (AxNull)], 4 alleles at Glu-B1-1 [Glu-B1-1a (Bx7), 1b (Bx7*), 1d (Bx6), 1ac (Bx6.8)], and 5 alleles at Glu-B1-2 [Glu-B1-2a (By8), 2b (By9), 2o (By8*), 2s (By18*), and 2z (By20*)] were revealed. In total, 16 allele combinations were observed. Molecular markers are particularly helpful in distinguishing the wheat Glu-A1a and Glu-A1b alleles from the rye Glu-R1a and Glu-R1b alleles in triticale genotypes, respectively, as well as subunits Bx7 from Bx7* and By8 from By8*, which could not be distinguished by SDS-PAGE. Novel glutenin subunits By18* and By20* (unique to triticale) were identified. HMW glutenin subunit combinations of Polish triticale cultivars, earlier identified by SDS-PAGE analyses, were verified by PCR-based DNA markers. Rapid identification of wheat Glu-1 alleles by molecular markers can be an efficient alternative to the standard separation procedure for early selection of useful triticale genotypes with good bread-making quality.     

Analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species

Considerable progress has been made in understanding the structure, function and genetic regulation of high-molecular-weight (HMW) glutenin subunits in hexaploid wheat. In contrast, less is known about these types of proteins in wheat related species. In this paper, we report the analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species, Aegilops umbellulata (UU) and Aegilops caudata (CC). SDS-PAGE analysis demonstrated that, for each of the four Ae. umbellulata accessions, there were two HMW glutenin subunits (designated here as 1Ux and 1Uy) with electrophoretic mobilities comparable to those of the x- and y-type subunits encoded by the Glu-D1 locus, respectively. In our previous study involving multiple accessions of Ae. caudata, two HMW glutenin subunits (designated as 1Cx and 1Cy) with electrophoretic mobilities similar to those of the subunits controlled by the Glu-D1 locus were also detected. These results indicate that the U genome of Ae. umbellulata and the C genome of Ae. caudata encode HMW glutenin subunits that may be structurally similar to those specified by the D genome. The complete open reading frames (ORFs) coding for x- and y-type HMW glutenin subunits in the two diploid species were cloned and sequenced. Analysis of deduced amino acid sequences revealed that the primary structures of the x- and y-type HMW glutenin subunits of the two Aegilops species were similar to those of previously published HMW glutenin subunits. Bacterial expression of modified ORFs, in which the coding sequence for the signal peptide was removed, gave rise to proteins with electrophoretic mobilities identical to those of HMW glutenin subunits extracted from seeds, indicating that upon seed maturation the signal peptide is removed from the HMW glutenin subunit in the two species. Phylogenetic analysis showed that 1Ux and 1Cx subunits were most closely related to the 1Dx type subunit encoded by the Glu-D1 locus. The 1Uy subunit possessed a higher level of homology to the 1Dy-type subunit compared with the 1Cy subunit. In conclusion, our study suggests that the Glu-U1 locus of Ae. umbellulata and the Glu-C1 locus of Ae. caudata specify the expression of HMW glutenin subunits in a manner similar to the Glu-D1 locus. Consequently, HMW glutenin subunits from the two diploid species may have potential value in improving the processing properties of hexaploid wheat varieties.     

小麦HMW-G12亚基基因启动子克隆及序列分析

为了研究高分子量谷蛋白基因启动子在种子中的特异性表达,以小麦品种“东农7742”的基因组DNA为模板,根据已发表序列设计并合成引物,用PCR的方法克隆了小麦贮藏蛋白中高分子量谷蛋白12亚基基因的上游调控序列。序列测定结果表明：所克隆的启动子片段大小为424bp与Thomspon报道的序列比较,同源性为97.9%,有9个核苷酸发生了改变。推测的TATA box位于-27— -30bp,Prolamin-box位于-175— -181bp,认为该元件可能与转录速率的调控有关。     

Structural and genetical studies on the high-molecular-weight subunits of wheat glutenin

Summary The high-molecular-weight (HMW) subunits of glutenin from about 185 varieties were fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). About 20 different, major subunits were distinguished by this technique although each variety contained, with only a few exceptions, between 3 and 5 subunits. Further inter-varietal substitution lines to those already described (Payne et al. 1980) were analysed and the results indicate that all the HMW subunits are controlled by the homoeologous group 1 chromosomes. All hexaploid varieties studied except ‘NapHal’ contained two major subunits controlled by chromosome 1D. Their genes were shown to be tightly linked genetically for only four different types of banding patterns were observed. The nominal molecular weights determined after fractionation in 10% polyacrylamide gels were between 110,000 and 115,000 for the larger of the two subunits and between 82,000 and 84,000 for the smaller. One quarter of the varieties contained only one major HMW subunit controlled by chromosome 1B whereas the rest had two. The chromosome 1B subunits were the most varied and nine different banding patterns were detected. All the subunits had mobilities which were intermediate between those of the two chromosome 1D-controlled subunits. Only two types of HMW subunit controlled by chromosome 1A were detected in all the varieties examined; a single variety never contained both of these subunits and 40% of varieties contained neither. The chromosome 1A-controlled subunits had slightly slower mobilities in 10% gels than the largest HMW subunit controlled by chromosome 1D. About 100 single grains were analysed from each of five different crosses of the type (F₁ of variety A × variety B) × variety C. The results indicate that the genes on chromosome 1B which control the synthesis of subunits 6, 7, 13, 14 and 17 are allelic, as are the genes of the chromosome 1A-controlled subunits, 1 and 2.     

Induction of small-segment-translocation between wheat and rye chromosomes

A new approach to produce wheat-rye translocation, based on the genetic instability caused by monosomic addition of rye chromosome in wheat, is described. 1 283 plants from the selfed progenies of monosomic addition lines with single chromosome of inbred rye line R12 and complete chromosome complement of wheat cultivar Mianyang 11 were cytologically analyzed on a plant-by-plant basis by the improved C-banding technique. 63 of the plants, with 2n = 42, were found containing wheat-rye translocation or substitution, with a frequency of 4. 91% . Compared with the wheat parent, other 32 plants with 2n = 42 exhibited obvious phenotypic variation, but their com-ponent of rye chromosome could not be detected using the C-banding technique. In situ hybridization with a biotin-la-beled DNA probe was used to detect rye chromatin and to determine the insertion sites of rye segments in the wheat chromosomes. In 20 out of the 32 variant wheat plants, small segments of rye chromosomes were found being inserted into dif     

Role of the amino- and carboxy-terminal regions in the folding and oligomerization of wheat high molecular weight glutenin subunits

The high molecular weight glutenin subunits are considered one of the most important components of wheat (Triticum aestivum) gluten, but their structure and interactions with other gluten proteins are still unknown. Understanding the role of these proteins in gluten formation may be aided by analyses of the conformation and interactions of individual wild-type and modified subunits expressed in heterologous systems. In the present report, the bacterium Escherichia coli was used to synthesize four naturally occurring X- and Y-type wheat high molecular weight glutenin subunits of the Glu-1D locus, as well as four bipartite chimeras of these proteins. Naturally occurring subunits synthesized in the bacteria exhibited sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration properties identical to those of high molecular weight glutenin subunits extracted from wheat grains. Wild-type and chimeric subunits migrated in sodium dodecyl sulfate gels differently than expected based on their molecular weights due to conformational properties of their N- and C-terminal regions. Results from cycles of reductive cleavage and oxidative reformation were consistent with the formation of both inter- and intramolecular disulfide bonds in patterns and proportions that differed among specific high molecular weight glutenin species. Comparison of the chimeric and wild-type proteins indicated that the two C-terminal cysteines of the Y-type subunits are linked by intramolecular disulfide bonds, suggesting that the role of these cysteines in glutenin polymerization may be limited.     

Characterization of Low Molecular Weight Glutenin Subunit Gene Representing Glu-B3 Locus of Indian Wheat Variety NP4

Low molecular weight (LMW) glutenin subunits represent major part (30%) of storage proteins in wheat endosperm and determine the quality of dough. Despite their importance few LMW glutenin genes have been characterized so far and none from Indian wheat variety. In the present investigation PCR technique was employed to characterize LMW-GS gene representing Glu-B3 locus from Indian bread wheat cultivar NP4. The deduced protein sequence coded by Glu-B3 locus of LMW-GS gene from NP4 showed the presence of regular structure of the repetitive domain with varying numbers of glutamine (Q) residues and the presence of 1^st cysteine residue within the repetitive domain at 40^th position in mature polypeptide. Such structure might increase and stabilize the gluten polymer through intermolecular interactions of the large numbers of glutamine side chains and cysteine residues for intermolecular disulphide bond formation leading to stronger dough quality of NP4. Moreover, Glu-B3 specific primers could also be used for identifying 1BL/1RS translocation in addition to amplifying LMW glutenin genes. There was no amplification in 1B/1R translocation lines as short arm of wheat was replaced by short arm of rye chromosome in these lines. Such information can be useful in wheat improvement for dough properties for better chapati and bread quality.     

Development of genetically modified wheat to assess its dough functional properties

End-use functionality of bread wheat depends mainly on the protein content, the presence of particular subunits of high and low molecular weight glutenin, the ratio of high molecular weight to low molecular weight glutenin subunits, and the ratio of glutenin to gliadin. The exact contribution of each of these factors to end-use functionality is still largely unknown. Transgenic plants can allow these factors to be studied within a particular background thus contributing to our understanding of end-use functionality. Two Canadian wheat lines, one of them containing high molecular weight glutenin subunits (HMW-GS) coded by all three Glu-1 loci and one line null at all three loci were assessed for dough rheological properties and bread and tortilla-making properties. Protein composition of the flours were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size exclusion high performance liquid chromatography, and sedimentation test. Proteins in the samples were fractionated and the proportions of monomeric proteins, soluble glutenin, and insoluble glutenin were quantified. Functionality of the flours were characterized by small-scale methods such as the 2 g mixograph, 10 g farinograph, and micro-extension testing. End-use quality was evaluated by small-scale bread and tortilla production. Mixograph development time and mixograph peak height were much higher for the lines containing HMW-GS. The lines null for HMW-GS showed no resistance to extension. Lines null for HMW-GS produced 'brick'-like bread. Tortilla prepared from the null lines had poor rollability and lower puncture force. The results showed very strong dependencies of quality on the presence of HMW-GS.     

Isolation of wheat-rye 1RS recombinants that break the linkage between the stem rust resistance gene SrR and secalin.

Chromosome 1R of rye is a useful source of genes for disease resistance and enhanced agronomic performance in wheat. One of the most prevalent genes transferred to wheat from rye is the stem rust resistance gene Sr31. The recent emergence and spread of a stem rust pathotype virulent to this gene has refocused efforts to find and utilize alternative sources of resistance. There has been considerable effort to transfer a stem rust resistance gene, SrR, from Imperial rye, believed to be allelic to Sr31, into commercial wheat cultivars. However, the simultaneous transfer of genes at the Sec-1 locus encoding secalin seed storage proteins and their association with quality defects preclude the deployment of SrR in some commercial wheat breeding programs. Previous attempts to induce homoeologous recombination between wheat and rye chromosomes to break the linkage between SrR and Sec-1 whilst retaining the tightly linked major loci for wheat seed storage proteins, Gli-D1 and Glu-D3, and recover good dough quality characteristics, have been unsuccessful. We produced novel tertiary wheat-rye recombinant lines carrying different lengths of rye chromosome arm 1RS by inducing homoeologous recombination between the wheat 1D chromosome and a previously described secondary wheat-rye recombinant, DRA-1. Tertiary recombinant T6-1 (SrR+ Sec-1-) carries the target gene for stem rust resistance from rye and retains Gli-D1 but lacks the secalin locus. The tertiary recombinant T49-7 (SrR- Sec-1+) contains the secalin locus but lacks the stem rust resistance gene. T6-1 is expected to contribute to wheat breeding programs in Australia, whereas T49-7 provides opportunities to investigate whether the presence of secalins is responsible for the previously documented dough quality defects.     

Isolation and characterization of wheat-rye recombinants involving chromosome arm 1DS of wheat

Summary The introgression of genetic material from alien species is assuming increased importance in wheat breeding programs. One example is the translocation of the short arm of rye chromosome 1 (1RS) onto homoeologous wheat chromosomes, which confers disease resistance and increased yield on wheat. However, this translocation is also associated with dough quality defects. To break the linkage between the desirable agronomic traits and poor dough quality, recombination has been induced between 1RS and the homoeologous wheat arm IDS. Seven new recombinants were isolated, with five being similar to those reported earlier and two havina new type of structure. All available recombinantsw ere characterized with DNA probes for the loci Nor-R1, 5SDna-R1, and Tel-R1. Also, the amount of rye chromatin present was quantified with a dispersed rye-specific repetitive DNA sequence in quantitative dot blots. Furthermore, the wheat-rye recombinants were used as a mapping tool to assign two RFLP markers to specific regions on chromosome arms 1DS and 1RS of wheat and rye, respectively.     

Characterisation of two gene subunits on the 1R chromosome of rye as orthologs of each of the Glu-1 genes of hexaploid wheat

The visco-elastic properties of bread flour are firmly associated with the presence or absence of certain HMW subunits coded by the Glu-1 genes. Identifying allelic specific molecular markers (AS-PCR) associated with the presence of Glu-1 genes can serve as a valuable tool for the selection of useful genotypes. This paper reports the use of primers designed from nucleotide sequences of the Glu-D1 gene of wheat (AS-PCR for Glu-D1y10) that recognise and amplify homologous sequences of the Glu-R1 gene subunits of rye. The primers amplify the complete coding regions and provided two products of different size in rye, in wheats carrying the substitution 1R(1D) and in rye-wheat aneuploid lines carrying the long arm of chromosome 1R. The location, the molecular characterisation of these sequences and their expression during grain ripening seem to demonstrate that the amplification products correspond to structural genes encoding the high-molecular-weight (HMW) glutenins of rye. The homology of the rye gene to subunits encoding HMW glutenins in wheat was confirmed by Southern blots and sequencing. The amplification-products were cloned, sequenced and characterised, and the sequences compared with the main glutenin subunits of wheat and related species. Further, an RT-PCR experiment was performed using primers designed from the sequence of both amplified products. This assay demonstrated that both sequences are expressed in endosperm during grain ripening. The results of these analyses suggest that both gene subunits correspond to x- and y-type genes of the Glu-R1 locus of rye. Received: 11 December 2000 / Accepted: 17 April 2001     

Mitotic illegitimate recombination is a mechanism for novel changes in high-molecular-weight glutenin subunits in wheat-rye hybrids

Wide hybrids can have novel traits or changed expression of a quantitative trait that their parents do not have. These phenomena have long been noticed, yet the mechanisms are poorly understood. High-molecular-weight glutenin subunits (HMW-GS) are seed storage proteins encoded by Glu-1 genes that only express in endosperm in wheat and its related species. Novel HMW-GS compositions have been observed in their hybrids. This research elucidated the molecular mechanisms by investigating the causative factors of novel HMW-GS changes in wheat-rye hybrids. HMW-GS compositions in the endosperm and their coding sequences in the leaves of F(1) and F(2) hybrids between wheat landrace Shinchunaga and rye landrace Qinling were investigated. Missing and/or additional novel HMW-GSs were observed in the endosperm of 0.5% of the 2078 F(1) and 22% of 36 F(2) hybrid seeds. The wildtype Glu-1Ax null allele was found to have 42 types of short repeat sequences of 3-60 bp long that appeared 2 to 100 times. It also has an in-frame stop codon in the central repetitive region. Analyzing cloned allele sequences of HMW-GS coding gene Glu-1 revealed that deletions involving the in-frame stop codon had happened, resulting in novel ～1.8-kb Glu-1Ax alleles in some F(1) and F(2) plants. The cloned mutant Glu-1Ax alleles were expressed in Escherichia coli, and the HMW-GSs produced matched the novel HMW-GSs found in the hybrids. The differential changes between the endosperm and the plant of the same hybrids and the data of E. coli expression of the cloned deletion alleles both suggested that mitotic illegitimate recombination between two copies of a short repeat sequence had resulted in the deletions and thus the changed HMW-GS compositions. Our experiments have provided the first direct evidence to show that mitotic illegitimate recombination is a mechanism that produces novel phenotypes in wide hybrids.