Isolation of low-molecular-weight glutenin subunit genes from wild emmer wheat (Triticum dicoccoides)

Three low-molecular-weight glutenin subunit (LMW-GS) genes, designated LMW-Td1, LMW-Td2 and LMW-Td3, were isolated from wild emmer wheat (Triticum dicoccoides), which is the tetraploid progenitor of common wheat (T. aestivum). The complete nucleotide sequence lengths of LMW-Td1, LMW-Td2 and LMW-Td3 are 858, 900 and 1062 bp, respectively. LMW-Td1 and LMW-Td3 can encode proteins with 284 and 352 amino acid residues, respectively, whereas LMW-Td2 is a putative pseudogene due to the presence of 3 inframe stop codons in its C-terminal domain. The deduced protein sequences of the 3 genes share the same typical polypeptide structures with known LMW-GS genes containing 8 cysteines in the mature protein domains. LMW-Td1 was clearly distinguished from all known LMW-GS genes, and considered as a novel LMW-GS gene. Two hydrophobic motifs (i.e. PIIIL and PVIIL) were observed in the repetitive domain of LMW-Td3. Sequence comparison indicates that sequences of the 3 LMW-GS genes from this study are strongly similar to known LMW-GS genes. Our phylogenetic analysis suggests that LMW-Td1 and LMW-Td2 are homologous with genes on chromosome 1A, and LMW-Td3 is closely related to genes on chromosome 1B.     

Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii,Ae. triuncialis and Ae. cylindrica,and their molecular organization

Background and Aims Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U^tU^tC^tC^t) and Ae. cylindrica (D^cD^cC^cC^c) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting.Methods The flow karyotypes obtained after the analysis of 4'',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA.Key Results FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C^t, T6U^tS.6U^tL-5C^tL, 1C^c and 5D^c could be sorted with purities ranging from 66 to 91 %, while the remaining chromosomes could be sorted in groups of 2–5. This identified a partial wheat–C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C–2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected.Conclusions The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat.     

Functional properties of a new low-molecular-weight glutenin subunit gene from a bread wheat cultivar

Some allelic forms of low-molecular-weight glutenin subunit (LMW-GS) can greatly influence the end-use of wheat flours, understanding the function of each allele of LMW-GS is important to wheat quality breeding. A LMW-GS gene XYGluD3-LMWGS 1(AY263369) has been cloned from bread wheat cultivar Xiaoyan 6. The deduced protein contained nine cystine residues, one more than that in all other LMW-GSs reported previously, indicating that it is either a new gene or a new allele of a known LMW-GS gene. In this study, the gene was expressed in E. coil in large scale for the testing of its functional property. Reactive Red 120-Agarose resin was used efficiently to purify the expressed LMW-GS proteins from bacteria, with the lactic acid–sodium lactate buffer (pH 4.5) which contained low concentration SDS as elution solution. The purified protein (belonging to the LMW-m family, MW about 35 KDa) was supplemented into a base flour, the results of 10 g dough mixing test indicated that incorporation of the LMW-GS increased the strength of the dough, with significant increases in mixing time (MT) and peak width (PW), and decrease in breakdown in resistance (RBD) compared with the control. In addition, the dough with incorporation of the LMW-GS had more glutenin macropolyeric protein than the control, suggesting that the LMW-GS participated in forming larger glutenin polymers, and greatly contributed to dough strength. The changes in mixing parameters and the amount of glutenin macropolyeric protein were related to the quantity of incorporating subunits.     

Molecular characterization and genomic organization of low molecular weight glutenin subunit genes at the Glu-3 loci in hexaploid wheat (Triticum aestivum L.)

In this study, we report on the molecular characterization and genomic organization of the low molecular weight glutenin subunit (LMW-GS) gene family in hexaploid wheat (Triticum aestivum L.). Eighty-two positive BAC clones were identified to contain LMW-GS genes from the hexaploid wheat ‘Glenlea’ BAC library via filter hybridization and PCR validation. Twelve unique LMW glutenin genes and seven pseudogenes were isolated from these positive BAC clones by primer-template mismatch PCR and subsequent primer walking using hemi-nested touchdown PCR. These genes were sequenced and each consisted of a single-open reading frame (ORF) and untranslated 5′ and 3′ flanking regions. All 12 LMW glutenin subunits contained eight cysteine residues. The LMW-m-type subunits are the most abundant in hexaploid wheat. Of the 12 LMW-GS, 1, 2 and 9 are i-type, s-type and m-type, respectively. The phylogenetic analysis suggested that the LMW-i type gene showed greater differences to LMW-s and LMW-m-type genes, which, in turn, were more closely related to one another. On the basis of their N-terminal sequences, they were classified into nine groups. Fingerprinting of the 82 BAC clones indicated 30 BAC clones assembled into eight contigs, while the remaining clones were singletons. BAC end sequencing of the 82 clones revealed that long terminal repeat (LTR) retrotransposons were abundant in the Glu-3 regions. The average physical distance between two adjacent LMW-GS genes was estimated to be 81 kb. Most of LMW-GS genes are located in the d-genome, suggesting that the Glu-D3 locus is much larger than the Glu-B3 locus and Glu-A3 locus. Alignments of sequences indicated that the same type (starting with the same N-terminal sequence) LMW-GS genes were highly conserved in the homologous genomes between hexaploid wheat and its donors such as durum wheat and T. tauschii. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

四川小麦地方品种AS1643中低分子量谷蛋白基因的克隆及其蛋白质的二级结构预测

根据小麦低分子量谷蛋白基因保守区序列设计引物P1/P2, 采用PCR法对四川小麦地方品种AS1643的基因组DNA进行扩增, 获得1条约900 bp的片段, 分离、纯化后连接到载体pMD18-T上, 对筛选阳性克隆测序, 获得1个低分子量谷蛋白基因LMW-AS1643(GenBank登录号: EF190322), 其编码区长度为909 bp, 可编码302个氨基酸残基组成的成熟蛋白。序列分析结果表明, LMW-AS1643具有典型的低分子量谷蛋白基因的基本结构, 其推导氨基酸序列与其它已知的LMW-GS相比, 最高相似性为93.40%。生物信息学分析表明, 在LMW-AS1643低分子量谷蛋白中, 无规则卷曲含量最高, 为67.90 %, 其次是a-螺旋, 占30.46 %, b-折叠含量最少, 为1.64 %。     

Characterization of the HMW glutenin subunits from Aegilops searsii and identification of a novel variant HMW glutenin subunit

High molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe a more detailed characterization of the HMW glutenin subunits from Aegilops searsii, which is diploid and contains the S^s genome related to the S genome of Aegilops speltoides and the A, B and D genomes of hexaploid wheat. SDS-PAGE experiments revealed two subunits (one x and one y) for each of the nine Ae. searsii accessions analyzed, indicating that the HMW glutenin subunit gene locus of Ae. searsii is similar to the Glu-1 locus found in wheat in containing both x and y genes. The primary structure of the four molecularly cloned subunits (from two Ae. searsii accessions) was highly similar to that of the previously reported x and y subunits. However, in one accession (IG49077), the last 159 residues of the x subunit (1S^sx49077), which contained the sequence element GHCPTSPQQ, were identical to those of the y subunit (1S^sy49077) from the same accession. Consequently, 1S^sx49077 contains an extra cysteine residue located at the C-terminal part of its repetitive domain, which is novel compared to the x-type subunits reported so far. Based on this and previous studies, the structure and expression of the Glu-1 locus in Ae. searsii is discussed. A hypothesis on the genetic mechanism generating the coding sequence for the novel 1S^sx49077 subunit is presented.     

Production and characterization of a transgenic bread wheat line over-expressing a low-molecular-weight glutenin subunit gene

The end-use properties, and thus the value, of wheat flours are determined to a large extent by the proteins that make up the polymeric network called gluten. Low molecular weight glutenin subunits (LMW-GS) are important components of gluten structure. Their relative amounts and/or the presence of specific components can influence dough visco-elasticity, a property that is correlated with the end-use properties of wheat flour. For these reasons, manipulation of gluten dough strength and elasticity is important. We are pursuing this goal by transforming the bread wheat cultivar Bobwhite with a LMW-GS gene driven by its own promoter. Particle bombardment of immature embryos produced several transgenic lines, one of which over-expressed the LMW-GS transgene. Southern blots confirmed that the transgene was integrated into the wheat genome, although segregation analyses showed that its expression was sometimes poorly transmitted to progeny. We have determined that the transgene-encoded LMW-GS accumulates to very high levels in seeds of this line, and that it is incorporated into the glutenin polymer, nearly doubling its overall amount. However, SDS sedimentation test values were lower from the transgenic material compared to a non transgenic flour. These results suggest that the widely accepted correlation between the amount of the glutenin polymers and flour technological properties might not be valid, depending on the components of the polymer.     

小麦近缘种低分子量麦谷蛋白亚基基因Glu-B3克隆及系统发育分析

发掘小麦近缘种低分子量麦谷蛋白基因, 可为小麦品质改良提供更多的基因资源。文章利用Glu-B3位点特异性标记LB1F/LB1R、LB2F/LB2R、LB3F/LB3R和 LB4F/LB4R, 对普通小麦B染色体组的7个可能供体近缘种, 即硬粒小麦(T. durum)、栽培二粒小麦(T. dicoccum)、野生二粒小麦(T. dicoccoides)、拟斯卑尔脱山羊草(Ae. speltoides)、高大山羊草(Ae. longissima)、西尔斯山羊草(Ae. searsii)和双角山羊草(Ae. bicornis)共20份材料进行PCR扩增, 克隆小麦近缘种中GluB3-1、GluB3-2、GluB3-3和GluB3-4基因的等位变异, 并对Glu-B3位点基因进行系统发育分析。共获得16个新等位变异, 其中GluB3-1基因的新等位变异1个, 命名为GluB3-16, 其推导氨基酸分子量为39.2 kDa; GluB3-3的新等位变异有3个, 分别命名为GluB3-35、GluB3-36和GluB3-37, 其推导氨基酸分子量为44.5 kDa(GluB3-36)或44.6 kDa(GluB3-35和GluB3-37); GluB3-4的新等位变异12个, 分别命名为GluB3-46、GluB3-47、GluB3-48、GluB3-49、GluB3-410、GluB3-411、GluB3-412、GluB3-413、GluB3-414、GluB3-415、GluB3-416和GluB3-417, 其推导氨基酸分子量变化在38.6(GluB3-414)~ 42.5 kDa(GluB3-413)之间; 16个新等位变异都包含单一的完整开放阅读框, 具有低分子量麦谷蛋白亚基的典型结构。文章进一步拓展了低分子量麦谷蛋白基因资源, 揭示不同Glu-B3基因的进化过程不完全相同, 为有效地利用小麦近缘种材料和转基因育种提供了新的基因资源。     

Characterization of low-molecular-weight glutenin subunit genes of Aegilops section Sitopsis and comparative analysis with those of wheat (Triticum aestivum L.) and some Aegilops species

Journal of Genetics -     

普通小麦1BL—1RS K,V型雄性不育体系育性恢复的研究

对１ＢＬ－１ＲＳ Ｋ,Ｖ型雄性不育系及其保持素与中国春及其第一部分同源群染色体全部６个缺－四体杂种Ｆ１的育性恢复进行了研究。结果表明：Ｋ型杂种的育笥恢复主要受１ＢＳ上Ｒｆｖ１基因的控制;而Ｖ杂种则受Ｒｆｖ１的１Ｄ染色上育性恢复基因的共同控制;在保持１Ｄ正常剂量的情况下,使恢复系中载有Ｒｆｖ１的１Ｂ染色体（片段）加倍,如１Ａ缺体－１Ｂ四体能使Ｋ,Ｖ型杂种1Ｆ的育性完全恢复。     

Production and morphology of the hybrids Aegilops kotschyi x Secale cereale and Ae. biuncialis x S. cereale

Hybrids between Aegilops kotschyi and Ae. biuncialis with Secale cereale were synthesized. Five Ae. kotschyi and four Ae. biuncialis accessions, as well as one inbred and four self-compatible forms of Secale cereale were used for crossing. The hybrids were produced directly from cultured embryos or through embryo callus culture. Sixty hybrids, 11 involving Ae. kotschyi and 49 Ae. biuncialis, had a stable somatic chromosome number 2n = 3x = 21. The plants showed good vegetative vigour and tillering capacity. Morphologically the hybrids were intermediate between their parents and completely sterile. In vitro propagation of Ae. kotschyi and Ae. biuncialis x S. cereale hybrids revealed that their capacity for callus production and plantlet regeneration - varies.     

Characterization of low-molecular-weight-glutenin subunit genes from the D-genome of Triticum aestivum,Aegilops crassa,Ae. cylindrica and Ae. tauschii

Twenty low-molecular-weight-glutenin subunit (LMW-GS) gene sequences from the D-genome from Aegilops crassa (2n = 4x = 28), Ae. cylindrica (2n = 4x = 28), Ae. tauschii (2n = 2x = 14) and Triticum aestivum (2n = 6x = 42) were obtained using five sets of specific allele primer pairs. Only the sequences of the first primer pair were complete coding sequences (cds) of LMW-GS, and had 305, 304, 306 and 305 LMW-m amino acid residues in Ae. crassa, Ae. cylindrica, Ae. tauschii and T. aestivum, respectively. The repetitive domain and repeat motif numbers of all LMW glutenin subunits showed eight conserved cysteine residues that lead to the same functional activity in different genome. Based on DNA and predicted protein sequences, phylogenetic trees for all sets of sequences were drawn. At the DNA level, the species closest to T. aestivum for the second, third, fourth and fifth set of sequences were Ae. cylindrica, Ae. tauschii and Ae. crassa, respectively. At the protein level, the species closest to T. aestivum based on the first, second and fifth set of sequences were Ae. cylindrica, Ae. crassa and Ae. crassa, respectively. For other sets of sequences, bread wheat proved to be a distinct species. The LMW-GS gene sequences have been recorded in the GenBank with accession numbers JQ726549–JQ726568JQ726549JQ726550JQ726551JQ726552JQ726553JQ726554JQ726555JQ726556JQ726557JQ726558JQ726559JQ726560JQ726561JQ726562JQ726563JQ726564JQ726565JQ726566JQ726567JQ726568.     

Molecular cloning and characterization of four novel LMW glutenin subunit genes from Aegilops longissima, Triticum dicoccoides and T. zhukovskyi

This paper reports cloning and characterisation of four novel low-molecular-weight glutenin subunit (LMW-GS) genes (designated as TzLMW-m2, TzLMW-m1, TdLMW-m1 and AlLMW-m2) from the genomic DNA of Triticum dicoccoides, T. zhukovskyi and Aegilops longissima. The coding regions of TzLMW-m2, TzLMW-m1, TdLMW-m1 and AlLMW-m2 were 1056 bp, 903 bp, 1056 bp and 1050 bp in length, encoding 350, 300, 350 and 348 amino acid residues, respectively. The deduced amino acid sequences showed that the four novel genes were classified as LMW-m types and the comparison results indicated that the four genes had a more similar structure and a higher level of homology with the LMW-m genes than the LMW-s and -i types genes. However, the first cysteine residue's positions of TzLMW-m2, TdLMW-m1 and AlLMW-m2 were different from the others. Moreover, AlLMW-m2, TdLMW-m1 and TzLMW-m2 all possessed a longer repetitive domain, which was considered to be associated with good quality of wheat. The secondary structure prediction revealed that the content of beta-strand in AlLMW-m2 and TdLMW-m1 exceeded the positive control, suggesting that AlLMW-m2 and TdLMW-m1 should be considered as candidate genes that may have positive effect on dough quality. In order to investigate the evolutionary relationship of the novel genes with the other LMW-GSs, a phylogenetic tree was constructed. The results lead to a speculation that AlLMW-m2, TdLMW-m1 and TzLMW-m2 may be the middle types during the evolution of LMW-m and LMW-s.     

Isolation and characterization of five novel high molecular weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrica

Analysis by SDS-PAGE of total protein fractions from single seeds of Aegilops cylindrica (genomes C and D) and Triticum timopheevi (genomes A and G) showed the presence of three bands corresponding to high molecular weight subunits of glutenin (HMW subunits) in the former and two major bands and a minor band corresponding to HMW subunits in the latter. Three Ae. cylindrica and two T. timopheevi HMW subunit gene sequences, each comprising the entire coding region, were amplified by polymerase chain reaction (PCR) and their complete nucleotide sequences determined. A combination of N-terminal amino acid sequencing of the proteins identified by SDS-PAGE and alignments of the derived amino acid sequences of the proteins encoded by the PCR products identified the Ae. cylindrica HMW subunits as 1Cx, 1Cy and 1Dy, and the T. timopheevi HMW subunits as 1Gx, 1Ax and 1Ay. It was not clear whether or not a 1Gy HMW subunit was present in T. timopheevi. The PCR products from Ae. cyclindrica were derived from 1Cy and 1Dy genes and a silent 1Dx gene containing an in-frame internal stop codon, while those from T. timopheevi were derived from 1Ax and 1Ay genes. The 1Cx, 1Gx and 1Gy sequences were not amplified successfully. The proteins encoded by the five novel genes had similar structures to previously characterized HMW subunits of bread wheat (Triticum aestivum). Differences and similarities in sequence and structure, and in the distribution of cysteine residues (relevant to the ability of HMW subunits to form high M_r polymers) distinguished the HMW subunits of x- and y-type and of each genome rather than those of the different species. There was no evidence of a change in HMW subunit expression or structure resulting from selective breeding of bread wheat. The novel 1Ax, 1Ay, 1Cy and 1Dy HMW subunits were expressed in Escherichia coli, and the expressed proteins were shown to have very similar mobilities to the endogenous HMW subunits on SDS-PAGE. The truncated 1Dx gene from Ae. cylindrica failed to express in E. coli, and no HMW subunit-related protein of the size predicted for the truncated 1Dx subunit could be identified by immunodetection in seed extracts.     

Molecular characterization and phylogenetic analysis of a novel glutenin gene (Dy10.1t) from Aegilops tauschii.

A novel y-type high molecular mass glutenin subunit (HMM-GS) possessing a mobility that is slightly slower than that of the subunit Dy10 obtained by SDS-PAGE, named Dy10.1t, in the wild wheat Aegilops tauschii was identified by 1- and 2-dimensional gel electrophoresis, capillary electrophoresis, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The gene encoding the HMM subunit Dy10.1t was amplified with allele-specific PCR primers, and the amplified products were cloned and sequenced. The coding domain of the Dy10.1t subunit gene consisted of 1980 bp encoding a protein of 658 residues with an M rs of 68 611 Da, which was similar to the M rs determined by MALDI-TOF-MS. The deduced amino acid sequence indicated that Dy10.1t subunit displayed a greater similarity to the Dy12 subunit, differing by only 8 amino acid substitutions. Six coding region single-nucleotide polymorphisms were discovered in the Dy10.1t gene by multiple alignments (1 per 330 bp), 1 in the N-terminal domain and the others in the central repeats. Five of them resulted in residue substitutions, whereas 3 created enzyme site changes. The homology and neighbour-joining trees constructed from code domain sequences of 20 x- and y-type glutenin genes from different Triticum species separated into 2 halves, which corresponded to the x-type and y-type HMM glutenin alleles. Phylogenetic analysis revealed that the Glu-1 gene duplication event probably occurred at about 16.83 million years ago, whereas the divergence times of A, B, and D genomes within x-type and y-type halves were before 7.047 and 10.54 million years ago, respectively.     

Pollen proteins after two-dimensional gel electrophoresis and pollen morphology of the amphiploids Aegilops kotschyi and Ae. variabilis with Secale cereale

Proteins from pollen of parent forms and amphiploids Aegilops variabilis ×Secale cereale and Ae. kotschyi×S. cereale, obtained by in vitro propagation or colchicine treatment of F₁ hybrids, were subjected to a study by two-dimensional (2-D) electrophoresis. Qualitative and quantitative diversities of protein patterns were revealed for the amphiploid pollen. The majority of peptides found in the parent forms were also present in the patterns of the amphiploid pollen; however, some of the parent-form-peptides were not expressed and proteins characteristic only of the amphiploids appeared. In the 2-D combined protein pattern obtained for the parent forms, amphiploids Ae. variabilis × S. cereale produced pollen with a poorer spectrum of proteins. In amphiploid 408B, obtained from treated the F₁ generation with colchicine, the 2-D pattern revealed the presence of less than 50% of the proteins recorded for the parent forms. Pollen grain morphology was studied under a scanning microscope. The structure and shape of exines differed from those of the parents. In the parent forms the pollen grains had only one pore, while in amphiploid pollen, one, two or three pores were observed. Possible explanations for the differences in the 2-D patterns of amphiploids and their parent forms (impoverishment of the protein spectrum and appearance of new peptides) are (1) somaclonal variation and mutagenic activity of colchicine, (2) suppression of structural genes, (3) activity of regulators and (4) translocations. Pollen grains with two or even three pores could appear as a result of the independent activity of the genes from three amphiploidal genomes. Received: 6 March 2001 / Accepted: 26 June 2001     

Characterization of low-molecular-weight glutenin subunit Glu-B3 genes and development of STS markers in common wheat (Triticum aestivum L.)

Low-molecular-weight glutenin subunit (LMW-GS) Glu-B3 has a significant influence on the processing quality of the end-use products of common wheat. To characterize the LMW-GS genes at the Glu-B3 locus, gene-specific PCR primers were designed to amplify eight near-isogenic lines and Cheyenne with different Glu-B3 alleles (a, b, c, d, e, f, g, h and i) defined by protein electrophoretic mobility. The complete coding regions of four Glu-B3 genes with complete coding sequence were obtained and designated as GluB3-1, GluB3-2, GluB3-3 and GluB3-4. Ten allele-specific PCR markers designed from the SNPs present in the sequenced variants discriminated the Glu-B3 proteins of electrophoretic mobility alleles a, b, c, d, e, f, g, h and i. These markers were validated on 161 wheat varieties and advanced lines with different Glu-B3 alleles, thus confirming that the markers can be used in marker-assisted breeding for wheat grain processing quality. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. L. H. Wang and X. L. Zhao contributed equally to this study.