Analysis of diversity of Russian and Ukrainian bread wheat (Triticum aestivum L.) cultivars for high-molecular-weight glutenin subunits

The allelic diversity of high-moleculat-weght glutenin subunits (H WIGS) in Russian and Ukrainian bread wheat cultivars was analyzed. The diversity of spring wheat cultivars for alleles of the Glu-1 loci is characterized by medium values of the polymorphism index (polymorphism information content, PlC), and in winter wheats it varies from high at the Glu-A1 locus to low at the Glu-D1 locus. The spring and winter cultivars differ significantly in the frequencies of alleles of the glutenin loci. The combination of the Glu-A1b, Glu-B1c, and Glu-D1a alleles prevails among the spring cultivars, and the combination of the Glu-A1a, Glu-B1c, and Glu-D1d alleles prevails among the winter cultivars. The distribution of the Glu-1 alleles significantly depends on the moisture and heat supply in the region of origin of the cultivars. Drought resistance is associated with the Glu-D1a allele in the spring wheat and with the Glu-B1b allele in the winter wheat. The sources of the Glu-1 alleles were identified in the spring and wheat cultivars. The analysis of independence of the distribution of the spring and winter cultivars by the market classes and by the alleles of the HMWGS loci showed a highly significant association of the alleles of three Glu-1 loci with the market classes in foreign cultivars and independence or a weak association in the Russian and Ukrainian cultivars. This seems to be due to the absence of a statistically substantiated system of classification of the domestic cultivars on the basis of their quality.     

Multiplex PCR identification of wheat HMW glutenin subunit genes by allele-specific markers

Wheat bread-making quality is closely correlated with composition and quantity of gluten proteins, in particular with high-molecular weight (HMW) glutenin subunits encoded by the Glu-1 genes. A multiplex polymerase chain reaction (PCR) method was developed to identify the allele composition of HMW glutenin complex Glu-1 loci (Glu-A1, Glu-B1 and Glu-D1) in common wheat genotypes. The study of multiplex PCR to obtain a well-balanced set of amplicons involved examination of various combinations of selected primer sets and/or thermal cycling conditions. One to three simultaneously amplified DNA fragments of HMW glutenin Glu-1 genes were separated by agarose slab-gel electrophoresis and differences between Ax1, Ax2* and Axnull genes of Glu-A1 loci, Bx6, Bx7 and Bx17 of Glu-B1, and Dx2, Dx5 and Dy10 genes of Glu-D1 loci were revealed. A complete agreement was found in identification of HMW glutenin subunits by both multiplex PCR analysis and SDS-PAGE for seventy-six Polish cultivars/strains of both spring and winter common wheat. Rapid identification of molecular markers of Glu-1 alleles by multiplex PCR can be an efficient alternative to the standard separation procedure for early selection of useful wheat genotypes with good bread-making quality.     

Analysis of diversity of russian and Ukrainian bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) cultivars for high-molecular-weight glutenin subunits

The allelic diversity of high-moleculat-weght glutenin subunits (HMWGS) in Russian and Ukrainian bread wheat cultivars was analyzed. The diversity of spring wheat cultivars for alleles of the Glu-1 loci is characterized by medium values of the polymorphism polymorphism information content (PIC), and in winter wheats it varies from high at the Glu-A1 locus to low at the Glu-D1 locus. The spring and winter cultivars differ significantly in the frequencies of alleles of the glutenin loci. The combination of the Glu-A1b, Glu-B1c, and Glu-D1a alleles prevails among the spring cultivars, and the combination of the Glu-A1a, Glu-B1c, and Glu-D1d alleles prevails among the winter cultivars. The distribution of the Glu-1 alleles significantly depends on the moisture and heat supply in the region of origin of the cultivars. Drought resistance is associated with the Glu-D1a allele in the spring wheat and with the Glu-B1b allele in the winter wheat. The sources of the Glu-1 alleles were identified in the spring and wheat cultivars. The analysis of independence of the distribution of the spring and winter cultivars by the market classes and by the alleles of the HMWGS loci showed a highly significant association of the alleles of three Glu-1 loci with the market classes in foreign cultivars and independence or a weak association in the Russian and Ukrainian cultivars. This seems to be due to the absence of a statistically substantiated system of classification of the domestic cultivars on the basis of their quality.     

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.     

Characterization of the genes coding for the high molecular weight glutenin subunits in Lophopyrum elongatum

In order to reveal the evolution of Glu-E1 loci of Lophopyrum elongatum (Host) A. L?ve and find novel alleles for wheat quality breeding, four Glu-1 alleles were isolated and characterized via genomic PCR, from this wheat-related species. Of them, 1Ex2 and 1Ey2 were novel alleles, which differ from all the previously known HMW-GS alleles of L. elongatum. Two alleles 1Ex1 and 1Ey2, which contain intact open reading frames, have been successfully expressed in E. coli. The expressed proteins showed similar electrophoresis mobility with the candidate high molecular weight glutenin subunit bands found in seeds. Sequence alignment indicated that proteins encoded by the novel alleles showed similar primary structure with those of wheat and other wheat-related grasses, however, they possess some unique modifications in their own structure. For example, the number of residues in the N-terminal domain is different from those of wheat, an irregular tripeptide present between two nonapeptide motifs and a unique cysteine position in the repetitive region. Phylogenetic analyses using N-terminal conserved sequences showed that 1Ex2 was homologous to those from the D genome; but 1Ey2 was homologous to a y-type allele 1Ky from the K genome. The evolution relationship of Glu-E1 alleles and the possible utilization of the alleles in wheat breeding are discussed.     

Expression analysis and physical mapping of low-molecular-weight glutenin loci in hexaploid wheat (Triticum aestivum L.).

Gliadins and glutenins are storage proteins important in determining the bread-, noodle-, and pasta-making quality of wheat. Glutenins consist of HMW and LMW subunits. The Glu-A3, Glu-B3, and Glu-D3 loci on the short arms of chromosomes 1A, 1B, and 1D, respectively, are the major loci for LMW glutenins. To construct physical maps of the Glu-3 loci, a set of 24 high-density filters representing a 3.1x genome coverage hexaploid wheat BAC library was screened by hybridization using a probe made of 3 LMW glutenin sequences. After 2 rounds of hybridization, a subset of 536 BAC clones were selected and fingerprinted. Three developing seed cDNA libraries were also constructed. A total of 5000-6000 ESTs were generated from each library, assembled into contigs and searched by homology for LMW glutenin sequences. In total, 90 full-length LMW glutenin sequences were found to cluster into 8 distinct groups representing at least 21 different LMW glutenin subunits. A set of 24 pairs of PCR primers was designed from these groups and used as markers on the BAC clones. The combined fingerprinting and marker data were used to build the physical maps using FPC software. A total of 91 contigs comprising 254 clones were obtained and 282 clones remained singletons.     

Characterisation and marker development for low molecular weight glutenin genes from<Emphasis Type="Italic"> Glu-A3</Emphasis> alleles of bread wheat (<Emphasis Type="Italic">Triticum aestivum.</Emphasis> L)

PCR was used to amplify low-molecular-weight (LMW) glutenin genes from the Glu-A3 loci of hexaploid wheat cultivars containing different Glu-A3 alleles. The complete coding sequence of one LMW glutenin gene was obtained for each of the seven alleles Glu-A3a to Glu-A3g. Chromosome assignment of PCR products using Chinese Spring nulli-tetrasomic lines confirmed the amplified products were from chromosome 1A. All sequences were classified as LMW-i-type genes based on the presence of an N-terminal isoleucine residue and eight cysteine residues located within the C-terminal domain of the predicted, mature amino acid sequence. All genes contained a single uninterrupted open reading frame, including the sequence from the Glu-A3e allele, for which no protein product has been identified. Comparison of LMW glutenin gene sequences obtained from different alleles showed a wide range of sequence identity between the genes, with between 1 and 37 single nucleotide polymorphisms and between one and five insertion/deletion events between genes from different alleles. Allele-specific PCR markers were designed based on the DNA polymorphisms identified between the LMW glutenin genes, and these markers were validated against a panel of cultivars containing different Glu-A3 alleles. This collection of markers represents a valuable resource for use in marker-assisted breeding to select for specific alleles of this important quality-determining locus in bread wheat.Communicated by P. Langridge     

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.     

小麦高分子量谷蛋白亚基Glu-B1位点沉默基因的克隆与序列分析

二粒小麦（Triticum turgidum L．var．dicoccoides）具有极其丰富的遗传多样性,是栽培小麦品种改良的巨大基因库。在高分子量谷蛋白基因的组成上,它具有许多栽培小麦不存在的变异类型,在Glu—B1位点上的变异更大。我们利用种子贮藏蛋白的SDS—PAGE方法从原产于伊朗的二粒小麦材料PI94640中观察到缺失Glu—B1区的高分子量谷蛋白亚基。利用Glu-1Bx基因保守序列设计PCR引物,对该材料的总DNA扩增,获得了X型亚基编码基因（Glu-1Bxm）的全序列,其全长为3442bp含1070bp的启动子区。序列比较发现,Glu-1Bxm在启动子区序列与Glu—1Bx7的最为相似。而在基因编码区,我们发现Glu—1Bxm仅编码212个氨基酸,由于开放阅读框中起始密码子后第637位核苷酸发生了点突变,即编码谷酰胺的CAA突变为终止密码TAA,可能直接导致了该高分子量谷蛋白亚基的失活,这是我们在小麦Glu—B1位点基因沉默分子证据的首次报道。将Glu—1Bxm全序列与Glu—B1位点其他等位基因进行了系统树分析,发现Glu—1Bxm是较为古老的类型。本文还对该特异高分子量谷蛋白亚基变异类型对品质遗传改良研究的意义进行了讨论。     

Dynamics of the evolution of orthologous and paralogous portions of a complex locus region in two genomes of allopolyploid wheat

Two overlapping bacterial artificial chromosome (BAC) clones from the B genome of the tetraploid wheat Triticum turgidum were identified, each of which contains one of the two high-molecular-weight (HMW) glutenin genes, comprising the complex Glu-B1 locus. The complete sequence (285 506 bp of DNA) of this chromosomal region was determined. The two paralogous x-type ( Glu-1-1 ) and y-type ( Glu-1-2 ) HMW-glutenin genes of the complex Glu-B1 locus were found to be separated by ca. 168 000 bp instead of the 51 000 bp separation previously reported for the orthologous Glu-D1 locus of Aegilops tauschii, the D-genome donor of hexaploid wheat. This difference in intergene spacing is due almost entirely to be the insertion of clusters of nested retrotransposons. Otherwise, the orientation and order of the HMW glutenins and adjacent genes were identical in the two genomes. A comparison of these orthologous regions indicates modes and patterns of sequence divergence, with implications for the overall Triticeae genome structure and evolution. A duplicate globulin gene, found 5' of each HMW-glutenin gene, assists to tentatively define the original duplication event leading to the paralogous x- and y-type HMW-glutenin genes. The intergenic regions of the two loci are composed of different patterns and classes of retrotransposons, indicating that insertion times of these retroelements were after the divergence of the two wheat genomes. In addition, a putative receptor kinase gene near the y-type HMW-glutenin gene at the Glu-B1 locus is likely active as it matches recently reported ESTs from germinating barley endosperm. The presence of four genes represented only in the Triticeae endosperm ESTs suggests an endosperm-specific chromosome domain.     

A novel chimeric low-molecular-weight glutenin subunit gene from the wild relatives of wheat Aegilops kotschyi and Ae. juvenalis: evolution at the Glu-3 loci

Four LMW-m and one novel chimeric (between LMW-i and LMW-m types) low-molecular-weight glutenin subunit (LMW-GS) genes from Aegilops neglecta (UUMM), Ae. kotschyi (UUSS), and Ae. juvenalis (DDMMUU) were isolated and characterized. Sequence structures showed that the 4 LMW-m-type genes, assigned to the M genome of Ae. neglecta, displayed a high homology with those from hexaploid common wheat. The novel chimeric gene, designed as AjkLMW-i, was isolated from both Ae. kotschyi and Ae. juvenalis and shown to be located on the U genome. Phylogentic analysis demonstrated that it had higher identity to the LMW-m-type than the LMW-i-type genes. A total of 20 single nucleotide polymorphisms (SNPs) were detected among the 4 LMW-m genes, with 13 of these being nonsynonymous SNPs that resulted in amino acid substitutions in the deduced mature proteins. Phylogenetic analysis demonstrated that it had higher identity to the LMW-m-type than the LMW-i-type genes. The divergence time estimation showed that the M and D genomes were closely related and diverged at 5.42 million years ago (MYA) while the differentiation between the U and A genomes was 6.82 MYA. We propose that, in addition to homologous recombination, an illegitimate recombination event on the U genome may have occurred 6.38 MYA and resulted in the generation of the chimeric gene AjkLMW-i, which may be an important genetic mechanism for the origin and evolution of LMW-GS Glu-3 alleles as well as other prolamin genes.     

Allelic variations in <Emphasis Type="Italic">Glu-1</Emphasis> and <Emphasis Type="Italic">Glu-3</Emphasis> loci of historical and modern Iranian bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) cultivars

Proline and glutamine-rich wheat seed endosperm proteins are collectively referred to as prolamins. They are comprised of HMW-GSs, LMW-GSs and gliadins. HMW-GSs are major determinants of gluten elasticity and LMW-GSs considerably affect dough extensibility and maximum dough resistance. The inheritance of glutenin subunits follows Mendelian genetics with multiple alleles in each locus. Identification of the banding patterns of glutenin subunits could be used as an estimate for screening high quality wheat germplasm. Here, by means of a two-step 1D-SDS-PAGE procedure, we identified the allelic variations in high and low-molecular-weight glutenin subunits in 65 hexaploid wheat (Triticum aestivum L.) cultivars representing a historical trend in the cultivars introduced or released in Iran from the years 1940 to 1990. Distinct alleles 17 and 19 were detected for Glu-1 and Glu-3 loci, respectively. The allelic frequencies at the Glu-1 loci demonstrated unimodal distributions. At Glu-A1, Glu-B1 and Glu-D1, we found that the most frequent alleles were the null, 7 + 8, 2 + 12 alleles, respectively, in Iranian wheat cultivars. In contrast, Glu-3 loci showed bimodal or trimodal distributions. At Glu-A3, themost frequent alleles were c and e. At Glu-B3 the most frequent alleles were a, b and c. At Glu-D3 locus, the alleles b and a, were the most and the second most frequent alleles in Iranian wheat cultivars. This led to a significantly higher Nei coefficient of genetic variations in Glu-3 loci (0.756) as compared to Glu-1 loci (0.547). At Glu-3 loci, we observed relatively high quality alleles in Glu-A3 and Glu-D3 loci and low quality alleles at Glu-B3 locus.     

Development and validation of a PCR-based marker assay for negative selection of the HMW glutenin allele <Emphasis Type="Italic">Glu-B1-1d</Emphasis> (<Emphasis Type="Italic">Bx-6</Emphasis>) in wheat

Polymorphisms between the coding sequences of high-molecular-weight (HMW) glutenin x-type genes at the Glu-1 locus were used to amplify Glu-1B x-type-specific PCR fragments. PCR analysis in a wheat cultivar subset carrying different Glu-1B x-type alleles resulted in PCR fragments that differed in size for Glu-B1-1d (B-x6) and non-Glu-B1-1d (B-x6) genotypes. Subsequent sequencing analysis revealed a 15-bp in-frame insertion in the coding regions of all Glu-B1-1d (B-x6) genotypes which allowed the development of a B-x6-specific PCR assay for high-throughput allele sizing by ion-pair reversed-phase high-performance liquid chromatography. The assay was validated in a set of 86 German wheat cultivars, and genotyping data unequivocally verified the presence of HMW glutenin subunits GLU-B1-1D (Bx-6) + GLU-B1-2A (By-8) by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These results demonstrate that the PCR assay can be applied for the detection and negative selection of the poor breadmaking quality Glu-B1-1d (B-x6) alleles in wheat breeding programs.     

Generation of high frequency of novel alleles of the high molecular weight glutenin in somatic hybridization between bread wheat and tall wheatgrass

Somatic hybridization between bread wheat and tall wheatgrass (Agropyron elongatum) has generated fertile introgression progenies with novel combinations of high molecular weight glutenin subunits (HMW-GS). Most of these novel HMW-GS alleles were stably inherited. Sixteen HMW-GS sequences were PCR amplified from three introgression progeny lines and sequenced. The alignment of these sequences indicated that five, probably derived from point mutations of the parental genes, whereas eight likely represent the product of replication slippage. Three Glu-1Ay sequences appear to have lost the transposon presented in the parental gene. Two subunits carry an additional cysteine residue, which may be favorable to the quality of end-use product. We demonstrate that novel HMW-GS alleles can be rapidly generated via asymmetric somatic hybridization. Heng Liu and Shuwei Liu have contributed equally to the work.     

High Molecular Weight Glutenin Subunit Composition of Indian Wheats and Their Relationships with Dough Strength

One hundred and seventy two wheat varieties including twenty-five durum wheat cultivars were evaluated for high molecular weight glutenin subunit (HMW-GS) composition using SDS-PAGE. The relationship between HMW-GS and sedimentation tests for dough strength was studied. Three alleles were present at the Glu-A1 locus, eight at Glu-B1 and two at Glu-D1 in bread wheat. The data indicated the prevalence of the Glu-A1b allele (63.5%) at the Glu-A1 and Glu-D1a (71.4%) at Glu-D1 loci. Three alleles, namely Glu-B1b (30.61%), Glu-B1c (25.85%) and Glu-B1i (34.00%) represented about 90% of the alleles at Glu-B1 locus. The combination of Glu-A1b, Glu-B1i and Glu-D1d alleles exhibited highest dough strength as measured by sedimentation value in comparison to other combinations (p<0.001). However, this combination was present only in 7% of the samples evaluated. In durum wheat, the null allele (Glu-A1c) was observed more frequently (76%) than the Glu-A1b allele (24%). Glu-B1f and Glu-B1e alleles represented equally (32% each). Protein subunits 13+16 and 6+8 were found correlated positively (p<0.05) with improved dough strength as compared to subunit 20 in durum wheat. This information can be a valuable reference for designing breeding programme for the improvement of bread and pasta making quality of bread and durum wheats, respectively in India.     

Molecular Characterization of a HMW Glutenin Subunit Allele Providing Evidence for Silencing of x-type Gene on Glu-B1

Understanding the molecular structure of high-molecular-weight glutenin subunit (HMW-GS) may provide useful evidence for the study on the improvement of quality of cultivated wheat and the evolution of Glu-1 alleles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) shows that the subunits encoded by Glu-B1 were null, named 1Bxm, in a Triticum turgidum var. dicoccoides line PI94640. Primers based on the conserved regions in wheat HMW-GS gene promoter and coding sequences were used to amplify the genomic DNA of line PI94640. The PCR products were sequenced, and the total nucleotide sequence of 3 442 bp including upstream sequence of 1 070 bp was obtained. Compared with the reported gene sequences of Glu-1Bx alleles, the promoter region of the Glu-1Bxm showed close resemblance to 1Bx7. The Glu-1Bxm coding region differs from the other Glu-1Bx alleles for a deduced mature protein with only 212 residues, and a stop codon (TAA) at 637 bp downstream from the start codon was present, which was probably responsible for the silencing of x-type subunit genes at the Glu-B1 locus. Phylogenetic tree based on the nucleotide sequence alignment of HMW glutenin subunit genes showed that 1Bxm was the most ancient type of Glu-B1 alleles, suggesting that the evolution rates are different among Glu-1Bx genes. Further study on the contribution of the unique silenced Glu-B1 alleles to quality improvement was also discussed.     

小麦新品种(系)Glu-1位点等位基因变异研究

应用SDS-PAGE技术分析了40份小麦新品种(系)的高分子量麦谷蛋白亚基等位基因变异。在Glu-1位点共检测到10种变异类型,其中Glu-Al位点有3种类型：Null、1、26 ,Glu-B1位点有5种类型：7 8、7 9、14 15、7、17 18,Glu-D1位点有2种类型：2 12、5 10;Null(54．3％)、7 8(51．4％)和2 12(62．9％)分别是Glu-Al、Glu-B1和Glu-D1位点上的主要亚基变异类型。另外,在2份材料的Glu-B1和Glu-D1位点各检测到1个新的亚基,分别命名为1By8．1和1Dx5^ 。Glu-1位点的Nei‘s遗传变异指数平均为0,5648,Glu-B1的遗传多样性最高,Glu-D1最低。供试小麦材料Glu-1位点的HMW-GS组合共有17种类型,以(Null,7 8,2 12)组合为主要类型,占31．4％;有9种亚基组合类型分别只在1份材料中出现,占26.1％。结果表明,这些小麦新品种(系)存在着丰富的亚基组合类型。     

Types and rates of sequence evolution at the high-molecular-weight glutenin locus in hexaploid wheat and its ancestral genomes

The Glu-1 locus, encoding the high-molecular-weight glutenin protein subunits, controls bread-making quality in hexaploid wheat (Triticum aestivum) and represents a recently evolved region unique to Triticeae genomes. To understand the molecular evolution of this locus region, three orthologous Glu-1 regions from the three subgenomes of a single hexaploid wheat species were sequenced, totaling 729 kb of sequence. Comparing each Glu-1 region with its corresponding homologous region from the D genome of diploid wheat, Aegilops tauschii, and the A and B genomes of tetraploid wheat, Triticum turgidum, revealed that, in addition to the conservation of microsynteny in the genic regions, sequences in the intergenic regions, composed of blocks of nested retroelements, are also generally conserved, although a few nonshared retroelements that differentiate the homologous Glu-1 regions were detected in each pair of the A and D genomes. Analysis of the indel frequency and the rate of nucleotide substitution, which represent the most frequent types of sequence changes in the Glu-1 regions, demonstrated that the two A genomes are significantly more divergent than the two B genomes, further supporting the hypothesis that hexaploid wheat may have more than one tetraploid ancestor.     

Genetic Diversity of Gli-1, Gli-2 and Glu-1 Alleles in Sichuan Wheat Landraces

Genetic diversity at Gli-1, Gli-2 and Glu-1 loci was investigated in 89 Sichuan wheat ( Triticum aestivum L.) landraces by using acid polyacrylamide gel electrophoresis (APAGE) and SDS-PAGE. In these landraces, a total of 32 gliadin and 3 high-molecular-weight (HMW) glutenin patterns were observed. In total, 14, 15 and 5 alleles were identified at Gli-1, Gli-2 and Glu-1, respectively. At each locus, the alleles in higher frequency were Gli-A1a (89%), Gli-B1 h (46%), Gli-D1a (65%), Gli-A2a (64%), Gli-B2j (45%), Gli-D2 a (48%), Glu-A1c (99%), Glu-B1b (99%) and Glu-D1a (100%). The Nei's genetic variation index (H) of Sichuan wheat landraces was 0.3706, varying from 0 to 0.7087. The highest genetic diversity was found at Gli-B2 locus, while the lowest was found at Glu-D1 . The genetic diversity at Gli loci was higher than that of Glu-1 loci among these landraces, but it was much lower than that of modern wheat cultivars. These results indicated a narrow genetic base of Sichuan wheat landraces. In this study, “Chengdu-guangtou” had the identical gliadin and HMW-glutenin patterns with “Chinese Spring”, further supporting the proposal that “Chinese Spring” is a strain of “Chengdu-guangtou”.     

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.