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Characterization of High-Molecular-Weight Glutenin Subunits and Their Coding Genes from Aegilops umbellulata
Abstract
The high-molecular-weight (HMW) glutenin subunits and their coding genes from Aegilops umbellulata Zhuk. (UU, 2n = 2x = 14) were characterized using SDS-PAGE analysis and molecular approaches. SDS-PAGE analysis showed that the 1 Ux subunits from four different accessions possessed electrophoretic mobilities close to, or slower than, that displayed by the 1Dx2.2 subunit of common wheat. The electrophoretic mobilities of the 1 Uy subunits were generally similar to those shown by the 1Dy subunits of common wheat. The complete open reading frames of the 1 Ux and 1 Uy genes were amplified by PCR and subsequently cloned and sequenced. Amino acid sequence comparisons suggested that the primary structure of the 1 Ux and 1 Uy subunits were identical to that of published HMW glutenin subunits from related species. Phylogenetic analysis indicated that the HMW glutenin subunits of Ae. umbellulata were most closely related to those encoded by the D genome of Triticeae.
... A pair of HMW-GS and their coding genes at Glu-U1 locus was characterized and cloned (Liu et al., 2002). Some Glu-U1 genes were transferred from Ae. umbellulata to the Chinese Spring wheat variety (Islam-Faridi, 1988). ...
Low molecular weight glutenin subunits (LWM-GSs) play a crucial role in determining wheat flour processing quality. In this work, 35 novel LMW-GS genes (32 active and three pseudogenes) from three Aegilops umbellulata (2 n = 2x = 14, UU) accessions were amplified by allelic-specific PCR. We found that all LMW-GS genes had the same primary structure shared by other known LMW-GSs. Thirty-two active genes encode 31 typical LMW-m-type subunits. The MZ424050 possessed nine cysteine residues with an extra cysteine residue located in the last amino acid residue of the conserved C-terminal III, which could benefit the formation of larger glutenin polymers, and therefore may have positive effects on dough properties. We have found extensive variations which were mainly resulted from single-nucleotide polymorphisms (SNPs) and insertions and deletions (InDels) among the LMW-GS genes in Ae. umbellulata . Our results demonstrated that Ae. umbellulata is an important source of LMW-GS variants and the potential value of the novel LMW-GS alleles for wheat quality improvement.
Background:
Wheat-related genomes may carry new glutenin genes with the potential for quality improvement of breadmaking. In this study, we estimated the gluten quality properties of the wheat line CNU609 derived from crossing between Chinese Spring (CS, Triticum aestivum L., 2n = 6x = 42, AABBDD) and the wheat-Aegilops umbellulata (2n = 2x = 14, UU) 1U(1B) substitution line, and investigated the function of 1U-encoded low molecular weight glutenin subunits (LMW-GS).
Results:
The main quality parameters of CNU609 were significantly improved due to introgression of the 1U genome, including dough development time, stability time, farinograph quality number, gluten index, loaf size and inner structure. Glutenin analysis showed that CNU609 and CS had the same high molecular weight glutenin subunit (HMW-GS) composition, but CNU609 carried eight specific 1U genome-encoded LMW-GS. The introgression of the 1U-encoded LMW-GS led to more and larger protein body formation in the CNU609 endosperm. Two new LMW-m type genes from the 1U genome, designated Glu-U3a and Glu-U3b, were cloned and characterized. Secondary structure prediction implied that both Glu-U3a and Glu-U3b encode subunits containing high α-helix and β-strand contents that could benefit the formation of superior gluten structure.
Conclusion:
Our results indicate that the 1U genome has superior LMW-GS that can be used as new gene resources for wheat gluten quality improvement.
Seven genes encoding glutenin subunits that present in Agropyron elongatum (Host) Nevski were cloned by PCR analysis and named AgeloG1 to AgeloG7. The complete open reading frames (ORFs) of the seven genes were amplified with primers special for high-molecular-weight (HMW) glutenin subunit genes and subsequently cloned and sequenced. Five of them were completely sequenced, and the other two (AgeloG1 and AgeloG4) were sequenced at the two ends only. Comparison of amino acid sequences suggested that the primary structure of the subunits encoded by the seven genes was very similar to that of y-type HMW glutenin subunits published from wheat, though four of them (AgeloG4, AgeloG5, AgeloG6 and AgeloG7) were shorter than 1.8 kb. Phylogenetic analysis of the five completely sequenced genes and those subunit genes of Triticum aestivum L. (AABBDD), Aegilops tauschii Coss. (DD), Aegilops caudata L. (CC), Secale cereale L. (RR) and Aegilops umbellulata Zhuk. (UU) indicated that the AgeloG2 was most closely related to 1Dy; the AgeloG3 was to 1By; the AgeloG5 AgeloG6 and AgeloG7 were to 1Ay.
Using degenerate oligonucleotide primers and genomic PCR reactions, the complete coding region sequences of two 1Ay high molecular weight (HMW) glutenin subunit genes were amplified from Triticum urartu accessions that showed differential expression of the 1Ay HMW glutenin subunits in their seeds. The coding sequence amplified from the accession that expressed the 1Ay gene (Tu1Ay-e) was highly homologous to that of known y type HMW glutenin subunit genes. Consequently, the primary structure of the protein translated from the coding sequence of Tu1Ay-e was identical to that of previously published y type subunits. Bacterial expression of the coding sequence of Tu1Ay-e produced a polypeptide identical to the 1Ay subunit extracted from seeds, indicating that the cloned sequence was an accurate representation of the original coding region of Tu1Ay-e. In contrast, the coding region sequence amplified from the accession that did not express 1Ay subunit contained three in-frame premature stop codons. Based on past findings on silenced HMW glutenin subunit genes, we conclude that the presence of in-frame premature stop codon(s) is an important feature of the silenced lAy gene (Tu1Ay-s) in T. urartu The potential value of the active A1y gene in improving the end use quality of common wheat and the mechanism underlying A1y gene silencing are discussed.
The Triticeae species Australopyrum retrofractum (genome WW) produces a single high molecular weight glutenin subunit (HMW-GS) in its endosperm. However, degenerate PCR amplification of its genome DNA revealed the presence of two related HMW-GS sequences, each consisting of an open reading frame. One of these (Glu-W1-2) has not previously been reported. Here, we sequenced Glu-W1-2 and showed that it encodes the same type of HMW-GS as Glu-W1-1, although its overall product length was much shorter, because the number of certain repetitive motifs was lower in its central region. Both A. retrofractum HMW-GSs have a unique repetitive motif, which differentiates them from other known x- and y-type subunits present in Triticeae species. We suggest that A. retrofractum must have diverged from the main Triticeae lineage prior to the Glu-1 duplication event which led to the evolution of the x- and y-type genes.
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.
The genetic control of major wheat endosperm proteins by homoeologous group 1 chromosomes has been studied by two-dimensional polyacrylamide gel electrophoresis. The control of at least 15 distinct protein subunits or groups of protein subunits has been allocated to chromosomes 1A, 1B and 1D of Chinese Spring wheat from the analysis of grains of aneuploid genotypes. In addition, six protein subunits have been shown to be controlled by chromosome 1C(u) of the related species, Aegilops umbellulata, from studies of wheat lines carrying disomic substitutions of 1C(u) chromosomes. On the basis of protein subunit patterns, chromosome 1C(u) is more closely related to chromosome 1D of wheat than to chromosomes 1A or 1B.
The seed proteins of 'Chinese Spring' wheat stocks which possess single chromosomes from other plant species related to wheat have been separated by gel electrophoresis in the presence of sodium dodecyl sulphate. Marker protein bands have been detected for both arms of barley chromosome 5, chromosome E (= 1R) and B (= 2R) of rye, chromosomes A,B (= 1C(u)) and C (= 5C(u)) of Aegilops umbellulata and chromosomes I and III of Agropyron elongatum. These studies, and previous findings, indicate that chromosome 5 of barley, chromosome 1R of rye, chromosome I of Ag. elongatum and possibly chromosome 1C(u) of Ae. umbellulata are similar to chromosomes 1A, 1B and 1D in hexaploid wheat in that they carry genes controlling prolamins on their short arms and genes controlling high-molecular-weight (apparent molecular weight greater than 86,000) seed protein species on their long arms. These findings support the idea that all these chromosomes are derived from a common ancestral chromosome and that they have maintained their integrity since their derivation from that ancestral chromosome.
Two high-molecular-weight subunit (HMWS) glutenin genes from the A and B genomes of the hexaploid bread wheat Triticum aestivum L. cv Cheyenne have been isolated and sequenced. Both of these genes are of the high Mr class (x-type) of HMW glutenins, and have not been previously reported. The entire set of six HMW genes from cultivar Cheyenne have now been isolated and characterized. An analysis of the Ax and Bx sequences shows that the Ax sequence is similar to the homoeologous gene from the D genome, while the Bx repeat structure is significantly different. The repetitive region of these proteins can be modelled as a series of interspersed copies of repeat modifs of 6, 9, and 15 amino acid residues. The evolution of these genes includes single-base substitutions over the entire coding region, plus insertion/deletions of single or blocks of repeats in the central repetitive domain.
A standard karyotype and a generalized idiogram of Triticum umbellulatum (syn. Aegilops umbellulata, 2n = 2x = 14) was established based on C-banding analysis of ten accessions of different geographic origin and individual T. umbellulatum chromosomes in T. aestivum - T. umbellulatum chromosome addition lines. Monosomic (MA) and disomic (DA) T. aestivum - T. umbellulatum chromosome addition lines (DA1U = B, DA2U = D, MA4U = F, DA5U = C, DA6U = A, DA7U = E = G) and telosomic addition lines (DA1US, DA1UL, DA2US, DA2UL, DA4UL, MA5US, (+ iso 5US), DA5UL, DA7US, DA7UL) were analyzed. Line H was established as a disomic addition line for the translocated wheat - T. umbellulatum chromosome T2DS·4US. Radiation-induced wheat - T. umbellulatum translocation lines resistant to leaf rust (Lr9) were identified as T40 = T6BL·6BS-6UL, T41 = T4BL·4BS-6UL, T44 = T2DS·2DL-6UL, T47 = 'Transfer' = T6BS·6BL-6UL and T52 = T7BL·7BS-6UL. Breakpoints and sizes of the transferred T. umbellulatum segments in these translocations were determined by in situ hybridization analysis using total genomic T. umbellulatum DNA as a probe.
TheAegilops genus contains species closely related to wheat. In common with wheat,Aegilops species accumulate high molecular weight (HMW) glutenin subunits in their endospermic tissue. In this study, we investigated
the composition of HMW glutenin subunits in four multiploidAegilops species using SDS-PAGE analysis. Furthermore, by working withAe. ventricosa, we established an efficient genomic PCR condition for simultaneous amplification of DNA sequences coding for either x-or
y-type HMW glutenin subunits from polyploidAegilops species. Using the genomic PCR condition, we amplified and subsequently cloned two DNA fragments that may code for HMW glutenin
subunits inAe. ventricosa. Based on an analysis of the deduced amino acid sequences, we concluded that the two cloned sequences encode one x- and one
y-type of HMW glutenin subunit, respectively.
A comparative genetic map of Aegilops umbellulata with wheat was constructed using RFLP probes that detect homoeoloci previously mapped in hexaploid bread wheat. All seven
Ae. umbellulata chromosomes display one or more rearrangements relative to wheat. These structural changes are consistent with the sub-terminal
morphology of chromosomes 2 U, 3 U, 6 U and 7 U. Comparison of the chromosomal locations assigned by mapping and those obtained
by hybridization to wheat/Ae. umbellulata single chromosome addition lines verified the composition of the added Ae. umbellulata chromosomes and indicated that no further cytological rearrangements had taken place during the production of the alien-wheat
aneuploid lines. Relationships between Ae. umbellulata and wheat chromosomes were confirmed, based on homoeology of the centromeric regions, for 1 U, 2 U, 3 U, 5 U and 7 U. However,
homoeology of the centromeric regions of 4 U with wheat group-6 chromosomes and of 6 U with wheat group-4 chromosomes was
also confirmed, suggesting that a re-naming of these chromosomes may be pertinent. The consequences of the rearrangements
of the Ae. umbellulata genome relative to wheat for gene introgression are discussed.
The high-molecular-weight (HMW) glutenin subunit composition of seven species from the Cylindropyrum and Vertebrata sections of the Aegilops genus was studied using SDS-PAGE and Western blot analysis. Two subunits were detected in Ae. caudata and three in Ae. cylindrica. In both species, subunits showing electrophoretic mobility similar to that of 1Dx2 were present. Western blot analysis using a monoclonal antibody (IFRN 1602) specific for the 1Ax and 1Dx subunits of bread wheat showed that the 1Dx-like subunit of Ae. caudata gave only a weak reaction. This indicates that Ae. caudata expresses subunits which are more distantly related to the 1Dx subunits. Two subunits were detected in each of the 60 accessions of Ae. tauschii, including several 1D(t)x subunits showing different electrophoretic mobilities from those of the 1Dx subunits commonly found in bread wheat. All of the 1Dtx subunits reacted strongly with IFRN 1602, confirming their close relationship to the 1Dx subunits of bread wheat. Three subunits were found in Ae. crassa (6 x), four in Ae, ventricosa and Ae. juvenalis and five in Ae. vavilovii. In these four species, the subunits that showed electrophoretic mobility similar, or close, to that of 1Dx2 all reacted with IFRN 1602. In addition, Ae. ventricosa contained a subunit showing electrophoretic mobility slower than that of 1Dx2.2, which also reacted with IFRN 1602. These results suggest that the D-genome component in the multiploid Aegilops species express at least one HMW glutenin subunit that is structurally related to the 1Dx subunits of bread wheat.
The theory of pivotal-differential evolution states that one genome of polyploid wheats remains stable (i.e., pivotal) during evolution, while the other genome or genomes may become modified (i.e., differential). A proposed mechanism for apparent modification of the differential genome is that different polyploid species with only one genome in common may exchange genetic material. In this study, we analyzed a set of sympatric and allopatric accessions of tetraploid wheats with the genomic constitutions UM and UC. The U genome of these species is from Triticum umbellulatum and is considered to be the pivotal genome. The M and C genomes, from T. comosum and T. dichasians, respectively, are considered to be the differential genomes. Low copy DNA was analyzed using "sequence tagged site" primer sets in the polymerase chain reaction, followed by digestion with restriction enzymes. Genetic similarity matrices based on shared restriction fragments showed that sympatric accessions of different U genome tetraploid species did not tend to share more restriction fragments than did allopatric accessions. Thus, no evidence for introgression was found. Analysis of the diploid progenitor species showed that the U genome was less variable than the M and C genomes. Additionally, comparison of diploid and polyploid species using genome-specific primer sets suggests a possible polyphyletic origin for T. triunciale and T. machrochaetum. Thus, our results suggest that the differential nature of the M and C genomes may be the result of variability introduced by the diploid progenitors and not the result of frequent introgression events after formation of the polyploid.