X-type Genes Research Articles

New insights into the evolution of wheat avenin-like proteins in wild emmer wheat (Triticum dicoccoides)

Fifteen full-length wheat grain avenin-like protein coding genes (TaALP) were identified on chromosome arms 7AS, 4AL, and 7DS of bread wheat with each containing five genes. Besides the a- and b-type ALPs, a c type was identified in the current paper. Both a and b types have two subunits, named x and y types. The five genes on each of the three chromosome arms consisted of two x-type genes, two y-type genes, and one c-type gene. The a-type genes were typically of 520 bp in length, whereas the b types were of 850 bp in length, and the c type was of 470 bp in length. The ALP gene transcript levels were significantly up-regulated in Blumeria graminis f. sp. tritici (Bgt)-infected wheat grain caryopsis at early grain filling. Wild emmer wheat [(WEW), Triticum dicoccoides] populations were focused on in our paper to identify allelic variations of ALP genes and to study the influence of natural selection on certain alleles. Consequently, 25 alleles were identified for TdALP-bx-7AS, 13 alleles were identified for TdALP-ax-7AS, 7 alleles were identified for TdALP-ay-7AS, and 4 alleles were identified for TdALP-ax-4AL Correlation studies on TdALP gene diversity and ecological stresses suggested that environmental factors contribute to the ALP polymorphism formation in WEW. Many allelic variants of ALPs in the endosperm of WEW are not present in bread wheat and therefore could be utilized in breeding bread wheat varieties for better quality and elite plant defense characteristics.

Molecular characterization and functional properties of two novel x-type HMW-GS from wheat line CNU608 derived from Chinese Spring × Ae. caudata cross

Two novel HMW-GS, designed as 1Dx2s and 1Dx2f in the wheat line CNU608 derived from hybrids between Chinese Spring (CS) and Ae. caudata were identified. Quality analysis showed that the introgression of 1Dx2s and 1Dx2f subunits led to significant improvement of dough strength and breadmaking quality. The complete encoding sequences of 1Dx2s and 1Dx2f subunits were isolated and cloned. 1Dx2s and 1Dx2f genes encode 831 and 808 amino acid residues, respectively. Their presence and authenticity were confirmed through Escherichia coli expression, Western blotting and tandem mass spectrometry identification. The subunit 1Dx2s had an octapeptide deletion (ELQELQER) in the N-terminal domain compared to 1Dx2 while the subunit 1Dx2f was encoded by a chimeric gene originated from 1Dx2 and 1Cy genes. Particularly, 1Dx2f had an extra cysteine residue from 1Cy subunit at position 730 as well as longer repetitive domain and higher glutamine content, indicating its potential values for wheat gluten quality improvement. Both 1Dx2s and 1Dx2f subunits were simultaneously expressed in the derived line, suggesting that one x-type gene duplication event at the Glu-D1 locus occurred. A putative origin of both 1Dx2s and 1Dx2f genes indicated that they could be originated from one octapeptide deletion and two unequal cross-over events.

New insights into high-molecular-weight glutenin subunits and sub-genomes of the perennial crop Thinopyrum intermedium (Triticeae)

Intermediate wheatgrass (Thinopyrum intermedium) is a perennial crop that possesses desirable agronomic traits and provides environmental services, e.g., reducing soil erosion, nitrate leaching and inputs of energy and pesticide. Thus, intermediate wheatgrass is currently being domesticated as a perennial grain crop. However, the genetic information for molecular breeding is quite limited. Here we report a molecular analysis of high-molecular-weight glutenin subunits (HMW-GS) in intermediate wheatgrass using gene cloning and protein biochemistry. Five HMW-GS genes were isolated from individual intermediate wheatgrass plants: two x-type genes TiHGS1 and TiHGS4, and three y-type genes TiHGS2, TiHGS3 and TiHGS5. Among them, TiHGS5 was novel and possessed an additional cysteine residue at the N-terminal domain or repetitive domain. Sequence alignments showed that TiHGS1 and TiHGS2 genes shared high identities (>96%) with the Glu-1Dx and Glu-1Dy genes, respectively, in common wheat and Aegilops species, TiHGS3 with HMW-GS genes from Dasypyrum or Pseudoroegneria, and TiHGS4 and TiHGS5 with HMW-GS genes from Thinopyrum elongatum. This work provides substantial new insights into the gene compositions and protein profiles of HMW-GS in intermediate wheatgrass, and also gives evidence about the genome components of intermediate wheatgrass.

Molecular characterization of four HMW glutenin genes from Heteranthelium piliferum C. E. Hubbard and Henrardia persica (Banks et Solander) Hochstetter

High molecular weight (HMW) glutenin genes Qy and Oy were isolated and characterized. Compared with previously reported HMW genes, they showed modifications in all domains. Qy genes had an extra glutamine and 105 residues in the N terminal, two irregular tandem repeats of P–GQQGQQVYYPTAPQQ in the repetitive domain, and had the same short peptide LAAQLPAMCRLE as in x-type genes in the C-terminal. Besides these modifications, the 11 aa peptides, GSYYPGQASPQQ, conserved in the repetitive domain in all genes, were absent in Qy. Oy genes with 103 residues, had the short IPTLLS peptide found in x-type genes in the N terminal, and had unique tandem repeats of PGQGQ-RQQGYYPTSQQQ and GQQGQQ in the repetitive domain, and shared the short peptide LAAQLPAMCRLE with those of x-type genes in the C-terminal. Three of four y gene ORFs were successfully expressed in bacteria. In the phylogenetic tree based on the N- and C-terminal sequences, Qy and Oy genes clustered with y and x genes, respectively. This suggested that genes Qy and Oy were not pure y genes, but also had some characteristics of x genes. These unique structures provide new information for understanding the origin and evolution of x and y genes as well as wheat quality improvement.

Characterizing HMW-GS alleles of decaploid Agropyron elongatum in relation to evolution and wheat breeding

Bread wheat quality is mainly correlated with high molecular weight glutenin subunits (HMW-GS) of endosperm. The number of HMW-GS alleles with good processing quality is limited in bread wheat cultivars, while there are plenty of HMW-GS alleles in wheat-related grasses to exploit. We report here on the cloning and characterization of HMW-GS alleles from the decaploid Agropyron elongatum. Eleven novel HMW-GS alleles were cloned from the grass. Of them, five are x-type and six y-type glutenin subunit genes. Three alleles Aex4, Aey7, and Aey9 showed high similarity with another three alleles from the diploid Lophopyrum elongatum, which provided direct evidence for the Ee genome origination of A. elongatum. It was noted that C-terminal regions of three alleles of the y-type genes Aey8, Aey9, and Aey10 showed more similarity with x-type genes than with other y-type genes. This demonstrates that there is a kind of intermediate state that appeared in the divergence between x- and y-type genes in the HMW-GS evolution. One x-type subunit, Aex4, with an additional cysteine residue, was speculated to be correlated with the good processing quality of wheat introgression lines. Aey4 was deduced to be a chimeric gene from the recombination between another two genes. How the HMW-GS genes of A. elongatum may contribute to the improvement of wheat processing quality are discussed.

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.

DHPLC scoring of a SNP between promoter sequences of HMW glutenin x-type alleles at the Glu-D1 locus in wheat.

The promoter regions of HMW glutenin x-type genes at the Glu-D1 locus were surveyed for SNPs within a subpopulation of German bread wheat cultivars. On the basis of the promoter sequences of HMW glutenin subunit genes Glu-A1-x1, Glu-A1-x2, Glu-B1-x1, Glu-B1-x7, Glu-D1-x2, and Glu-D1-x5, an amplification refractory mutation system assay was designed to selectively amplify Dx-specific PCR fragments. Comparative sequence analysis among seven Glu-D1-x2 and seven Glu-D1-x5 wheat cultivars only confirmed a G-A transition in the promoter sequence to be a true polymorphism. SNP scoring by DHPLC of 95 German bread wheat cultivars, with the exception of cv. Anemos, showed that the transition completely agreed with the presence of HMW glutenin subunits 1Dx5 + 1Dy10 in SDS-PAGE. Therefore, the developed DHPLC assay is suitable for high-throughput genotyping to assist the selection of HMW glutenin genes in wheat quality breeding programs.

PCR analysis of x- and y-type genes present at the complex Glu-A1 locus in durum and bread wheat

Genes (x-type) corresponding to different high-molecular-weight glutenin subunits encoded at the Glu-A1 locus present in bread- and durum-wheat cultivars have been selectively amplified by the polymerase chain reaction (PCR). DNA fragments corresponding to an unexpressed x-type gene were also amplified. As unexpressed y-type genes may or may not contain an 8-kb transposon-like insertion, two different sets of primers were designed to obtain amplification of DNA fragments corresponding to these genes. Amplified DNA fragments were also digested with restriction enzymes. The digestion patterns of amplified fragments corresponding to unusual x-type subunits showed similarities with genes encoding the most common subunits 2* and 1. The unexpressed amplified x-type gene showed a restriction pattern similar to the one obtained with the allelic gene encoding high-molecular-weight glutenin subunit 1; homologies were also found within the repetitive region of the linked y-type genes. On the basis of these observations it is postulated that an ancestral active x-type gene, most likely corresponding to subunit 1, was silenced following the insertion of the 8-kb transposon-like fragment into the linked y-type gene.

Two distinct immunoglobulin heavy chain isotypes in a primitive, cartilaginous fish,Raja erinacea

Immunoglobulin heavy chain genes in Raja erinacea (little skate) are organized in clusters consisting of VH, DH, JH segments and CH exons (1). An immunoglobulin heavy chain mu-like isotype that exhibits 61-91% nucleotide sequence identity in coding segments to the Heterodontus francisci (horned shark) mu-type immunoglobulin is described. The overall length of the mu-type clusters is approximately 16 kb; transmembrane exons (TM1 and TM2) are located 3 to CH exon 4 (CH4). In three of four TM-containing genomic clones, a significant deletion is present in TM1. A second isotype of Raja immunoglobulin heavy chain genes has been detected by screening a spleen cDNA library with homologous Raja VH- and CH1-specific probes complementing the respective regions of the mu-like isotype. Weak hybridization with VH-specific probes and no discernable hybridization with C mu-specific probes were considered presumptive evidence for a second immunoglobulin isotype that nominally is designated as X-type. The Vx region of the X-type cDNA is approximately 60% identical at the nucleotide (nt) level to other Raja VH segments and thus represents a second VH family. Putative Dx and Jx sequences also have been identified. The constant region of the X-type immunoglobulin heavy chain gene consists of two characteristic immunoglobulin domains and a cysteine-rich carboxy terminal segment that are only partially homologous with the mu-like isotype. Genomic Southern blotting indicates that the V and C segments of both immunoglobulin heavy chain isotypes are encoded by complex multigene families. Vx- and different Cx-specific probes hybridize to different length transcripts in northern blot analyses of Raja spleen RNA suggesting that the regulation of expression of the X-type genes may involve differential RNA processing.