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

Abstract

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.

Figure 1.—

PCR amplification products from cDNA of three Aegilops species. Lanes 1–3: PCR product from Ae. kotschyi, Ae. neglecta, and Ae. juvenalis with allele-specific-PCR primer 1 + 2. Lanes 4–6: the same materials with primer 3 + 4. Lane 7: 1 kb plus DNA marker.

Figure 2.—

Comparison of deduced amino acid sequences of 15 LMW-GS genes. The mature protein sequence was divided into five domains, namely: I, N-terminal domain; II, repetitive domain; III, cysteine-rich region; IV, glutamine-rich region; and V, C-terminal conservative region. The same sequences and deletions with the AnLMW-m1 subunit are indicated by dots and dashes, respectively. The cysteine residues are represented by a box. The shaded area indicates the DR present in both LMW-m- and LMW-i-type subunits.

Figure 3.—

SDS–PAGE and West blotting detection of induced fusion proteins in different expression vectors. (a) AnLMW-m1, AnLMW-m2, and AnLMW-m3 genes without signal sequences were expressed in pGEX-4T-2 plasmid. The fusion proteins, including GST-tag with ∼26 kDa, are shown. Lane 1: protein maker. Lane 2: CK (empty vector). Lanes 3–5: the fusion proteins of AnLMW-m1, AnLMW-m2, and AnLMW-m3 subunits are indicated by arrows. (b) AnLMW-m1, AnLMW-m2, and AnLMW-m3 genes with signal sequences amplified by primer 5 + 6 were expressed in pET-30a plasmid. The fusion proteins included an additional sequence coding His-tag with ∼840.86 Da of six amino acid residues in the downstream sequence of the insertion site of the pET-30a vector. Lane 1: CK (empty vector). Lanes 2–4: AnLMW-m1, AnLMW-m2, and AnLMW-m3 subunits indicated by arrows. (c) Western blotting detection of the fusion protein of the AnLMW-m1 gene expressed in E. coli. Lanes 1 and 2 are the induced proteins from bacterial medium, which were transformed with the positive and negative pET-30a plasmids, respectively. Lane 3 is the expressed protein (arrow) strongly hybridizing to the anti-His Tag mouse monoclonal antibody, but without any signal to bacterium.

Figure 4.—

Homology tree constructed with complete sequences to show the relationships among LMW-GS and other prolamin genes from different Triticum species and barley (Hordeum vulgare L.). The LMW-GS genes cloned in this study are in boldface type.

Figure 5.—

Several hypotheses on the genetic mechanisms that may generate the chimeric gene. (a) Holliday model (single-stranded invasion mode). Red and blank frame represented LMW-i- and LMW-m-type genes, respectively. Two nicks took place in two non-sister chromatids in synaptonemal complex arrowed in 1. Subsequently, Holliday junction and the branch migration were produced and are indicated by single arrow in 2. As the result of the resolution of the branch in the LMW-i- and LMW-m-type genes in 3, the chimeric gene was formed as indicated with a black arrow in 4. (b) Double-stranded break-repair pathway. Double strands were broken in the same sites of the LMW-i-type gene and digested from 5′ to 3′ as arrowed in 1. Subsequently, a bare single strand intruded into the other chromatids and partnered with one of the chromatids, and the other chromatid was used as template to synthesize a new sequence as shown in 2–4. After the resolution of the branch, the new chimeric gene was produced as arrowed in 5. (c) Illegitimate recombination mechanism. Through the DR coding for nonapeptide (QQPPFSQQQ), the intrastrand recombination occurred in the U genome, and then the novel chemeric gene was generated by crossing over between LMW-i- and LMW-m-type genes. This illegitimate recombination event was estimated to have occurred ∼6.38 (±1.62) MYA.