Development of a new marker system for identifying the complex members of the low-molecular-weight glutenin subunit gene family in bread wheat (Triticum aestivum L.)

Abstract

Low-molecular-weight glutenin subunits (LMW-GSs) play an important role in determining the bread-making quality of bread wheat. However, LMW-GSs display high polymorphic protein complexes encoded by multiple genes, and elucidating the complex LMW-GS gene family in bread wheat remains challenging. In the present study, using conventional polymerase chain reaction (PCR) with conserved primers and high-resolution capillary electrophoresis, we developed a new molecular marker system for identifying LMW-GS gene family members. Based on sequence alignment of 13 LMW-GS genes previously identified in the Chinese bread wheat variety Xiaoyan 54 and other genes available in GenBank, PCR primers were developed and assigned to conserved sequences spanning the length polymorphism regions of LMW-GS genes. After PCR amplification, 17 DNA fragments in Xiaoyan 54 were detected using capillary electrophoresis. In total, 13 fragments were identical to previously identified LMW-GS genes, and the other 4 were derived from unique LMW-GS genes by sequencing. This marker system was also used to identify LMW-GS genes in Chinese Spring and its group 1 nulli-tetrasomic lines. Among the 17 detected DNA fragments, 4 were located on chromosome 1A, 5 on 1B, and 8 on 1D. The results suggest that this marker system is useful for large-scale identification of LMW-GS genes in bread wheat varieties, and for the selection of desirable LMW-GS genes to improve the bread-making quality in wheat molecular breeding programmes.

Fig. 1

Sequence alignment of the deduced proteins of 11 active LMW-GS genes in Xiaoyan 54. a Schematic diagram showing the structure of a typical LMW-GS as deduced from their encoding genes. b Multiple alignments of the amino acid sequences deduced from 11 active LMW-GS genes in Xiaoyan 54 (Dong et al. 2010). The s- and m-type subunits, with serine and methionine as the first amino acids of the mature LMW-GS (ACY08809, ACY08812–ACY08819), contain a signal peptide (Sig), an N-terminal domain (N-ter), a repetitive domain (Rep) and a C-terminal domain (C-ter), which is further divided into three regions: C-terminal I (C-ter I), C-terminal II (C-ter II) and C-terminal III (C-ter III). The two i-type subunits (ACY08810 and ACY08811) lack the N-terminal domain (enclosed with broken lines), starting directly with the repetitive domain after the signal peptide. The group of numbers (200, 238, 150, 137, 140, 134, 182, 199, 184, 197 and 230) represents the length of the sequences covering the signal peptide, the N-terminal domain, the repetitive domain and part of the C-terminal I region

Fig. 2

Design of the conserved primers based on nucleotide sequence conservation of the LMW-GS genes in Xiaoyan 54. Based on the conserved sequences of LMW-GS genes, three sets of conserved primers were designed: LMWGS1, LMWGS2 and LMWGS3. LMWGS1 comprised LMWGS1,2F and LMWGS1R. LMWGS2 contained the pair of primers LMWGS1,2F and LMWGS2R, whereas LMWGS3 comprised the three pairs of conserved primers LMWGS3a (LMWGS3aF and LMWGS3aR), LMWGS3b (LMWGS3bF and LMWGS3bR) and LMWGS3c (LMWGS3cF and LMWGS3cR). All of the conserved primers were matched to the sequences flanking the repetitive sequences. Specifically, all the forward primers were located in the sequences encoding the signal peptide or the N-terminal domain, while the reverse primers were located in the sequences encoding the C-terminal I region

Fig. 3

Analysis of polymerase chain reaction (PCR) products amplified from Xiaoyan 54 with the conserved primers. a Electrophoresis of the PCR products amplified from Xiaoyan 54 with the conserved primers in an agarose gel. M DNA ladder Marker III (200, 500, 800, 1,200, 2,000, 3,000 and 4,500 bp; Tiangen Biotech Co., Ltd.). b Electropherograms showing capillary electrophoresis separation of the DNA fragments amplified from Xiaoyan 54 with the conserved primers. The horizontal axis shows the size of the detected DNA fragments, while the vertical axis displays the intensity of the signal (i.e. the concentration of DNA fragments in the PCR products). The orange peaks match the standard DNA fragments in the GeneScan 1200 LIZ size standard, while the blue ones representing the DNA fragments in the PCR products, should match theoretically with the LMW-GS genes in Xiaoyan 54. The numbers on the horizontal axis represent the size of the corresponding peak in the GeneScan 1200 LIZ size standard (orange). In Xiaoyan 54, 14 blue peaks (DNA fragments) could be detected with the conserved primer LMWGS1, 16 with LMWGS2 and 16 with LMWGS3 (7 with LMWGS3a, 6 with LMWGS3b and 3 with LMWGS3c). The data shown are representative of four independent sets of PCR amplifications and DNA fragment analyses

Fig. 4

Electropherograms displaying the patterns of the DNA fragments detected in Chinese Spring and its nulli–tetrasomic lines with the marker system. a Identification of the LMW-GS genes in Chinese Spring with the developed marker system. b Determination of the location of the DNA fragments with the Chinese Spring nulli–tetrasomic lines N1AT1D, N1BT1D and N1DT1B. The horizontal and vertical axes are the same as those described in Fig. 3b. In Chinese Spring, 15 blue peaks (DNA fragments) could be detected with the conserved primer LMWGS1, 14 with LMWGS2 and 16 with LMWGS3 (7 with LMWGS3a, 7 with LMWGS3b and 2 with LMWGS3c). The data collected from three sets of conserved primers indicated that 12 blue peaks (DNA fragments) were detected in N1AT1D, 12 in N1BT1D and 9 in N1DT1B. However, DNA fragment 684 (marked with red arrows) could be detected in all three nulli–tetrasomic lines. All data provided are representative of four independent sets of PCR amplifications and analyses using the Applied Biosystems 3730 DNA Analyzer