Rapid and Easy High-Molecular-Weight Glutenin Subunit Identification System by Lab-on-a-Chip in Wheat (Triticum aestivum L.)

Abstract

Lab-on-a-chip technology is an emerging and convenient system to easily and quickly separate proteins of high molecular weight. The current study established a high-molecular-weight glutenin subunit (HMW-GS) identification system using Lab-on-a-chip for three, six, and three of the allelic variations at the Glu-A1, Glu-B1, and Glu-D1 loci, respectively, which are commonly used in wheat breeding programs. The molecular weight of 1Ax1 and 1Ax2* encoded by Glu-A1 locus were of 200 kDa and 192 kDa and positioned below 1Dx subunits. The HMW-GS encoded by Glu-B1 locus were electrophoresed in the following order below 1Ax1 and 1Ax2*: 1Bx13 ≥ 1Bx7 = 1Bx7OE > 1Bx17 > 1By16 > 1By8 = 1By18 > 1By9. 1Dx2 and Dx5 showed around 4-kDa difference in their molecular weights, with 1Dy10 and 1Dy12 having 11-kDa difference, and were clearly differentiated on Lab-on-a-chip. Additionally, some of the HMW-GS, including 1By8, 1By18, and 1Dy10, having different theoretical molecular weights showed similar electrophoretic mobility patterns on Lab-on-a-chip. The relative protein amount of 1Bx7OE was two-fold higher than that of 1Bx7 or 1Dx5 and, therefore, translated a significant increase in the protein amount in 1Bx7OE. Similarly, the relative protein amounts of 8 & 10 and 10 & 18 were higher than each subunit taken alone. Therefore, this study suggests the established HMW-GS identification system using Lab-on-a-chip as a reliable approach for evaluating HMW-GS for wheat breeding programs.

Association of High-Molecular-Weight Glutenin Subunits with Grain and Pasta Quality in Spring Durum Wheat (Triticum turgidum spp. durum L.)

“…Due to the difficulty in distinguishing Bx7 and Bx7OE at SDS-PAGE electropherogram, differentiation is possible using MALDI-TOF-MS technology [52], Lab-on-a-Chip [21], highperformance liquid chromatography (RP-HPLC) [53], or with the use of more accessible classical PCR markers or KASP technology markers with primers developed at the 7OE locus [22,31,44,54]. When considering the frequencies of the Glu-B1al and Glu-B1b alleles obtained in the present study separately, it is obvious that the former (55.1%) has an advantage over the latter (2.9%) in the studied spring durum wheat collection of NCC.…”

“…Improvements in gluten strength are most likely associated with the replacement of the Glu-B1e allele (Bx20 + By20) with the Glu-B1b (Bx7 + By9) and Glu-B1d (Bx6 + By8) high-molecularweight glutenins (HMW-GS), the positive effect of low-molecular-weight glutenin (LMW-GS) subunits of the LMW-2 group, and the γ-45 subunit of gliadin associated with high firmness and viscoelasticity of pasta [11][12][13]15,[18][19][20]. Identifying alleles (including novel ones) with a positive impact on quality and implementing them into the breeding process is facilitated by classic methods of protein (SDS-PAGE) and molecular genetic analysis (PCR), as well as modern approaches such as Lab-on-a-Chip [21], KASP [22,23], and GWAS-based markers [24,25].…”

Synergizing breeding strategies via combining speed breeding, phenotypic selection, and marker-assisted backcrossing for the introgression of Glu-B1i in wheat

“…Garnet (IT 205495) was used as the donor parent ( McCallum and DePauw, 2008 ). The target gene of Garnet was Glu-B1i , located on chromosome 1B, which enhances bread quality by encoding the high-molecular-weight glutenin subunit (HMW-GS) 17 + 18 in wheat ( Payne et al., 1987 ; Jang et al., 2017 ; Guo et al., 2019 ; Shin et al., 2020 ; Guzmán et al., 2022 ). Jokyoung is a leading bread wheat cultivar in Korea that harbors Glu-B1b , which encodes the HMW-GS 7 + 8 ( Jang et al., 2017 ).…”

“…HMW-Gs were extracted from the flour and identified using a lab-on-a-chip (Agilent 2100 bioanalyzer & protein 230 kit, Agilent Technologies, Waldbronn, Germany), following the series of processes proposed by Shin et al ( Shin et al., 2020 ).…”

“…To analyze flour quality, the OYT and AYT lines were milled using a Brabender Quadurmat Junior mill (CW Brabender Instruments, Inc., South Hackensack, NJ, USA) and a Buhler MLU 202 laboratory mill (Bühler AG, Uzwil, Switzerland), respectively. HMW-Gs were extracted from the flour and identified using a lab-on-a-chip (Agilent 2100 bioanalyzer & protein 230 kit, Agilent Technologies, Waldbronn, Germany), following the series of processes proposed by Shin et al (Shin et al, 2020).…”