Allelic variations of HMW-GS and LMW-GS and quality analysis in Yannong series wheat cultivars/derivative lines

Introduction Gluten quality is one of the most important traits of the common wheat (Triticum aestivum L.). In Chinese wheat production, Yannong series cultivars/derivative lines possess unique characteristics and play an important role in both yield and quality contribution. Methods To dissect their genetic basis of the gluten quality, in this study, allelic variations of high-molecular-weight glutenin subunit (HMW-GS) and low-molecular-weight glutenin subunit (LMW-GS) in 30 Yannong series wheat cultivars/derivative lines and three check cultivars were evaluated using the allele-specific molecular markers, and six crucial quality indexes were also further measured and analyzed. Results The results demonstrated that the frequencies of HMW-GSs By8, Dx5+Dy10 and Dx5+Dy10+Dy12 in these 30 genotypes and three check cultivars accounted for 87.9%, 24.2% and 9.1%, respectively. For the allelic variations of LMW-GSs, Glu-A3a, Glu-A3b, Glu-A3c, Glu-A3f, and Glu-A3g were identified in 18, 9, 13, 11, and 2 genotypes, respectively; Glu-B3d, Glu-B3g and Glu-B3f were identified in 13, 23 and 4 genotypes, respectively. Notably, Yannong 999, containing By8 + Dx5 + Dy10, and Jinan 17 containing By8 + Dy12 both meet the national standard for high-quality wheat and belong to the category of first-class high-quality strong gluten wheat. Discussion These findings can provide reference for wheat quality improvement and popularization in the production.


Introduction
Wheat (Triticum aestivum L.) is an important food crop that provides about 20% of the calories for human consumption in the world (Wang et al., 2023;Han et al., 2024a).Protein is one of the organic compounds in wheat seeds, which accounts for 10%-12% of seed weight (Goesaert et al., 2005).The glutenin proteins play a key role in determining the quality of the wheat flour (Jin et al., 2015;Wang et al., 2018).They can be divided into four types based on the solubility: albumin dissolving in water and diluted buffer; globulin soluble dissolving in salt solution; gliadins dissolving in 70%-90% ethanol, and glutinin dissolving in dilute acid or alkali (Shwry et al., 1995).Under normal condition, albumin and globulin are considered as the metabolic proteins, accounting for about 15% of the glutenin proteins; whereas the remaining gliadins and glutinin are referred as the storage proteins, accounting for approximate 85%.It is reported that gliadin proteins mainly influence dough viscosity and extensibility, whereas the glutenin proteins were mainly involved in the process of dough cohesiveness and elasticity; and their relative proportions determine the distinct characteristics of the wheat gluten (Biesiekierski, 2017;Chen et al., 2018;Gao et al., 2021).
In wheat breeding and production, the quality trait is complex which is often influenced by multiple quality indexes, such as crude protein content, wet gluten content, water absorption, stability time, maximum resistance, stretch area, and bulk density (Ooms and Delcour, 2019).The content and composition of the glutenin proteins are important in determining various quality traits (Weegels et al., 1996).According to the molecular weight, wheat glutenin could be divided into high-molecular-weight glutenin subunits (HMW-GSs) and low-molecular-weight glutenin subunits (LMW-GSs), accounting for 7%-15% and 20%-35% of the storage protein, respectively (Peng et al., 2022).The diversified allelic variations of HMW-GS and LMW-GS play a crucial role in determining different processing quality of wheat flour, particularly affecting gluten strength and dough extensibility (Nagamine et al., 2000;Goesaert et al., 2005;Gao et al., 2016;Sherman et al., 2018;Jiang et al., 2019).
HMW-GSs are encoded by the Glu-1 locus located on the long arms of homoeologous Group I chromosomes 1A, 1B and 1D, which were designated as Glu-A1, Glu-B1 and Glu-D1, respectively (Payne et al., 1980).There are two closely linked genes at each locus: x-type and y-type subunits, and the molecular weight of x-type is higher than that of y-type (Shewry et al., 2003).Theoretically, there are six HMW-GSs in common wheat, however, 3-5 loci were usually expressed because of allelic variations and gene silencing (Yu et al., 2019).Different types of HMW-GSs have different impacts on the gluten quality (Jiang et al., 2019;Li et al., 2019).It was reported that variations in Glu-D1 provided a greater contribution than that in Glu-B1 or Glu-A1 (Payne et al., 1980).The LMW-GSs are encoded by Glu-3 locus that mapped on the short arms of homoeologous Group I chromosomes 1A, 1B and 1D, which were designated as Glu-A3, Glu-B3 and Glu-D3, respectively (Jackson et al., 1996).The LMW-GSs in 222 wheat genotypes were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a total of 20 LMW-GS alleles were identified, including six alleles a-f at Glu-A3 locus, nine alleles a-i at Glu-B3 locus, and five alleles a-e at Glu-D3 locus (Jackson et al., 1996).Due to the similar migration or overlap of LMW-GS alleles in SDS-PAGE detection, accurate identification of the LMW-GS is extremely difficult.With the development of molecular markers, the gene-specific markers were developed at Glu-A3 and Glu-B3 loci (Wang et al., 2009;2010), but not at Glu-D3 locus due to the rarely variations of the alleles (Zhao et al., 2006;Dai et al., 2020).Previous studies also showed that HMW-GS can explain 18%-55% of the variations in gluten strength and elasticity, while LMW-GS can explain 20% (Gianibelli et al., 2001;Li et al., 2019).For example, the HMW-GSs 1Ax1, 1Ax2*, 1Bx17 + 1By18, and 1Dx5 + 1Dy10 have been widely regarded as the high-quality subunits or subunit combinations, with positive effects on gluten strength and elasticity (Payne et al., 1987;Rasheed et al., 2019).The Glu-A3b, Glu-B3b, and Glu-D3e of LMW-GSs are the alleles that mainly contribute to wheat gluten strength (Gupta and acrichie, 1991).
Yannong series wheat cultivars, developed by Shandong Yantai Academy of Agricultural Sciences (Yantai, China), have unique characteristics in Chinese wheat production due to the unique ecological and climatic conditions in Yantai, China.Their promotion areas have reached 39.69 million hm 2 in production (Liu et al., 2019).Lots of wheat cultivars have been developed using Yannong series cultivars as parents.For instance, the backbone parent Youbaomai is the first semi-dwarf high-yield cultivar with a yield exceeding 7,500 kg/hm 2 in China; Yannong 15, as a high-yield and high-quality wheat cultivar, has been used in production more than 40 years; Yannong 999, a super-high yield and strong-gluten wheat cultivar, produced 12,255 kg/hm 2 in the high-yield establishment of wheat and created the highest record of wheat yield in Shandong province and the highest winter wheat yield record in the national acceptance test (Xin et al., 2019).
To dissect the genetic basis of the gluten quality in Yannong series wheat cultivars/derivative lines, this study investigated the allelic variations of three key loci Glu-1, Glu-A3, and Glu-B3 affecting gluten quality using molecular markers, measured their main quality related indexes, such as protein content, wet gluten content, development time and stability time, and also explored the relationship between the allelic variations and index of gluten quality.This study could provide insights in wheat quality improvement and popularization in the production.

Plant materials and field trials
Thirty Yannong series wheat cultivars/derivative lines and three check cultivars were provided by Shandong Yantai Academy of Agricultural Sciences, Yantai, China.All these genotypes were sown at Yantai National Crop Variety Regional Test Station (37 °65′59″N, 120 °47′01″E) from 2022 to 2023 in a randomized complete block design with three replicates, and among them, wheat cultivars Chinese Spring, Jimai 22 and Jinan 17 which carried subunits By8 and Dy12 at Glu-B1 and Glu-D1 locus, respectively, were used as positive controls.Each cultivar/line was planted as a plot with three rows (1.5 m length and 0.25 m between rows) and 30 seeds per row.The field management was the same as the local field production at Yantai National Crop Variety Regional Test Station.After normal maturity, each wheat genotype was separately harvested.After the grains moisture were less than 13%, phenotypes of the quality traits were gained with three replicates for each genotype.

Quality index determination
The wet gluten content, protein content, and stability time, water absorption, development time, and sedimentation value traits were set as the key factors which significantly influenced wheat quality.These six crucial quality indexes of the 30 Yannong series wheat cultivars/lines and three check cultivars were determined using the near-infrared instrument Infratec TM 1241 (Foss, Denmark) to scan NIR spectra.WinISI II v1.50 (InfraSoft International LLC, 2000) software was used to find out the final reading (Irshad et al., 2023).All the experiments were repeated three times.

Descriptive statistics and correlation analysis
The phenotype and genotype data were analyzed using the SPSS 19.0 software (IBM, Chicago, United States).Descriptive statistics were employed to assess the variability of the examined parameters (i.e., the means, maximum, minimum, and standard deviations).Coefficients of variation (CV) also were calculated as part of the analysis of variation.

Index of the gluten quality traits
For these 30 Yannong series wheat genotypes and three check cultivars, the protein content ranged from 11.1% to 19.2%; wet gluten content from 27.9% to 38.9%; development time from 2.7 to 8.8 min, and stability time from 3.8 to 12.5 min, respectively.Water absorption and sedimentation value ranged from 50.8 to 64.8 mL/g and 26.3-41.2mg, respectively.The variance analysis revealed that these indexes were significantly different among 30 Yannong series wheat genotypes and three check cultivars (Figure 3; Table 3).Notably, Yannong 999 with the protein content 17.3%, wet gluten content 36.2%,water absorption 58.6 mL/g, development time 8.6 min, and stability time 11.6 min, and Jinan 17 with these indexes of 19.2%, 38.9%, 63.1 mL/g, 8.8 min and 12.5 min, both meet the national standards for high-quality wheat and belong to the category of first-class high-quality strong gluten wheat.

Relationship between the genotypes and phenotypes
The Dx5 + Dy10 composition has been identified as an elite alleles composition for improving bread-making quality.In the present study, 18 of 30 Yannong series wheat cultivars or derivative lines carried Dx5 + Dy10 composition, indicating it will be necessary to strengthen positive selection for the Dx5 + Dy10 composition for the genetic improvement of high-quality wheat cultivars.Wheat cultivars Yannong 999, Jinan 17, Yannong 377 and Yannong 199 were identified to possess Glu-B3f locus for LMW-GS, and their protein and wet gluten contents were more than 15% and 35% (the first-class high-quality gluten standard), respectively, suggesting their elite gluten characteristics (Figure 3; Table 3).Additionally, the stability time of these genotypes containing Dx5 locus for HMW-GS and Glu-B3g locus for LMW-GS were longer than the others, such as Yannong 999 (11.6 min),Yannong 199 (9.3 min),and Yannong 5158 (7.8 min).So, they meet the secondclass high-quality gluten standard of 7.0 min.

Discussion
For a long time, wheat breeding mainly focused on improving the yield, and hence neglected the pursuit of developing high quality cultivars, leading to the disconnect between wheat supply and demand and the lacking coexistence of high yield and high quality.Therefore, strengthening the breeding and production ability of high-quality wheat cultivars is of great significance for improving food security, solving food conflicts, and enhancing people's sense of happiness.In China, Yannong series wheat cultivars have unique characteristics, such as super-high yield and the harmonious improvement between the yield and quality traits.For instance, Yannong 999, one of the representatives of the Yannong series wheat cultivars, concurrently possesses elite superhigh yield and high quality performance, which reached the Chinese strong gluten wheat standard for two consecutive years in the regional test of the national Huang Huai southern winter water group.To explore genetic basis of the high quality in Yannong series wheat cultivars and their derivative lines, molecular markers for the related genes to quality were used to detect their HMW-GSs and LMW-GSs, and we also combined with the main quality phenotype indexes to dissect the grain quality traits.Determining the alleles composition conferring the quality traits could contribute to both wheat quality breeding and popularization using Yannong series wheat cultivars/derivative lines.
In wheat grain quality traits, although HMW-GS accounts for only 10% of the storage protein content, it is a key factor affecting wheat processing quality (Payne et al., 1987;Zhang et al., 2009).Previous studies proved that the Glu-D1 locus has the greatest effect on the processing quality of the wheat flour, while Glu-B1 and Glu-A1 have relatively less effect.The different protein subunit compositions showed diversified effects on the processing quality of wheat flour.Among them, 1, 2*, 5 + 10 are high-quality protein subunits or subunit compositions for improving the quality of gluten (Liu et al., 2005;Lu et al., 2017); 2 +  Frontiers in Genetics frontiersin.org08 Sun et al. 10.3389/fgene.2024.1465540common subunit composition in 123 regional and modern bread wheat cultivars, accounting for 63.4%, and 5 + 10 subunit composition associated with strong gluten bread wheat were found in 22 wheat cultivars, accounting for only 17.8%.Besides, 2.1 + 12, 2 + 12′, and 2 + 12* subunit compositions were also found among these wheat cultivars (Sönmez et al., 2023).Meanwhile, a previous study showed that Glu-B1 had the highest effect on the variations for the gluten, dough and end-use quality traits, whereas Glu-A1 and Glu-D3 had the lowest impact.The Glu-D1 locus had a strong impact on gluten strength but its contribution to either SDS-Sedimentation volume, gluten extensibility and bread loaf volume was minimal (Guzmán et al., 2022).Jin et al. (2015) found that HMW-GS has a positive effect on dough strength, percentage of insoluble gluten aggregates, and total score of bread.Wheat genotypes containing subunits By9, Bx17, and Dx5 have the highest dough strength and percentage of insoluble gluten aggregates; the genotypes carrying Ax1, Ax2, and Dx5 have the highest total score for the bread.In this present study, the frequencies of subunit compositions Dx5 + Dy10 and Dx5 + Dy10 + Dy12 in 30 Yannong series wheat genotypes and three check cultivars accounted for 24.2% and 9.1%, respectively.Further analysis revealed significant greater ratings for stability time among wheat cultivars carrying Dx5 + Dy10, which was consistent with the report by Jin et al. (2015).Despite an increasing trend in the selection frequency of Dx5 + Dy10 in recent years, there is still potential for further improvement.Notably, Yannong 999 containing subunit composition By8 + Dx5 + Dy10 and Jinan 17 with subunit composition By8 + Dy12 both meet the national standard for high-quality wheat and belong to the category of firstclass high-quality strong gluten wheat.Therefore, they can be popularized in large area for high quality wheat production, and also used as elite breeding parents for high quality wheat breeding.
In analyzing the constitution of the protein subunits, there are certain difficulties in using traditional SDS-PAGE to analyze LMW-GS in hexaploid common wheat: firstly, there are more allelic variations in LMW-GS; secondly, LMW-GS exhibits similar electrophoretic mobility or overlap with alcohol content proteins (Jackson et al., 1996).With the development of molecular markers, a set of STS markers, including seven alleles (Glu-A3a-g) and nine alleles (Glu-B3a-i) have been successively developed (Wang et al., 2009;Wang et al., 2010).Meanwhile, six alleles at Glu-D3 locus have not been developed the detected markers due to their small variations (Zhao et al., 2006;Dai et al., 2020).Using these markers, LMW-GS variations in 343 wheat cultivars from Xinjiang, China were analyzed, and two new types at the Glu-A3 locus, named Glu-A3new1 and Glu-A3new2, two new types at the Glu-B3 locus, named Glu-B3new3 and Glu-B3new4, were identified (Dai et al., 2020).In the present study, we revealed the allelic variations of LMW-GS in 30 Yannong series wheat genotypes and three check cultivars.At the Glu-A3 locus, Glu-A3a, Glu-A3b, Glu-A3c, Glu-A3f, and Glu-A3g were identified in 18, 9, 13, 11 and two wheat cultivars/ lines, respectively.At the Glu-B3 locus, Glu-B3d, Glu-B3g and Glu-B3f were identified in 13, 23 and four genotypes, respectively.Glu-A3d, Glu-A3e, Glu-B3a, Glu-B3b, Glu-B3c, Glu-B3e, and Glu-B3h were absent in all the tested 33 genotypes.These information are valuable for the popularization of these genotypes and breeding improvement using these genotypes as parents.
In the present study, allelic variations of Glu-1, Glu-A3, Glu-B3, and six crucial quality indexes were identified in 30 Yannong series wheat cultivars/derivative lines and three check cultivars.However, the quality traits are complex which are often influenced by multiple quality indexes (Ooms and Delcour, 2019).In future, more quality related genes should be analyzed to identify the genetic basis of the high quality in Yannong series wheat cultivars/derivative lines, such as wheat yellow pigment, polyphenol oxidase activity, and grain (LOX) activity.Additionally, during the process of wheat highquality breeding, more attention could be paid to combine different high quality subunits, evaluate the impact of different combinations on wheat quality and other breeding traits, and finally, find the optimal combinations for high-quality breeding.

Conclusion
In conclusion, the present study determined the allelic variations of Glu-1, Glu-A3, and Glu-B3, measured the quality related traits, and explored the relationship between allelic combinations and quality performance in 30 Yannong series wheat cultivars/ derivative lines and three check cultivars.Therefore, this study could provide reference information for modern wheat quality improvement and popularization in the production.formal analysis, investigation, methodology, resources, software, validation, visualization, and writing-review and editing.DW: data curation, formal analysis, investigation, methodology, resources, software, validation, visualization, and writing-review and editing.JL: data curation, formal analysis, investigation, methodology, validation, visualization, and writing-review and editing.TY: formal analysis, investigation, methodology, validation, visualization, and writing-review and editing.WL: formal analysis, investigation, methodology, validation, visualization, and writing-review and editing.NY: formal analysis, investigation, methodology, validation, visualization, and writing-review and editing.XX: formal analysis, investigation, methodology, validation, visualization, and writing-review and editing.LL: conceptualization, funding acquisition, project administration, validation, visualization, and writing-review and editing.YJ: conceptualization, project administration, validation, visualization, writing-original draft, and writing-review and editing.PM: conceptualization, project administration, validation, visualization, and writing-review and editing.

Funding
The author(s) declare that financial support was received for the research, authorship, and/or publication of this article.This research was financially supported by the research and application of rapid and accurate breeding technology system for wheat (2023CXPT016), and National Modern Wheat Industry Technology System, Yantai Comprehensive Experimental Station project(CARS-03-62).

TABLE 1
Molecular markers for the detection of high and low molecular weight glutenin subunits.

TABLE 2
Distribution of high and low molecular weight glutenin subunits in 30 Yannong wheat cultivars/derivative lines and three check cultivars Chinese Spring, Jinan 17 and Jimai 22.

TABLE 3
Quality indexes of 30 Yannong wheat cultivars/derivative lines and three check cultivars Chinese Spring, Jinan 17 and Jimai 22.