Analysis of Dynamic Accumulation of Three Types of Glutenin Subunits and Their Content in Relation to Sedimentation Value in Common Wheat

The biosynthetic time and accumulations of A-, B-, and C-type glutenin subunits in 7 winter wheat cultivars with different quality （strong, medium, weak gluten） were analyzed by SDS-PAGE. The results showed that no glutenin subunit was observed within 8 d after anthesis. Parts or all A-, B-, and C-type subunits appeared around day 12 in different cultivars. Other A-, B-, and C-type subunits appeared gradually. The accumulation of A-, B-, and C-type subunits fluctuated before maturity. The results of analysis of correlation between the ratios of A/T （total content of glutenin subunits）, A/C, AJ （B＋C）, （A＋B）/C, and （A＋B）/T and SDS-sedimentation value suggested that they were more significant. The negative correlation between the ratio of （B＋C）/T and SDS-sedimentation value was more significant, and the correlations between the ratio C/T and the SDS-sedimentation value were significantly negative.

Characterization of High-Molecular-Weight Glutenin Subunits and Their Coding Genes from Aegilops umbellulata

The high-molecular-weight (HMW) glutenin subunits and their coding genes from Aegilops umbellulata Zhuk. (UU, 2n = 2x = 14) were characterized using SDS-PAGE analysis and molecular approaches. SDS-PAGE analysis showed that the 1Ux subunits from four different accessions possessed electrophoretic mobilities close to, or slower than, that displayed by the 1Dx2.2 subunit of common wheat. The electrophoretic mobilities of the 1Uy subunits were generally similar to those shown by the 1Dy subunits of common wheat. The complete open reading frames of the 1Ux and 1Uy genes were amplified by PCR and subsequently cloned and sequenced. Amino acid sequence comparisons suggested that the primary structure of the 1Ux and 1Uy subunits were identical to that of published HMW glutenin subunits from related species, Phylogenetic analysis indicated that the HMW glutenin subunits of Ae. umbellulata were most closely related to those encoded by the D genome of Triticeae.

Allelic Variation and Genetic Diversity at HMW Glutenin Subunits Loci in Yunnan, Tibetan and Xinjiang Wheat

Allelic variation and genetic diversity at HMW glutenin subunits loci, Glu-A1, Glu-B1and Glu-D1 were investigated in 64 accessions of three unique wheats of western Chinausing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Two HMWglutenin patterns (i.e., null, 7+8, 2+12 and null, 7, 2+12) in 34 Yunnan wheataccessions, 3 HMW glutenin patterns (i.e., null, 7+8, 2+12; null, 6+8, 2+12 andnull, 7+8, 2) in 24 Tibetan accessions and 1 HMW glutenin pattern (null, 7, 2+12) in6 Xinjiang wheat accessions were found. The Tibetan accession TB18 was found to be witha rare subunit 2 encoded by Glu-D1. A total of 4 (i.e., Glu-A1c, Glu-B1a, Glu-B1b andGlu-D1a), 5 (i.e., Glu-A1c, Glu-B1d, Glu-B1b, Glu-D1a and Glu-D1) and 3 alleles (i.e.,Glu-A1c, Glu-B1a and Glu-D1a) at Glu-1 locus were identified among Yunnan, Tibetan andXinjiang unique wheat accessions, respectively. For Yunnan wheat, Tibetan wheat andXinjiang wheat, the Neis mean genetic variation indexes were 0.1574, 0.1366 and 0,respectively, which might indicate the higher genetic diversity at HMW glutenin subunitsloci of Yunnan and Tibetan wheat accessions as compared to that of Xinjiang wheataccessions. Among the three genomes of hexaploid wheats of western China, the highestNeis genetic variation index was appeared in B genome with the mean value of 0.2674,while the indexes for genomes A and D were 0 and 0.0270, respectively. It might bereasonable to indicate that Glu-B1 showed the highest, Glu-D1 the intermediate and Glu-A1 always the lowest genetic diversity.

Composition and Content of High-Molecular-Weight Glutenin Subunits and Their Effects on Wheat Quality

Sedimentation values, flour glutenin macropolymer (GMP) contents, composition and contents of high-molecular-weight (HMW) glutenin subunits (GS) of 233 flour samples were determined. Our data indicated that subunit 1 occurred more frequently at Glu-A1 , subunit pair 7 + 8 at Glu-B1 and 2 + 12 at Glu-D1. The significant relationships between Glu-1 quality score and total HMW glutenin content, sedimentation value and GMP content suggested that the composition of HMW-GS affects wheat quality strongly. Moreover, the total content of HMW-GS was correlated with certain quality parameters more significantly. Relationship between subunit 5 + 10 content and breadmaking quality was better than others, but 2 + 12, 7 + 8, 7 + 9 and 4 + 12 also correlated with certain quality parameters significantly. The contents of total HMW-glutenin, x-type subunits and y-type subunits related with sedimentation value, flour GMP content, and Glu-1 quality score more strongly than that of individual subunit or subunit pair. The flour GMP content, with excellent correlation to sedimentation value, total contents of HMW glutenin, x- and y-type subunits and many other quality parameters, could be an ideal indicator of breadmaking quality at earlier generations for breeding purpose for its simple procedure and small scale.

High-Molecular-Weight Glutenin Subunit Composition of Chinese Wheat Germplasm

The objective of the present study was to characterize the high molecular glutenin subunits （HMW-GS） composition and the presence of 1B/1R translocation in newly developed wheat （Triticum aestivum L.） germplasm, which have one or more traits that are useful in wheat improvement. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis （SDS-PAGE） and acid polyacrylamide-gel electrophoresis （A-PAGE） were used to detect HMW-GS composition and the presence of 1B/1R wheat-rye （Secale cereale L.） chromosome translocation in the wheat germplasm. Bread-making quality scores of these lines were determined. A high level of variations in HMW-GS encoded by Glu-1 locus was observed. Sixteen major HMW-GS, with 30 combinations, were detected. The percentage of cultivars with more than two desirable subunits was 38.7%. Thirteen cultivars had bread-making quality scores of 10 in combination with one or two desirable agronomical traits, such as high-yield potential, dwarfing stem, resistance to diseases, and/or tolerance to abiotic stress. Sixty-eight （36.6%） cultivars possessed 1B/1R translocation. The newly developed germplasm with HMW-GS for good quality can be promising resources for improving bread-making quality of wheat.

Effect of HMW-GS 5+10 on Quality Parameters in Four Leading Wheat Cultivars

HMW-GS 5 + 10 was introduced to four major wheat cultivars Kehan9, Kefeng3, Longmai20 and Kenda4 through 5 or 6 consecutive backcrosses. No significant difference in protein content and dry gluten content was observed between cultivars containing 5 + 10 and 2 + 12 or 3 + 12 (P>0.1). In cultivars containing HMW-GS 5 + 10, the ratio of wet gluten content to dry gluten content was 2. 9 - 5.0% (P<0. 01) lower than the ratio from the cultivars containing 2 + 12 or 3 + 12, the ratio of Zeleny sedimentation volume to dry gluten content was 4. 5 - 13. 4% (P<0.05) higher. The degree of softening in cultivars having 5 + 10 was 15 -25 FU (P<0. 01) lower, while the maximum resistance was 82 - 193 EU (P<0. 05) higher. Backcrosses with biochemical marker assisted selection in the improvement of quality in wheat cultivars was discussed.

Allelic Variation and Genetic Diversity at Glu-1 Loci in Chinese Wheat (Triticum aestivum L.) Germplasms

Wheat processing quality is greatly influenced by the seed proteins especially the high molecular weight glutenin subunit (HMW-GS) components, the low molecular weight glutenin subunit (LMW-GS) components and gliadin components. Genes encoding the HMW-GS and LMW-GS components were located on the long arms and the short arms of homoeologous group 1 chromosomes, respectively. HMW-GS components in 5 129 accessions of wheat germplasms were analyzed systematically, including 3 459 landraces and 1 670 modern varieties. These accessions were chosen as candidate core collections to represent the genetic diversity of Chinese common wheat ( Triticum aestivum) germplasms documented and conserved in the National Gene Bank. These candidate core collections covered the 10 wheat production regions in China. In the whole country, the dominating alleles at the three loci are Glu-A1b (null), Glu-B1b (7 + 8), and Glu-D1a (2 + 12), respectively. The obvious difference between the land race and the modern variety is the dramatic frequency increase of alleles Glu-A1a (1), Glu-B1c (7 + 9), Glu-B1h (14 + 15), Glu-D1d (5 + 10) and allele cording 5 + 12 subunits in the later ones. In the whole view, there is minor difference on the genetic(allelic)richness between the landrace and the modern variety at Glu-1, which is 28 and 30 respectively. However, the genetic dispersion index (Simpson index) based on allelic variation and frequencies at Glu-A1, Glu-B1 and Glu-D1 suggested that the modern varieties had much higher genetic diversity than the landraces. This revealed that various isolating mechanisms (such as auto-gamous nature, low migration because of undeveloped transposition system) limited the gene flow and exchange between populations of the landraces, which led up to some genotypes localized in very small areas. Modern breeding has strongly promoted gene exchanges and introgression between populations and previous isolated populations. In the three loci, Glu-Bl has the highest genetic diversity , then Glu-D1, while Glu-A1 always keeps the lowest genetic diversity. In the landrace, the three regions with the highest allelic richness are Huanghuai Winter Wheat Region, Northwest Spring Wheat Region and Southwestern Winter Wheat Region. For the bred varieties, the highest allelic richness existed in Southwest Winter Wheat Region, Huanghuai Winter Wheat Region, Low & Middle Branch Winter Wheat Region of Yangtze River. Introduction and utilization of foreign varieties in cross breeding has had great effects on the allelic components and frequency of the three loci, which greatly affected the genetic dispersion index. This has made 'population' of the modern variety quite different from that of the landrace.

Allelic Variation of Glu-A1, Glu-B1 and Glu-Dl in Chinese Released Wheat Varieties in the Last 50 Years

: The high-molecular-weight glutenin subunit (HMW-GS) components from about 85 varieties were fractionated by SDS-PAGE, including 22 corner stone breeding parents, 45 varieties taking important role in Chinese wheat production and 18 varieties with good bread making quality. The 22 corner stone breeding lines carry either null (c) or l(a) summits on Glu-Al loci. Five types are detected on Glu-Bl, which are 7 + 8(b), 7 + 9 (c), 14 + 15 (h), 17 + 18 (i), 6 + 8 (d). 7 + 8 and 7 + 9 are the two major types. Six types have been detected on Glu-Dl, which are 2 + 12 (a), 2 + 10 (e), 5 + 10 (d), 4 + 10 (j), 4 + 12 (c), 2 + 11 (g). 2 + 12 and 2 + 11 are the two major types. In the corner stone breeding lines, early premium from American carries 5 + 10, St2422/464 from Italy carries the 14 + 15. In addition, two relatively essential varieties, Mara and Alondra carry 5 + 10. In the 45 commercialized varieties, sowed more than 666 000 ha (10 million mu) annually, only Yangmai 5 conveys the 5 + 10 on Glu-Dl, and Xiaoyan 6 and Yumai 7 carry the 14 + 15 on Glu-B1. Four varieties, Funo, Nongda 139, Zhengzhou 683 and Fan 6 convey the 17 + 18 on Glu-B1. The 18 bread wheat varieties recommended by the Ministry of Agriculture in 1992 could be classified into two groups, 5 + 10 and 14 + 15. Zhongzuo 8131 and its selections are the typical genotype of 5 + 10. Genes coding the two subunits are from either Yecora F-70 or IRN68-181. Xiaoyan 6 and its derivative varieties are the typical genotype of 14 + 15. The genes coding 17 + 18 subunits in Chinese varieties were derived from Funo or its selections. These results basically reflect allelic changes on Glu-1 in Chinese wheat varieties in the past 50 years. It is also proven that HMW-GS components may have great diversity between lines from the same cross, which causes great difference on baking quality.

A genetic evidence of chromosomal fragment from bridge parent existing in substitution lines between two common wheat varieties

Locating of important agronomic genes onto chromosome is helpful for efficient development of new wheat varieties. Wheat chromosome substitution lines between two varieties have been widely used for locating genes because of their distinctive advantages in genetic analysis, compared with the aneuploid genetic materials. Apart from the substituted chromosome, the other chromosomes between the substitution lines and their recipient parent should be identical, which eases the gene locating practice. In this study, a set of chromosome substitution lines with cv. Wichita （WI） as the recipient parent and cv. Cheyenne （CNN） as the donor parent were studied for the composition of high molecular weight glutenin subunits （HMW-GS） as well as a range of agronomic important traits. Results revealed that the substitution lines of WI（CNN5D）, WI（CNN6A） and WI（CNN7B） had higher plant heights than the two parents of WI and CNN, and WI（CNN3D） had later maturity than the parents. By sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS） analysis, a substitution line WI（CNN5B） was found to contain different HMW-GS patterns from its two parents, in which 1 By9 was replaced by 1 By8 on chromosome 1BL. Simple sequence repeat （SSR） analysis confirmed that the variation on 1BL in WI（CNN5B） was originated from Chinese Spring （CS）. It is concluded that chromosomal fragments from bridge material and donor parent were quite often retained in intracultivaral chromosome substitution lines except the substituting chromosomes.

Development of a wheat material with improved bread-making quality by overexpressing HMW-GS 1S^(l)x2.3^(*)from Aegilops longissima

Wheat bread-making quality can be improved by use of high-molecular-weight glutenin subunits(HMW-GSs)from wild relatives.Aegilops longissima is a close relative of wheat that contains a number of HMW-GS-encoding genes including 1S^(l)x2.3^(*).In this study,transgenic wheat lines overexpressing 1S^(l)x2.3^(*)were obtained by Agrobacterium-mediated transformation and used to investigate the genetic contribution of 1S^(l)x2.3^(*)to wheat flour-processing quality.The 1S^(l)x2.3^(*)transgene was stably inherited and expressed over generations.Expression of 1S^(l)x2.3^(*)increased the relative expression of 1Dx2 and 1Dy12 and reduced that of 1By18 during grain development.In general,integration of 1S^(l)x2.3^(*)stimulated the accumulation of endogenous HMW-GSs and low-molecular-weight glutenin subunits in wheat kernels,greatly increasing the glutenin:gliadin ratio and resulting in faster formation of protein bodies in the endosperm during grain development.A wheat material with improved flour-making quality was developed in which 1S^(l)x2.3^(*)improved wheat bread-making quality.

Evaluation of Emmer Wheat Genetic Resources Aimed at Dietary Food Production

Emmer wheat cultivated by organic farmers is used as a component of some bio （organic） food products. Its positive influence on consumer health is caused by grain composition. With the aim of selecting varieties or landraces for their possible further use we tested selected emmer wheat accessions maintained in the Czech Gene Bank. In the set of 8 emmer wheat accessions, the main grain components, bread making characteristics and contents of health supporting chemical substances such as total dietary fibre content and its components, content of total polyphenols plus catechin and ferulic acid contents, vitamins of the B group and E plus total content of carotenoids were evaluated by standard methods. Tests of bread making quality confirmed a very well known fact that emmer wheat grain is much more suitable for other purposes as whole grain mixtures, cereal pure, etc. than for bread preparation. The results indicate the possibilities to select emmer wheat genotypes differing in grain composition and containing compounds with positive effects on human health. Among the tested emmer wheat accessions the Rudico variety had a complex of positive characteristics such as content of total dietary fibre, total polyphenol content with prevailing catechin and the highest amount of B group vitamins such as B 1, B2, niacin, pantothenic acid and B6.

Isolation and Molecular Characterization of High Molecular Weight Glutenin Subunit Genes 1Bx13 and 1By16 from Hexaploid Wheat

The high molecular weight glutenin subunit （HMW-GS） pair 1Bx13＋1By16 are recognized to positively correlate with bread-making quality; however, their molecular data remain unknown. In order to reveal the mechanism by which 1By16 and 1Bx13 creates high quality, their open reading frames （ORFs） were amplified from common wheat Atlas66 and Jimai 20 using primers that were designed based on published sequences of HMW glutenin genes. The ORF of 1By16 was 2220bp, deduced into 738 amino acid residues with seven cysteines including 59 hexapeptides and 22 nanopeptides motifs. The ORF of 1Bx13 was 2385bp, deduced into 795 amino acid residues with four cysteines including 68 hexapeptides, 25 nanopeptides and six tripepUdes motifs. We found that 1By16 was the largest y-type HMW glutenin gene described to date in common wheat. The 1By16 had 36 amino acid residues inserted in the central repetitive domain compared with 1By15. Expression in bacteria and western-blot tests confirmed that the sequence cloned was the ORF of HMW-GS 1By16, and that 1Bx13 was one of the largest 1Bx genes that have been described so far in common wheat, exhibiting a hexapeptide （PGQGQQ） insertion in the end of central repetitive domain compared with 1Bx7. A phylogenetic tree based on the deduced full-length amino acid sequence alignment of the published HMW-GS genes showed that the 1By16 was clustered with Glu-1B-2, and that the 1Bx13 was clustered with Glu-1B-1 alleles.

Cloning and Expression of Low Molecular Weight Glutenin Genes from the Chinese Elite Wheat Cultivar ＂Xiaoyan 54＂

The low molecular weight （LMW） glutenln subunlts account for 40% of wheat gluten protein content by mass and these proteins are considered to significantly affect dough quality characteristics. Five new full-length LMW glutenln genes （designated LMW-5, LMW-7, LMW-42, LMW-58, and LMW-34） were isolated from the Chinese elite wheat cultivar ＂Xlaoyan 54＂ by PCR amplification of genomlc DNA using a pair of degenerate primers designed from the conserved sequences of the N- and C-terminal regions of published LMW glutenln genes. Deduced amino acid sequence analysis showed that LMW-5 belongs to the LMW-i type genes and that the other four belong to LMW-m type genes. Sequence comparisons revealed that point mutations occasionally occurred in signal peptide and N-terminus domains and often existed in domain III and domain V. Small insertions and deletions are represented in the repetitive domain. There is a stop codon after amino acid position 110 In the repetitive domain of LMW.34, indicating that It is a pseudogene. The other four genes have complete open reading frames and the putative mature regions of these genes were subcloned Into pET-30a expression vector and successfully expressed in Escherlchla coll. Protein sodium dodecyl sulfate-polyacrylamlde gel electro- phoresls analysis showed that all proteins expressed in E. coil by the four genes could be related to B-group LMW glutenln subunits of wheat.

Molecular Characterization of Two Silenced y-type Genes for Glu-B1 in Triticum aestivum ssp.yunnanese and ssp.tibetanum

The high molecular weight glutenin subunits （HMW-GSs） are a major class of common wheat storage proteins. The breadmaking quality of common wheat flour is influenced by the composition of HMW-GSs. In the present study, two unexpressed 1 By genes from Triticum aesitvum L.ssp.yunnanese AS332 and T. aesitvum ssp.tibetanurn AS908 were respectively cloned and characterized. The results indicated that both of the silenced 1By genes in AS332 and AS908 were 1Byg. In contrast to previously reported mechanisms for silenced genes lAx and lay, which was due to the insertion of transposon elements or the presence of premature stop codon via base substitution of C→T transition in trinucleotides CAA or CAG, the silence of 1By9 genes was caused by premature stop codons via the deletion of base A in trinucleotide CA.A, which lead to frameshift mutation and indirectly produced several premature stop codons （TAG） downstream of the coding sequence.

Creating Wheat Germplasm for High Quality Breeding by Monosomic Backcrossing

By crossing bread wheat cultlvar GC8901 with the 1D monosonlc line of Xiaoyan No. 6 and backcrosslng the offsprlng with the Xlaoyan No. 6 1D monosonlc llne for 5 years, high-molecular-welght glutenin subunlts 1Dx5＋1Dy10 from GC8901 have been transferred Into wheat cultivar Xiaoyan No. 6. The BC5F1 offspring lines had been detected by using methods of cytology, marker, molecular marker and six elite single plants with high molecular-welght glutenin subunlts： lAx1, 1Bx14＋1 By15, 1Dx5＋1 Dy10 were Identified. Those lines have high-yleld potential with better agronomic characters and have been used In high quality wheat breeding processes as well.