Abstract: The HMW glutenin subunit (HMW-GS) allelic variations of two hundred and twenty nine Tibetan wheat landraces were analyzed by using SDS-PAGE analysis. Seventeen different allelic variations were detected in the evaluated accessions, which were three alleles at the Glu-A1 locus, nine alleles at the Glu-B1 locus and five alleles at the Glu-D1 locus, respectively. Two novel HMW-GS, designated as 5** at the Glu-D1 in As1243 and 6** at the Glu-B1 locus in As1510, were screened out. Based on the present results and previous results, it was suggested that the HMW-GS combinations null, 7+8, 2+12, is the predominate types in all the Chinese wheat landraces. It was noteworthy that 5+10, the generally accepted HMW-GS pairs endowing wheat with good bread making quality at Glu-D1, could be detected in Tibetan wheat landraces, which have been found very rare in other Chinese wheat landraces. Additionally, the rare subunit combinations 2+10 and 2.1+10.1 were respectively found in Tibetan wheat landrace.
INTRODUCTION
The main storage proteins in the wheat seed endosperm comprise glutenins and gliadins. The glutenins include two different components, high molecular weight glutenin subunits (HMW-GS) and low molecular weight glutenin subunits (LMW-GS) (Lawrence and Shepherd, 1981; Wim and Delcour, 2002). The numbers and combinations of the functional HMW-GS have a profound influence on the end use quality of wheat flours (Shewry et al., 1995; Shewry and Halford, 2002; Wim and Delcour, 2002). In hexaploid wheat (Tritium aestivum L, AABBDD, 2n = 6x = 42), there are three different loci, designated as Glu-A1, Glu-B1 and Glu-D1, respectively, encoding for HMW-GS on each of the 1AL, 1BL and 1DL chromosome arms (Lawrence and Shepherd, 1981; Wim and Delcour, 2002). Each locus displays allelic variation that causes differences in protein composition and thus results in differences in baking quality processing among wheat cultivars (Branlard and Dardevet, 1985; Payne et al., 1987, 1988; Luo et al., 2001). Usually, each locus encodes one x and one y type HMW-GS. Owing to gene silencing, there are three to five HMW-GS expressed in hexaploid wheat in most cases, that is two by Glu-D1, one or two by Glu-B1 and one or zero by Glu-A1 loci, by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Payne and Lawrence, 1983).
Wheat landrace have many desirable characters and traits that are valuable for wheat breeding. Introduction of these desirable characters and traits into advanced wheat lines has been the practicable way to improve wheat yield, resistance to biotic and abiotic stress as well as processing quality. China has some endemic wheat, including the Sichuan white wheat complex (Triticum aestivum L.), the Tibetan semi-wild weedrace (T. aestivum var. tibetanum (Shao) Yen et J.L. Yang), Yunnan hulled wheat (T. aestivum concv. Yunnanense King ex Yen and J.L. Yang), Xinjiang rice wheat (T. petropavlovskyi Udacz. and Migusch.) (Shao et al., 1980; Dong et al., 1981; Yen et al., 1988). The HMW-GS compositions of Sichuan white wheat complex, Xinjiang rice wheat and Tibetan semi-wild weedrace wheat have been investigated, which showed a low level of diversity for HMW-GS (Wei et al. 2000, 2001). Besides these endemic wheats, there are many other bread wheat landraces. For example, In Tibet, besides the semi-wild weedrace wheat, which has special characters, such as the hulled glume and spike disarticulation, there are many bread wheat landraces. However, there is no information on HMW-GS composition for these wheat landraces.
The objective of this study was to detect the allelic variation of HMW-GS in 229 accession of Tibetan bread wheat landrace using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The information provided in present study will be valuable for the development of breeding strategies to improve bread-making quality.
MATERIALS AND METHODS
Two hundred and twenty nine Tibetan hexaploid wheat landraces were investigated for HMW-GS compositions (Table 1). All of these landraces were collected from Tibetan in 1988 with the financial support of IBPGR, FAO and UN and conserved in the Triticeae Research Institute of Sichuan Agricultural University, Sichuan, China.
| Table 1: | Materials used in this study |
| As is the code by Triticeae research institute, Sichuan Agriculture University | |
To determine the electrophoretic mobility of each HMW-GS by SDS-PAGE, standards of Chinese Spring (-, 7+8, 2+12), Chuanyu 12 (1,7+8, 5+10), Longfumai 1(2*, 7+9, 5+10), Xiaoyan 6(-, 14+15, 2+12) and Chuannong16 (1, 20, 5+10) that included the specific subunits in the landraces were used. These landraces were analyzed by SDS-PAGE on December 2006 at Triticeae Research Institute of Sichuan Agricultural University according to the procedure of Yan et al. (2002). Half of the wheat grains were ground into powder and added HMW-GS extraction buffer with 0.625 M Tris-HCl buffer (pH 6.8) containing 2% (w/v) SDS, 10% (v/v) glycerol and 2 % β-mercaptoethanol, 0.002% (w/v) bromophenol blue and were shaken four times and left at room temperature for 2 h. The suspension was heated at 95°C for 5 min. The supernatant solution was obtained by centrifugation at 6000x g for 5 min and a 10 μL of the supernatant solution was loaded into a sample well of the SDS-PAGE gel. The electrophoresis was performed at 20 mA by constant current for about 2 h until the tracking dye (bromophenol blue) migrated off the gel. The gels were stained with 0.01% (W/V) Coomassie Blue R250 for 30 min.
During the scoring of the HMW-GS for each accession, the nomenclature system for allelic variation at Glu-A1, Glu-B1 and Glu-D1 loci by Payne and Lawrence (1983) was adopted. The HMW Glu-1 quality scores were determined the same as that by Payne et al. (1987).
RESULTS
For the 229 Tibetan wheat accessions (Table 1), a total of 19 different combinations (Table 2) and 17 different allele variants (Table 3) (three alleles at the Glu-A1 locus, nine alleles at the Glu-B1 locus and five alleles at the Glu-D1 locus, respectively) were detected in the evaluated accessions. Two types of novel x-type HMW-GS alleles, one at the Glu-B1 locus in As1510 and the other at the Glu-D1 locus in As1243, were screened out. We designated the novel x-type HMW-GS in Glu-B1 of As1510, the electrophoretic mobility of this subunit situated between HMW-GS Bx6 and Bx7, as 6**. We also designated the novel x-type HMW-GS in the Glu-D1 of As1243, the electrophoresis mobility of this subunit was faster than that of Dx5, as 5**(Fig. 1).
At the Glu-A1 locus, two active HMW-GS, 1 and 2*, were detected. The null type gene Glu-A1c, encoding no HMW-GS on chromosome 1A, was observed at a very high frequency of 94.32% in the 229 Tibetan wheat accessions.
| Table 2: | Frequencies for HMW glutenin subunit compositions of 229 Tibetan wheat |
| ?Represent the quality scores of some HMW glutenin subunit allelic locus has not assigned, so the total quality scores could not be accounted | |
| Table 3: | Frequencies for HMW glutenin subunit at Glu-A1, Glu-B1 and Glu-D1, respectively |
| Fig. 1: | Two novel high molecular weight glutenin subunits in Tibetan wheat, 1. Chinese Spring 2. Chuanyu12 3. As1243 4. As1513 5. As1510 6. Chinese Spring |
The two types of the active HMW-GS 1 and 2* were found at a very low frequency of 4.80% and 0.88%, respectively (Table 3), indicating that the predominate HMW-GS type at this locus is the null type.
Among the three loci of Glu-B1, Glu-A1 and Glu-D1, Glu-B1 showed highest variations. There were nine alleles at the Glu-B1 locus. i.e., 7+8, 7, 6+8, 21, 17+18, 6**+8, 7+9, 20 and 22. The predominate HMW-GS type at Glu-B1 locus was 7+8 (at a frequency of 84.28%).
In the Glu-D1 locus, the predominate HMW-GS type was 2+12 (at a frequency of 94.32%). The remaining other four HMW-GS were 2+10, 5+10, 2.1+10.1 and 5**+10, at low frequencies of 2.18, 1.75, 1.31 and 0.43%, respectively. Though HMW-GS combination 2+12 was the predominant HMW-GS type at this locus; the frequency of the other four HMW-GS type were different from the other investigation. It is interesting that, HMW-GS 5+10, the generally accepted subunit pair with good baking quality in wheat flour, is very rare in other Chinese wheat landraces but can be detected in Tibetan common wheat landraces. Similarly, The HMW-GS 2+10 were found to be very rich in Aegilops tauschii, D genome progenitor of the bread wheat, but very rare in bread wheat. However, this study indicated that 2+10 existed in Tibetan wheat landraces. Similarily, subunits 2.1+10.1, mainly occur in Aegilops tauschii and are very rare in common wheat (Lagudah and Halloran, 1988), but are also found in Tibetan wheat landraces.
From the HMW-GS composition at the three loci Glu-A1, Glu-B1 and Glu-D1, the predominate HMW-GS composition was null, 7+8, 2+12. In one hundred and seventy-five accessions the subunit composition had a frequency of 76.42%. The quality score for most Tibetan wheat lines was 6, but two accessions have the quality score of 10. Some accessions could not assigned a total quality score, because some HMW-GS, such as 6**+8, 2+10. 21, 22, 2.1+10.1, 20, 5**+10, do not have a generally accepted quality score.
DISCUSSION
Based on the investigation of the HMW-GS composition in 229 accession of Tibetan wheat landraces and previous studies (Wei et al., 2000, 2001; Zhang et al., 2002; Wang et al., 2005), it is suggested that HMW-GS combination null, 7+8, 2+12 is the predominate type in all of the Chinese wheat landraces. However, there were differences in Sichuan bread wheat, showing a percentage up to 97.8% (87 out of 89 accessions), which was much higher than that of Tibetan landrace wheat, in which the percentage is 76.42% (175 out of 229 accessions).
In the three different loci encoding for HMW-GS in the hexaploid wheat, the number of variations showed in all of the Chinese wheat landraces differently from each other. The common is that Glu-B1 detected most variations than both Glu-A1 and Glu-D1 did. However, the types detected at the same HMW-GS locus in different Chinese endemic wheat are not the same. In Glu-A1, the Tibetan wheat landraces showed all the three types (null, 1, 2*), while Sichuan white wheat complex and Tibetan semi-wild weedrace wheat detected HMW-GS null and 1 and Yunnan hulled wheat and Xinjiang rice wheat showed only the null type at this locus (Wang et al., 2005; Wei et al., 2000, 2001). In Glu-B1, the Tibetan wheat landrace detected nine variations, while Yunnan hulled wheat, Xinjiang rice wheat and Tibetan weedrace wheat detected three, four and three types, respectively (Wei et al., 2000, 2001).
In Glu-D1, Tibetan wheat showed five variations, while other Chinese endemic wheat showed only 2+12 at this locus except for Xinjiang rice wheat. It is very interesting that both Xijiang rice wheat and Tibetan wheat can showed the HMW-GS 5+10, the one with good baking quality but this is rare in wheat landraces (Wei et al., 2000, 2001; Zhang et al., 2002) and 2.1+10.1, the one very rare in common wheat and absent in other Chinese endemic wheat is rich in Aegilops tauschii (Lagudah and Halloran, 1988).
As a whole, it can be conclude that Tibetan wheat has the richest polymorphism in HMW-GS composition among all of the Chinese endemic wheat, including Sichuan white complex, Xijiang Rice wheat, Yunnan hulled wheat and Tibetan weedrace wheat.
ACKNOWLEDGMENTS
This research was partially support by the National Natural Science Foundation of China (30370882, 30671272), the Foundation for the Author of National Excellent Doctoral Dissertation of China (200458) and Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, IRT0453), China, the National High Technology Research and Development Program of China (863 program 2006AA10Z179 and 2006AA10Z1F8) and the Youth Foundation of Sichuan Province, China (04ZQ026-040).