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Materials and methods

Plant materials: In recent 50 years, more than 120 wheat cultivars had been cultivated in Sichuan (Yu 1998). The genetic diversity among 40 elite Sichuan wheat cultivars (Table 1), which had once popularized above 66,700 ha per year, were evaluated in this study. Electrophoresis: According to the procedure described by Yang et al. (1992), esterase isozymes were extracted from young shoots of 5 day-old seedling and fractionated by polyacrylamide gel electrophoresis.

Gliadin proteins were extracted from single seeds with a solution of 70% (V/V) ethanol and 0.01% (W/V) methyl green, and fractionated by a standard acid-polyacrylamide gel electrophoresis (APAGE) at pH#3.1 according to the procedure of Cooke (1987). DNA extraction and PCR amplification: Genomic DNA was extracted from young leaves following the procedure described by Sharp et al. (1988). The primers used as random primers in the PCR were purchased from Operon Technologies. The PCR volume was 25 maicroliter and contained 10 ng genomic DNA as template, 1U Taq DNA polymerase, 100 nM primer, 100　 microM each of dATP dCTP, dGTP and dTTP, and 1 x PCR buffer. DNA amplification was performed in a Biometra UNO II DNA Thermal Cycler programmed for 45 cycles of 1 min at 94C, 1 min at 36C, 2 min at 72C. PCR products were separated on 1.0% agarose gels and visualized by ethidium bromide staining. RAPD data scoring and analysis: For each cultivar x primer combination, the presence (1) or absence (0) of an amplified fragment was treated as an independent character without consideration of the quantitative aspects of the results, i.e. band intensity. The data matrix was then used to calculate genetic similarity index (GS)

　　　　　GS=2Nij/(Ni+Nj)

where Nij is the number of RAPD bands in common between genotypes i and j, and Ni and Nj are the total number of RAPD bands observed for genotypes i and j, respectively. Based on the 1--GS matrix, a dendrogram showing the genetic relationships between genotypes was constructed by a UPGMA method (Sneath and Sokal 1973) using computer software NTSYS (Rohlf 1993).

Results and discussion

Esterase variations: Only seven esterase patterns were observed among 40 Sichuan wheat cultivars, while 32 out of 40 cultivars (80%) had identical esterase pattern. The seven different zymograms were described in Fig. 1. The esterase zymograms were similar with each other. Esterase pattern I was the most frequent pattern appeared in Sichuan wheat cultivars. A total of 16 esterase bands, including seven polymorphic bands, were, observed in seven zymograms. In the seven polymorphic bands, four bands were strong stained, and three bands were faint. Yang et al. (1992) also reported that the esterase variations among Sichuan wheat landraces were much low. These results suggested that low level of esterase variability was present in Sichuan wheats. Gliadin variations: Fig. 2 shows the representative gliadin patterns of some Sichuan wheat cultivars. There were 38 different gliadin patterns among 40 Sichuan wheat cultivars. Thirty-six cultivars (90%) had unique gliadin pattern, while Fan 6 and Fan 7, and Mianyang 19 and Mianyang 20 had identical gliadin patterns. Fan 7 was the selected line from Fan 6 (Yen 1999), and also Mianyang 20 from Mianyang 19 (Yu 1998), and therefore it is reasonable that they had identical gliadin patterns.

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