INTRODUCTION
Two electrophoretic band patterns for beta-amylase activity have been reported in barley (Nielsen and Johansen, 1986, Forster et al., 1991, Evans et al., 1997) and malt (Allison 1973, Allison and Swanston, 1974, Evans et al., 1997). The barley pattern being transformed into the more basic malt pattern by limited proteolytic cleavage of the carboxy-terminal end of the beta-amylase protein (Lundgard and Svensson, 1987, Evans et al., 1997). Determination of the beta-amylase band pattern has been shown to be useful in predicting diastatic power in the progeny of crosses between band pattern types (Swanston, 1980, Evans et al., 1995) and for the estimation of beta-amylase activity by ELISA (Evans et al., 1997). The present study applies our beta-amylase isoelectric focusing (IEF) procedure to determine the IEF band pattern of varieties that had previously been assessed by other electrophoretic procedures.
MATERIAL
Authentic samples of barley for each variety was obtained from the Australian Winter Cereals Collection, Tamworth, Australia and from the Waite Collection of barley varieties.
METHOD
IEF was performed using ultra thin (0.4 mm) polyacrylamide gels containing 5.5 % carrier ampholytes cast onto Gel bond PAG support film (FMC Products). Individual components of the IEF gel were; 1.8 ml glycerol, 3.3 ml 30 % acrylamide, 0.33 ml pH 4-6.5 ampholytes, 0.66 ml pH 4.5-5.4 ampholytes, 0.025 ml TEMED, 0.05 ml 10 % ammonium persulfate, 11.8 ml water. For malt samples the ampholytes were 0.5 ml pH 4.5-5.4 and 0.5 ml pH 5-7. Gels were degassed in a sonicator bath. The catholyte was 0.2 M histidine and the anolyte 0.04 M glutamic acid. Crude extracts were prepared from 20 mg flour or malt, extracted with 1.0 ml buffer (1% glycine, 143 mM beta-mercaptoethanol) for 30 minutes. Extracts were spun down and 5 ul loaded 3 cm from the anode. IEF was performed on an LKB Multiphor II electrophoresis unit at 10 deg.C with light paraffin oil providing even contact between the cooling plate and the gel. Gels were prefocussed for 20 min at 5 W constant power. IEF was performed for 2.5 hours or 4000 volt hours at 10 W constant power. Beta-amylase activity was detected by starch staining (Guerin et al., 1992).
RESULTS AND DISCUSSION
Only two characteristic barley beta-amylase IEF band patterns, Bmy1-Sd1 and -Sd2, can be recognised in 50 varieties tested in Figure 1 (Click here to view) and in all other barley varieties tested thus far (Table 1, 2). These IEF band patterns are controlled by two co-dominant alleles that segregate in the ratio 1:2:1 (Allison, 1973, Evans et al., 1995). The IEF pattern of the heterozygote Bmy-Sd1/2 is a composite of the Bmy-Sd1 and -Sd2 patterns (Evans et al., 1995, 1997). The assignment of variation for beta-amylase band pattern has in the past had at least three different systems of nomenclature, despite varieties being distributed similarly between two patterns regardless of the procedure used (Table 1). Evans et al., 1997 suggested a new and unifying nomenclature for the two alleles that give rise to the electrophoretic patterns for beta-amylase in barley and malt, which are based on the gene expressed and the first description of the electrophoretic banding patterns. Table 1 shows that the Bmy1-Sd1 band pattern type is equivalent to the Sd1 malt pattern (Allison 1973, Allison and Swanston, 1974), the Bmyl1 Ar barley pattern (Nielsen and Johansen, 1986) and the b-amy 1b pattern (Forster et al., 1991) with only five conflicts in assignment from 30 varieties analysed. The Bmy1-Sd2 band pattern type is equivalent to the Sd2 malt pattern (Allison 1973, Allison and Swanston, 1974), the Bmyl1 Br barley pattern (Nielsen and Johansen, 1986) and the b-amy 1a pattern (Forster et al., 1991), with only five conflicts in assignment from 40 varieties analysed. These differences can probably be explained by outcrossing, miss-labelling or varietal heterozygosity that have occurred in the intervening time period. Further testing of the beta-amylase band pattern with our method identified the band pattern types of an additional 65 varieties (Table 2). We conclude that there are two characteristic IEF band pattern types that are common to cultivated barley.
Table 1. Comparison of beta-amylase band pattern types exhibited on IEF in this study with published reports for beta-amylase typing.
The samples were obtained from the Australian Winter Cereals Collection, Tamworth.
Variety
|
IEF
beta-amylase allele
|
Allison
(1973) and
Allison & Swanston (1974)
|
Nielsen
& Johansen (1986)
|
Forster
et al. (1991)
|
|---|---|---|---|---|
| Source
|
Barley
|
Malt
|
Barley
|
Barley
|
| Alexis
|
Bmy1-Sd1
|
-
|
-
|
b-amy
1b
|
| Aramir
|
Bmy1-Sd1
|
-
|
Bmyl1
Ar
|
-
|
| Atem
|
Bmy1-Sd1
|
-
|
-
|
b-amy
1b
|
| Asse
|
Bmy1-Sd1
|
-
|
-
|
-
|
| Balder
|
Bmy1-Sd1
|
Sd1
|
-
|
-
|
| Bente
|
Bmy1-Sd1
|
-
|
Bmyl1
Ar
|
-
|
| Blenheim
|
Bmy1-Sd1
|
-
|
-
|
b-amy
1b
|
| Carina
|
Bmy1-Sd1
|
-
|
Bmyl1
Ar
|
-
|
| Charlottetown
80
|
Bmy1-Sd1
|
Sd1
|
-
|
-
|
| Dandy
|
Bmy1-Sd1*
|
-
|
-
|
b-amy
1a
|
| Digger
|
Bmy1-Sd1
|
-
|
-
|
b-amy
1b
|
| Feebar
|
Bmy1-Sd1
|
Sd1
|
-
|
-
|
| Georgie
|
Bmy1-Sd1
|
-
|
Bmyl1
Ar
|
-
|
| Gerda
|
Bmy1-Sd1
|
Sd1
|
-
|
-
|
| Golf
|
Bmy1-Sd1
|
-
|
-
|
b-amy
1b
|
| Keystone
|
Bmy1-Sd1
|
Sd1
|
-
|
-
|
| Lami
|
Bmy1-Sd1
|
-
|
Bmyl1
Ar
|
-
|
| Magnum
|
Bmy1-Sd1
|
-
|
Bmyl1
Ar
|
-
|
| Morgenrot
|
Bmy1-Sd1*
|
Sd2
|
-
|
-
|
| Natsha
|
Bmy1-Sd1
|
-
|
-
|
b-amy
1b
|
| Olli
|
Bmy1-Sd1
|
Sd1
|
-
|
-
|
| Pirkka
|
Bmy1-Sd1
|
Sd1
|
-
|
-
|
| Plumage
Archer
|
Bmy1-Sd1*
|
Sd2
|
-
|
-
|
| Regatta
|
Bmy1-Sd1*
|
-
|
-
|
b-amy
1a
|
| Triumph
|
Bmy1-Sd1
|
-
|
-
|
b-amy
1b
|
| Vada
|
Bmy1-Sd1
|
Sd1
|
-
|
-
|
| Vantage
|
Bmy1-Sd1
|
Sd1
|
-
|
-
|
| Vantmore
|
Bmy1-Sd1
|
Sd1
|
-
|
-
|
| Vega
|
Bmy1-Sd1*
|
-
|
Bmyl1
Br
|
-
|
| Zephyr
|
Bmy1-Sd1
|
Sd1
|
-
|
-
|
| Akka
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Birka
|
Bmy1-Sd2
|
-
|
Bmyl1
Br
|
-
|
| Betzes
|
Bmy1-Sd2
|
Sd2
|
-
|
b-amy
1a
|
| Boreham
Warrior
|
Bmy1-Sd2
|
Sd2
|
-
|
|
| Bussell
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Camague
|
Bmy1-Sd2
|
-
|
-
|
b-amy
1a
|
| Cambrinus
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Carlsberg
II
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Conquest
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Corniche
|
Bmy1-Sd2
|
-
|
-
|
b-amy
1a
|
| Doublet
|
Bmy1-Sd2
|
-
|
-
|
b-amy
1a
|
| Emir
|
Bmy1-Sd2
|
-
|
Bmyl1
Br
|
-
|
| Gerbel
|
Bmy1-Sd2
|
-
|
Bmyl1
Br
|
-
|
| Glacier
AC38
|
Bmy1-Sd2*
|
Sd1
|
-
|
-
|
| Golden
Promise
|
Bmy1-Sd2
|
Sd2
|
-
|
b-amy
1a
|
| Harry
|
Bmy1-Sd2
|
-
|
Bmyl1
Br
|
-
|
| Hassan
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Herta
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Hi
Proly
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Ingrid
|
Bmy1-Sd2*
|
Sd1
|
-
|
-
|
| Julia
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Koru
|
Bmy1-Sd2
|
-
|
Bmyl1
Br
|
-
|
| Kristina
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Maris
Baldric
|
Bmy1-Sd2
|
Sd2
|
-
|
b-amy
1b*
|
| Maris
Mink
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Midas
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Mona
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Montcalm
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Pallas
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Prisma
|
Bmy1-Sd2
|
-
|
-
|
b-amy
1a
|
| Scotch
Bere
|
Bmy1-Sd2*
|
Sd1
|
-
|
-
|
| Sherpa
|
Bmy1-Sd2
|
-
|
-
|
b-amy
1a
|
| Sonja
|
Bmy1-Sd2
|
-
|
Bmyl1
Br
|
-
|
| Sultan
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Tyne
|
Bmy1-Sd2
|
-
|
-
|
b-amy
1a
|
| Tyra
|
Bmy1-Sd2
|
-
|
Bmyl1
Br
|
-
|
| Universe
|
Bmy1-Sd2*
|
Sd1
|
-
|
-
|
| Wisa
|
Bmy1-Sd2*
|
Sd1
|
-
|
-
|
| Ymer
|
Bmy1-Sd2
|
Sd2
|
-
|
-
|
| Zenit
|
Bmy1-Sd2
|
-
|
-
|
-
|
Table 2: Additional lines screened for beta-amylase type by IEF.
Bmy1-Sd1
|
Bmy1-Sd2
| |
|---|---|---|
| Bearpaw Bomi Caminant Cheri Dera Ellice Franklin Galaxy Harrington Kaputar Kiltra Kustaa Kymppi Maltine Fr Manley Monarch Pokko Shannon Tallon TR 306 Sloop (WI 2875)
|
2EBYT
23 Abyssinia 403046 Abyssinia 403047 Arapilies Bandulla Barque (WI-2868) Cantala Chebec Chieften Clipper CM 72 CYMMYT42002 Dampier Ethiopia 183 Fergie Forrest Fuji Nijo Galleon Gilbert Gimpel Halycon
HE 3631
|
Haruna
Nijo Igri Ketch KMBR 52 Lara Malebo Mazurka Moondyne Morrell Namoi O'Conner Piroline Pomo Proctor Rubin Sahara (3771/1) Schooner Skiff Steptoe Stirling Tweed
Weeah
|
REFERENCES
Allison, M.J. (1973) Genetic studies on the b-amylase isozymes of barley malt. Genetica 44 1-15.
Allison, M.J. and Swanston, J.S. (1974) Relationships between b-amylase polymorphisms in developing, mature and germinating grains of barley. J. Inst. Brew. 80: 285-291.
Evans, D.E., Lance, R.C.M., Eglinton, J.K., Logue, S.J. and Barr, A.R. (1995) The influence of beta-amylase isoform pattern on beta-amylase actvity in barley and malt. In "Proc. 45th Aust. Cereal Chem. Conf., Adelaide, September 10-14" pp 357-354.
Evans, D.E., MacLeod, L.C., Eglinton, J.K., Gibson, C.E., Zhang, X., Wallace, W., Skerritt, J.S. and Lance, R.C.M. (1997) Measurement of beta-amylase in malting barley (Hordeum vulgare L.). Part 1: Development of a quantitative ELISA for beta-amylase. J. Cereal Sci. In press.
Forster, B.P., Thompson, D.M., Watters, J. and Powell, W. (1991) Water-soluble proteins of mature barley endosperm genetic control, polymorphism, and linkage with b-amylase and spring winter habit. Theor. Appl. Genet. 81: 787-792.
Guerin, J.R., Lance, R.C.M. and Wallace, W.W. (1992) Release and activation of barley beta-amylase by malt endopeptidases. J. Cereal Sci. 15: 5-14.
Nielsen, G. and Johansen, H.B. (1986) Proposal for the identification of barley varieties based on the genotypes for 2 hordein and 39 isoenzyme loci of 47 reference varieties. Euphytica 35: 717-728.
Lundgard, R. and Svensson, B. (1987) The four major forms of barley beta amylase, purification, characterisation and structural relationship. Carlsberg Res. Commun. 52: 313-326.
Swanston, J.S. (1980) The use of electrophoresis in testing for high diastatic power in barley. J. Inst. Brew. 86 : 81-83.
