ß-amylase polymorphism in barley is detected by PCR with an arbitrary primer
V.P. Netsvetaev¹, R.N. Kalendar'², and Yu.M. Sivolap²
¹Biochemical Genetics Laboratory and ²Gene Engineering Department,
Plant Breeding and Genetics Institute, Odessa-36, 270036, Ukraine

Using primer P6 (GAG CAA GTT CAG CCT GG) and PCR procedures, differential amplification of a DNA fragment was demonstrated in registered barley cultivars (Sivolap and Kalendar'¹,1994). To map the DNA fragment named f915, which has a molecular weight of about 915 b (Fig. 1), a self-pollinated population from the cross F->inf. Odessky 115 X Golf was studied. Maximum likelihood formulas developed to calculate recombination frequencies and information per individual for similar crosses were described by Netsvetaev and Sozinov, 1984). Chromosomes 1, 3, and 4 of the parents were variable for isozymes at the Est5, Est1, and Bmy1 loci (esterase 5, esterase 1, and ß-amylase 1, respectively).

The discontinuous system of polacrylamide gel (PAAG) electrophorosis was used to separate the esterase isozymes (Netsvetaev, 1992). The tray buffer, pH 8.3, included Tris, EDTA, and boric acid. PAAG electrophoresis for ß-amylase was accomplished with the tris-glycine system, pH 8.3 (Netsvetaev, 1993). Esterase extract from 7 to 8 day-old seedlings was obtained in a 0.024 M sodium bicarbonate. Mature dry seeds were used for extraction of ß-amylase. Flour from the endosperm was poured into a solution consisting of 0.024 M sodium bicarbonate, 0.01 M dithiothreitol, 0.6 M sucrose, and bromophenol blue for coloring. After centrifugation, the supernatant was used for electrophoretic separation of ß-amylase isozymes.

DNA was obtained from 7 to 8 day-old seedlings. The leaf blade from one seedling was crushed with a glass rod in an Eppendorf tube that contained 0.5 ml of lysing solution (0.1 M Na3EDTA, 10 mM tris-HCl pH 8.5, 100 mM NaCl, 1% SDS, 0.1% Triton X-100, and 100 µg/ml proteinase K). For protein destruction, the samples were treated with this solution for 2 hours at 55°C. Peptides were dissolved in volumes of a chloroform-ispropenthanol mixture. The DNA was precipitated with a 0.5 volume of isopropanol. The PCR was conducted according with the method described by Sivolap and Kalendar' (1994). The reaction volume was 20 µl and contained 16.6 mM (NH/4/)/2/SO/4/, 4 mM MgCl/2/, 0.2 mM each dNTP, 0.01% w/w Tween-20, 57 mM Tris-HCl (pH 8.8), 200 nM primer P6, 20 ng DNA, and 1 unit Taq polymerase. Samples were overlaid with light mineral oil Bajol F (Serva). The first cycle of PCR was carried out under the following conditions: 2 min. at 94°C, 2 min. at 42°C, and 2 min. at 72°C. Conditions for cycles 2 to 4 were 1 min. at 94°C, 2 min. at 42°C, and 2 min. at 72°C. Cycles 5 to 37 had the parameters: 1 min. at 94°C, 1.5 min. at 52°C, and 2 min. at 72°C. This was followed by a final incubation at 72°C for 7 min. The PCR products were electrophoresed in 2% agarose gel.

The genotypes of the parents were: Odessky 115 - Est1Ca, Est5Pi, Bmy1Ar, and absence of the PCR 915B fragment (f915); Golf - Est1Pr, Est5Te, Bmy1Br, and presence of f915. Data for linkage estimates between genetic and molecular markers in F->inf. Odessky 115 X Golf lines are summarized in Table 1. Since no recombinants were found, the f915 locus showed complete linkage with the ß-amylase locus, Bmy1. The other genetic factors were inherited independently of f915 (Table). Thus, the f915 locus is located at (or near) the Bmy1 locus in the long arm of chromosome 4. Presence of the f915 fragment was associated with the slow ß-amylase 1 isozyme (Fig. 2). which is determined by the Bmy1Br allele.

References

Netsvetaev, V.P. 1992. Identification of barley leaf esterases 11, 12 and their control. Genetika 28:105-119 [In Russian].

Netsvetaev, V.P. 1993. Location of the ß-amylase locus (Bmy1) in chromosome 4 of barley. Cytology and Genetics, Kiev 27:74-78. [In Russian].

Netsvetaev, V.P., and A.A. Sozinov. 1984. Location of a hordein G locus, Hrd G,on chromosome 5 of barley. BGN 14:4-6.

Sivolap, Yu.M., and R.N. Kalendar'. 1994. PCR with arbitrary primers for detection of genetic polymorphism in barley. Genetika (In press) [In Russian].

Table 1. Linkage data between PCR fragment (f915) and the Bmy1, Est1, and Est5 loci in a cross of barley cultivars (F->inf. Odessky 115 X Golf).

_______________________________________________________________
Genetic factors  No. of F->inf. families  
_______________  _____________________
                 Pheno-               Total       Recombination
A     X     B    types   BB    bb       n    X²L        %                
_______________________________________________________________
Bmy1   X  f915    AA     65      0   
                  aa      0     75    140  140.0    0.0 ± 0.3     

Est1   X  Est5    AA     32     23
                  aa     16     22     93    2.4    Independent     

Est1   X  f915    AA     25     34
                  aa     22     16     97    2.3    Independent     

Est5   X  f915    AA     21     30
                  aa     26     20     97    2.3    Independent  
_______________________________________________________________

Figure 1. Electrophoresis of the PCR products with P6 primer in the parents (P1, P2) and F->inf. Odessky 115 X Golf lines of a barley: 1 - P2, 2 - L71, 3 - L72, 4 - L73, 5 - L74, 6 - L75, 7 - L76, 8 - P1; M - M13 mp8 cutted Csp6I(=RsaI).


Figure 2. Polyacrilamide-gel (pH 8,3) zymograms of ß-amylase from mature barley seed in the parents (P1, P2) and F->inf. Odessky 115 X Golf lines: I - P2, 2 - L71, 3 - L72, 4 - L73, 5 - L74, 6 - L75, 7 - L76, 8 - P1.