DEKALB, ILLINOIS

Northern Illinois University

 Comparison of maize RFLP probes --M. A. Johns, C. A. Hennelly, A. P. Ryan and S. L. Morrow In the course of looking for RFLP markers linked to various disease resistance genes, we have used many of the Brookhaven National Lab's probes on several common inbred lines. We have found that, in our hands, the probes vary in quality, and we are aware that other labs have had similar experiences. Although it is not possible to give a detailed description of all of our data, we give here a list of the probes we have tried, our estimate of the quality of the resulting bands, and whether the inbred we used showed any polymorphism.

In pilot studies, we found that EcoR1 gave the greatest number of polymorphisms (at least, of the reasonably priced enzymes), and all of the probes were tested on EcoR1-digested genomic DNA. Inserts were separated from the plasmid vector DNA by digestion of mini-prep plasmid DNA with PstI, electrophoresis, and then electro-elution of the insert bands. About 0.1 ug of insert DNA was labeled by random priming for each blot. We used Zeta-probe membrane (Bio-rad) and hybridized overnight at 42 C in a solution of 10% dextran sulfate, 50% formamide, 1% SDS, 3 x SSPE, 1x Denhardt's and 50 ug/ml denatured salmon sperm DNA. The blots were then washed in 6x SSPE, 0.25% SDS for 1 hr at room temperature, then in 1x SSPE, 1% SDS for 1 hr at 42 C, and finally in 0.1x SSPE, 1% SDS for 1 hr at 65 C. We have found that results are improved for some probes with a final wash at even higher stringency, in 0.05x SSPE, 0.15% SDS at 65 C. The high stringency wash is 13.4 mM Na+; in comparison, we have successfully removed all of the probes using a 65 C wash that is 5.2 mM Na+. Thus, we do not believe that further increases in the stringency of wash are likely to help much.

Table 1. RFLP probes and alleles detected.
 
probe rating
Alleles
 probe rating
Alleles
    Oh43 W153R B37     Oh43 W153R B37
                   
chromosome 1
chromosome 6
5.62 C
0
0
1
 6.29 C
0
0
1
8.05 D
--
--
--
 15.37 B
0
1
1
10.38 C
0
1
0
 5.47 B
0
1
0
5.59 B
0
1
1
chromosome 7
15.18 C
0
0
1
 15.40 A
0
0
1
8.10 D
--
--
--
 15.21 A
1
1
1
7.25 C
0
1
0
 4.24 D
--
--
--
8.29 A
0
1
0
 13.24 C
0
0
0
6.32 B
0
1
2
 8.32 B
1
0
1
chromosome 2
 8.37 A
0
0
 0
8.45 B
1
0
2
 14.07 A
1
1
1
12.36 C
0
0
1
 8.39 D
--
--
--
8.04 D
--
--
--
 16.06 C
0
0
1
12.09 C
1
0
1
 8.44 C
0
0
1
chromosome 3
chromosome 8
8.15 C
1
1
1
 9.11 A
1
1
0
8.35 A
0
0
0
 10.39 C
0
0
0
6.06 A
0
1
2
 9.44 A
1
1
0
5.37 D
--
--
--
 9.08 D
--
--
--
8.01 B
0
1
0
 8.26 D
--
--
--
6.16 A
0
1
0
 12.30 B
1
1
2
3.18 A
0
0
0
 10.12U D
--
--
--
chromosome 4
chromosome 9
5.46 D
--
--
--
 3.06 B
0
0
0
15.27 C
0
0
0
 5.10 A
0
0
0
15.45 B
0
0
1
 7.13 B
0
0
1
7.65 A
0
1
2
 5.04 D
--
--
--
10.05 A
0
1
2
 8.17 B
0
1
2
5.67 A
0
0
0 
chromosome 10
chromosome 5
 10.17 D
--
--
 --
8.33 D
--
--
--
 3.04 D -- -- --
6.25 A
0
0
0
 10.13 D -- -- --
7.56 A
0
0
0
 7.49 D -- -- --
5.02 D
--
--
--
          
6.10 D
--
--
--
          
6.22 C
0
0
0
          
10.06 C
0
0
0
          
7.43 C
0
0
0
          
4.36 C
0
0
0
          
7.71 C
0
0
0
          
5.71 B
0
0
0
          
5.40 C
0
0
0
          
5.24 C
0
1
0
          
Ratings: A = single clear band; B = several clear bands; C = bands obscured by high background; D = too faint or too much background to see.

Alleles: All A619 alleles are "0"; others are listed in increasing order.

We examined several examples (disease-resistant conversion lines) of the inbreds A619, B37, Oh43, and W153R. Not counting differences specific to individual conversion lines, we found that A619 and Oh43 differed at 8 of the 51 scorable loci, reflecting the high percentage of Oh43 that went into A619 initially. All other pairs of lines differed at 17 to 25 loci. The data are shown in Table 1. The allele numbers are assigned so the A619 allele is always "0" and then each different allele is given a different number. The numbers do not reflect the position of the bands on the blot.

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors

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