CHESTNUT HILL, MASSACHUSETTS
Boston College

Molecular markers of anther culture-derived plants
--Y. C. Ting

In last year's Maize Genetics Cooperation Newsletter, I reported studies on the mutants cur and cfm, curling tassel and chromosome fusion at meiosis respectively, produced by maize anther culture in vitro. Since then, different studies on the progeny plants of maize anther culture-derived microspore plants were carried out. The procedures followed were RAPD (random amplification of polymorphic DNA).

Williams et al. (Nucl. Acids Res. 18:6531-6535, 1990) reported findings of polymorphic DNA in maize and other eukaryotic as well as prokaryotic organisms. They employed single primers without previous knowledge of their sequence to amplify segments of genomic DNA. The products were reproducible and could be used for physical mapping.

For the present studies, genomic DNA was extracted by following the protocols of Dellaporta et al. (Plant Mol. Biol. Rep. 1:19-21, 1983). For example, instead of using the crude preparation as directed in the PCR protocol, the preparation was subjected to further purification by the following treatment with RNase. The untreated DNA was removed from the original preparation and transferred into TE buffer in a total volume of 400 µl. Five µl of RNase (10 mg/µl) was added to the solution, which was kept at 37 C for four hr or overnight. The DNA was repeatedly precipitated with NH4OAc three times with the following components: one v. of DNA, 0.5 v. of NH4OAc (7.5 M) and two v. EtOH (100%), then washed with 70% EtOH three times. The DNA was dried at room temperature and resuspended in TE (total 400 µl). Then, the concentration of DNA was estimated with a spectrophotometer (1.0 OD260 = 50 µg DNA; the sample was diluted in TE for 2-2 dilution; for example, 10 µl ---- 1.0 ml).

PCR procedure was carried out in a Perkins-Elmer Cetus Thermal Cycler with the following specifications for each amplification: template DNA (genomic), 1.37 µg; 5' primer 500 ng; 10 x PCR reaction buffer, 10 µl; 10 x dNTP (2 mM), 10 µl; Taq polymerase (Boehringer) 5 µ, 0.5 µl; with H2O to make up a final volume of 100 µl. Then a few drops of mineral oil were added to the mixture. The cycler was programmed for 40 cycles; for each cycle it took 1 min at 94 C for denaturation of the template DNA, 2 min at 45 C for annealing, and 3 min at 72 C for extension of the primer action. The amplification products were analyzed with agarose gel electrophoresis prepared with 1.2% agarose. Each lane was loaded with 40 µl aliquots. The gel was stained with ethidium bromide, viewed under ultraviolet lamp and photographed with Polaroid film 655. Tubes containing all of the above components except genomic DNA templates were used as a check for all the primers employed. Molecular weight standard was the lambda DNA digested with BstEII.

The employment of eight primers revealed a certain number of polymorphisms of the amplified DNA sequences, which varied from 1370 to 100 bp in size. The number of products (bands) obtained with a single primer ranged from one to five. Figure 1 depicts the results for three of the primers. These products, or molecular markers, represent the sequence variations in the genomic DNA of different populations. They are dominant and scorable, and caused by base changes in the primer binding sites or by chromosome alterations within the amplified sequence during culturing. If a maize hybrid and its parents were analyzed with RAPD procedures, the markers appeared to be inherited only from one parent. Amplification of alternate alleles has not yet been found. For primer BCP-4, a band of size 270 bp was present in parental "D and K" (Dan-San 91 and King Huang 13 respectively), and progeny populations of "K", but absent from both progenies of "D" (Table 1). In the same table, it is apparent that polymorphic DNAs were manifested by the different primers. These polymorphisms demonstrated the occurrence of mutations. Thus, they constitute one more piece of evidence supporting the previous conclusion that maize anther culture per se is mutagenic.

Table 1. Polymorphism of genomic DNA of maize anther culture derived progeny plants and their parents revealed by amplification of DNA segments with arbitrary primers, "D" and "K" designate maize varieties. Under "D" and "K", "number 1" indicates parents; 2 and 3, progenies. Nomenclature of primers, BC means Boston College. "P-numeral" numeral means primer code number; "-number" means molecular weights of the products (bands) in base pairs. "+" means present; "0" absent.
 
 
Varieties
 
D
K
Primers
1
2
3
1
2
3
BCP-4-27 0
+
0
0
+
+
+
BCP-4-25 0
+
+
0
+
+
+
BCP-4-22 4
0
0
0
+
+
+
BCP-4-11 4
0
0
0
0
+
+
BCP-4-10 0
+
0
+
+
+
+
BCP-5-30 0
+
0
0
0
0
0
BCP-5-27 0
0
+
+
+
+
+
BCP-5-25 0
+
0
+
+
+
+
BCP-5-22 4
+
0
+
+
+
+
BCP-5-11 4
+
0
0
0
0
0
BCP-6-25 0
+
0
+
+
0
0

Arbitrarily primed RAPDs are molecular markers which are transmitted as genes (mutations). Thus, the procedures of RAPD can add one more category of genes to the existing ones for physical mapping of maize linkage. The protocols involved are simple, inexpensive and rapid. They can be carried out easily without going through hybridization and radioactivity. It is conceivable that RAPD technology will play an important role in the future genetic research of maize and other organisms (credits are due to Dr. Gabin Lazar, Department of Molecular Biology, MGS, Boston, MA, for his skillful help in the PCR of this study).

Figure 1. Polymorphisms detected by RAPD markers in maize anther culture-derived progeny plants and their parental individuals. Top: different primers used for three sets of plants. Bottom: D designates Dan-San 91 line and K, King Huang 13 line. In each set, the first lane contains parental DNA from D or K. The second and third lanes contain DNA from the progenies. Numbers to the left side indicate molecular size specified by the standard.


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