MOSCOW, RUSSIA

Institute of Plant Physiology

RAPD markers variability in maize somaclones produced from inbred A188 --Osipova, ES, Dolgykh, YI, Shamina, ZB, Gostimsky, SA Random amplified polymorphic DNA (RAPD) markers have been used to study genetic variation among somaclones produced from inbred A188. These somaclones differed from the original line in several inherited morphological and biochemical traits (Dolgykh et al., MNL 65:89, 1991; Khavkin et al., MNL 67:84, 1993; Dolgykh, MNL 73:70, 1999 ). The clones investigated represented two independently produced groups of plants: R11, R14, R27 and R54, which had been regenerated from scutellar callus after cultivation in vitro for two months; and R105, R106, R107 and R119 which had been regenerated after cultivation in vitro for eight months.

Sixteen 10-base primers were used to amplify genomic DNA extracted from leaves of three week old plantlets (Table 1).

Table 1. Sequences of 10-base primers used.
 
Primer Sequence Primer Sequence
318 5-CGG-AGA-GCG-A-3 QR-5 5-CGG-CCC-CGG-C-3
340 5-GAG-AGG-CAC-C-3 B-1 5-CGT-CGT-TAC-C-3
450 5-CGG-AGA-GCC-C-3 B-2 5-GTC-CTC-AGT-G-3
QR-1 5-CGG-TCA-CTG-T-3 B-3 5-GCA-GAC-TGA-G-3
QR-2 5-CGG-CCA-CTG-T-3 B-4 5-TCT-TAG-TGC-C-3
QR-3 5-CGG-CCC-CTG-T-3 B-5 5-GAC-AGT-AGC-A-3
QR-4 5-CGG-CCC-CGG-T-3 B-6 5-CTT-GGA-TGG-A-3
10 5-AGG-CGG-GTA-C-3 11 5-AGG-CGG-GAA-C-3

All primers generated from 2 to 17 markers, which varied in size from 200 to 2000 bases.

RAPD analysis did not detect any DNA polymorphism among the individual plants of original inbred A188 (Fig. 1), confirming a high level of inbreeding. At the same time multiplicity qualitative and quantitative differences were found between RAPD profiles A188 and somaclones. One type of the primers, for example QR-1, QR-3, B-2 and B-4, generated an individual spectrum of markers for each somaclone (Fig. 2). All genotypes examined, including A188, could be distinguished on the basis of their RAPD profiles with these primers. RAPD profiles produced by other primers (QR-2, QR-4, B-1, 10, 11, 318 and 340) included both polymorphic and monomorphic bands. The primers of the third type (QR-5, B-3, B-5, B-6 and 450) generated common markers for the somaclones of one group. For example, the RAPD spectrum of clones R11, R14, R27 and R54 produced using the primer QR-5 contained a band of about 870 bases, which was absent in RAPD profiles of A188 and other somaclones. The clones R14, R27 and R54 displayed three specific light amplification products with this primer (Fig. 3). Both groups of somaclones could be distinguished one from the other and from the line A188 with the primer B-5 (Fig. 4). The similarity of RAPD profiles of regenerated plants inside one group seems to be conditioned by common origin .

These results demonstrate that the RAPD technique can be applied for elucidation of genetic polymorphism of regenerated plants.

Figure 1. RAPD profiles of individual plants of inbred A188 generated by primer 11.

Figure 2. Individual spectrum of markers for each somaclone with primer QR-1.

Figure 3. Specific RAPD bands for the somaclones R11, R14, R27 and R54 generated by primer QR-5.

Figure 4. Group-specific RAPD profiles generated by primer B-5.


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