The deviations of the somaclones from the initial line A188 at the molecular level have been determined by RAPD analysis (Osipova et al., MNL 74:52, 2000). The main objective of this investigation was to reveal some unique RAPD- and ISSR-fragments specific both for every group of somaclones and for individual regenerants, and to transform these fragments into SCAR markers (Sequence Characterized Amplified Region). To extend a choice of the specific markers, 28 RAPD and 10 ISSR primers were synthesized. Some polymorphism was found using 14 RAPD and 6 ISSR primers. The fragments specific for 1) all the somaclones, 2) individual somaclones, 3) each of the regenerant groups, were revealed among the products of amplification.

To quantify RAPD and ISSR polymorphism, the data obtained were constructed as a matrix of the binary character states. There the presence or absence of amplified fragments of a certain size in RAPD and ISSR patterns was considered as state 1 or 0 respectively. Index 1 was conferred to the amplified fragments revealing with high intensity and steadily repeating in all the experiments. Jacquard coefficient was used to calculate the matrices of differences based on the matrix of states. The difference between the somaclones and their initial line varied from 6.5 to 23%. The diversity among plants of the second group (R105-R119) was 5-12%, whereas the plant deviation from each other was only 2-6% in the first group (R11-R54).

Based on the matrix of differences, a dendrogram was constructed by the neighbor-joining method (Fig. 1). This dendrogram reflected the diversity between the RAPD and ISSR patterns of nine subjects studied with the usage of 24 primers with respect to 161 binary traits comprising presence or absence of the amplified fragments. The initial line A188 was used as a root in the dendrogram.

Two somaclone clusters could be marked in the constructed dendrogram. One of them was quite similar to the first group of regenerants and involved families R11, R14, R27 and R54. The other cluster involved lines R105, R106, R107 and R119 forming the second group. Somaclones R105-R119 were more distant both from the initial line and from each other. The results obtained completely corresponded to the morphological data, according to which, the second group of somaclones, regenerated after the longer-term subculturing, displayed the greater range of variation. This fact confirmed the assumption that accumulating mutations had resulted from the lengthening of subculturing. Thus, the investigation carried out using the random primers resulted in revealing somaclonal deviations both from each other and from the initial line. Also, some somaclone specific fragments were found. These fragments' specificity was examined in 4-8 individual R1 plants for every somaclone.

Six polymorphic amplified fragments, five RAPD and one ISSR, were used to create SCAR markers. All the regenerants carried a 1050-bp fragment amplifying with the QR-2 primer, which was absent in the A188 line (Fig. 2). The common band for the first somaclone group was found with the M10 primer and for the second group, with the Q-20 primer. Primers OPC-09, NO-15 and Leb-10 revealed some amplified fragments, characteristic for individual somaclones (Table 1).

Table 1. Polymorphic fragments selected for cloning.
 

Primer	Nucleotide sequence	Total fragment quantity	Polymorphic fragment quantity	Somaclones with polymorphic fragment	Polymorphic fragment size  bp
QR-2	CGG-CCA-CTG-T	11	1	All somaclones (R11-R119)	1050
M-10	(CA)6AGG	9	1	First group (R11-R54)	860
Q-20	TCG-CCC-AGT-C	10	1	Second group (R105-R119)	860
OPC-09	CTC-ACC-GTC-C	9	1	188, R106	670
NO-15	CAG-CGA-CTG-T	7	1	R105, R107	420
Leb-10	AGC-CGC-AGC-T	10	1	R105	920

The specific fragments were cloned in vector pGem-T and their terminal regions were sequenced. Based on the nucleotide sequence, primer pairs of 20-30 bp in length were selected using the OLIGO computer program and synthesized to obtain SCAR markers. To optimize the conditions of amplification, the annealing temperature for each pair was determined experimentally. Using the SCAR primers obtained we confirmed polymorphism for five of the six fragments (Table 2). Fragment Leb-10 with SCAR primers amplified both in all the somaclones, and in the initial A188 line.

The inheritance of six SCAR-fragments was examined in the R2 generation of each somaclone. In some cases, we examined the R3 and R4 generations and, also, F1 and F2 of R27xA188 and R105xA188 hybrids. The dominant inheritance of these fragments was established.

Besides the expected fragment, characteristic only for somaclones in R1, the heavier amplified fragment was revealed in the A188 line by using a pair of SCAR QR-2 primers (Fig. 3). In this case, the deletion probably took place in the genome of the initial line A188 as a result of somaclonal variability. But the final conclusion could only be done after comparing the sequences of these fragments. Investigating F1 hybrids (R27xA188) and (R105xA188) with QR-2-primers, both amplified fragments were found (Fig. 4a). These data supposed co-dominance. The following segregation was found in the F2: 8 plants only comprised a light fragment, 8 plants only comprised a heavier one, and 19 genotypes synthesized both fragments. That result corresponded approximately to a 1:2:1 ratio (Fig. 4b).

Studying SCAR Leb-10 inheritance, it was found that the fragment was present in all plants of line A188 investigated, whereas the segregation and sometimes the loss of this marker were noticed in R1, R2 and F1 somaclones. Thus, although SCAR Leb-10 is not unique for R105, as it had been supposed earlier, it can distinguish all the somaclones from the initial line in accordance with the inheritance pattern.

The segregation in the R1-R4 generations was found for fragments OPC-09, NO-15 and Leb-10. This indicated heterozygosis of the regenerated plants. SCAR Q-20, M-10 and QR-2 fragments were present in all the plants investigated in the R1-R4 and F1 generations. The segregation according to the dominant pattern of inheritance was only determined in the F2. That result is supposed to be due to the homozygous nature of the regenerants.

Thus, the results of this investigation confirmed the genetic nature of the regenerant variability, and also, several molecular markers were produced both for maize line A188 and for all the somaclones, each group of the somaclones and individual genotypes.

Figure 1. Dendrogram based on the genetic differences between the initial A188 line and A188-derived somaclones constructed using the NJ method.
Dj- Jacquard distance, I and II � two clusters of somaclones.

Figure 2. DNA fragments amplifying with RAPD QR-2 primer.
Note: M - marker (123 bp DNA Ladder) "GIBCO BRL"

Figure 3. DNA fragments amplifying with SCAR QR-2 primer.
Note: M - marker (123 bp DNA Ladder) "GIBCO BRL"

Figure 4a. DNA fragments amplifying with SCAR QR-2 primer in F1 hybrid R105xA188.
Note: 1-4 � individual plants; C- - negative control (instead of DNA-sample, some water was added); C+ - positive control (cloned specific fragment); M - marker (123 bp DNA Ladder) "GIBCO BRL"

Figure 4b. DNA fragments amplifying with SCAR QR-2 primer in F2 hybrid R105xA188.
Note: 1-35 � individual plants; C- - negative control (instead of DNA, water was added); C+ - positive control (cloned specific fragment); M - marker (123 bp DNA Ladder) "GIBCO BRL"
 
 

Table 2. SCAR primers, synthesizing polymorphic fragments
 

Primer	Nucleotide sequence	Somaclones comprising SCAR fragment	Fragment size	Annealing temperature
QR-2	5'-CGG-CCA-CTG-TCT-AGT-GCT-AA-3' 5'-CGG-CCA-CTG-TAC-CTA-GAT-TTT-3'	A188 All somaclones	1300 1050	54/55
M-10	5'-CAA-AAT-CAG-AGC-AAC-AAT-ACG-CAC-ACA-AGT-3' 5'-CAC-ACA-GGT-TCA-CAT-TAA-TAT-AAA-T-3'	First group (R11-R54)	840	54/55
Q-20	5'-TGT-TCC-AAG-AAA-AAG-GAA-TCG-AAC-TGC-TTG-3' 5'-AAC-GGA-TGC-GCT-AAC-GTT-TTC-CTC-TTG-CAG-3'	Second group (R105-R119)	840	56/57
OPC-09	5'-CTC-ACC-GTC-CAA-ATC-AAG-GG-3' 5'-CTC-ACC-GTC-CCA-GTG-CAC-T-3'	188, R106	660	58
NO-15	5'-ACC-TTC-CAT-GAT-TCA-TTC-CAT-TGC-TTC-TAG-3� 5'-ACT-ATT-CTT-ATA-TTT-GAA-ATT-TGA-A-3'	R105, R107	250	54/55
Leb	5'-TGT-ATA-GAC-TCA-TCA-AAA-GCC-TGG-ACC-CAT-3' 5'-CAG-AGT-GGT-CCC-GAT-GCA-TGG-GTC-TCC-GAG-3'	A188 and all somaclones	900	58

 

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