Institute of Plant Physiology and Biochemistry
Institute of Cytology and Genetics
Krasnodar Research Institute for Agriculture
AFLP analysis of the DNA in subline A344-CP5
--Baschalkhanov, SI, Mashnenkov, AS, Kobzev, VF, Katyshev, AI, Konstantinov, YM
The maize subline A344-CP5 causes heterosis in the hybrid including the initial line A344 (Mashnenkov, NGCNL, 1982, 56: 92-98). The aim of the present work was to study genetic polymorphism in an A344-CP5 subline, an A344 inbred line and their hybrid using amplified fragment length polymorphism (AFLP) markers.
DNA was extracted from individual Z. mays shoots (70-100 mg of fresh tissue) using the modified CTAB method (Doyle and Doyle, Phytochem. Bull. 19:11–15, 1987). The DNA preparations were treated with RNAse E (Sigma) and extracted with chloroform. After ethanol precipitation, the DNA pellets were resuspended in 50 μl of 10 mM Tris-HCl, pH 7.5. Then DNA was quantified by agarose gel electrophoresis.
AFLP reactions were carried out according to Vos et al. (Nucleic Acids Res. 23:4407, 1995) with minor modifications.
The template DNA was digested with EcoRI and Tru9I (an isoshizomer of MseI). Then EcoRI and MseI adapters were ligated onto corresponding sticky ends. First, PCR was performed with primers complementary to the ligated adapters with no selective nucleotides. Selective PCR was carried out with primers posessing three selective bases each. Eight combinations of selective primers were used: M-CTG + E-ACG, M-CTG + E-AGC, M-CTG + E-ACC, M-CTG + E-AGG, M-CAG + E-ACG, M-CAG + E-AGC, M-CAG + E-ACC, M-CAG + E-AGG where M and E are sequences of the MseI and EcoRI adapters respectively. The selective PCR products were radiolabeled by direct incorporation of α-32P-dATP (20 μCi per reaction). The resulting AFLP fragments were resolved on a sequencing gel and visualized by autoradiography.
It is known that 150 bands are sufficient for reliable estimates of genetic similarity (Pejic et al., Theor. Appl. Genet. 97:1248–1255, 1998). In our AFLP analysis we detected 258 bands (Fig. 1). This allowed us to reliably distinguish the subline A344-CP5 from inbred line A344 and their hybrid. In the case of the hybrid, a significant number of unique bands were revealed. Such results could be explained, presumably, by substantial DNA rearrangements in the hybrid genome. Thus, apart from the existence of sufficient genetic similarity of two parent inbred lines, crossing between them is accompanied by serious genomic DNA reorganization. We suggest that such DNA reorganization possibly is one of the important factors for the realization of the heterotic effect.