Direct amplification of the conserved sequences of MADS-box genes in A188 inbred and two of its somaclones
--Zabrodina, MV, Karyagina, AS, Khavkin, EE

Somaclones of the A188 inbred manifest heritable modifications in inflorescence architecture, including the decreased number of kernel rows per ear and the increased numbers of tassel branches and kernels per row (Dolgykh Y.I., this MNL issue). The mapping positions for these traits often collate with the loci for homeotic genes (Khavkin and Coe, MNL 72:60, 1998). It is presumable that somaclonal variation directly affects homeotic genes in control of inflorescence development.

DNA fragments produced by amplifying genomic DNAs and cDNAs were compared by size. Genomic DNA was extracted from the seedling leaves of A188 inbred and its somaclones, R27 and R105. Degenerated primers for amplifying MADS-box region of MADS-box genes were constructed using the consensus for zag1--zag5, zmm1, zmm2, and zap1 (Genbank accessions L18924, X80206, L46397, L46399, L46398, X81199, X81200, L46400). Degenerate primers for amplifying K-box sequence were designed separately for three subgroups of MADS-box genes, agamous, agamouslike6, and apetala1. The clones of zag1, zmm2, zag5 and zap1a, kindly provided by Drs. R. J. Schmidt and B. A. Ambrose (University of California-San Diego), served as the references.

Amplification of the MADS-box sequence in leaf and clone DNAs produced a single DNA band of predicted size. Amplification of the K-box sequences of agamous, agamouslike6, and apetala1 genes in leaf DNA produced correspondingly four, two, and one DNA bands, some similar in size to the products of clone amplification and some considerably larger. The size of the products of clone amplification matched the sizes predicted from the published sequences.

Each DNA band observed could comprise more than one product of amplification of related sequences. It is, however, clear that A188 and somaclones did not differ in the size of any of the DNA fragments produced by amplifying leaf DNA with each particular pair of primers. Apparently, no substantial inserts into the conserved regions of MADS-box genes resulted from somaclonal variation.
 


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