Variation of genomic DNA methylation in the nitrate reductase gene of sibling tobacco (Nicotiana tabacum) cultivars.

To better understand genomic DNA methylation in sibling plant cultivars, methylation-sensitive amplification polymorphism analysis was used to investigate two sibling tobacco cultivars, Yunyan85 and Yunyan87, and their two parents, K326 and Yunyan No. 2. Differences in the degree of genomic DNA methylation were found among the four tobacco cultivars. Compared with parents, the two sibling cultivars had fewer methylated sites. Twenty-nine methylation-sensitive amplification polymorphism fragments that exhibited methylation alteration in the four tobacco cultivars were recovered and sequenced. BLAST (nucleotide BLAST) searches showed that two of the 29 sequences have 99% similarity with nucleotides 1442-1694 of the nia-1 gene and the other 27 sequences contain GC, CAAT or TATA box. The nitrate reductase genes from Yunyan87, K326 and Yunyan No. 2 were found to be identical; however, the third intron of the nitrate reductase gene from Yunyan85 was different compared to the third introns of Yunyan87, K326 and Yunyan No. 2. We conclude that methylation alteration of promoter regions could be responsible for the different phenotypes in tobacco and that introns of the nitrate reductase gene can vary as a result of intra-species crossing in tobacco.