Wang, R., Huang, H., Lin, Y., Chen, Q., Liang, Z. and Wu, T. 2014. Genetic and gene expression analysis of dm1, a dwarf mutant from Cucurbita maxima Duch. ex Lam, based on the AFLP method. Can. J. Plant Sci. 94: 293–302. dm1, a dwarf mutant from Cucurbita maxima (Duch. ex Lam) by natural mutation, showed distinct dwarf phenotypes such as shorter vines and fewer and shorter internodes. Genetic analysis indicated that the dm1 mutation was recessive, and the dwarfing character was controlled by a single locus. DNA-AFLP analysis showed that a fragment (MCAG/ETT) was linked with the dwarfing character of dm1 and that the fragment contained 152 base pairs (bp). It was investigated in F2 populations of dm1 and vine plants, and the genetic distance between the MCAG/ETT fragment and dwarf gene in dm1 was 11.2 cM, calculated by JoinMap 3.0 software. In addition, the result of cDNA-AFLP analysis showed that there were 52 differential transcript derived fragments (TDFs) found between dm1 and vine plants. Only four TDFs, A16T12, A16T9, A6T14 and A6T16, were expressed stably and specifically in dm1 plants in subsequent investigation. The four fragments share 71, 79, 87 and 79% nucleic acid sequence similarity with the complete coding sequence of Arabidopsis thaliana histidine kinase 3 (AHK3) mRNA, nucleic acid sequence of Vitis vinifera dihydroflavonol-4-reductase-like (DFRL), nucleic acid sequence of Glycine max histone-lysine N-methyltransferase ATX4-like and nucleic acid sequence of Arabidopsis thaliana histidinol dehydrogenase (HDH), respectively. Bioinformatics analysis indicated that AHK3, DFRL and HDH were respectively related to Cytokinin signaling, indole acetic acid signaling and Ni accumulation, which played important roles in plant growth, so the expression of the four TDFs may contribute to form dwarfism in dm1.
One-step purification of nucleic acid for gene expression analysis via Immiscible Filtration Assisted by Surface Tension (IFAST)