Map-Based Cloning of FON4
Map-Based Cloning of FON4
2010/08/17 12:39:03
The FLORAL ORGAN NUMBER4 Gene Encoding a Putative Ortholog of Arabidopsis CLAVATA3 Regulates Apical Meristem Size in Rice
Huangwei Chu, Qian Qian, Wanqi Liang, Changsong Yin, Hexin Tan, Xuan Yao, Zheng Yuan, Jun Yang, Hai Huang, Da Luo, Hong Ma and Dabing Zhang
Plant Physiology, 2006, 142: 1039-1052
The fon4 locus was first mapped to a region between simple sequence repeat marker PSM415 (5’-CTCCCTCCTGCTCGTTTTCTC-3’; 5’-ACCTAGTTAGGTAGCGCCCAT-3’) and RM6094 on the long arm of chromosome 11 by using 96 F2 plants of fon4-1 and Guang-lu-ai 4 (spp. indica). Then, by using 2,100 F2 plants, the FON4 locus was narrowed to a region between two insertion/deletion markers, CH1142 (5’-TGTAGCTCAGAGGTGCTGTGT-3’; 5’-TGCTTGGTGGCAATCGT-3’) and CH1143 (5’-CAAAAATGAGTACACTCCCCTT-3’; 5’-TCATCACACCATCACCCATAC-3’), which were designed as described previously (Shen et al., 2004).
We then used the amino acid sequences of CLV1, CLV2, and CLV3 of Arabidopsis (Arabidopsis thaliana) as queries, and carried out tBLASTn (Altschul et al., 1997) searches in the rice genome database. The open reading frame of the putative gene was predicted by using the GeneMark gene prediction program (Lukashin and Borodovsky, 1998; Lomsadze et al., 2005). Mutations in fon4-1, fon4-2, and fon4-3 were determined by PCR amplification and sequence analysis using the primers designed based on the rice genome sequence. cDNA of FON4 was amplified and total RNAs were purified from young panicles by reverse transcription-PCR by using the FON4-specific primers: FP1 (5’-GTGTGTTTGCTTGACATGGGCCG-3’) and RP1 (5’-GATTTGCACCGTCCGTCCGTTC-3’). The nucleotide sequence for the cDNA of FON4 can be found in GenBank (accession no. DQ836359) and the gene structure of FON4 was deduced by comparing the sequence of cDNA and genomic sequence. A 2,969-bp DNA fragment containing the coding region, 1,912 bp of the sequence upstream of the start codon and 412 bp downstream of the stop codon, was amplified from genomic DNA and cloned into a binary vector pCAMBIA 1301, then transformed into fon4-1 by Agrobacterium-mediated transformation (Hiei et al., 1994).