Map-Based Cloning of KAM2

Map-based cloning of the KAM2 gene was performed essentially as described previously (Tamura et al., 2005). We used codominant cleaved amplified polymorphic sequence markers (Konieczny and Ausubel, 1993) and simple sequence length polymorphism markers (Bell and Ecker, 1994). Twenty recombinants of the F2 progeny were screened for the kam2 phenotype for rough mapping. The position of the KAM2 gene was located in the middle of chromosome 2. For fine-scale mapping, DNA was isolated from 220 plants of the F2 progeny. Nucleotide sequences were determined from both strands using an ABI Prism Big Dye Terminator Cycle Sequence Reaction kit (Applied Biosystems) and a DNA sequencer (model 3100-Avant Genetic Analyzer; Applied Biosystems).

From: Kentaro Tamura, Hideyuki Takahashi, Tadashi Kunieda, Kentaro Fuji, Tomoo Shimada and Ikuko Hara-Nishimura. (2007). Arabidopsis KAM2/GRV2 Is Required for Proper Endosome Formation and Functions in Vacuolar Sorting and Determination of the Embryo Growth Axis. The Plant Cell, 19: 320-332

Map-Based Cloning of PDV1

The pdv1-1 mutation was mapped with molecular markers based on a cleaved amplified polymorphic sequence (Konieczny and Ausubel, 1993) and simple sequence length polymorphisms (Bell and Ecker, 1994). We used some markers listed on The Arabidopsis Information Resource (TAIR; http://www.Arabidopsis.org ); other markers were designed based on polymorphisms listed at TAIR (http://www.Arabidopsis.org/Cereon ) in the Monsanto SNP and Ler Sequence Collection. The pdv1-1 homozygous mutant was crossed with Landsberg erecta wild-type plants to generate a mapping population. Analyses using 48 F2 progeny with the pdv1-1 phenotype showed that the mutation is located in a region of 0.7 Mb on chromosome 5 (between polymorphisms SGCSNP18686 and SGCSNP220). Using 500 F2 plants, we fine-mapped the pdv1-1 mutation to a region of 65 kb that includes 12 genes (between polymorphisms CER437355 and SGCSNP18689). Of these 12 genes, 6 were amplified from pdv1-1 and sequenced; a mutation was found in At5g53280. At5g53280 was then amplified from pdv1-2 plants, and a different mutation was found in the same gene.

From: Shin-ya Miyagishimaa , John E. Froehlichb and Katherine W. Osteryounga. (2006) PDV1 and PDV2 Mediate Recruitment of the Dynamin-Related Protein ARC5 to the Plastid Division Site. The Plant Cell, 18: 2517-2530

Map-Based Cloning of FON4

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 RNA s 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).

From: 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. (2006) The FLORAL ORGAN NUMBER4 Gene Encoding a Putative Ortholog of Arabidopsis CLAVATA3 Regulates Apical Meristem Size in Rice. Plant Physiology, 142: 1039-1052