Isolating Of The Arabidopsis Mutant Sam9-6 And Mapping Of The Target Gene

An plant coded 9-6 with distinct novel phenotype as follows was isolated during the screening of Arabidopsis 46 T-DNA insert mutants : develops slowly, both sprout and bloom later than wild-type plant; reduced organ in cubage, smallish laminae and petiole, dumpy mature legumen and dwarfism; SAM（shoot apical meristem） terminates prematurely and lateral meristem finished prematurely; determinate inflorescence; darken-coloured laminae; fascicled leaves. The fact that the phenotypic change is controlled by a monogenic recessive mutation was revealed by genetics research. The mutant was designated as sam9-6 because of its defective SAM. kanamycin antibiosis and co-segregation analysis between T-DNA insert and the novel morphology proved that T-DNA insertion is not responsible for the mutation, tfll was found much similar to sam9-6 in phenotype, but the PCR amplification with specific primers TFL1-F/TFL1-R and sequence analysis revealed that there is no mutation TFL sequence of sam9-6, proved that the target gene is not TFL. Constructed a population of 200 F₂ individuals for first-pass mapping, 50 homozygous mutants was identified based on phenotype and DNA of which was prepared and analyzed by PCR amplification with 22 SSLP markers which spaced roughly every 20 centiMorgan （cM） apart on the five chromosomes of Arabidopsis, and the target gene was found to co-segregate with the two markers, CIW6 and CIW7, which are both in the fourth chromosome of Arabidopsis, none of the additional 20 markers has obvious linkage with the target gene, that is the target gene is in the fourth chromosome of Arabidopsis. The result above illuminated further that T-DNA insertion is not responsible for sam9-6 （the insertion locus AT1G18960 is in the first chromosome）and the target gene is not the known gene TFL （in the fifth chromosome）. A larger F₂ population with ultimately 1000 plants for fine-mapping was planted and 200 homozygous mutants was isolated. The marker F24G24 which is between markers CIW5 and CIW6 and 5.8cM away from CIW6 and the marker FCA5 which is between markers CIW6 and nga1107 and 0.7cM away from CIW6 were used subsequently, and the target gene was delimited to a 1470Kb DNA fragment between the two markers, F24G24 and CIW6. The new markers, F7L13, F25124-18 and T26M18, which are between F24G24 and CIW6 were used later, and the target gene was delimited to a 650K DNA fragment between the two markers, F7L13 and T26M18, finally.