| Types and frequencies of natural mutations in plants and their underlying mechanisms are poorly understood. Genome stability and its evolution yet did not all known. Tobacco cultivars TI1473, Yellow Pryor and Samsun have the resistance gene N against TMV at different loci in their genome, while TG34is a cultivar with a transgenic N gene. All three tobacco genotypes were crossed with P50, a homozygous transgenic tobacco with N’s avirulence gene from TMV. Their hybrids died10days after germination due to hypersensitive response. All survivals must have loss of function mutations for the resistance gene N or the avirulence gene P50. The survivals were analyzed to study the mutation type and rate of the N gene or the transgenic P50gene.Approximately200,000Fl hybrids were obtained from a cross between Ti1473and transgenic P50. A total of182seedlings survived after germination. TMV inoculation discovered31mutants with loss of the N function, and marker genotyping indicated that they were authentic hybrids. Therefore, the N gene in Ti1473has a rate of1/6400for loss of function. Among the31mutants, one was caused by point mutation, which is located in the fourth exon of the N gene resulting amino acid change from lle to Met. The other mutants had lost the entire N gene sequences. Further analysis found that the deletions spanned a region corresponding to the region between3.48Mb and4.67Mb from the telomere of the short arm of chromosome11in tomato.Approximately520,000F1hybrids were obtained from a cross between transgenic tobacco TG34and transgenic tobacco P50. A total of480seedlings survived10days after germination.113mutants were found with loss of the N function after TMV inoculation. The loss-of-function mutation rate of N gene in TG34is1/4600. All mutants had deleted the entire N. The flanking sequence of the transgenic insert in tobacco was obtained using the FPNI-PCR method, and BLAST search suggest that it is located on the chromosome10in tomato genome. T-DNA vector right border sequence was got by TAIL-PCR. The transgenic donors of N gene and P50gene are the same tobacco cultivar SRI. No markers could be developed to distinguish the tobacco TG34and P50except the mark of N gene and P50gene. The result showed N gene with inserted vector were all deleted.A total of800,000F1hybrids between YP and transgenic P50were obtained. TMV inoculation discovered89mutants with loss of the P50function, and marker genotyping also indicated that they were authentic hybrids. Therefore, the P50gene in YP has a rate of1/8900for loss of function.All mutants had deleted the entire P50. Further analysis showed the left border of T-DNA had a tandem repeat of the same vector. Repeatedly try did not get the right border sequence and preliminary thought P50gene with inserted vector were all deleted.In this study, the interaction between disease resistance gene N and its avirulence gene (P50) from TMV was used to study genome stability and investigated the loss of function mutation rates. The data provide further insights on the mutation of genome stability of high plants. |
