Identification Of The Mutants Of TMV Resistance Gene N And Studies On Genome Stability Of Tobacco

More than half of plant species are polyploids,and most current diploid species have experienced ancient whole genome duplications(polyploidization).The role of polyploidization in plant divergence and speciation is a key question in plant evolution.It is believed that polyploidization may increase mutation rate and promote speciation.However,mutations in plants are too low to be detected using current technology.Identification of mutations and their frequencies in plant genome and understanding the evolutionary characteristics of polyploids are major challenges for studies on plant evolution.In this study,we used a high throughput strategy to identify a large number of mutants with loss-of-function mutation of the resistance gene N in tobacco(Nicotiana tabacum),and provide insights on the stability of the allotetraploid genome.The main results are as following:(1)Identification of loss-of-function mutation of the resistance gene N against tobacco mosaic virus(TMV).A TMV-susceptible tobacco genotype with transgenic avirulence gene P50 was crossed with a N-containing resistance genotype,and all F1 hybrids have both the N gene and its corresponding avirulence gene and therefore triggers programed cell death.Consequently,most F1 hybrids died a few days after germination.A F1 seedling survived only when the N gene or the P50 gene lost its function.(2)Using above high throughput system,more than 1,400,000 F1 hybrid seeds were screened,and 2,134 surviving seedlings were shown to lose the resistance function of the N gene,with a mutation rate of 1/6,600.PCR amplification using N-gene specific primers had PCR products from 14 mutants,but not from the other mutants,suggesting deletion of the entire N gene in most mutants.Screening the mutant using markers on the N-located chromosome(N-chromosome)showed that some mutants lost the entire N-chromosome with a frequency of 1/15,000.The loss of N-chromosome was supported by genome in situ hybridization(GISH).On the other hand,the partial loss of the N-chromosome had a frequency of 1/12,000.(3)A mutant(N160)with partial loss of the N-chromosome was randomly chosen to investigate its underlying genetic mechanism.Screening the mutant using a series of markers on the N-chromosome delimited the boundary of the deletion to a 36 bp region.Sequences replacing the deleted region were obtained using FPNI method.Sequence analysis suggested the deleted region was replaced by its homeologous sequences.Therefore,the N gene in mutant N160 was lost due to homeologous recombination between the N-chromosome and its homeologous chromosome(called NH-chromosome).The homeologous recombination resulted in a chimeric chromosome,which was verified using GISH.(4)Homeologous recombination will cause a skewed segregation ratio in the F2 population derived from selfing the F1 mutant hybrid.Markers from the NH-chromosome may have an expected ratio of 4:11:1 in such F2 population.Indeed,such ratio was found for marker NHC-1 in F2 population derived from N160,a confirmed mutant with homeologous recombination.Five additional mutants were chosen to investigate the NHC-1 marker in their derived F2 populations.As expected,the marker showed distorted segregation ratio,indicating homeologous recombination.Therefore,the majority,if not all,mutants with partial loss of the N-chromosome were caused by homeologous recombination,with an estimated frequency of at least 1/12,000(note,some homeologous recombination events would not be detected using our strategy)(5)The frequency of homeologous recombination in a region was found to be not correlated with the frequency of homologous recombination in the same region,indicating different mechanisms involved in these two types of recombinations.(6)The homeologous recombination or the loss of the N-chromosome significantly decreased the viability of the homozygous mutants.In summary,2,134 mutants with loss-of-function of the N gene were detected in tobacco in this study.The loss of function of the N gene was mainly caused by homeologous recombination or chromosome loss,and their frequencies were much higher than that of point mutation or indels.Furthermore,each event of homeologous recombination or chromosome loss would change 106-108 nucleotides,in comparison of change of only one or a few nucleotides for point mutations and indels.Interestingly,homeologous recombination or chromosome loss occurs exclusively in polyploids since such mutations are inevitably lethal for diploids.We hypothesize that the genomes of polyploids are much more unstable than those of diploids.The extremely high rate of mutations might push polyploids to extinction.On the other hand,high frequency of mutations may generate a large number of diversity for polyploidy,which may occasionally outperform their corresponding diploid in special niche and promote speciation.