Studies On Genome Stability Of Allotetraploid Tobacco And Mechanism Of The Red Color On Abaxial Leaf Surfaces Of Gynura Bicolor

This study includes two parts:The first part is about tobacco genome unstability due to frequent segmental deletions and chromosome losses,and the second part is about the mechanism underlying the red color on the abaxial leaf surfaces of Gynura bicolor.1.Unstable allotetraploid tobacco genome due to frequent segmental deletion and chromosome loss The ultimate sources for evolution are mutations in the genomes of a species.There are several different types of mutations,including point mutations,insertions and deletions,translocations,inversions and chromosome losses,which may generate phenotypic diversity for a species.Another source of diversity is caused by recombinations,such as unequal crossovers and homeologous recombinations.Plants have multiple mechanisms to safeguard the genome such as proofreading and DNA repair.Consequently,the mutation rate for plant genomes is so low that it is difficult to be detected using current technique.In this dissertation,a novel high throughput method was applied to identify the loss-of-function mutations for a resistance gene and its avirulent gene.The main results are as follows:?1?The resistance protein in plants interacts with the avirulence protein from the pathogen directly or indirectly,triggering program cell death called hypersensitative response?HR?.In this study,the avirulent gene?P50?from tobacco mosaic virus?TMV?was transformed to a tobacco genotype SR1 which is susceptible to TMV.The transgenic plant?P50?was crossed with a TMV resistant tobacco genotype with resitance gene N.The F₁ hybrids have both the resistance gene N and its corresponding avirulence gene P50.As expected,most F₁ hybrids died with a week after germination.Only plants with loss-of-function mutations of either the N gene or the P50 gene can survive.The surviving plant had a loss-of-function mutation of the N gene if it did not show HR response to TMV inoculation.Otherwise,the surviving plant had a loss-of-function mutation in the P50 gene.Analyzing the surviving plants can estimate the types and frequencies of spontaneous mutations occurred in tobacco genome.?2?Using manual emasculation,more than 14,000,000 F₁ seeds were obtained.F₁ seeds were germinated in petri dish.Most of the seedlings died within a week after germination.We screened the surviving plants through TMV inoculation and PCR amplification.A total of 2,134 surviving plants were shown to have loss-of-function mutations in the N gene,with a mutation rate of 1/6,600.Interestingly,the transgenic P50 gene has similar mutation rate?1/6,500?.This study will focus on the sequence analysis of the N gene and the mechanism for the loss-of-function mutations of the P50 gene.?3?A total of 261 P50 mutants were randomly chosen to investigate their genetic events underlying the loss of function of the P50 gene.Among them,23 had lost the P50 gene due to rearrangement within the transgenic insert;105 lost a chromosome segment spaning the locus where P50 was inserted,with a estimated frequency of 1/16,000;the other 133 mutants lost the entire chromosome where the P50 gene was inserted,with an estimated frequency of 1/13,000?Except the mutations within the P50 insert,the other two types of mutations considerably decreased the viability of the mutants,as indicated by the decreased frequency of the homozygotes of the mutations in F₂ populations derived from selfing the F₁ mutants.However,the chromosome in which the P50 gene was inserted did not pair with its homeologous chromosome since no evidence of homeologous recombination was detected for this chromosome,in striking contrast to the chromosome where the N gene was located.?4?Mutant P237,which had segmental deletion on the chromosome where P50 was inserted,was chosen to analyze its genetic event underlying the loss-of-function of the P50 gene.A series of primers were designed on the chromosome and used to screen the mutant P237.The two deletion boundaries were delimited to a region of less than 1 kb,and the join?repair?region was PCR amplified and sequenced.Squence analysis showed that the flanking sequences of the deletion have no homology and the deletion was believed to be repaired through non-homologous end join?NHEJ?.?5?Above screening identified 9 N mutants with point mutations,and 5 with short insertion and deletions.Based on this data,the frequency of indels in the tobacco genome was estimated to be 5.3�10^-11per site per generation.We also mutagenized the pollens from Samsun?NN?using EMS,followed by above procedure to identify N mutants.Six additional N mutants caused by point mutations were obtained.Analysis of the 15 N mutants caused by point mutations suggests that amino acid change at several domains may cause the loss-of-function for the N gene.2.The mechanism for the red color of abaxial leaf surfaces of Gynura bicolor G.bicolor is a perennial herb in the Compositae family.Its abaxial leaf surface is red due to the accumulation of anthocyanin in its epidermal cells.The mechanism for such phenotype remains unknown.In this study,the mechanism of red color of abaxial surfaces of G.bicolor was investigated through transcriptome analysis.The main results are as follows:?1?First,RNAs were extracted from the epidermal cells of both adaxial and abaxial surfaces of G.bicolor and G.divaricate,the latter with green color on both leaf surfaces.The four RNAs were sequenced using Illumina technology.The RNA sequences from each species were de novo assembled,and 189,196 unigenes were obtained for G.bicolor and 147,686 unigenes for G.divaricate.?2?A total of 3,588 genes were differentially expressed between the abaxial and adaxial surfaces of G.bicolor leaves,which considerably outnumbered the differentially expressed genes?DEGs?in G.divaricate?514?.There were 417 common DEGs between the two species,including genes that are known to be specifically expressed in abaxial of plant leaves,such as AS2,ARF4,KAN and YABBY2.These common DEGs might not be associated with accumulation of anthocyanin specifically on the abaxial surfaces and were excluded for further study.?3?There were 3,171 DEGs specific to G.bicolor,including 4 genes from the anthocyanin biosynthesis pathway:F3'H,CHS,DFR and LDOX,suggesting that the red color was due to the upregulation of the anthocyanin biosynthesis pathway.?4?The activation of the anthocyanin biosynthesis pathway might be caused by the upregulation of one transcription factor that regulates multiple genes in the pathway.The transcription factors MYB,bHLH and WD40 were systematically analyzed.A bHLH encoding gene was found highly expressed in the epidermal cells of abaxial surfaces of G.bicolor but not in other tissues or in G.divaricate.The bHLH encoding gene is homologous to bHLH42 gene involved in Anthocyanin biosynthesis in Arabidopsis.We hypothesize that the bHLH encoding gene is responsible for the excessive accumulation of anthocyanin in the epidermal cells in the abaxial surfaces of G.bicolor.