Tea 18s-26s Of Rdna Polymorphism And Evolution

This thesis is focus on the polymorphism and evolution pattern of the 18S-26S rRNA gene family within the genome of tea(Camellia sinensis) and two relative species.The intergenic spacer(1GS) of the 18S-26S rRNA gene of tea,and regions near the 3'end of 26S rRNA gene and the 5' end of 18S rRNA gene of tea and two relative species have been cloned and sequenced,respetively.Results are as follows:1) The IGS of tea has a high degree of polymorphism.Eight kinds of sequences were obtained from the genome of tea,of which the lengths are from 916 bp to 2882 bp.These sequences not only differ in length,but also in structure.In obtained sequences,two sequences lost part near the 3'end of 26S rDNA,and one sequence lost the NTS region completely.2) Five kinds of subrepeat were found in the IGS sequences of tea,with 25 bp, 70 bp,76 bp,87 bp,and 223 bp in length,respectively.Different IGS sequences have different combination of subrepeats and different repetition times of subrepeats. Considering the order of arrangement and the phylogenetic tree of subrepeats,we found the evidence of unequal crossover in the repeated sequence region.3) In the external transcribed spacer(ETS) of tea rRNA gene,the putative promoter,which is related to the transcription of 18S rRNA gene, TATA(G)TA(N)GGGG motif were not found.However,a motif,TGAGTKGTA, conserved highly in many flowering plants belonging to different families was detected in the ETS sequences.4) The 26S rRNA gene and 18S rRNA gene have a high degree of polymorphism,too.Of 164 26S rDNA sequences cloned randomly from tea,the haplotypes are 147.The proportion of the hapiotypes is 89.6%.The proportion of 18S rRNA gene haplotypes is almost equal to this value(89.2%).5) There are not only functional genes but also pseudo genes in the sequences of 26S rRNA gene.Under standard PCR conditions,83.00%of cloned sequences are pseudo genes,and the possibility of pseudo genes is still as high as 55.88%using RTPCR. Phylogenic analysis shows lots of pseudo genes of tea have been existed for a long period of time,but many of which could still express,though these expressed rRNA could not be splicing normally.6) When PCR amplification,the position of primer pairs and reaction conditions could affect the type and proportion of copies in PCR products.For example,if primer pair 26S-IGS-1/26S-IGS-2 was used to amplify the 26S rDNA,only 22.06% of 136 sequences are functional genes,but,about half of obtained sequences are functional genes when primer pair nc26S12/3331rev was used.Moreover,without DMSO in PCR contents,only 4.10%of obtained 26S rDNA sequences are functional genes,but this rate rises to 71.25%,about 17 times of the former,if DMSO was added into PCR contents.7) Using the polymorphism of ITS within and between the species of Genus Camellia,three primer pairs were designed.Together with other SSR primer pairs from the chloroplast DNA and nuclear DNA,we successfully identified the authenticity of hybrids between golden camellia and other species of Camellia.The above results will be helpful for us to understand the evolution pattern of rDNA gene family better.It also has the prospects for a wide range of applications in the classification and germplasm identification of Camellia,as well as the conservation and utilization of the wild resources of Camellia.