Genetic Relationship For Tea Plant And Its Closely Related Species Based On Chloroplast DNA Sequences

Chloroplast genome, one of three plant genetic systems, has a character of single-copy andmaternally inherited. Its recombinant and intraspecific variation occurs rarely, while its evolutionaryroute is independent for it is undisturbed by missing gene, overlapping gene and pseudogenes. What’smore, Chloroplast genome’s nucleotide substitution rate is moderate, while its evolutionary rates ofnon-coding region and coding region are different, so that it can be used to study the evolution’srelationship of different taxa. So far, chloroplast genomes from422kinds of plants have beensequenced.Tea plant (Camellia sinensis (L.) O. Kuntze), originating from China, is one of the importantwoody crops of China. In this paper, extraction methods of tea plant’s cpDNA (chloroplast DNA,cpDNA) were studied. We sequenced and analyzed the chloroplast genome sequence of ‘Longjing43’(C. sinensis cv. Longjing43), and then do a phylogenetic analysis of different species of Camelliaphylogenetic using the chloroplast DNA fragments. The main contents and results of this paper are asfollowing:1. We extracted cpDNA of tea plant by sucrose density gradient centrifugation, Percoll densitygradient centrifugation and High-salt low-pH methods, then compared their results. As a result, thedensity gradient is not easy to maintain using sucrose density gradient method to extract tea plantcpDNA, and it is impossible to separate the undamaged chloroplast and damaged chloroplasts bycentrifuges of normal levels. While the density gradient of Percoll density gradient centrifugationmethod is easy to maintain and had a better result during separating of chloroplasts layer. Itsshortcomings are the width of chloroplasts layer and quantity of cpDNA. The developed High-saltlow-pH method had the best results. The value of A260/A280was between1.8and1.9. It had a completesingle electrophoretic band, and a six of cpDNA compared with Percoll density gradient method.2. We obtained the chloroplast genome sequence of ‘Longjing43’ by high-throughput sequencingtechnology. According to bioinformatics analysis, the length of Longjing43’s chloroplast genome is157,096bp, length of large single copy region (LSC) is86,653bp, length of small single copy region(SSC) is18,283bp，length of inverted repeat sequences (IRS) is26,080bp. We Successfully annotated133genes,86protein-coding genes,39tRNA genes and8rRNA genes.3. We finished amplification, sequencing, comparative analysis of chloroplast’s ycf15、psbA andtrnL-trnF gene sequences among98accessions from13Camellia species. As a result, ycf15genesequence has no mutation sites and is highly conserved in the selected material, while it is a littledifferent with the result of the psbA gene sequence. Insertions and deletions occurred among multi-locusof trnL-trnF intergenic region sequence, as well as base substitutions. Its evolutionary rate is greaterthan gene ycf15and gene psbA. It can be used for research of different kinds of genetic relationshipbetween Camellia plants. Three cpDNA gene sequences has less informative positions among all the56C.sinensis cultivars, showing a high degree of intraspecific sequence conservation.