The Research Of Evolution And Phylogeny Of Chinese Wild Grapes Based On Chloroplast Genome Sequencing

Conventional methods were difficult to isolate chloroplast and extract cpDNA.In order to explore a simple and fast method to isolate chloroplast and extract cpDNA mature leaves collected from several Chinese wild grape species were used in this study.Although the method was capable of isolating chloroplast of several Chinese wild grape species,this method was not suitable for all Chinese wild grape species.In order to elucidate evolution and phylogeny of Chinese wild grape species total genomic DNA was extracted from 19 Chinese wild grape species which included 20 strains,3 strains of North American species,1 variety of V.vinifera and 1 variety V.rotundifolia.Then total genomic DNA was sequenced by Illumina HiSeq PE150.Biology information technology assembled the total chloroplast genome sequence.Analysis and comparison of chloroplast genomic characteristics of twenty complete chloroplast genome proceeded by related software.The main results of this study were as follows:1.The modified high-salt low-pH method obtained higher concentration and less impurity of chloroplast comparing with the column plant chloroplast DNAout.And the cpDNA was extremely high-quality and pure.As a result,the cpDNA extracted by the modified high-salt low-pH method meet the demand of subsequent chloroplast genome sequencing.So the modified high-salt low-pH method was more suitable for isolating chloroplast and extracting cpDNA.2.The values of OD₂₆₀/OD₂₈₀ and OD₂₆₀/OD₂₃₀ of the total genomic DNA which extracted from 19Chinese wild grape species which included 20 strains,3 strains of North American species,1 variety of V.vinifera and 1 variety of V.rotundifolia meet the requirements of building library and sequencing.Furthermore,the agarose gel electrophoresis showed that the stripe was bright and consistent,and it did not show impurity band.The results indicated that the qualities and reliabilities of sequencing data were extremely high.The average base sequencing error rate was 0.03%.The Q20?%?were both more than95%and the Q30?%?were both more than 90%.3.This study assembled 20 complete and 5 incomplete grape chloroplast genomes.This study analyzed and compared the characteristics of 20 complete grape chloroplast genomes.The results showed that the size of chloroplast genome and ycf1 gene which stretched the border of LSC and IRB,trnN gene which was located in IRA and rpl23 gene which was located in IRB showed different constraction and expansion in V.betulifolia comparing with other Chinese wild grape species.The types of SSR which included mononucleotide repeat,dinucleotide repeats,trinucleotide repeats,tetranucleotide repeats and pentanucleotide repeats existed in all 20 experimental materials.In addition,hexanucleotide repeats were existed only in V.chunganensis and V.erythrophylla.The phylogenetic tree supported that Vitis included Subgen.Muscadinia Planch and Subgen.Euvitis Planch.And V.vinifera,North American species and East Asian populations were also distinguished very well.The phylogenetic tree based on 19 Chinese wild grape species showed that black spine grape branched earliest,so it was the most primitive.Tangweiputao and white spine grape were different from black spine grape.And they existed foreign blood.In addition,Tangweiputao,white spine grape,V.chungii,V.amurensis,V.yeshanensis,V.chunganensis and V.betulifolia were more evolutionary.