Identification Of Closelyrelated Species In Camellia Using The DNA Barcoding Technique

The Camellia genus containing about 280 species is the only genus with great commercial values in the family Theaceae.The products of this genus such as seed-oil,ornamental plants and different kinds of tea are popular in the current market.The classification of species in Camellia genus using molecular methods may have potential power in finding cryptic species,rearing new variety and controlling the trade involving counterfeit.DNA barcoding is regarded as a promising tool for species-level identification marked by short,standardized DNA sequences.In this study,we tested the value of widely used barcoding markers(rbc L,mat Kand trn H-psb A)on 64 Camellia species and compared with those of other genera in different family downloaded from NCBI,and the pairwise distances,aligned length,variable sites were calculated.The main results are listed as follows:1 The extraction of genomic DNACamellia plant tissue contains large amount of secondary metabolism substance,such as sugar,polyphenols,tannin,protein.During the extraction of plant DNA,these kinds of substances are irreversibly combined with DNA and making DNA samplespolluted.Total DNA was extracted for each sample using a modified cetyltrimethylammonium bromide(CTAB),and it was proved having good effects on plants of the genus Camellia DNA extraction.2 Sequencing and data analysisRbcL showed 100% in amplification and 95.31% in sequencing.Mat K performed100% in amplification and 95.31% in sequencing.Trn H-psb A showed 100% success rates in PCR amplification and 95.31% in sequencing.In all,61 rbc L,64 mat Kand 62 trn H-psb A sequences for 16 sects genera of Camellia species were submitted to Gen Bank.The aligned length of trn H-psb A was 491 bp with the most variable sites(156).3 Identification of species using single sequenceThe identification efficiency of each single sequence as rbcL,mat K and trnH-psbA is respectively 29.51%,10.94%,32.26%.Using Eurya chinensis,Cleyera japonica,Ternstroemia gymnanthera,Prunus persica,Indigofera aspalathoides,Ficus pumila and Punica granatum as outgroup,the identification rate among genera of mat K and trn H-psb A region reachs 87.50%.4 Identification of species using combined sequencesAmong combined sequences,mat K + trn H-psb A performed the highest identification rate(36.07%),the results of discrimination were similar between combined sequences and single sequences.However,it is found the consistency of rbc L,mat Kand trn H-psb A region in Camellia genus is pretty high.Sect Stereocarpus Pierre Sealy,Sect Thea L Dyer,Sect Chrysantha Chang,Sect Camellia L Dyer and Sect Longissima Chang have high rate of variation and can be successfully identified.