| Iridis Tectori Rhizoma(Chuanshegan)is the rhizome of the plant Iris tectorum Maximowicz of Iridaceae.It is a traditional Chinese medicine.Since Pharmacopoeia of People’s Republic of China(version 2005),Iridis Tectori Rhizoma has been recorded in its own part distinguished from Belamcandae Rhizoma(Shegan).In addition,Iridis Tectori Rhizoma had been used as a substitute of Belamcandae Rhizoma in Chinese ancient dynasties.Meanwhile,Belamcandae Rhizoma(Shegan)is the rhizome of the plant Belamcanda chinensis(Linnaeus)Redoute of Iridaceae.The latest result of market survey shows that the main adulterants of Belamcandae Rhizoma and Iridis Tectori Rhizoma are the rhizomata from the plant 1.dichotoma and 1.japonica.Moreover,Iris species are widely distributed in gardens as horticultural ornament plants,due to their flowers with gorgeous and various colors and appearances.Therefore,plenty of researches have been conducted on flower breeding,cultivation,and improvement of agronomic characters of Iris plants.However,there are few studies in species identification and phylogenetic relationships of the original plant of Iridis Tectori Rhizoma and Belamcandae Rhizoma and its adulterants and related species.For accurate and secure in clinic therapy,in current study,the researches had conducted on the original plant of Iridis Tectori Rhizoma and its adulterants and related species about decipherment of Chuanshegan and Shegan in Chinese Ancient Literature,identification analysis among I.tectorum and its related species by DNA barcoding sequences ITS2 and psbA-trnH,and comparison analysis based on complete chloroplast genomes and insights into plastid phylogenomic of four Iris species.Clarified the medicinal usage of Chuanshegan and Shegan in ancient period,by deciphering Chuanshegan and Shegan in Chinese Ancient Literature.Tackled fast accurate identification in some Iris species,by using DNA barcoding ITS2 and psbA-trnH sequences among I.tectorum and its related species within B.chinensis.Distinguished related species and discussed phylogenetic relationships among I.tectorum and it related species within B.chinensis,by comparing analysis based on complete chloroplast genomes and insights into plastid phylogenomic of four Iris species.The results of decipherment of Chinese Ancient Literature show that:1)The producing areas of original plant of Shegan had distributed in nationwide,and the original plant of Chuanshegan had distributed in modern provinces,Henan,Shanghai,Jiangsu,Zhejiang,Anhui,Jiangxi,Hubei,Hunan,Guangdong.Guangxi,Fujian,Sichuan and so forth.2)The original plant of Shegan was B.chinensis,the original plant of Chuanshegan was I.tectorum,and the original plant of Baihuashegan was I.japonica f.pallescens.3)Sheng Nong’s herbal classic had recorded Shegan and Chuanshegan,until Ming dynasty in Compendium of Materia Medica,the two drugs could not be distinguished independently.4)Shegan drugs had been collected in lunar March 3rd.The primary processing of Shegan drugs had contained dried in the sun,dried in the shade,removed the peel and dried in the sun.Processing drugs had recorded in Leigong Treatise on the Preparation,soaked in rice washing water for a whole night,fished it out,boiled with bamboo leaves for 8 hours,fished it out,dried in the sun.The results of identification analysis among I.tectorum and its related species by DNA barcoding sequences ITS2 and psbA-trnH shed light on that:1)The ITS2 sequences of all ingroup and outgroup samples(168 samples from 35 species)had been amplified with a 100%success rate,but success rate of sequenced were 73.8%.The lengths were ranged from 228-275 bp,and their aligned length was 310 bp.The numbers of variable sites among ITS2 were 178 at 57.4%percent.2)The psbA-trnH sequences of all ingroup and outgroup samples(168 samples from 35 species)had been amplified with a 100%success rate,but success rate of sequenced were 89.9%.The lengths were ranged from 339-625 bp,and their aligned length was 684 bp.The numbers of variable sites among psbA-trnH were 194 at 28.4%percent.3)The identification efficiencies of K2P-based genetic distance analysis were 51.9%(ITS2)and 34.4%(psbA-trnH).However,the identification efficiencies of NJ tree-based analysis were 55.6%of ITS2 and 34.4%of psbA-trnH.Although some Iris species fast accurate identification have being tackled,neither ITS2 nor psbA-trnH sequences could thoroughly distinguish I.tectorum,B.chinensis,I.dichotoma,and I.japonica,and absolutely discriminate other related Iris species.The results of comparison analysis based on complete chloroplast genomes and insights into plastid phylogenomic of four Iris species reveal that:1)The four CP genomes structures displayed a typical quadripartite circular.The entire length of four complete CP genomes is 153,253 bp,(I.tectorum),152,443 bp,(I.dichotoma),153,658 bp,(I.japonica),and 153,736 bp,(B.chinensis),respectively,and their GC contents correspond to 37.89%,37.85%,37.87%,and 37.85%,respectively.2)Meanwhile,a total of 133 genes were identified in each genome.In the CP genomes of I.tectorum,I.dichotoma,and B.chinensis,86 protein-coding genes(CDS),38 tRNA genes,8 rRNA genes,and one pseudogene(ycf1)were identified,while in CP genome of I.japonica,85 CDS,38 tRNA genes,8 rRNA genes,and two pseudogenes(rps19 and ycf1)were identified.3)A total of 59,58,42 and 56 simple sequence repeats(SSRs)were detected in the complete CP genomes of I.tectorum,I.dichotoma,I.japonica,and B.chinensis,and the SSRs are mostly composed of A/T mononucleotide units.Besides,a total of 38,43,34 and 67 long repeats were identified in the CP genomes of I.tectorum,I.dichotoma,I.japonica,and B.chinensis.4)Comparative analysis among four genomes observed more highly divergent regions in intergenic spacers(IGSs).Three highly variable regions,ndhFrpl32,rps15-ycf1,and rpl16 were screened combining mVISTA and nucleotide diversity values(Pi).The rpl16 could to be a potential specific molecular marker for the identification of Iris species based on NJ-tree analysis.5)Phylogenetic analysis by using maximum likelihood(ML)method supports that I.tectorum is a sister of I.japonica.In subgenus Pardanthopsis,B.chinensis clustered into one branch with several I.domestica confirmed that B.chinensis is a synonym of I.domestica,and I.domestica as a sister of I.dichotoma.6)Complete CP genomes,as a super-barcode,could clearly separate four species,I.tectorum,I.dichotoma,I.japonica,and B.chinensis. |
