| China is one of the origins and distribution’ centers for Pueraria plants,where there are about 11 species(9 species and 2 varieties).Among them,Pueraria lobata(Willd)Ohwi and Pueraria thomsonii Benth are the most important and widely used Pueraria plants.Their dried roots were used as Chinese herbal medicine,and be called Ge-Gen in Chinese before 2005.The main active isoflavones in Ge-Gen are puerarin and daidzein.Modern pharmacological research has shown that puerarin can be used to treat diabetes and vascular hypertension.Daidzein can be used to decrease blood alcohol levels.Further research found that the pharmacological effect and clinical effect of P.lobata roots were better than that of P.thomsonii roots.Therefore,in the 2005 edition of the Chinese Pharmacopoeia,the roots of P.lobata and P.thomsonii were distinguished,and named Ge-Gen and Fen-Ge in Chinese,respectively.With the emphasis on health and illness,Ge-Gen and Fen-Ge were used widely in our lives.However,the appearance characteristics of these two species are very similar,and it is difficult to distinguish Fen-ge from Ge-gen,when as Chinese Herbal Pieces,which will affect the identification of Ge-gen and Fen-ge,leading to the inconsistent efficiency of the medicine products.In this study,total DNA was construction from the leaves of P.lobata and P.thomsonii,and established DNA library,respectively,for the first time.After sequencing by Illumina HiSeq X-ten platform,these sequencing reads were analyzed by a series of bioinformatics softwares,and assembled into the complete chloroplast genome.Result showed that both chloroplast genomes of P.lobata and P.thomsonii display typical cyclic quartic structure,which contains a long single copy region(LSC),a short single copy region(SSC),and two copies of inverted repeat regions(IRA,IRB).In P.lobata chloroplast genome the regions of LSC,SSC and IR are 84,089 bp,17,992 bp,25,656 bp in length,respectively.While in P.thomsonii chloroplast genome the regions of LSC,SSC and IR are 84,162 bp,17,999 bp and 25,640 bp in length,respectively.The GC contents in the region of LSC,SSC and IR in P.lobata are 32.87%,28.90%and 41.87%,respectively.The GC contents in the region of LSC,SSC and IR in P.thomsonii are 32.86%,28.90%and 41.88%,respectively.Through comparative genome analysis,we found that the difference between the chloroplast genomes of P.lobata and P.thomsonii distributed mostly in the non-coding regions,and occasionlly in the protein-coding region.Then,primers were designed to amplify the high-variation region of the two species,and 6 molecular markers were identified which could help to identify P.lobata and P.thomsonii quickly and effectively.At the same time,the commonly used plant Emei-Ge was identified,and it was found this species may be a kind of P.thomsonii.In addition,we identified the small RNAs(sRNAs)mapping to the chloroplast genome of P.lobata preliminarily.The majority of the mapped sRNAs range from 18 to 20 nt in length and the sum of their clean reads account for about 61%of the clean reads of total sRNAs.There were two clusters of sRNAs abundant in chloroplast rRNA loci,located in the inverted repeats regions(IRa and IRb)of the chloroplast genome,which were similar to melon and other species,indicating that the distribution of sRNAs in chloroplast was conservative.Finally,phylogenetic analysis was carried out by using chloroplast genomes of P.lobata,Pthomsonii and 42 published legume species,using Nicotiana tabacum and Arabidopsis thaliana as outgroup taxa.Results showed that the P.lobata and P.thomsonii were clustered in a small branch in the legumes,and the near relatives were soybean and cowpea plants.Taken together,all the results in this study will laid the foundation for the identification of these two species. |
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