Genetic Diversity And Cultivated Origin Of Rehmannia Glutinosa Based On Molecular Evidence

Rehmannia glutinosa(Gaert.)Libosch.ex.Fisch is a perennial herb of Rehmannia belonging to Scrophulariaceae,which is widely distributed in China.The cultivar of R.glutinosa is one of the four famous “Huai medicines” and has a long history of cultivation.At present,studies on genetic diversity of R.glutinosa are mainly focused on different cultivars of R.glutinosa or different individuals in some wild populations of R.glutinosa.There is a lack of comparative population genetics on cultivars and wild populations of R.glutinosa.There are also some disputes about the origin place,cultivation history and origin mode of R.glutinosa cultivar(especially“Huai medicines”cultivar of R.glutinosa).Based on ISSR molecular marker technique,sequencing,assembly,annotation and phylogenetic construction of chloroplast genome of cultivar and wild populations of R.glutinosa,we analysed the the genetic diversity and genetic relationship of cultivar and wild population of R.glutinosa and compared their genetic diversity differences.In order to explore cultivated origin of R.glutinosa,we also choosed the chloroplast gene sequences with high variation and nuclear ITS gene,and constructed the phylogenetic tree of cultivar and wild population of R.glutinosa(including wild population in traditional and authentic production areas of R.glutinosa).The main results are as follows:1)Genetic diversity analysis on cultivar and wild population of Rehmannia glutinosa based on ISSR markers106 samples including cultivar and wild population of R.glutinosa and its relative species were studied by ISSR-PCR markers.Seven ISSR primers generated85 loci of which 83 loci were polymorphic.The percentage of polymorphic bands(PPB)of all samples was 97.65%.Nei’s genetic diversity index(H)and Shannon’s information index(I)were 0.2659 and 0.4125.The percentage of polymorphic bands(PPB)of cultivar of R.glutinosa was 30.59%.The percentage of polymorphic bands(PPB)of wild population of R.glutinosa in He Nan province was 83.53%.The results of ISSR analysis reveal that the level of genetic diversity between wild populations of R.glutinosa is higher than that within cultivar populations of R.glutinosa.The genetic diversity among wild populations of R.glutinosa in He Nan province is higher than other region,which is consistent with authentic producing areas of R.glutinosa in this area.The relationships of wild population of R.glutinosa were not correlate to their geographical distribution pattern.2)Comparative study of chloroplast genomes between cultivars and wild populations of Rehmannia glutinosaThe chloroplast genome of 4 cultivars of R.glutinosa(6 samples)and 22 wild populations R.glutinosa were sequenced using the MGISEQ-2000PE150 sequencing platform in Hua Da.Genomics(Shenzhen,China).The chloroplast genome sequences were annotated and assembled using Geneious 11.0.3 and Geneq-Annotation of Organellar(https://chlorobox.mpimp-golm.mpg.de/geseq.html).The chloroplast genome sequences of 16 samples of R.glutinosa species and Triaenophora shennongjiaensis were downloaded from NCBI database.Phylogenetic tree of cultivars and wild populations R.glutinosa was constructed by using phylo Suitev l.2.1software.The chloroplast genomic differences between cultivars and wild populations of R.glutinosa were compared.The results showed that the chloroplast genomic structure of both cultivars and wild populations of R.glutinosa had a typical Four section structure,including a large single copy region(LSC),a pair of reverse repeat regions(IR)and a short single copy region(SSC).All of them had 133 genes,including 88 protein coding genes(CDS),8 r RNA genes and 37 t RNA genes.The total length of chloroplast genomes on 22 wild populations of R.glutinosa ranged from 153,240-153,852 bp,and GC content 37.9-38.0%.The chloroplast genomes of 6 cultivars with R.glutinosa were 153,499 bp,and the GC content was 37.9%.Using Triaenophora shennongjiaensis as the outgroup,we constructed the phylogenetic tree of chloroplast genome sequence,CDS region,LSC and SSC sequence and chloroplast gene spacer by phylo Suite vl.2.1 software.the results show that‘Huai’ cultivar and wild population of R.glutinosa in Wenxian County of Henan Province and other cultivar of R.glutinosa in NCBI,were clustered into a monophyletic clade with 88-99% support.Chloroplast genome data support that wild populations in Wenxian County of Henan Province was involved in the origin of cultivars of R.glutinosa3)Origin of cultivar of Rehmannia glutinosa based on chloroplast sequences and nuclear ITS gene.Based on the above chloroplast genome study,we expanded the sampling of wild populations of R glutinosa(115 samples including cultivars and wild populations of R.glutinosa and its relative species).We conducted phylogenetic analyses of the combined sequences of one nuclear DNA(ITS)region and six chloroplast DNA gene regions(trn H-psb A、trn S-trn G、rpo B-trn C、trn P-psa J、rpoc1、petb)using Triaenophora shennongjiaensis as the outgroup.Phylogenetic tree showed that the wild populations of R.glutinosa in different provinces were clustered in different branches,and there was no phenomenon that all the wild populations in a single province were clustered in same clade.It indicated that the relationships of wild population of R.glutinosa were not correlate to their geographical distribution pattern.The cultivar and the wild populations of R.glutinosa in Wenxian County,Henan Province(XZ1710)and Xiangfen,Shanxi Province(XZ19218)gathered in one clade,with 100% support.Then,they are clustered with wild population of R.glutinosa in Wuyi,Hebei(XZ19185)in a monophyletic clade,with 75% support.This indicated that they were closely related to each other.The Wenxian county of Henan Province and Xiangfen county of Shanxi Province are the main traditional and authentic production areas of R.glutinosa.Wuyi county of Hebei Province is subordinate to Hengshui(called Jizhou in ancient times in Song Dynasty,it used to be a traditional and authentic production areas of R.glutinosa).Therefore,we speculate that wild populations of R.glutinosa in Wenxian county of Henan Province(XZ1710),Xiangfen County of Shanxi Province(XZ19218)and Wuyi County of Hebei Province(XZ19185)may be involved in the origin of cultivar of R.glutinosa.