Multiplex Genetic Diversity And Phylogenetic Analyses Of The Cultivated Panax Populations

Ginseng（Panax Ginseng C.A.Mey）is one of the precious Chinese medicinal materials.It is a perennial herb,and has been lauded as"the King Among the Hundreds of Herbs".The dried rhizome of ginseng contains a variety of active chemical components,including ginsenosides,proteins,amino acids,vitamins,polysaccharides,flavonoids,inorganic elements,organic acids and so on.Panax is a small genus in the family Araliaceae,and according to the species classification,it is composed of Panax ginseng and Panax quinquefolium.According to the ginseng cultivation environment,ginseng can be divided into wild ginseng,forest ginseng and garden ginseng.Korea ginseng is the ginseng produced in Korea peninsula.At present,due to the unrestrained deforestation,wild resources of ginseng are constantly depleting while the demand is increasing rapidly,which broadens its artificial cultivation year by year,and also results in quite a few problems,such as shoddy,unstable quality,seed provenance confusion,and inadequate protection of the germplasm in authentic producing regions,which seriously affect the quality of ginseng medicinal materials.In order to provide better technical support for the ginseng industry development,further optimize and improve the approbation of ginseng varieties,strength its protection,quick and accurate identification of ginseng germplasm resources by use of DNA molecular marker barcoding combined with producing areas,is not only essential parts to establish the quality and safety control system of Chinese medicinal materials,but also effective means for ensuring the safety and quality,for the prevention of loss and protection of resources.In this research,after preliminary comparison of the shape and size of seeds of different populations of Panax,some morphological traits of ginseng at different stages of growth and development were observed to authenticate the materials.Subsequently the internal transcribed spacer（ITS）and the intergenic spacer（IGS）of the r DNA of the34 cultivated populations of Panax were PCR amplified,sequenced and analyzed by means of bioinformatics;and then the expressed sequence tag-simple sequence repeats（EST-SSR）were PCR amplified and phylogenetically analyzed;and finally the four key enzyme genes in the biosynthesis pathway of ginsenosides,i.e.3-hydroxy-3-methylglutaryl-Co A reductase（HMGR）,cytochrome P450（CYP450）,squalene epoxidase（SE）and farnesyl pyrophosphate synthase（FPS）,were cloned by walking techniques,and the molecular fingerprint markers were identified from the introns,exons as well as the deduced amino acid sequences.Phylogenetic relationships of each gene among the 34 populations was also revealed and analyzed.The main research results are as follows:1 Preliminary observation on some agronomic traits of different populations of PanaxThe seed shape,size,growth and developmental morphology of the cultivated Panax populations were observed to authenticate the material.In this section,through the observation of Panax seed collected,it was found that P.quinquefolium seeds were larger than those of P.ginseng;the seed surface of P.quinquefolium has no wrinkles and is rough whereas the seed surface of P.ginseng is wrinkled and relatively smooth.The root material of forest ginseng were relatively special in that the reed rhizome（the basal or residue part of the stem in the rhizome）is longer than that of Panax quinquefolium and the garden ginseng,and its reed bowl（concave-shaped stem residue in the rhizome）bigger.The growth and development of ginseng takes a long period of time（3-5 years）,and at each stage of the growth and development,the requirements for water and light are very strict,and at the emergence stage,the requirements for soil and climate are very high.2 Genetic diversity analysis of internal transcribed spacer（ITS）in Panax populationsIn this section,the internal transcribed spacers（ITS）of the collected 34 cultivated populations of Panax were PCR amplified,sequenced,and multiple aligned for the genetic diversity analysis.Results showed that the 5.8S r DNA regions of the P.ginseng C.A Meyer and P.quinquefolium were 162bp and 160bp respectively;the discriminating fingerprints occurring in ITS1,5.8S and ITS2 regions can be used for the molecular identification of P.ginseng and P.quinquefolium.The specific SNP fingerprints were at the same four nucleotide sites:（ITS1）A ¹⁴³---（5.8S）A/G ²⁶²---（ITS2）C ⁴⁴¹---（ITS2）T⁴⁵² for P.ginseng,and（ITS1）C ¹⁴³---（5.8S）G ²⁶²---（ITS2）T ⁴⁴¹---（ITS2）C ⁴⁵² for P.quinquefolium.The phylogenetic tree constructed from the ITS2 regions showed that all the P.quinquefolium,all the Korean gingseng,and the garden gingseng and the wild gingseng cluster in 3 separate clades respectively;and the phylogenetic tree based on all the 29 cultivated populations of P.ginseng showed that most of the garden gingseng cluster together,indicating that there is some distance between the garden gingseng and other gingseng.3 Genetic diversity analysis of ribosomal intergenic spacer sequences（IGS）in Panax populationsThe ITS sequences are commonly used for inter-specific identification,while IGS sequences are mostly used for intra-specific identification due to the higher-than-ITS evolving rate within species.Therefore,the IGS regions were cloned from 34 cultivated populations of Panax,and the genetic diversity was studied in this section.It was found that,of the well-defined 24 IGS sequences from 24 populations of Panax,the non-transcribed spacer（NTS）regions were genetically more diverse,while the external transcribed spacer（ETS）regions relatively conserved.And 10 potentially new IGS sequences from 10 populations of Panax have been detected yet to be defined.Phylogenetic relationships based on the well-defined 24 ETS,NTS,the entire IGS sequences,and the 10 potentially new IGS sequences detected from the 10 cultivated Panax populations were also revealed and analyzed.4 Genetic diversity analysis of EST-SSR markers of different populations of PanaxSSR molecular markers are characterized by high polymorphism,good repeatability,co-dominance,abundance in quantity,simplicity in technique,and strong specificity.SSR molecular markers include the genomic SSR and expressed sequence tag-SSR（EST-SSR）.The latter is a more convenient and quicker method to find SSR loci from expressed sequence tag library,and has been exploited and fruitful in some studies.In this section,the genetic diversity of the cultivated Panax populations based on EST-SSR markers was studied by using the software Powermarker for polymorphism detection,and clustering analysis of the amplified results.Results showed that up to 10primer pairs out of the screened 24 could discriminate P.ginseng from P.quinquefolium populations,and also found are the specific marker bands for Korea ginseng populations and the discriminatory marker bands for HLJJX,L-JLTHQH,R-HG,and S-JLBS.Clustering results showed that the genetic distance within the populations of P.ginseng was generally within 0.3,while that between P.quinquefolius and P.ginseng was more than 0.4,indicating that the populations of P.quinquefolius and P.ginseng were relatively genetically distant.These results well explained the genetic relationships among the populations of Panax,and further proved the reliability of EST-SSR molecular markers in genetic diversity analysis of the cultivated Panax populations.5 Cloning and genetic diversity analysis of key enzyme genes for active ingredient biosynthesis of the cultivated Panax populationsBased on the genomic gene sequences of Panax retrieved from NCBI for the four key enzymes,HMGR,CYP450,SE and FPS in ginsenoside synthesis,the degenerated homologous primers were designed,and the corresponding genomic gene sequences of34 populations of Panax were cloned in overlapping segments by walking technique,sequences obtained spliced,and their sequence structures were compared for the first time.Specific SNP molecular tracing fingerprint markers were identified from the intron and exon sequences of these genes;the deduced amino acid sequence variation sites were also identified and analyzed from the spliced coding regions.Results showed that,based on its abundant SNP fingerprints,the HMGR gene could discriminate all the34 cultivated populations of Panax,CYP450 and SE genes could distinguish P.quinquefolius and P.ginseng populations,and FPS gene could distinguish 19populations of Panax.Results from the analyses of the deduced amino acid sequences found that there are 10 amino acid variation sites in Panax HMGR.P.quinquefolius and P.ginseng differ only in one site in CYP450 at site 165,the former being Ser and the later being Thr;there are 4 amino acid differences in SE between P.quinquefolius and P.ginseng,and P.quinquefolius and P.ginseng differ only in one site in FPS at site 341,with the formal being Glu and the later being Gln.Moreover,the clustering based on the gene sequences and deduced amino acid sequences were also performed and phylogenetic relationships of the Panax populations revealed.This research for the first time,mined out molecular markers from the functional genes involved in the biosynthesis of ginsenocides and successfully used for the genetic identification of the cultivated Panax populations.The establishment of the multiplex genetic markers,genetic diversity,and phylogenetic relationships in this research,from ITS,IGS,EST-SSR,and the 4 key enzyme genes（HMGR,CYP450,SE and FPS in ginsenocide biosynthetic pathway）for the cultivated Panax populations enriched the content and the genetic means for the the molecular identification,genetic diversity,and phylogenetic studies of Panax populations,and laid solid foundations for further related and in-depth investigations.