Phylogenetic And Varietal Identification Of Yam Based On Chloroplast Molecular Lineage Geography

Which we usually called Chinese yam is the underground rhizome of Dioscorea polystachya Turcz.It is in great demand as a medicine and food homologous Chinese medicine.At the same time,D.polystachya is a typical representative of multi-variety sources of traditional Chinese medicine and asexual propagation of large-scale cultivation of medicinal plants.Variety factors are the primary factors affecting the stability of Chinese medicines.The cultivation of vegetative propagation is an important factor leading to the decline of genetic diversity.Therefore,identify the germplasm sources and genetic relationship of different varieties of D.polystachya,analysis the genetic diversity and genetic structure of D.polystachya,and establish an objective and effective method for identification of D.polystachya.These above have important practical significance for regulating the circulation and clinical effective and sustainable utilization of D.polystachya.Therefore,based on extensive field investigations and market inspections,this study fully understands the current status and morphological characteristics of D.polystachya resources.based on comprehensive chloroplast genome analysis of six different genotypes of D.polystachya.Three chloroplast DNA fragments were screened,and 385 D.polystachya samples from 31 populations in 15 provinces were amplified,sequenced,and analyzed by genetic evolution.The main contents are as follows:1.Based on the sampling survey of different populations of D.polystachya,to observe the resources status.The market survey and field survey were combined to compare the morphological characteristics of the 44 populations in 16 provinces in China.The result shows that,it is obviously regional for D.polystachya's distribution and a variety of local specialties have been formed.It is the most widely circulated with D.polystachya.cv.Tiegun.According to the stem wing(edge),it can be divided into D.polystachya and D.alata.There are 12 groups to be further classified according to leaf shape,root morphology and root cross section.The variety and distribution of D.polystachya are wide.Wild resources are only destroyed in parts of the south.The quality of the cultivars is high and the wild resources are not widely concerned.The morphological characteristics of different cultivars are evident in stems,leaves and roots.Therefore,we can make a simple identification from that.2.In order to develop more effective genetic markers to assess the genetic diversity of D.polystachyas and to identify varieties,this study has developed whole chloroplast genome sequences as a new molecular genetic marker method from six wild D.polystachyas in different provinces.Ultraviolet generator was used to randomize the DNA samples into 400-600 bp lengths.Using the NEBNext(?)UltraTM DNA Library Prep Kit,an Illumina double-end DNA library with a 500-bp insertion size was established according to various criteria.Sequence the chloroplast gene by Illumina HiSeq X 10(2 × 150 bp).The analysis found that the chloroplast genome of D.polystachya is a typical circular tetrad structure consisting of two single-copy regions(large single-copy region LSC and small single-copy region SSC)and two inverted repeat regions.Through the comparative analysis of the chloroplast genomes of 6 D.polystachya,a total of 141 single nucleotide polymorphisms(SNPs)and 70 simple repeats(SSRs)were discovered,including 24 polymorphic simple repeats.43 common indels and 5 small inversions were found,too.This experimental study shows that the whole genomic sequence of chloroplast can be used as a reliable and effective molecular marker to reveal the genealogical structure of D.polystachya,the degree of genetic variation of wild populations,and the identification of different cultivars.3.Based on the molecular phylogeography,we study the genetic diversity and genetic structure of D.polystachya.Explore the genealogy of D.polystachya to provide basis for the classification and resource protection.This study conducted DNA extraction,PCR amplifying and sequencing of cpDNA regions(trnH-psbA,trnL-trnF matK).The sequencing results were corrected by software ContigExpress and alighed by software BioEidt.Then we analysed the quantity and the frequency of different haplotypes with the sofrware DnaSP.The evolution tree-NJ constructed by Mega software.After that we used Network software to construct single-fold Network evolution diagram.After accepted the GST and NST we drew geographic distribution of the haplotypes by software ArcMap.The results showed that there were 15 provinces include 31 populations and 385 individual,they have 22 variation sites and 10 haplotypes.HAP2 is a wide-cloth single-type.Gst=0.869,Nst=0.931.The diversity of D.polystachya is mainly in different population.Evolution tree-NJ results show that D.polystachya and D.alata,cultivated D.polystachya and wild D.polystachya,D.polystachya.cv.Xishi,D.polystachy a,cv.Ximao,D.polystachya.cv.Xiaobaizui and Other D.polystachya varieties,D.polystachya.cv.Foshou in Qichun Hubei province and Other D.polystachya varieties,they can be distinguished from the molecular level.Cultivated D.polystachya mainly originated from Yuntaishan Henan,Cultivated D.alata mainly originated from Yuanzhou Jiangxi.We should protect the abundant genetic diversity population of Chinese yam,such as Nanjing Jiangsu province.We should protect other groups with unique haplotypes too,such as Yuntaishan Henan province,Qichun Hubei province,Minhou Fujian province,and other original population,to protect the genetic diversity of D.polystachya.