The Study In Genetic Diversity And Development SCAR Markers Of Xihuangcao

Xihuangcao is a Chinese medicinal materials in Lingnan region.The who le herbs of Rabdosia lophanthoides(Buch.-Ham.ex D.Don)H.Hara、R.lophan thoides var.graciliflora(Benth.)H.Hara and R.serra(Maxim.)H.Hara w ere used as Xihuangcao in《Guangdong Chinese Materia Medicine Criterion》.Isodon lophanthoides var.gerardianus was used as Xihuangcao in《Yunnan C hinese Materia Medicine Criterion》.They mainly distribute in Lingnan area s such as Yunnan,Guangxi,Guangdong and Fujian province.They have the ef feet of clearing away heat evil and promoting diuresis,cooling blood elim inating stasis,so they are used for treating acute jaundice hepatitis,ac ute cholecystitis,dysentery and enteritis.Xihuangcao as the main raw ma terial in many Chinese patent medicines such as Xiaoyan Lidan Tablets and Danshitong Capsules,thus the market demand is increasing quickly.There are two species and two varieties in《Chinese Pharmacopoeia》and the provincial standards of traditional Chinese medicinal materials,folk people use the plants with similar external morphological characteristics,such as Rabdosia stracheyi(Benth.Ex Hook.f.)Hara and Rabdosia.nervosa(Hems1.)C.Y.Wu et H.W.Li,as the medicinal materials of Xihuangcao.Xihuangcao’s origin is not inconsistency and homonymous in commercial medicinal materials led to unclear sources of Xihuangcao.The disordered trading market of medicinal materials lead to the quality of medicinal materials is unstable.The patient’s demand for medication has been seriously affected.Therefore,it is necessary to accurately identify the plants that can be used as Xihuangcao,so as to provide a basis for the stability and control of the quality of medicinal materials.Wild germplasm resources of Xihuangcao is decreasing in Lingnan area and the cultivation industry is growing quickly.However,the problems of unclear germplasm always exist in the growth of Xihuangcao.It is urgent to develop the collection,identification,collation and evaluation of germplasm resources to provide basis and guidance for the selection of good germplasm and lay a foundation for the research of breeding of good varieties.The paperis have great significance to the sustainable development of Xihuangcao medicine industry.ObjectiveThis study was intended to study on germplasm resources of Xihuangcao and analysis of genetic distance and structure among different germplasm types.At the same time,This study explore the genetic differentiation and genetic relationship to lay foundation for the evaluation of Xihuang germplasm Resources.The paper also study Phenotypic traits and coefficient of genetic similarity to explore whether Isodon lophanthoides var.gerardianus and Rabdosia lophanthoides var.graciliflora should merged into Rabdosia lophanthoides.This study also develop ISSR-SCAR marker molecular identification system for the original plant of Xihuangcao to provide scientific basis.Methods1.Study on phenotypic diversity of Xihuangcao germplasm resourcesNine phenotypic character of Rabdosia serra、Rabdosia lophanthoides var.graciliflora and Isodon lophanthoides var.gerardianus were measured,and their phenotypic diversity was analyzed by SPSS22.0.2.Analysis on genetic diversity of Xihuangcao germplasm resources by ISSR markerThe optimal ISSR-PCR reaction system was established by setting different gradients of the factors affecting ISSR-PCR.The primers were preliminarily screened using Rabdosia serra’DNA as template and second screened using Rabdosia lophanthoides’DNA as template.The annealing temperature was determined according to its Tm.The DNA of 358 Xihuangcao germplasm resources from 33 populations was amplified by the established reaction system.The DNA of 358 Xihuangcao germplasm resources from 33 populations was amplified by the established reaction system.The genetic diversity and cluster analysis of Xihuangcao germplasm resources from different populations were carried out by using POPGENE 32 and NTSYSpc 2.10s software.3.Analysis on genetic diversity of Xihuangcao germplasm resources by SCoT markerThe optimal SCoT-PCR reaction system was established by setting different gradients of the factors affecting SCoT-PCR.The primers were preliminarily screened using Rabdosia serra’DNA as template and second screened using Rabdosia lophanthoides’ DNA as template.Screening annealing temperature between 45 and 55℃.The DNA of 358 Xihuangcao germplasm resources from 33 populations was amplified by the established reaction system.The genetic diversity and cluster analysis of Xihuangcao germplasm resources from different populations were carried out by using POPGENE 32 and NTSYSpc 2.10s software.4.Development of SCAR markers for Xihuangcao originAmplification with 13 primers screened by ISSR markered of Rabdosia serra、Rabdosia lophanthoide、Rabdosia lophanthoides var.graciliflora、Rabdosia stracheyi and Rabdosia.nervosa and ISSR specific bands of four species and one variety were transformed into SCAR markers.Results1.Study on phenotypic diversity of Xihuangcao germplasm resourcesAmong the 9 phenotypic characters of Rabdosia serra,the length-width ratio of leaf had significantly different among populations,and the other 8 phenotypic character had high significantly different.The two characters with the largest CV were thousand-grain weight and leaf area,the CV of the length-width ratio of leaf、length of seed-like fruitand length-width ratio of seed-like fruit were less than 10%,The results showed that the diversity was low and stability was high of the three characters.The three principal components extracted contributed 85.7%to the variation of Rabdosia serra’s 9 phenotypic characters,among them,thousand-grain weight and length-width ratio of leaf were not extracted.9 phenotypic characters of Rabdosia lophanthoides var.gracili flora and Rabdosia lophanthoides var.gerardianus had significantly different among populations.The largest CV of variation was leaf area,and the CV of length of seed-like fruit and length-width ratio of seed-like fruit were less than 10%,which indicated that the two characters were relatively stable.The three principal components extracted contributed 81.79%to the variation of Rabdosia lophanthoides var.gracili flora and Rabdosia lophanthoides var.gerardianus s 9 phenotypic characters,among them,thousand-grain weight and length-width ratio of leaf were not extracted.2.Analysis on genetic diversity of Xihuangcao germplasm resources by ISSR markerThe best reaction system of ISSR-PCR was established.Thirteen primers with clear amplification bands and good polymorphism were screened from 100 ISSR primers.A total of 491 bands were amplified by 13 primers,of which 490 were polymorphic.The results showed that the average of observed number of alleles(Na)is 1.9980,effective alleles(Ne)is 1.2508,Nei’s genetic diversity index(H)is 0.1719,and the Shannon’s information index(I)is 0.2906,polymorphic loci was 490,and the percentage of polymorphic loci(PPB)is 99.8%.The total genetic diversity(Ht)is 0.1704,the intra-population genetic diversity(Hs)is 0.0276.The genetic differentiation coefficient(Gst)is 0.8381.The analysis of eight Rabdosia serra show that Ht is 0.2716,Hs is 0.0891,Gst is 0.6718,Nm*is 0.0966,indicating that 67.18%of genetic differentiation existed among populations,and 32.82%of genetic differentiation existed in populations,The genetic differentiation among populations was higher than that within populations,and the results of AMOVA analysis were consistent with the results.The genetic differentiation of 14 Rabdosia lophanthoides var.graciliflora and 4 Isodon lophanthoides var.gerardianus showed that Ht is 0.2393,Hs is 0.0465,Gst is 8055,Nm*is 0.1207.The differentiation mainly existed among populations,which was consistent with the results of AMOVA analysis.14 Rabdosia lophanthoides var.gracili flora and 4 Isodon lophanthoides var.gerardianus were divided into two groups for AMOVA analysis.The results showed that 14.27%of the genetic variation occurred among species and 85.73%of the genetic variation occurred within species.3.Analysis on genetic diversity of Xihuangcao germplasm resources by SCoT markerThe best reaction system of SCoT-PCR was established.8 primers with clear amplification bands and good polymorphism were screened from 36 SCoT primers.A total of 272 bands were amplified by 8 primers,of which 270 were polymorphic.The results showed that the average of observed number of alleles(Na)is 1.9926,effective alleles(Ne)is 1.3275,Nei’s genetic diversity index(H)is 0.2057,and the Shannon’s information index(I)is 0.3300,polymorphic loci was 270,and the percentage of polymorphic loci(PPB)is 99.26%.The total genetic diversity(Ht)is 0.2060,the intra-population genetic diversity(Hs)is 0.0469.The genetic differentiation coefficient(Gst)is 0.7722.The analysis of eight Rabdosia serra show that Ht is 0.2301,Hs is 0.0914,Gst is 0.6028,Nm*is 0.3295,indicating that 60.28%of genetic differentiation existed among populations,and 39.72%of genetic differentiation existed in populations,The genetic differentiation among populations was higher than that within populations,and the results of AMOVA analysis were consistent with the results.The genetic differentiation of 14 Rabdosia lophanthoides var.graciliflora and 4 Isodon lophanthoides var.gerardianus showed that Ht is 0.2253,Hs is 0.0767,Gst is 6594,Nm*is 0.2582.The differentiation mainly existed among populations,which was consistent with the results of AMOVA analysis.14 Rabdosia lophanthoi des var.graciliflora and 4 Isodon lophanthoides var.gerardianus were divided into two groups for AMOVA analysis.The results showed that 7.25%of the genetic variation occurred among species and 92.75%of the genetic variation occurred within species.4.Development of SCAR markers for Xihuangcao originThe specific bands of ISSR amplification of Rabdosia lophanthoides、Rabdosia stracheyi、Rabdosia serra and Rabdosia nervosa were transformed into SCAR markers,which and could identified four species of plants specifically.ConclusionThere were obvious phenotypic differences among different populations of Rabdosia serra、Rabdosia lophanthoides var.gracili flora and Isodon lophanthoides var.gerardianus,the phenotypic diversity was abundant.The germplasm resources of Xihuangcao have high genetic diversity.The inter-population differentiation of Rabdosia serra、Rabdosia lophanthoides var.gracili flora and Isodon lophanthoides var.gerardianus is larger than that of intra-population differentiation,which indicated that the genetic variation mainly existed among populations.So the protection strategy of germline resources should collect more populations for resource protection.The specific bands of Rabdosia lophanthoides、Rabdosia stracheyi、Rabdosia serra and Rabdosia nervosa were transformed into SCAR markers,which and could identified four species of plants specifically.