Geoherbalism Characteristics And SCAR Authentication Of Ophiopogon Japonicus Produced In Zhejiang And Sichuan Province Based On Chemical And Genetic Analysis

[Objective]To clarify the chemical and genetic characteristics of O.japonicus produced in Zhejiang(ZMD)Sichuan(CMD),as well as the environmental and genetic factors affecting the geoherbalism characteristics of O.japonicus,and to establish an identification method of different O.japonicus germplasms,so as to lay the foundation for the quality evaluation,conservation,and rational utilization of O.japonicus.[Method](1)Liquid chromatography-mass spectrometry(LC-MS)was used to determine the contents of 8 chemical components in ZMD and CMD,and principal component analysis and one-way analysis of variance were used to clarify the chemical characteristics of O.japonicus from different regions.(2)ISSR molecular marker and cpDNA sequences were used to analyze the genetic diversity and genetic structure of O.japonicus from different origins.(3)The main ecological factors influencing the accumulation of chemical components in O.japonicus were determined through chemical-ecological correlation analysis by SPSS software,and ISSR loci with high correlation with chemical components were screened out by Tassel software through the association analysis between ISSR loci and 8 chemical components of O.japonicus individuals.(4)The unique SCAR markers of ZMD were developed and verified based on ISSR markers.[Results](1)The results of statistical analyses based on the chemical composition content in O.japonicus from different origins showed that both ZMD and CMD contain flavonoids and saponins.The contents of ophiopogonin C’ and ophiopogonin D’ were higher in Sanmenproduced ZMD(373.31±85.252 μg/g;970.55±77.932 μg/g)than that in Cixi-produced ZMD(52.18±7.551 μg/g;19.5±3.425 μg/g)and CMD(84.42±8.632 μg/g;300.14±24.629 μg/g),the content of methylophiopogonanone B was higher in Cixi-produced ZMD(468.37±27.303 μg/g)than that in Sanmen-produced ZMD(215.15±19.078 μg/g)and CMD(146.89±10.205 μg/g),and the content of methylophiopogonone A was higher in CMD(22.72±1.77 μg/g)than that in Cixi-produced ZMD(2.95±0.234 μg/g)and Sanmen-produced ZMD(14.68±1.743 μg/g).(2)Genetic diversity analysis of O.japonicus from different origins based on ISSR molecular marker and cpDNA sequences showed that the genetic diversity of O.japonicus had declined a lot after domestication(ISSR:H=0.2319,I=0.3560;cpDNA:hT=0.521,hS=0.030,VT=0.842,VS=0.067).The UPGMA clustering based on ISSR showed that Cixi-produced ZMD,Sanmen-produced ZMD,and CMD were clustered into three branches,and the phylogenetic tree based on cpDNA showed that ZMD and CMD were clustered into two branches respectively,indicating that there was some genetic differentiation between ZMD and CMD.Phylogeographical analysis based on cpDNA indicated that Sichuan was the origin of cultivated O.japonicus in this study.(3)Correlation analysis between chemical components and ecological factors showed that mean temperature of wettest quarter was the major ecological factor affecting the content of ruscogenin and ophiopogonin C’;annual mean temperature,mean temperature of wettest quarter and annual precipitation were the main ecological factors affecting the content of ophiopogonin D’;and temperature annual range,temperature seasonality,max temperature of warmest month and so on were the main ecological factors affecting the accumulation of methylophiopogonanone A and methylophiopogonanone B.Association analysis showed that the difference in genetic material among the germplasms of Cixi-produced ZMD,Sanmen-produced ZMD,and CMD was one of the reasons for the variation in chemical quality among the three,and indicated that among the 31 ISSR loci which were highly related to 8 chemical components in O.japonicus,7 loci were correlated with diosgenin content,5 with ruscogenin,6 with ophiopogonin C’,2 with ophiopogonin D,3 with ophiopogonin D’,6 with methylophiopogonanone A,2 with methylophiopogonanone B,1 with methylophiopogonone A,and 5 loci were common for some traits.(4)A total of 39 pairs of SCAR primers were designed and synthesized based on 19 specific bands of ZMD.While the PCR amplification results indicated that there was a pair of SCAR primers specific to Cixiproduced ZMD.[Conclusion]This study revealed the chemical content differences between ZMD and CMD based on content determination and statistical analysis using LC-MS.The genetic diversity and genetic structure of O.japonicus were analyzed to elucidate the genetic differentiation between ZMD and CMD using ISSR molecular markers and cpDNA sequences.The ecological and genetic factors affectting the formation and accumulation of chemical components in O.japonicus were clarified through the correlation analysis of chemical-ecological-genetic between ZMD and CMD.Finally,a pair of SCAR primers that was specific to ZMD produced in Cixi was designed.