| In recent years,with the comprehensive social,economic and ecological development,the demand for authentic,high-quality original herbs is accelerating,and the construction of standardised famous-region drug has been rapidly promoted.Min County produces Angelicae Sinensis Radix,specially called “Mingui”,which is the bulk,authentic and commonly used characteristic Traditional Chinese medicine in Gansu Traditional Chinese medicine industry.And the area with Min County as the core is the main production area of A.sinensis,known as the “hometown of A.sinensis in China”.However,after thousands of years of application and hundreds of years of artificial cultivation,the germplasm resources of the “Mingui” cultivation group have become mixed in genetic background and reduced in resistance.The prevalence of soil-borne diseases is increased,which directly affects the quality and commercial properties of the original medicinal materials,and becoming a serious problem that restricts the healthy development of the “Mingui” industry.In order to solve many of the current problems,this study is based on the quality evaluation of germplasm resources of cultivated populations of Individual pure lines of “Mingui”,and carries out systematic work to comprehensively evaluate the current quality of “Mingui” cultivated populations in terms of medicinal active substances,genetic background and germplasm purity.It is proposed to construct an evaluation system for the identification,screening and application of high-quality “Mingui” germplasm.At the same time,association markers for key quality factors were analysed and the genetic background was clarified,so as to provide strong support for the benign development of “Mingui” industry.1.Three batches of samples F-1 to F-3 from the mulch cultivation and three batches of samples L-1 to L-3 from the open field cultivation at different stages of growth and development in the trial area,with a total of 129 plants.The main phenotypic indicators and the content of the two signature medicinal substances ferulic acid and ligustilide were determined.The effects of mulching on the growth and development of “Mingui”and the changes in the signature substances were analyzed,and the degree of variation in the important indicators during the growth period was studied.Under different cultivation patterns,there were differences in the accumulation of the signature effective material during the same growth period and the growth rate of different growth periods.Compared with open field cultivation,mulching significantly increased the dry weight of “Mingui” roots and increased the content of ligustilide.Differences in phenotypic traits were found between individual samples of mulched at different growth stages,while the content of the two signature medicinal components was more stable and characteristic.2.Three batches of F-2,F-3 and L-3 were selected,with a total of 70 “Mingui”single roots from different cultivation modes in the experimental area.The HPLC fingerprints of each batch of “Mingui” were constructed.The contents of alcohol-soluble extractives of each batch of original medicinal materials were determined and compared.Four data analysis methods(correlation analysis,PCA,factor analysis and OPLS-DA)were used to investigate the effect of cultivation patterns on key factors of the quality of the original medicinal materials.Eleven shared peaks were identified in the HPLC fingerprints of the three batches of F-2,F-3 and L-3 of the“Mingui” original medicinal materials,all with similarity above 0.9.The chemical composition of each batch was stable and similar.PCA was performed based on the peak areas of the shared peaks,and a total of four principal components were obtained.Among them,ferulic acid and ligustilide had the largest contribution values in the first and second principal components respectively,and played a dominant role in the formation of chemical quality.The results of the analysis of the content of the signature active ingredients showed that,the amount of ferulic acid in the three batches of the original medicinal materials met the standard of the “Chinese pharmacopoeia(2020)”(>0.05%)exceeded 80%.Analysis of the alcohol-soluble leachate content showed that batch F-3 had the highest mean value of 59.42% and the lowest coefficient of variation of 5.87%.Analysis of the commercial traits showed that the number of original medicinal materials reaching second grade and above was significantly higher cultivated with film mulching than cultivated in the open field.The quality correlation analysis of F-2,F-3 and L-3 batches of “Mingui” original medicinal materials showed that,the root dry weight was highly significantly and positively correlated with root length,number of branch roots,content of ferulic acid,ligustilide and alcohol-soluble extract.PCA and OPLS-DA showed that both methods could achieve the basic separation of the original medicinal materials of “Mingui” in different cultivation patterns.Among the six quality indicators,root dry weight and the content of ferulic acid were the two important factors that dominated the differences in the quality of materials in different cultivation patterns,which could be used as an important reference for evaluating the quality of “Mingui”.The results of factor analysis showed that root length and root dry weight contributed significantly to the phenotypic trait factors,and ferulic acid contributed the most to the chemical quality factors;the overall score results showed that the mulching cultivation had a higher overall score and was ranked more highly.3.Three batches of F-2,F-3 and L-3,with a total of 70 samples of “Mingui” from the experimental area were selected for ISSR molecular markers on single plant samples.The molecular level was used to investigate the causes of background “Mingui”cultivation population.Two primers,UBC-809 and UBC-808,were selected from 9ISSR primers and used to amplify the DNA of 70 single plants of “Mingui”.30 bands were amplified and 26 polymorphic loci were detected,with a polymorphism ratio of86.67%.The DNA fingerprinting analysis of the “Mingui” cultivation population showed that the specific fingerprints of the samples were significantly different,except for a few samples with consistent bands.The results indicated that the genetic background of the “Mingui” cultivation population in the experimental area was complex and in production was actually analysed in terms of population purity,for the actual phenomenon of unclear origin of germplasm resources and mixed genetic background within the cultivation population.4.Two batches of F-2 and F-3 of the original medicinal materials of “Mingui” were selected for the study and coupled with quality key factors and genetic association markers for analysis.The original medicinal materials of “Mingui” in the mulching mode with good quality were used as the main subject of the screening study.The content of the iconic medicinal substance ferulic acid was used as the reference index for the initial screening.Combined with ISSR molecular markers for re-screening and grouping,and then final screening was carried out within each group based on the quality index data.Samples of group 5(F-3-2 and F-3-3)with excellent overall quality and stable genetic traits were finally selected as the best material for the subsequent screening of “Mingui” germplasm resources,for innovative application and new variety selection.In this study,single plants of “Mingui” were used as the research objects.Through the study of the quality characteristics of “Mingui” populations in different cultivation modes and their associated genetic background,it is proposed to construct a system for the screening and application of excellent “Mingui”germplasm resources and to screen out high-quality “Mingui” monocots.It will provide ideas and model references for the gradual solution of many serious problems in the development of “Mingui” industry. |
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