| Objective:The genetic diversity of Cyclocarya Paliurus from 22 different habitats was analyzed by SCoT Molecular Marker technique to explore the genetic diversity and genetic relationship of Cyclocarya Paliurus from different habitats.The contents of three flavonoids(isoquercitrin,Quercitrin and Afudin)in Cyclocarya paliurus from 24 different habitats were determined by HPLC and their fingerprints were studied to evaluate the quality of Cyclocarya Paliurus from different habitats.Method:1.DNA extraction from Cyclocarya paliurus from different habitats was studied by using CTAB,improved CTAB method,improved kit method,improved kit method and SDS method.2.In this study,177 samples of Cyclocarya Paliurus were collected from Nanning,Longsheng Various Nationalities Autonomous County County,Hunan Province,Hubei Province,Jiangxi province,Anhui Province and Guizhou Province,the total genomic DNA of Cyclocarya Paliurus was extracted by kit method,and the SCoT-PCR reaction system of Cyclocarya Paliurus was optimized by single factor investigation The best primers were selected from 54 SCoT Primers(including 36 universal primers and 18 self-designed Primers),and the genetic diversity of Cyclocarya paliurus samples from different habitats was analyzed by SCoT Molecular Markers,the Best Primers were amplified by PCR and the PCR map was obtained by Gel electrophoresis.The genetic diversity of Cyclocarya Paliurus was analyzed by using POPGENE 1.32 and NTSYS 2.10 software.3.HPLC method with acetonitrile-0.1% phosphoric acid water as mobile phase for gradient elution,detection wavelength 256 nm,column temperature 35°C,volume flow rate 1m L/min,the contents of three flavonoids(isoquercitrin,Quercitrin and Afudin)in Cyclocarya Paliurus(batal.)Iljinsk from different habitats were determined,and the differences and changing trends of the contents of flavonoids in Cyclocarya paliurus from24 habitats were analyzed.4.HPLC method with acetonitrile-0.1%phosphoric acid as mobile phase gradient elution,Detection Wavelength300 nm,column temperature 30°C,volume flow rate 1m L/min,established fingerprint of Cyclocarya Paliurus from 24 different habitats,similarity analysis was carried out by fingerprint similarity system,the Cluster Analysis and principal component analysis of sample data were carried out by SPSS21.0.Results:1.SCoT Marker Analysis:(1)total DNA extraction: five methods of DNA extraction were studied,including DNA purity,yield,extraction time,DNA degradation consumption,etc.(2)the best 20 m L SCoT-PCR reaction system of Cyclocarya Paliurus was as follows: Primer concentration:0.5mmol/L,annealing temperature: 51°C and 54°C,cycle times: 34 cycles,agarose Gel concentration: 1.0%,electrophoresis voltage: 100 V.(3)SCoT Primer screening of Cyclocarya Paliurus: A total of 5 primers were screened from 54 primers,including 3 universal primers and 2 self-designed Primers.(4)scat primer screening heritage diversity analysis of Cyclocarya Paliurus(batal.)A total of 69 bands were amplified from 177 samples with 5 primers,of which 69 bands were polymorphic and the percentage of polymorphic(PPB)was 100%.The effective allele(Ne)was 1.4912,neis genetic diversity index(H)was 0.3017,Shannons information index(I)was 0.4662,which indicated that Cyclocarya paliurus population had very high genetic diversity and its genetic diversity was rich.The results showed that the genetic diversity of Cyclocarya Paliurus population was rich.The results of cluster analysis showed that the genetic relationship and differentiation of Cyclocarya Paliurus germplasm resources were affected by wild or cultivated GERMPLASM,and also affected by producing area..2.Content determination and analysis: The contents of three flavonoids(isoquercitrin,Quercitrin and Afudin)in 24 batches of Cyclocarya paliurus leaves were compared,the contents of isoquercitrin were0.8452～10.4928mg/g,0.9390～3.0786mg/g,0.2659～3.9694mg/g,and3.1588～14.9402 mg/g,respectively,the highest was cultivated in Xingan County of Guilin,the next was cultivated in Tonggu County,and the lowest was wild Cyclocarya Paliurus in Quanzhou County of Guilin,Guangxi.The total content was 3.1588 mgg.At the same time,by comparing the total content of the same producing area,it was found that the total content of cultivated product was higher than that of wild product.3.Fingerprint Analysis: In the fingerprints of Cyclocarya paliurus from different habitats,12 common peaks were identified and 4 flavonoids were identified as chlorogenic acid,Isoquercitrin,Quercitrin and Afudin The results of sample similarity analysis showed that(1)the similarity of Cyclocarya paliurus samples from other habitats was higher than 0.902,except that from Sangzhi County of Zhangjiajie and Shimen County of Changde of Hunan were 0.890 and 0.888respectively;(2)the RSD values of the relative retention time of the common peaks of Cyclocarya Paliurus from different habitats were small,which indicated that the chemical constituents of Cyclocarya paliurus from different habitats had good consistency,and the RSD values of the relative peak areas were large,which indicated that the contents of the chemical constituents of Cyclocarya Paliurus were different,(3)when the distance is 20,24 batches of Cyclocarya paliurus samples could be clustered into two categories: S18 and S19,and the other 22 samples could be clustered into another category.When the distance is 15,24 batches of Cyclocarya paliurus samples can be grouped into four categories.(4)the results of principal component analysis showed that the information of the first principal component mainly came from the peaks of 1,2,4(chlorogenic acid),5 and 6,and the information of the second principal component mainly came from the peaks of 3,8,10(Afudin)and 11,the information of the third principal component mainly comes from the peaks of 7(Isoquercitrin),9(Quercitrin)and 12.According to the principal component information of Cyclocarya paliurus leaves and the ranking results of its comprehensive scores,S3,S18 and S19 were higher in the comprehensive scores of principal components,which suggested that the quality of these habitats might be better,followed by S4,S7,S8,S11,S15 and S16 samples,the lower combined score of S10,S17,S20,S21,S22,S23 and S24 and the classification of the last group suggest that the quality of these regions may be more unstable.Conclusion: The results showed that the genetic diversity of Cyclocarya Paliurus germplasm resources from different habitats was high and there was some genetic differentiation.The genetic relationship among different samples was related to the origin,provenance(wild and cultivated)and other factors The method for the determination of Cyclocarya Paliurus(batal.)Iljinsk is simple,reproducible and reliable.It can be used as a reference for quality evaluation. |
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