Evaluation And Analysis Of Germplasm Resources Of Zanthoxylum Bungeanum Maxim Based On Metabolic Markers And EST-SSR Markers

Zanthoxylum bungeanum Maxim.is a plant belongs to the Zanthoxylum genus of the family Rutaceae.Z.bungeanum is an important economic forest in China.It is a homologous plant of food and medicine with high medicinal,edible and ecological value.Due to their unique flavor and physiological activities,alkylamides and flavonoids can be used as important markers for the evaluation of Z.bungeanum germplasm resources.But at present,the existing quality evaluation is mainly based on the appearance quality and volatile oil content.It is difficult to fully reflect the overall effect of Z.bungeanum.In this study,the technology of ¹H-NMR,RNA-Seq and EST-SSR molecular markers were used to evaluate and analyze the quality of 9 Z.bungeanum germplasm resources based on the metabolic markers of alkylamides and flavonoids.The main results were as follows:1.A ¹H-NMR method was established for the quantitative analysis of alkylamides and flavonoids for the first time.Pyrazine was as internal standard and the extract of 85%ethanol dissolved in CDCl₃ was analyzed by ¹H-NMR.The hydrogen proton（δ:6.33 ppm,triple peak）on the amide bond of alkylamides was used as the quantitative signal peak.Flavonoids was extracted with 85%ethanol then extracts by ethyl acetate and acetone.Flavonoids extracts were dissolved in CD₃COCD₃-d₆ and analyzed by ¹H-NMR.And 6’-H（δ7.40、δ7.57、δ7.62、δ8.01 ppm）were used as quantitative signal peaks.Methodological validation showed that the recovery rate of samples was over 97%,and its precision,stability and repeatability were good（RSD were less than 3%）.2.The contents of alkylamides and flavonoids in Z.bungeanum from different germplasms and months were analyzed by ¹H-NMR and HPLC,which were verified the validity of ¹H-NMR.The results showed that the optimum picking time of Hancheng Shizitou was July,Fengxian Dahongpao,Hancheng Chenjiagelao stingless,Hancheng Dahongpao,Hancheng stingless,Qin’an No 1 and Wudu Dahongpao were August,Fugu and Xinong stingless were September.3.The ¹H-NMR fingerprints of Z.bungeanum from different germplasms were constructed and analyzed by stoichiometry（SA,HCA,PCA and DA）.9 Germplasms of Z.bungeanum were divided into three groups:the first group consists of 3 groups,which were stingless group,was Hancheng Chen Jiagelao stingless,Hancheng stingless and Xinong stingless.The contents of alkylamides and flavonoids were higher and the quality were better.The second group consists of 5 groups which were Fengxian Dahongpao,Wudu Dahongpao,Hancheng Dahongpao,Qin’an No.1 and Hancheng Shizitou,the third group consists of 1group,which was Fugu,and the contents of alkylamides and flavonoids were lower.Comprehensive analysis showed that Hancheng stingless was the best germplasm.4.27 differentially synthesized genes related to flavonoids were further screened by RNA-Seq analysis combined with the screening of differentially synthesized genes of alkylamides in the early stage.Combined with the differential expression multiples of different genes,10 key genes were identified in Z.bungeanum:c100778.graph＿c0,c103584.graph＿c0,c103637.graph＿c0,c103908.graph＿c0,c107987.graph＿c0,c96219.graph＿c0,c96857.graph＿c0,c97975.graph＿c1,c98433.graph＿c1 and c99880.graph＿c0.The expression of key differentially expressed genes was verified by q RT-PCR.The results showed that the relative expression of the other 9 genes were consistent with the transcriptome results except c96857.graph＿c0.5.According to the differentially synthesized genes of alkylamides and flavonoids,EST-SSR primers were designed and developed to reveal the genetic diversity of Z.bungeanum from the molecular level.16 pairs of primer combinations were screened out from 36 pairs,and 94 bands were amplified.The polymorphism ratio was reached 98.94%.The results of genetic diversity analysis showed that the average ratio of polymorphic loci was 82.51%,Nei’s genetic diversity index（H）was 0.3494,and Shannon’s information index（I）was 0.6103.Comprehensive analysis showed that the genetic diversity level of Hancheng Dahongpao was the highest,and Fengxian Dahongpao was lowest.The genetic variation mainly occurred within the germplasm.UPGMA cluster analysis and PCo A cluster analysis classified 9 germplasms into three groups:the first group was Fengxian Dahongpao and Wudu Dahongpao;the second group was Xinong stingless,Hancheng Shizitou,Hancheng stingless and Hancheng Chen Jiagelao stingless;the third group was Fugu Z.bungeanum,Hancheng Dahongpao and Qin’an No.1.Germplasm genetic diversity was higher in the latter two groups.EST-SSR fingerprints showed that 9 Z.bungeanum germplasms could be distinguished by characteristic bands.6.To analyze the correlation between the contents of alkylamides and flavonoids in Z.bungeanum and its genetic diversity index.The results showed that the genetic diversity index of Z.bungeanum was negatively correlated with the contents of alkylamides and flavonoids.The correlation between the contents of alkylamides and the number of effective alleles was significant（Pearson correlation coefficient was-0.696）.The results showed that the difference of alkylamides contents were more significantly regulated by related expressed genes.