Based On UPLC/Q-TOF MS And AFLP Technology Assisted Polygala Tenuifolia Breeding Research

RATIONALES:Polygala tenuifolia, one of commonly used medicinal materials in China, has the efficacy of tranquilization, restoring normal coordination between heart and kidney, expectorant and detumescence. At present, the cultivated varieties of P. tenuifolia are various. The breeding of P. tenuifolia new varieties mainly refers to the breeding standards of crops, taken the production, plantation age and apparent agronomic traits as the main index of breeding. And the drug efficacy is the only index in the measurement of its quality, with the content of chemical compositions as the material basis. As the chemical composition content can not be directly observed, it is necessary to establish a correlation analysis of agronomic traits and the content of chemical components in P. tenuifolia, and to primarily explore the relationship between genetic difference and agronomic traits from the DN A molecular level for elaborating its genetic diversity and further guiding directional breeding.OBJECTIVE:1. To explore the relationship between chemical index component and agronomic traits and clearly propose the traditional varieties breeding index.2. To find out differences metabolites according to the different agronomic characters and provide a guide for directional breeding.3. To illustrate the genetic background of P. Tenuifolia via screening out the good polymorphism primers in P. tenuifolia, and analyzing the relationship of polymorphic bands and the agronomic traits.METHOD:1. The agronomic traits of 18 P. tenuifolia samples werre determined.2. The content of senegenin, Polygalaxanthone Ⅲ,3,6’-Disinapoly were analyzed by HPLC-UV technology.3. Based on UPLC/Q-TOF MS metabolomics technology, the content of difference metabolites in each agronomic trait was analyzed.4. Based on AFLP molecular markers, primers which can amply fragment with good polymorphic were screened, and polymorphic bands of 18 samples were analyzed.RESULTS:1. In this study, the six agronomic traits, namely, plant height, diameter, root length, root bifurcation height, number of lateral root and root weight, have different levels of genetic variation. And the agronomic traits of plant height, root bifurcation height, lateral root number, root weight have larger variation and exist significant difference. Correlation analysis of agronomic traits found that the diameter and root weight are of the largest correlation.2. The P. tenuifolia samples which the content of senegenin, Polygalaxanthone Ⅲ, 3,6’-Disinapoly achieved the standard of (pharmacopoeia of China (2010)》 is 1,3,4,7,8, 9,13,14,15 and 17. The content of senegenin and agronomic traits has no obvious correlation The content of 3,6’-Disinapoly is higher in longer root P. tenuifolia samples; The content of Polygalaxanthone Ⅲ is higher in P. tenuifolia samples which has shorter root, shorter root bifurcation height and more lateral root number.3. Using UPLC/Q-TOF MS technology,29 compounds were identified. By UPLC/Q-TOF MS metabonomics technology, differences metabolites of six agronomic traits were found. Differences metabolites which have larger contribution to the classification of six agronomic traits are mainly concentrated in Sibiricoses A6, Tenuifoliside B, Tenuifoliside A, Polygalaxanthone Ⅲ, Tenuifoliose B, Tenuifoliose A and Polygalasaponin xx Ⅷ these seven compounds. These compounds are closely related with the expression of P. tenuifolia agronomic traits. Via analyzing the common differences metabolites of six related agronomic traits showed, that the common differences metabolites in positive related agronomic traits had same change trends, and the common differences metabolites in negative related agronomic traits had opposite change trends.4. Basd on AFLP molecular marker,12 good polymorphism primers were screened in P. tenuifolia. Then 18.P tenuifolia samples were analyzed by the selected 12 primers, polymorphic bands of 184 were amplified, polymorphism bands ratio is 94.57%. Based on the One-way AN OVA,20 polymorphism bands were screened which is significantly associated with six agronomic traits. By AFLP molecular markers, we found that one gene can control multiple agronomic traits at the same time, while one agronomic trait can also be controlled by multiple genes. The result of 18 samples polymorphic bands clustering is consistent with genetic identity and genetic distance analysis. Agronomic traits clustering, UPLC/Q-TOF MS data clustering polymorphic bands clustering all divided 18 P. tenuifolia samples into three categories, but not identical. Through the analysis on the relationship between the compounds and polymorphism bands in P. tenuifolia, found that the formation of individual compounds could be regulated by multiple genes, while a gene may also adjust the synthesis route of multiple compounds. The conclusion revealed a complex correlation network, which conforms to the complexity of biological systems.CONCLUSION:The process of P. tenuifolia variety breeding, combined with the content of chemical composition and agronomic traits can guide the breeding more accurately. If the P. tenuifolia sample is used as the ingredient of Chinese medicine preparation, the sample 4,13 should be selected. If the P. tenuifolia sample s used for the production of Chinese medicine decoction pieces, the sample 1,9 should be selected. The result of UPLC/Q-TOF MS metabonomics showed that, the sample which has the higher content of the target compounds, its agronomic traits was screened for directional breeding. Using the study of AFLP molecular marker showed, strengthening the research on the genetic background of P. tenuifolia samples can help to solve the problem of confusion varieties from the source at the present stage. In a word, the application of UPLC/Q-TOF MS metabonomics technology and AFLP molecular marker provides a new method for breeding/P. tenuifolia varieties.