RESUMO
Objective: To clone and screen the stable internal reference genes from Dipsacus asper for qRT-PCR analysis correction, so as to provide a preliminary basis for future research on expression analysis and regulation mechanism of D. asper functional genes. Methods: The internal reference genes of Actin, Tubulin and GAPDH gene families were screened and cloned from D. asper transcriptome database. The D. asper plants from different origins, different tissues and different developmental stages were used to obtain expression information of each gene by qRT-PCR. The expression stability of each gene was analyzed by geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, and the best genes were synthetically evaluated and screened. Results: Ten core fragments for candidate internal reference genes were cloned, belonging to three gene families: Actin, Tubulin and GAPDH, with high homology among them. The results of stability analysis showed that the expression of DaACT103 was stable and relatively high in different regions and tissues, while the expression of DaTUB5 was stable and relatively low in different developmental stages. Conclusion:s DaACT103 and DaTUB5 are suitable as the internal reference genes for D. asper. DaACT103 is used as the internal reference gene with high abundances and DaACT105 is used as the internal reference gene with low abundances.
RESUMO
Objective: To study the genetic diversity of Dipsacus asper from different populations and provide a reference for the rational utilization of its germplasm. Methods: The genetic diversity of the 14 populations of D. asper was analyzed by SRAP molecular markers. Results: Ten pairs of primers produced 124 sites, among which 102 were polymorphic sites. The percentage of polymorphic loci (PPL) was 82.26%. The Nei's genetic diversity index (H) and the Shannon's information index (I) were 0.2800 and 0.4353, respectively. At the population level, PPL was 53.92%, H was 0.1212-0.2440, and I was 0.1796-0.3611. The genetic diversity values of the five populations were relatively high, and the populations had the characteristics of high altitude and microhabitat. Genetic differentiation coefficient (Gst) was 0.2930, gene flow (Nm) was 1.2064. Cluster analysis based on genetic similarity indicated that the 14 populations could be divided into three groups. Conclusion: The genetic diversity among the populations of D. asper was at relatively high level. The genetic variance of D. asper mainly existed within the populations. The high genetic diversity could be attributed to the geographical position (altitude) and climate, while geographic isolation (microhabitat) was another important factor for the genetic variance within the populations.