| Eucommia ulmoides Oliv. is a valuable medicinal woody plant endemic to China. Thethesis is composed of two parts:(1) establishment of ISSR-PCR reaction system, primersscreening and their applications to analysis of genetic diversity for E. ulmoides;(2) Sexidentification of E. ulmoides based on ISSRs.Inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) system wasestablished through orthogonal test for E. ulmoides. The total reaction volume is20.0μL,containing DNA(5-10ng·μL-1)2.0μL, dNTP (10mmol·L-1)0.25μL, primer (10μmol·L-1)0.7μL, Mg2+(25mmol·L-1)2.4μL, Taq DNA polymerase (5U·μL-1)0.2μL,10×PCRbuffer2.8μL, HPLC H2O11.65μL; Fourteen ISSR polymorphic primers were selected;the genetic diversity was analyzed based on the selected primers. A total of108polymorphic loci with distinct and repeatable bands were amplified, showing an effectivenumber of alleles per locus (ne) of1.505, an expected heterozygosity (He) of0.308, andShannon’s information index (I) of0.472. The results of the preliminary study revealed arelatively high level of genetic diversity, indicating that ISSRs are suitable markers foranalysis of genetic diversity in E. ulmoides, and will provide theoretic and technical basisfor the further population genetic study of E. ulmoides.The sex identification was carried out using male and female individuals of E.ulmoides based on established ISSR-PCR reaction system and amplification program.Results revealed a female-linked band of3000bp amplified using the primer UBC858,which was absent among the male individuals, indicating that ISSRs are useful markers forthe early sexual identification of E. ulmoides. The results of the prenent study will providereliable theoretic and practical support for optimal allocation of seedlings, for conservation,exchange and sustainable utilization of germplasm resources, and also for molecularbreeding of E. ulmoides. |
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