| Artemisia annua L.(Qinghao), which is the traditional Chinese medicine in China. Its active ingredient artemisinin is the only real effective drug for the treatment of falciparum malaria. The production of artemisinin in China depends mainly on wild resources, but existing wild resources can not meet the growing demand of market for artemisinin drug. In the study, wild populations and progeny genetic diversity and population genetic structure of A. annua were detected and compared. Using ISSR molecular marker in order to elucidate its level of genetic variation of the level and space distribution and genetic stability. At the same time, germplasm resources of A.annua was comprehensive evaluated combined with its effective components. It will provide theoretical reference for determining fine germplasm, breeding and utilization. The study results are as follows:1. The orthogonal design combined with single factor experiment was applied to optimize ISSR-PCR amplification system of A. annua, establish the best reaction system of A. annua, a system of25μL containing dNTPs0.20mmol/L, the primer0.80μmol/L Mg2+1.60mmol/L, Taq DNA polymerase1.00U, the template DNA80.00ng,10×Buffer2.50μL. The best ISSR-PCR procedures:94℃for5min; followed by38cycles of94℃for30s, annealing at50.0℃～57.8℃for45s and72℃for1.5min; re-extension at72℃; stored at4℃.2. Using13primers screened ISSR-PCR amplification of13A.annua wild populations were amplified117bands,107polymorphic loci, polymorphic loci of the species level (PPL) was91.45%, an average of69.62%of the population level of polymorphic loci (PPL), Nei’s diversity index (H) was0.3316, Shannon information index (I) was0.4999, indicating that wild A.annua had the higher level of genetic diversity, the coefficient of genetic differentiation (Gst) was0.1749,82.51%of the genetic variation within populations,17.49%of the different individuals in different populations, gene flow (Nm) was2.3585. The wild populations of Guangxi Yulin (YL) and Zhejiang Hangzhou (HZ) were as a key protected because its level of genetic diversity wer higher. We should collect more plant individuals as much as possible for when establishing germplasm of A. annua.3. The correlative analysis of geographical distance and genetic distance of A.annua wild populations, which results showed that the difference was not significant correlation. In addition, the artemisinin content of A. annua wild populations and genetic distance correlation was not significant.4. Compared with parental generation(wild populations), the progeny populations of A.annua genetic diversity parameters less than the wild populations. The difference between the progeny and parental of Zhengzhou(ZZ), and Yingde(YD) population were small and their genetic stability were better, but the artemisinin content far from the requirements of industrial production. Comprehensive comparison genetic stability and artemisinin content, We suggest that Tien population(TE) is the population for cultivated seed production followed by Dahua population(DH).5.13wild populations of A. annua could be clustered into two groups:Beihai populations(BH) was the first category separately; Guangxi Chongzuo(CZ), Guangxi Dahua(DH), Hunan Shaoyang(SY), Guangxi Rongan(RA), Guangxi Liuzhou(LZ), Guangxi Tien(TE), Henan Zhengzhou(ZZ), Guangxi Yangshuo(SY), Hunan Liling (LL), Guangxi Yulin(YL), Guangdong Yingde (YD) and Zhejiang Hangzhou(HZ) Poly were the second largest category. Dahua (DH) and Guangxi Chongzuo (CZ) populations had the closest kinship relation. |
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