Abstract：Aconitum carmichaeli is a perennial herb of Aconitum in family Ranunculace, and it can be radix aconiti as medicine. Reports about Aconitum drug poisoning incidents have been very common, which are mainly caused by failure to identify species of Aconitum effectively. Through the research on development of Aconitum microsatellite primer and genetic diversity, the research realized fast and accurate identification of genetic resources and fake A. carmichaeli, which provided scientific guarantee for protection, development and successive research on superior genetic resources of A. carmichaeli. By extracting DNA of A. carmichaeli, establishing genomic library of A. carmichaeli, and sequencing A. carmichaeli with Illumina high-throughput sequencing technology, the research acquired plenty of 100 bp short-sequence microsatellite fragments. After screening the microsatellite fragments, microsatellite library of A. carmichaeli was established. According to the results, through second-generation high-throughput sequencing technology of Illumina, 38 942 660 strips of 125 bp pairing sequence were acquired. Through quality trim, 200 386 strips of splicing sequence were obtained after assembly. After filtration and screening, 12 095 strips of sequence fragments being larger than 299 bp were acquired finally. The fragments with repeat segment being larger than 100 bp were selected from microsatellite library of A. carmichaeli for primer design of microsatellite, and 55 pairs of microsatellite primers were obtained. By the use of these 55 pairs of microsatellite primers, amplification and polypropylene gel electrophoresis were carried out for 80 individuals from 4 wild Aconitum populations to detect the polymorphism of microsatellite primers and genetic diversity of population. After detection, 14 pairs of Aconitum microsatellite primers with clear stripe and rich polymorphism were acquired. For the 14 pairs of Aconitum microsatellite primers, average number of allele (A) was 4.85, average observation heterozygosity (Ho) was 0.613, average expected heterozygosity (He) was 0.583 and PIC was 0.528. Four sibling species of A. carmichaeli were used for universality detection of microsatellite primers. It was found that the stripe of 11 pairs of microsatellite primers were clear, and they occupied 78.57% of total microsatellite primers. According to the results of universality detection of Aconitum microsatellite primers, most of the microsatellite primers were proved amplifiable in sibling species of A. carmichaeli. Through analysis of genetic diversity of 4 A. carmichaeli populations, it was found that A. carmichaeli showed high genetic diversity at species level. Besides, for the Aconitum populations, A was 3.25, HO was 0.612, He was 0.493, Shannon's information index (I) was 0.851 and Nei's genetic diversity index (h) was 0.505. Genetic differentiation between populations showed that certain genetic differentiation (FST=0.149) existed in 4 populations of A. carmichaeli and gene flow between populations (NM)=1.432 was limited. According to research results, 14 pairs of microsatellite primers developed by the research showed high polymorphism and good universality, which could be used for identification of genetic resources, analysis of genetic diversity and construction of DNA fingerprint of A. carmichaeli. From the perspective of species level, A. carmichaeli had rich genetic diversity, which verifies abundant heritable variation ability of A. carmichaeli from DNA level and explained strong evolution potentiality of A. carmichaeli. The study will offer scientific guarantee for identification of genetic resources and fake A. carmichaeli, protection, development and successive research on superior genetic resources of A. carmichaeli.
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