DNA Extraction From Noninvasive Samples And Isolation Of SSR Markers In Giant Panda

Giant panda is one of the famous endangered species in the world and also is a national treasure of China. Conservation researches for giant panda have received worldwide and considerable attention. Now giant panda confronts some serious problems, such as few individuals in population, confined distribution, lack of genetic diversity, close relative propagation and weak ability to procreate. Therefore, it is urgent to solve these problems and make proper breeding plans and improve the giant panda's ecological environments. SSR markers can be taken as the most efficient tool to analyze and solve these problems and are accepted by many researchers in the correlative fields. However, the development of SSR markers from giant panda is not enough and so far only 36 SSR markers have been isolated. In addition, it is so difficult for most researchers to obtain sufficient or even a small amount of the giant panda's genome DNA that molecular genetic study of giant panda can't be widely developed. So here, the technique of DNA extraction from noninvasive samples and isolation of SSR markers are developed by using these samples: blood, hair roots and faeces. The main results obtained are shown as follows:1. An improved method that facilitates the extraction of PCR-compatible faecal DNA from giant panda's faeces is described. The method involves a novel preprocessing step in DNA extraction. The faeces is washed two or three times with precooled acetone, which removes numerous potential PCR inhibitors, and then digested with proteinase K. The DNA is purified with phenol/chloroform. The faecal DNA obtained is sufficiently pure to support reliable amplification, and isapplied as template DNA to amplify a portion of the giant panda brain derived neurotrophic factor (BDNF) gene and mitochondrial cytochrome b gene. The sequenced results of PCR products confirm that the extracted DNA is from the giant panda. Comparison with the PCR products demonstrates that the faecal DNA extracted by the improved protocol is better than the faecal DNA extracted without acetone preprocessing.2. Giant panda's SSR markers are isolated from blood DNA by using two PCR-based methods, RAMS and MP-PCR. Three SSRs are isolated by using RAMS, but only one is perfect. Seventeen SSRs are isolated by using MP-PCR, but only three fragments provide the conserved regions flanking the repeat at both ends while the rest only provide the conserved regions at 5' or 3' end. Through comparing the two methods, a simple and rapid approach to isolate SSR markers from a small amount of hair DNA is generated. Also, four perfect SSR markers are isolated by using this approach.