| Population genetic studies with Microsatellite markers and PCR techniques using non-invasive sampling methods are commonly applied in modern wildlife management and conservation.However,risks usually exist when coming to scientific conclusions because of the DNA of low quality extracted from non-invasive samples.Firstly,this study was designed to evaluate the genotyping risks of 12 microsatelite loci common used in wild Amur tiger studies by comparison of the genotyping results of these loci with blood DNA and fecal DNA of 10 captive Amur tigers as amplification template,and the accumulative genotyping matching ratio(Rm)was used.Our researches showed that:1)Because of differentiations in chromosome positions and segment size,the genotyping risks of different microsatelite loci varied,so the times of PCR may also differ to get a reliable genotyping results.Moreover,wrong genotypings detected in our study may affect following population genetic analysis.2)Before applying fecal DNA into microsatelite based genetic analysis,to achieve a reliable genotyping result,DNA of higher quality,such as tissue or blood DNA should first be used as pilot studies to verify the reliability of the loci that are not specificly designed for the target species,and the quality of scats used in the pilot studies should be close to those collected in the field.3)Rm had better be used to evaluate the genotyping risks and determine the times of PCR needed,and this parameter can be further used in individual identification and population genetic analysis.Second,based on the work above,10 microsatelite loci were selected for genetic study with 58 suspected Amur tiger fecal samples collected from the wild in Northeast China,and the results are as followings:1)DNA was successfully extracted from all of the 58 fecal samples,and the Amur tiger species specific mitochondrial primers identified 50 of them to belong to Amur tigers.2)All of the 10 microsatelite loci were successfully amplified by different PCR times according to our pilot study.Totally,20 Amur tigers were identified from these 50 samples,and the accumulative individual identification probability is 99.9%.Of the 20 tigers identified,3 are females,11 are males and the sex information of other 6 is unknown because of failed amplification of tiger specific sex determination primers.3)We detected 38 alleles,alleles ranging from 2 to 7,with an average of 3.8.Allele frequencies ranged from 0.0109 to 0.9643;Ho ranged from 0 to 0.8293,averaging 0.4954;He ranged from 0.0701 to 0.7344,averaging 0.4445;PIC(polymorphism information content)ranged from 0.0665 to 0.6804,averaging 0.3869.4)Cumulative exclusion probability of paternity of 20 individuals was 94.21,and the MLH(multilocus heterozyosity)ranged from 0.2 to 0.6,with an average of 0.425.The LR-estimator(relation parameter)between two individuals ranged from-0.71 to 0.6.The individual No.112,which was far away from No.1,4 and 50 in geographic distance was also far away in genetic distance,and departed into two different clusters in the genetic relation tree.5)From this study,we see that the genetic diversity in wild Amur tiger population is still high and the population is under random mating pattern.However,we also find the signs of inbreeding and unbalanced sex ratio,so more effective management measures should be taken timely. |
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