Zhu Ying, Liu Hong-Yi, Yang Hai-Qiong, Li Yu-Dong, Zhang He-Min
Sichuan Nature Resources Science Academy, Chengdu, Sichuan, China.
Sichuan Province Laboratory for Natural Resources Protection and Sustainable Utilization, Chengdu, Sichuan, China.
PeerJ. 2017 May 23;5:e3358. doi: 10.7717/peerj.3358. eCollection 2017.
Fecal samples play an important role in giant panda conservation studies. Optimal preservation conditions and choice of microsatellites for giant panda fecal samples have not been established. In this study, we evaluated the effect of four factors (namely, storage type (ethanol (EtOH), EtOH -20 °C, 2-step storage medium, DMSO/EDTA/Tris/salt buffer (DETs) and frozen at -20 °C), storage time (one, three and six months), fragment length, and repeat motif of microsatellite loci) on the success rate of microsatellite amplification, allelic dropout (ADO) and false allele (FA) rates from giant panda fecal samples. Amplification success and ADO rates differed between the storage types. Freezing was inferior to the other four storage methods based on the lowest average amplification success and the highest ADO rates ( < 0.05). The highest microsatellite amplification success was obtained from either EtOH or the 2-step storage medium at three storage time points. Storage time had a negative effect on the average amplification of microsatellites and samples stored in EtOH and the 2-step storage medium were more stable than the other three storage types. We only detected the effect of repeat motif on ADO and FA rates. The lower ADO and FA rates were obtained from tri- and tetra-nucleotide loci. We suggest that freezing should not be used for giant panda fecal preservation in microsatellite studies, and EtOH and the 2-step storage medium should be chosen on priority for long-term storage. We recommend candidate microsatellite loci with longer repeat motif to ensure greater genotyping success for giant panda fecal studies.
粪便样本在大熊猫保护研究中发挥着重要作用。目前尚未确定大熊猫粪便样本的最佳保存条件以及微卫星的选择。在本研究中，我们评估了四个因素（即储存类型（乙醇（EtOH）、-20°C乙醇、两步储存介质、二甲基亚砜/乙二胺四乙酸/三羟甲基氨基甲烷/盐缓冲液（DETs）并在-20°C冷冻）、储存时间（一、三、六个月）、片段长度以及微卫星位点的重复基序）对大熊猫粪便样本微卫星扩增成功率、等位基因脱扣（ADO）率和假等位基因（FA）率的影响。不同储存类型的扩增成功率和ADO率存在差异。基于最低的平均扩增成功率和最高的ADO率（<0.05），冷冻不如其他四种储存方法。在三个储存时间点，EtOH或两步储存介质获得的微卫星扩增成功率最高。储存时间对微卫星的平均扩增有负面影响，储存在EtOH和两步储存介质中的样本比其他三种储存类型更稳定。我们仅检测到重复基序对ADO和FA率的影响。三核苷酸和四核苷酸位点的ADO和FA率较低。我们建议在微卫星研究中，不应将冷冻用于大熊猫粪便保存，长期保存应优先选择EtOH和两步储存介质。我们推荐具有较长重复基序的候选微卫星位点，以确保大熊猫粪便研究获得更高的基因分型成功率。
