Genotyping success of historical Eurasian lynx (Lynx lynx L.) samples

Historical samples, like tanned hides and trophy skulls, can be extremely important for genetic studies of endangered or elusive species. Selection of a sampling protocol that is likely to provide sufficient amount and quality of DNA with a minimum damage to the original specimen is often critical for a success of the study. We investigated microsatellite genotyping success of DNA isolated from three different types of Eurasian lynx historical samples. We analysed a total of 20 microsatellite loci in 106 historical samples from the endangered Dinaric lynx population, established from re-introduction of three pairs of lynx in 1973 from Slovakian Carpathians. Of the three tested sample types, turbinal bone and septum from the nasal cavity of the trophy skulls had the lowest percentage of samples successfully genotyped for all 20 microsatellite loci. Footpad samples, collected using a cork drill, exhibited better results in polymerase chain reaction amplification and genotyping than samples of footpad epidermis cut with a scalpel. We report simple and efficient sampling protocols, which could be widely applied for future studies utilizing historical samples.     

Improved noninvasive genotyping method: application to brown bear (Ursus arctos) faeces

We redesigned new microsatellite primers and one sex‐specific primer for amplification of faecal DNA from brown bears (Ursus arctos). We also combined a semi‐nested polymerase chain reaction (PCR) with a newly developed multiplex preamplification method in order to increase the quality of the amplified DNA fragments. In comparison with a conventional PCR approach, the genotyping error rate was substantially reduced and the amplification rate was increased. This new approach could be transposed to other species where conventional PCR methods experience low success due to limited DNA concentration and/or quality.     

Pooling hair samples to increase DNA yield for PCR

Hairs are useful non-invasive sources of DNA, but the DNA yield can be very small, thus promoting genotyping errors. Using multiple hairs can counter this problem, but may introduce multiple contributors to a sample if collected remotely. With microsatellite genotyping, samples representing multiple animals are obvious if three or more alleles are detected at any locus: these samples can then be removed from any analyses. However, some multiple-individual samples may have only one or two alleles at each of the loci examined. We investigated the probability of failing to identify mixed pooled samples by simulating pooled samples (10 000 replicates) from microsatellite data from the northern and southern hairy-nosed wombats (NHNW, Lasiorhinus krefftii; SHNW, L. latifrons), species with low and high genetic diversity respectively. The majority (81.7%) of the 40 000 simulated samples had three or four alleles, so were readily identified as mixed. In the remaining 1-or-2-allele SHNW samples, forensic science software (DNAMIX) correctly identified mixed versus single-individual samples for all cases when the probability of locus failure was low (P _(LF) = 0.1), and 99% of samples when locus failure was high (P _(LF) = 0.5). For NHNW however, the probability of failing to identify a mixed sample was too high for population size estimation (0.05), even when the probability of locus failure was low. In cases such as this, pooled samples may be adequate for less demanding tasks, such as estimation of allele proportions. However, for animal populations with at least average levels of genetic variation, pooling of samples could safely be utilised for most applications. This revised version was published online in July 2006 with corrections to the Cover Date.     

非损伤性取样研究进展

非损伤性取样即在不捕获、触及,甚至是未亲眼见到动物的情况下,收集不同形式的样品获取样品中的DNA。通过介绍各种类型非损伤性取样的研究进展,就该取样存在的问题及现有解决方法进行讨论,并在此基础上对非损伤性取样的研究和应用前景进行了分析,以期对进一步研究提供有价值的参考。     

Mitochondrial COII Introgression into the Nuclear Genome of Gorilla gorilla

Numts are nonfunctional mitochondrial sequences that have translocated into nuclear DNA, where they evolve independently from the original mitochondrial DNA (mtDNA) sequence. Numts can be unintentionally amplified in addition to authentic mtDNA, complicating both the analysis and interpretation of mtDNA-based studies. Amplification of numts creates particular issues for studies on the noncoding, hypervariable 1 mtDNA region of gorillas. We provide data on putative numt sequences of the coding mitochondrial gene cytochrome oxidase subunit II (COII). Via polymerase chain reaction (PCR) and cloning, we obtained COII sequences for gorilla, orangutan, and human high-quality DNA and also from a gorilla fecal DNA sample. Both gorilla and orangutan samples yielded putative numt sequences. Phylogenetically more anciently transferred numts were amplified with a greater incidence from the gorilla fecal DNA sample than from the high-quality gorilla sample. Data on phylogenetically more recently transferred numts are equivocal. We further demonstrate the need for additional investigations into the use of mtDNA markers for noninvasively collected samples from gorillas and other primates.