Nuclear DNA Fragments Research Articles

Reactive oxygen species in the normal and acutely injured liver

Reactive oxygen species in the normal and acutely injured liver

Molecular phylogeny of African hinged and helmeted terrapins (Testudines: Pelomedusidae: Pelusios and Pelomedusa)

Fritz, U., Branch, W. R., Hofmeyr, M. D., Maran, J., Prokop, H., Schleicher, A., Široký, P., Stuckas, H., Vargas‐Ramírez, M., Vences, M. & Hundsdörfer, A. K. (2010). Molecular phylogeny of African hinged and helmeted terrapins (Testudines: Pelomedusidae: Pelusios and Pelomedusa). —Zoologica Scripta, 40, 115–125.With 18 currently recognised species, Pelusios is one of the most speciose chelonian genera worldwide, even though the taxonomy of some species is contentious. Recent investigations suggested that the closely related, but morphologically distinct genus Pelomedusa is paraphyletic with respect to Pelusios, and that Pelomedusa consists of nine deeply divergent lineages. Using three mitochondrial and three nuclear DNA fragments (2054 bp mtDNA, 2025 bp nDNA), we examined for the first time the phylogeny of Pelusios by molecular means. Our analyses included all Pelusios species, except the probably extinct P. seychellensis, as well as the nine Pelomedusa lineages. The results showed that Pelusios and Pelomedusa are reciprocally monophyletic. Limited sampling of Pelusios species and homoplasy introduced by remote outgroups most likely explain the paraphyly of Pelomedusa in previous studies. The distinctiveness of most Pelusios species was confirmed, but none of the currently recognised species groups within Pelusios was monophyletic. In Pelusios rhodesianus and P. sinuatus distinct genetic lineages were discovered, suggestive of cryptic taxa. In contrast, the recognition of the weakly differentiated P. castaneus and P. chapini as full species is doubtful, as is the validity of the Malagasy and Seychellois subspecies of P. castanoides. GenBank sequences of P. williamsi were nested within P. castaneus, but the morphological distinctiveness of the two species makes it likely that the GenBank sequences (derived from a turtle from the pet trade) are misidentified. Divergence among the distinct genetic lineages of Pelomedusa equals or exceeds the differences among Pelusios species, supporting the view that Pelomedusa is a species complex.

Obtaining high-quality DNA from elusive small mammals using low-tech hair snares

Noninvasive sampling approaches are becoming increasingly important for enabling genetic studies of wildlife populations. While a number of methods have been described to noninvasively sample hair from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. Here we describe a novel and inexpensive noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We explore the quality of the sample by assessing PCR amplification success of mitochondrial and nuclear DNA fragments across four commercially available DNA isolation kits and two different quantities of hair in a factorial design. Additionally, we determined the sex of the individual samples using PCR–RFLP of ZFX/ZFY loci. We found that our snare is effective in obtaining hair that yield DNA of sufficient quality and quantity to successfully amplify a range of mitochondrial and nuclear fragment sizes. Specifically, we found the greatest success in amplifying mitochondrial DNA, nuclear microsatellites and ZFX/ZFY loci using at least 25 hairs as starting material and the DNA IQ™ system. The hair snares thus provide a cost-effective and minimally intrusive approach to sample elusive or rare small mammals. We anticipate that this approach will be useful to obtain samples for molecular studies of the ecology, evolution and conservation of small, elusive mammals.

High Gyrodactylus salaris infection rate in triploid Atlantic salmon Salmo salar

We describe an unusually high infection rate of Gyrodactylus salaris Malmberg in juvenile Atlantic salmon Salmo salar L. of Baltic Sea origin, which are generally believed to be more resistant to G. salaris than East Atlantic salmon populations. Based on analyses of mitochondrial (complete cytochrome oxidase 1 [CO1] gene, 1548 bp) and nuclear (ADNAM1, 435 bp; internal transcribed spacer [ITS] rDNA region, 1232 bp) DNA fragments, the closest relatives of the characterized Estonian G. salaris strain were parasites found off the Swedish west coast and in Raasakka hatchery, Iijoki (Baltic Sea, Finland). Analyses of 14 microsatellite loci of the host S. salarrevealed that approximately 40% of studied fish were triploids. We subsequently identified triploid Atlantic salmon of Baltic origin as more susceptible to G. salaris infection than their diploid counterparts, possibly due to compromised complement-dependent immune pathways in triploid salmon. This is in accordance with earlier studies that have shown elevated susceptibility of triploids to various viral or bacterial pathogens, and represents one of the first reports of increased susceptibility of triploid salmonid fish to an ectoparasite. However, further experimental work is needed to determine whether triploid Atlantic salmon is generally more susceptible to G. salaris compared to their diploid counterparts, irrespective of the particular triploidization method and population of origin.

Mapping of the nuclear matrix-bound chromatin hubs by a new M3C experimental procedure

We have developed an experimental procedure to analyze the spatial proximity of nuclear matrix-bound DNA fragments. This protocol, referred to as Matrix 3C (M3C), includes a high salt extraction of nuclei, the removal of distal parts of unfolded DNA loops using restriction enzyme treatment, ligation of the nuclear matrix-bound DNA fragments and a subsequent analysis of ligation frequencies. Using the M3C procedure, we have demonstrated that CpG islands of at least three housekeeping genes that surround the chicken α-globin gene domain are assembled into a complex (presumably, a transcription factory) that is stabilized by the nuclear matrix in both erythroid and non-erythroid cells. In erythroid cells, the regulatory elements of the α-globin genes are attracted to this complex to form a new assembly: an active chromatin hub that is linked to the pre-existing transcription factory. The erythroid-specific part of the assembly is removed by high salt extraction. Based on these observations, we propose that mixed transcription factories that mediate the transcription of both housekeeping and tissue-specific genes are composed of a permanent compartment containing integrated into the nuclear matrix promoters of housekeeping genes and a ‘guest’ compartment where promoters and regulatory elements of tissue-specific genes can be temporarily recruited.

Genetic Structure of Chorthippus parallelus (Orthoptera: Acrididae: Gomphocerinae) Populations in Anatolia: A Stable Rear Edge Population

Abstract Anatolian populations inhabit the southern latitudinal margin of species distribution ranges and therefore may be considered as “rear edge” populations. The genetic structures of such populations have critical importance in species responses to climatic change and are essential for long-term conservation genetics. Here, the genetic structure of Chorthippus parallelus (Zetterstedt) (Orthoptera: Acrididae: Gomphocerinae) populations in one of the main southern glacial refugium is investigated. Ten populations of C. parallelus from Anatolia have been studied by investigating single-copy nuclear DNA (Cpnl-1) fragment using polymerase chain reaction-single strand conformational polymorphism methods. The level of allelic number was high (total of 20 alleles in the locus, of which 11 were unique), but the level of gene flow among populations seemed to be low (FIS = 0.090). However, most populations were genetically diverse (HE > 0.5, A = 6.211, and ne = 5.774). The level of genetic differentiation among populations was high (FST = 0.330). No statistically significant correlation between genetic diversity and spatial distribution was observed. The analysis of molecular variation analysis indicated that a large proportion of genetic variation was due to differentiation among individuals within populations. Genetic drift was a more likely cause of differentiation among populations rather than geographical distance. These results suggest that a presence of a “stable rear edge” population in contrary to the center-periphery model.

Molecular phylogeny and systematics of the banana family (Musaceae) inferred from multiple nuclear and chloroplast DNA fragments, with a special reference to the genus Musa

Musaceae is a small paleotropical family. Three genera have been recognised within this family although the generic delimitations remain controversial. Most species of the family (around 65 species) have been placed under the genus Musa and its infrageneric classification has long been disputed. In this study, we obtained nuclear ribosomal ITS and chloroplast (atpB-rbcL, rps16, and trnL-F) DNA sequences of 36 species (42 accessions of ingroups representing three genera) together with 10 accessions of ingroups retrieved from GenBank database and 4 accessions of outgroups, to construct the phylogeny of the family, with a special reference to the infrageneric classification of the genus Musa. Our phylogenetic analyses elaborated previous results in supporting the monophyly of the family and suggested that Musella and Ensete may be congeneric or at least closely related, but refuted the previous infrageneric classification of Musa. None of the five sections of Musa previously defined based on morphology was recovered as monophyletic group in the molecular phylogeny. Two infrageneric clades were identified, which corresponded well to the basic chromosome numbers of x=11 and 10/9/7, respectively: the former clade comprises species from the sections Musa and Rhodochlamys while the latter contains sections of Callimusa, Australimusa, and Ingentimusa.

Sex in Penicillium: Combined phylogenetic and experimental approaches

We studied the mode of reproduction and its evolution in the fungal subgenus Penicillium Biverticillium using phylogenetic and experimental approaches. We sequenced mating type (MAT) genes and nuclear DNA fragments in sexual and putatively asexual species. Examination of the concordance between individual trees supported the recognition of the morphological species. MAT genes were detected in two putatively asexual species and were found to evolve mostly under purifying selection, although high substitution rates were detected at some sites in some clades . The first steps of sexual reproduction could be induced under controlled conditions in one of the two species, although no mature cleistothecia were produced. Altogether, these findings suggest that the asexual Penicillium species may have lost sex only very recently and/or that the MAT genes are involved in other functions. An ancestral state reconstruction analysis indicated several events of putative sex loss in the genus. Alternatively, it is possible that the supposedly asexual Penicillium species may have retained a cryptic sexual stage.

Systematics and phylogeography of a threatened tortoise, the speckled padloper

AbstractThis study investigated the systematics and phylogeography of a threatened tortoise of South Africa, the speckled padloper Homopus signatus. Sixty three specimens were collected from 17 localities that covered the distributional range of the two subspecies in western South Africa and a north‐eastern population that was recently discovered near Pofadder. The Pofadder sample could not be assigned to either subspecies based on morphology. The samples were sequenced for two partial mtDNA fragments, nicotinamide adenine dinucleotide dehydrogenase component four and cytochrome b, which yielded ∼1.1 kb, while a subset of the samples were sequenced for a 390 bp nuclear DNA (nDNA) fragment of prolactin. Phylogenetic analyses of mtDNA using minimum evolution, maximum parsimony and Bayesian inferences supported the monophyly of H. signatus and revealed that the Pofadder specimen was basal in the topology and sister to the remainder. The phylogenetic analyses did not support the recognition of two subspecies; there was statistical support for a Homopus signatus signatus clade but Homopus signatus cafer was not monophyletic. The nDNA analysis showed no difference between the subspecies and placed the Pofadder sample distant but not distinct from H. s. signatus. The mtDNA and the nDNA data suggest that the subspecies are invalid taxonomic units. The structure of the mtDNA network corresponded to the geographical distribution of populations. The north‐western populations formed one haplocluster, corresponding to H. s. signatus, whereas the south‐western populations formed three haploclusters, corresponding to H. s. cafer. The Pofadder sample was unconnected to the network. The morphology of the northern and southern morphotypes probably reflects selection for crypsis on the different substrate types of the regions, granites and sedimentary rocks, respectively. These results highlight that subspecies designations should be authenticated by molecular techniques because morphological plasticity can obfuscate phylogenetic relationships. We consider the western H. signatus populations as one taxonomic unit and recommend wider sampling of the Pofadder locality to clarify the taxonomic status of this lineage.

Achievements and Peculiarities in Studies of Ancient DNA and DNA from Complicated Forensic Specimens

Studies of ancient DNA specimens started 25 years ago. At that time short mitochondrial DNA (mtDNA) fragments were the main targets in ancient DNA studies. The last three years were especially productive in the development of new methods of DNA purification and analysis. Complete mtDNA molecules and relatively large fragments of nuclear DNA are the targets of ancient DNA studies today. Ancient DNA studies allowed us to study organisms that went extinct more than ten thousand years ago, to reconstruct their phenotypic traits and evolution. Ancient DNA analyses can help understand the development of ancient human populations and how they migrated. A new evolutionary hypothesis and reconstruction of the biota history have been re-created from recent ancient DNA data. Some peculiarities and problems specific to the study of ancient DNA were revealed, such as very limited amounts of DNA available for study, the short length of the DNA fragments, breaks and chemical modifications in DNA molecules that result in “postmortem” mutations or complete blockage of DNA replication in vitro. The same specific features of DNA analysis were revealed for specimens from complicated forensic cases that result in the lack of experimental data or interpretation problems.. Here, we list the specific features of ancient DNA methodology and describe some achievements in fundamental and applied research of ancient DNA, including our own work in the field.

Achievements and Peculiarities in Studies of Ancient DNA and DNA from Complicated Forensic Specimens

Studies of ancient DNA specimens started 25 years ago. At that time short mitochondrial DNA (mtDNA) fragments were the main targets in ancient DNA studies. The last three years were especially productive in the development of new methods of DNA purification and analysis. Complete mtDNA molecules and relatively large fragments of nuclear DNA are the targets of ancient DNA studies today. Ancient DNA studies allowed us to study organisms that went extinct more than ten thousand years ago, to reconstruct their phenotypic traits and evolution. Ancient DNA analyses can help understand the development of ancient human populations and how they migrated. A new evolutionary hypothesis and reconstruction of the biota history have been re-created from recent ancient DNA data. Some peculiarities and problems specific to the study of ancient DNA were revealed, such as very limited amounts of DNA available for study, the short length of the DNA fragments, breaks and chemical modifications in DNA molecules that result in "postmortem" mutations or complete blockage of DNA replication in vitro. The same specific features of DNA analysis were revealed for specimens from complicated forensic cases that result in the lack of experimental data or interpretation problems.. Here, we list the specific features of ancient DNA methodology and describe some achievements in fundamental and applied research of ancient DNA, including our own work in the field.

A new subspecies of Batagur affinis (Cantor, 1847), one of the world’s most critically endangered chelonians (Testudines: Geoemydidae)

Estuarine Batagur are among the most critically endangered chelonian species. We assess the taxonomic status of the recently discovered Cambodian relic population of Batagur by phylogenetic analyses of three mitochondrial (2096 bp) and three nuclear DNA fragments (1909 bp) using sequences from all other Batagur species and selected allied geoemydids. Furthermore, we calculated haplotype networks of the mitochondrial cytochrome b gene for Cambodian terrapins, B. affinis, B. baska, and B. kachuga and compare external morphology of estuarine Batagur populations. Genetically, Cambodian Batagur are closely related with, but distinct from B. affinis from Sumatra and the west coast of the Malay Peninsula. Morphologically, Cambodian Batagur resemble the distinctive B. affinis populations from the eastern Malay Peninsula that were not available for genetic study. We suggest that the Batagur populations from the eastern Malay Peninsula and Cambodia represent a new subspecies of B. affinis that once was distributed in estuaries surrounding the Gulf of Thailand (Batagur affinis edwardmolli subsp. nov.). Its patchy extant distribution is most probably the result of large-scale habitat alteration and century-long overexploitation. In addition, our phylogenetic analyses suggest repeated switches between riverine and estuarine habitats during the evolution of the extant Batagur species.

A combination of techniques proves useful in the development of nuclear markers in the newt genus Triturus

To increase the number of markers available for study of phylogeny and phylogeography in the newt genus Triturus, we developed and tested 59 primer pairs using three different techniques. Primers were obtained from published sources, by designing exon-primed intron-crossing primers and from randomly cloned anonymous nuclear DNA fragments. Successful polymerase chain reaction products were cloned and sequenced. Five fragments were successfully amplified and sequenced for six species of Triturus: intron 7 of the β-fibrinogen gene (βfibint7), third intron of the calreticulin gene (CalintC), the 11th intron of the α-subunit of the platelet derived growth factor receptor (PDGFRα) and two anonymous markers (Cri1 and Cri4). The average percentage species divergence across all the markers is low (c. 3%), compared to what has been found in mitochondrial DNA (25-30%).

Further specimens and phylogenetic position of the recently described leaf turtle species Cyclemys gemeli (Testudines: Geoemydidae)

We describe external morphology and habitat of ten specimens of Cyclemys gemeli, a recently discovered leaf turtle species from north-eastern India, previously known only from its incomplete holotype and photos of a live female. Further, we assess the phylogenetic position of C. gemeli using sequence data of the mitochondrial cytochrome b gene as well as of three nuclear DNA fragments (C-mos, Rag2 genes, intron 1 of R35 gene) and confirm its genetic distinctiveness. Mitochondrial data strongly suggest a sister group relationship of C. gemeli and C. fusca, another species occurring in Myanmar. According to our new records, the Naga Hills and the Arakan Mts could constitute the geographical divide between C. gemeli and C. fusca. Morphologically, C. gemeli resembles other dark-bellied Cyclemys species and determination by external morphology alone is quite difficult.

Persistence of Plant DNA Sequences in the Blood of Dairy Cows Fed with Genetically Modified (Bt176) and Conventional Corn Silage

To determine whether plant sequences, including transgenic sequences, are present in animal blood, we tested blood samples from Holstein cows fed with either Bt176 genetically modified corn or conventional corn. We used previously described sensitive real-time PCR assays targeting transgenic sequences (35S promoter and Bt176 specific junction sequence), a monocopy maize-specific sequence (ADH promoter), and two multicopy sequences from plant nucleus (26S rRNA gene) and chloroplast (psaB gene). The presence of Cry1A(b) protein in bovine blood samples was also tested using a sandwich ELISA kit. Our study shows the ability of plant nuclear and/or chloroplast DNA fragments to enter bovine blood circulation. However, maize nuclear DNA, both mono- and multicopy sequences, was less detected than chloroplast DNA, probably because the higher number of chloroplast copies and also possibly because nuclear DNA might be less protected by the nuclear membrane. Despite our data confirm the ability of small (ca.150 bp) plant DNA fragments to cross the intestinal barrier, we were unable to demonstrate clearly the presence of transgenic DNA or proteins in bovine blood. No sample tested positive with the two real-time PCR assays targeting transgenic sequences (35S promoter and Bt176 specific junction sequence). Only faint punctual positive results occurred randomly and were probably due to postsample collection or laboratory contamination or can be considered as artifact as they have never been confirmed. Our data highlight the difficulties to detect transgenic sequences in blood of dairy cows fed genetically modified corn (Bt176) silage. Those results show that in order to meet the consumers' demand of animals fed with GM products there is currently no cost-effective analytical procedure to replace documentary traceability.

Molecular phylogeny and divergence times of ancient South American and Malagasy river turtles (Testudines: Pleurodira: Podocnemididae)

The eight extant podocnemidid species are the last survivors of a speciose ancient group of turtles known to have existed since the Cretaceous. One species, representing the monotypic genus Erymnochelys, occurs on Madagascar; the remaining seven species are confined to South America ( Peltocephalus: one species; Podocnemis: six species). Phylogenetic relationships of all extant species were reconstructed from six mitochondrial (3385 bp) and six nuclear DNA fragments (4115 bp) in separate and combined analyses (Bayesian inference, Maximum Likelihood, Maximum Parsimony). In a total evidence approach for all concatenated genes, all methods yielded the same well-supported phylogenetic hypothesis for the three basal lineages. The Malagasy genus Erymnochelys is sister to the South American Podocnemis, and Peltocephalus constitutes the sister taxon to Erymnochelys+ Podocnemis. Within Podocnemis, P. unifilis+( P. erythrocephala+ P. lewyana) constitute a well-supported crown clade; P. sextuberculata, P. vogli, and P. expansa were revealed as successive sister taxa. According to Bayesian relaxed molecular clock calculations calibrated with fossil evidence, Peltocephalus originated during a period of the Late Cretaceous (∼86 mya), when a contiguous Gondwana landmass exclusive of Africa is likely to have still existed. The Late Cretaceous split between Erymnochelys and Podocnemis (∼78 mya) coincides with the supposed submergence of the land bridge between Madagascar and Antarctica+South America, suggesting that the origin of those genera is linked to this vicariant event. The extant Podocnemis species evolved from the Late Eocene (∼37 mya) to the Middle Miocene (∼15 mya), during a phase characterized by dramatic global cooling, aridification, and massive Andean uplift.

Species Diversity and Genetic Variation of Terrestrial Lichens and Bryophytes in A Boreal Jack Pine Forest of Central Canada

Little is known about species diversity and genetic structure of populations of lichens and bryophytes in the Sandilands Provincial Forest in Manitoba. This study investigated the current species diversity of lichens and bryophytes in the area. It also examined the population structure and gene flow in the lichen (Cladonia arbuscula) with the highest cover and second most frequent occurrence, and bryophyte (Pleurozium schreberi) with the highest frequency and percent ground cover. Analysis of Molecular Variance (AMOVA) of the presence and absence of group I introns in the fungal small subunit (SSU) nuclear ribosomal DNA (rDNA) and Restriction Fragment Length Polymorphisms (RFLP) of the algal Internal Transcribed Spacer (ITS) rDNA suggests that low levels of gene flow were occurring in the fungus while a single algal genotype was maintaining the lichen association. Similarly, Interspersed Simple Sequence Repeat (ISSR) markers of the bryophyte suggested low levels of gene flow. The examination of variation in both lichen symbionts and a bryophyte, dominant within the same habitat, is a novel aspect of this study. Both these widespread species, dominating the understory in mature jack pine forests, exhibited low levels of sexual reproduction.

Protective Effects of Scutellarin Against Cerebral Ischemia in Rats: Evidence for Inhibition of the Apoptosis-Inducing Factor Pathway

Scutellarin (Scu) is the major active principle (flavonoid) extracted from Erigeron breviscapus (Vant.) Hand-Mazz, a Chinese herbal medicine. In this paper, we investigated the effects of Scu on brain injury through the inhibition of AIF-mediated apoptosis induced by transient focal brain ischemia in rats. Rats were treated with Scu for 7 d and then subjected to cerebral ischemia/reperfusion (I/R) injury induced by a middle cerebral artery occlusion (MCAO). After 2 h of ischemia and 22 h of reperfusion, the infarct volume and the neurological deficit were determined by TTC staining and Longa's score. IN SITU end-labeling of nuclear DNA fragments (TUNEL) was employed to determine the degree of DNA fragmentation. NAD content and PARP activity in brain homogenate were determined. The expression of AIF in the nucleus was analyzed by Western blot. The present study showed that Scu significantly reduced the infarct volume and ameliorated the neurological deficit. An increase in the number of TUNEL-positive cells and a decrease in the NAD level were also observed after 2 h of ischemia and 22 h of reperfusion. At the same time, Scu (50 and 75 mg kg (-1), i. g.) treatment reversed brain NAD depletion and reduced DNA fragmentation. Scu also inhibited PARP overactivation and AIF translocation from the mitochondria to the nucleus following cerebral I/R. These findings suggested that the neuroprotective effects of Scu on brain ischemic injury-induced apoptosis might be associated with inhibition of PARP-dependent mitochondrial dysfunction and subsequent translocation of AIF.

Paradoxical Effect of Mitochondrial Respiratory Chain Impairment on Insulin Signaling and Glucose Transport in Adipose Cells

Adipocyte function is crucial for the control of whole body energy homeostasis. Pathway analysis of differentiating 3T3-L1 adipocytes reveals that major metabolic pathways induced during differentiation involve mitochondrial function. However, it is not clear why differentiated white adipocytes require enhanced respiratory chain activity relative to pre-adipocytes. To address this question, we used small interference RNA to interfere with the induction of the transcription factor Tfam, which is highly induced between days 2 and 4 of differentiation and is crucial for replication of mitochondrial DNA. Interference with Tfam resulted in cells with decreased respiratory chain capacity, reflected by decreased basal oxygen consumption, and decreased mitochondrial ATP synthesis, but no difference in many other adipocyte functions or expression levels of adipose-specific genes. However, insulin-stimulated GLUT4 translocation to the cell surface and subsequent glucose transport are impaired in Tfam knockdown cells. Paradoxically, insulin-stimulated Akt phosphorylation is significantly enhanced in these cells. These studies reveal independent links between mitochondrial function, insulin signaling, and glucose transport, in which impaired respiratory chain activity enhances insulin signaling to Akt phosphorylation, but impairs GLUT4 translocation. These results indicate that mitochondrial respiratory chain dysfunction in adipocytes can cause impaired insulin responsiveness of GLUT4 translocation by a mechanism downstream of the Akt protein kinase.

Quantitative PCR assessment of microsatellite and SNP genotyping with variable quality DNA extracts

In this study we developed eight quantitative PCR (qPCR) assays to evaluate the starting copy number of nuclear and mitochondrial DNA fragments ranging from 75 to 350 base-pairs in DNA extracts from Chinook salmon tissues with varying quality. Samples were genotyped with 13 microsatellite and 29 SNP assays and average genotyping success for good, intermediate, and poor quality samples was 96%, 24%, and 24% for microsatellite loci, and 98%, 97%, and 79% for SNPs, respectively. As measured by qPCR, good quality samples had a consistently high number of starting copies across all fragment sizes with little change between the smallest and largest size. In contrast, the intermediate and poor quality samples displayed decreases in starting copy number as fragment size increased, and was most pronounced with poor samples. Logistic regression of genotyping success by starting copy number indicated that in order to achieve at least 90% genotyping success, approximately 1,000 starting copies of nuclear DNA are necessary for microsatellite loci, and as few as 14 starting copies for SNP assays (but we recommend at least 50 copies to reduce genotyping error). While these guidelines apply specifically to Chinook salmon and the genetic markers included in this study, the principles are transferable to other species and markers due to the underlying process associated with template quantity and PCR amplification.