DNA extraction protocol for animal blood samples using the EZNA blood mini kit.

The E.Z.N.A. Blood DNA Mini Kit provides an easy and rapid method for the isolationof genomic DNA for consistent PCR and Southern analysis. Up to 250 μL fresh,frozen, or anticoagulated whole blood can be readily processed at one time. TheE.Z.N.A. Blood DNA Mini Kit can also be used for the preparation of genomic DNAfrom buffy coat, serum, plasma, saliva, buccal swabs, and other body fluids. TheE.Z.N.A. Blood DNA Kit allows for single or multiple simultaneous processing ofmultiple samples. There is no need for phenol/chloroform extractions, and timeconsuming steps are eliminated (e.g. precipitation using isopropanol or ethanol).Purified DNA obtained with the E.Z.N.A. Blood DNA Kit is ready for applications suchas PCR, restriction digestion, and Southern blotting

Methylation-based markers for the estimation of age in African Cheetah, Acinonyx jubatus

Age is a key demographic in conservation biology where individual age classes show diffuse differences in terms of important population dynamics metrics such as morbidity and mortality. Furthermore, several traits including reproductive potential show clear senescence with aging. Thus, the ability to estimate the ages for the individuals of a population as part of age class assignment is critical in understanding both the current population structure as well as in modelling and predicting the future survival of species. This study explored the utility of age-related changes in methylation for six candidate genes, EDARADD, ELOVL2, FHL2, GRIA2, ITGA2B, and PENK, to create an age estimation model in captive cheetah. Gene orthologues between humans and cheetah were retrieved containing a hundred CpG’s. Target regions were assayed for differential methylation and fragmentation patterns in fifty samples using mass array technology for a total of seventy-seven CpG clusters. Correlation analyses between CpG methylation and chronological age identified six CpG’s with an age relationship, of which four were hypomethylated and two were hypermethylated. Regression models, fitted for different combinations of CpG’s, indicated that age models using four and six CpG’s were most accurate, with the six CpG model having superior correlation and predictive power (R2 = 0.70, Mean Absolute Error = 25 months). This model was more accurate than previous attempts using methylation sensitive Polymerase Chain Reaction and performed similarly to models created using a candidate gene approach in several other mammal species, making methylation a promising tool of age estimation in cheetah

PCR Amplification of Clock and Adcyap1 genes with EmeraldAmp® GT PCR Master Mix in Avian species for polymorphism elucidation

This PCR protocol is used to amplify Clock and Adcyap1 gene regions in avian species which have previously shown polymorphisms, such as poly-Q runs, that correlated to migration phenology. It was tested and optimized in Woodlands kingfisher (Halcyon senegalensis) and Diederik cuckoos (Chrysococcyx caprius). The primers were designed based on those previously used by Johnson et al. (2007) and Steinmeyer et al. (2009) by comparing the relevant gene sequences for chickens (Galus galus) with several other available avian species to select primers that would account for the most common variations in primer regions, enabling more universal amplification. Individual clock gene sequences were retrieved from Genbank and aligned in BioEdit 7.2. Primers were then selected based on the annotated regions. The assay was designed using 25 μL (half) reactions of EmeraldAmp® GT PCR Master Mix, which is premixed with loading buffer for easy gel loading following PCR and does not require a long initial denaturation step (thereby shortening the run time). Gel electrophoresis was able to confirm successful amplification of a product ±280 bp long in both species. The same primers were subsequently used for sanger sequencing. A BLAST search of the resulting sequences confirmed the identity of the amplified regions

ABI Sanger Sequencing of Avian Clock genes to elucidate markers for Migration Phenology

This protocol follows up on "PCR Amplification of Clock genes with EmeraldAmp® GT PCR Master Mix in Avian species" and is intended to provide the next steps used in the sanger sequencing of the produced amplicons. This protocol uses ABI BigDye reagents (but suitable alternatives exist). The same primers used to produce the PCR products, of the respective clock genes, are used individually in a forward and reverse sequencing reaction. Cycling conditions mimic those used for PCR. Sequencing reactions are purified and subsequently resolved on an ABI Genetic Analyzer. The sequence read data was used in a BLAST search and confirmed to be the genes and regions of interest for all tested species

Designing an EpiTYPER bisulfite sequencing assay for age estimation in Acinonyx jubatus based on human orthologues

Age is key factor in animal ecology as it can be used to assign animals to important age classes, ranging from immature young to reproductive adults and eventual old age and fragility. Different groups contribute to different aspects that need to be considered when modeling current and future population dynamics as part of continued conservation efforts. Due to the need of an accurate molecular method for assigning age, several studies have explore various aspects of epigenetic clocks. Epigenetics is a collective term for mechanisms that modify DNA and DNA packaging, independent of genetic sequence. One widely studied epigenetic feature is DNA methylation; a process that adds a methyl group to the 5’ cytosine of Cytosine-Guanine pairs (CpG’s). Studies have revealed that within genes, nearly a third of all CpG sites are influenced by age. Given its consistency, the epigenetic clock is a promising avenue of chronological age prediction which has been illustrated in many human studies. This protocol illustrates how CpG's with known age-correlations from human studies can be used to (1) identify orthologous regions in other species and (2) design primers to assay differential methylation using EpiTYPER mass array technology.<br/

Phenotypic correlates between clock genes and phenology among populations of Diederik cuckoo, Chrysococcyx caprius

The Diederik cuckoo, Chrysococcyx caprius, is a small Afrotropical bird in the family Cuculidae. It is taxonomically related to 13 other species within the genus Chrysococcyx and is migratory in sub- Saharan Africa. It has a unique breeding behaviour of being a brood parasite: Breeding pairs lay their eggs in the nests of a host species and hatchlings expel the eggs of the host species. The aim of the present study was to investigate diversity in two circadian clock genes, Clock and Adcyap1, to probe for a relationship between genetic polymorphisms and their role in circannual timing and habitat selection (phenology) in intra- African migrants. DNA extracted from blood was used for the PCR amplification and sequencing of clock genes in 30 Diederik cuckoos. Three alleles were detected for Clock with similar genotypes between individuals from the Northern and Southern breeding ranges while 10 alleles were detected for Adcyap1, having shorter alleles in the North and longer alleles in the South. Population genetic analyses, including allele frequency and zygosity analysis, showed distinctly higher frequencies for the most abundant Clock allele, containing 10 polyglutamine repeats, as well as a high degree of homozygosity. In contrast, all individuals were heterozygous for Adcyap1 and alleles from both regions showed distinct differences in abundance. Comparisons between both clock genes and phenology found several phenotypic correlations. This included evidence of a relationship between the shorter alleles and habitat selection as well as a relationship between longer alleles and timing. In both instances, evidence is provided that these effects may be sex- specific. Given that these genes drive some of the synchronicity between environments and the life cycles of birds, they provide valuable insight into the fitness of species facing global challenges including climate change, urbanisation and expanding agricultural practices

Birds of a feather flock together:a dataset for Clock and Adcyap1 genes from migration genetics studies

Birds in seasonal habitats rely on intricate strategies for optimal timing of migrations. This is governed by environmental cues, including photoperiod. Genetic factors affecting intrinsic timekeeping mechanisms, such as circadian clock genes, have been explored, yielding inconsistent findings with potential lineage-dependency. To clarify this evidence, a systematic review and phylogenetic reanalysis was done. This descriptor outlines the methodology for sourcing, screening, and processing relevant literature and data. PRISMA guidelines were followed, ultimately including 66 studies, with 34 focusing on candidate genes at the genotype-phenotype interface. Studies were clustered using bibliographic coupling and citation network analysis, alongside scientometric analyses by publication year and location. Data was retrieved for allele data from databases, article supplements, and direct author communications. The dataset, version 1.0.2, encompasses data from 52 species, with 46 species for the Clock gene and 43 for the Adcyap1 gene. This dataset, featuring data from over 8000 birds, constitutes the most extensive cross-species collection for these candidate genes, used in studies investigating gene polymorphisms and seasonal bird migration.</p

Data from: Methylation-based markers for the estimation of age in African Cheetah, Acinonyx jubatus

&lt;p&gt;This is a dataset for methylation analyses in cheetah done by EpiTYPER mass array.&lt;/p&gt