A method to estimate relative telomere length in 27 taxonomically diverse avian species.

Telomeres are found at the ends of chromosomes and serve as a protective barrier to prevent damage during DNA replication. Telomeres shorten with each cell replication until they reach a critical length, at which point apoptosis occurs leading to mortality. Increased long-term stress has been shown to cause an increased rate of telomere loss (Boonekamp et al 2014). Previous studies have developed methods relating relative telomere length (RTL) and relate it to chronological age and/or life expectancy in avian species through the use of real-time quantitative polymerase chain reaction (qPCR). However, methods that work on multiple species have not yet been developed. A universal method to determine the relative telomere length across a broad range of species would require primers to amplify a control gene from genomic DNA (gDNA) that are highly conserved. The availability of such a primer set eliminates the need to design primer sets for each new species tested. Herein, we tested the well conserved 18S rRNA for this purpose. We isolated and tested red blood cell gDNA from 157 individuals in 27 different species covering12 phylogenetic orders (Galliformes, Anseriformes, Phoenicopteriformes, Eurypygiformes, Columbiformes, Cuculiformes, Gruiformes, Accipitriformes, Bucerotiformes, Coraciiformes, Psittaciformes, Passeriformes). Five sets of 18S primers were designed and tested for efficiency at the telomere annealing temperature (56°C) determined in previous studies. The relationship of relative telomere length and chronological age was examined in six species, each with 6 to 50 samples by calculating the coefficient of determination (R2). For the 27 species tested, the average qPCR efficiency of the 18S primers was 94.3% ± 5.9, with efficiencies ranging from 85.2% to 107.6%. R2 values were 0.07 in the nene (Branta sandvicensis), 0.003 in the alala (Corvus hawaiiensis), 0.17 in the Chilean flamingo (Phoenicopterus chilensis), 0.41 in the blue and gold macaw (Ara ararauna), 0.26 in the blue-bellied roller (Coracias cyanogaster) and 0.008 in the greater flamingo (Phoenicopterus roseus). Although we successfully developed a technique to determine RTL in a broad range of avian species, we did not find a relationship between chronological age and RTL for any of the six species, suggesting telomere dynamics in captive populations may be different than those found in the wild. This study provides a tool that can be used to measure the effects of long-term stress on relative telomere length in captive individuals from a broad range of avian species. Care managers can use this information when determining which individual is most suited for breeding or reintroduction.