Introduction

Telomeres, repetitive, non-coding sequences of DNA found at the tips of chromosomes, function to maintain chromosome and genome integrity during replication . Previous research has linked telomere length loss to aging and disease in various taxa . However, new evidence suggests changes in telomere length are influenced by the environment . Indeed, telomere length loss has been associated with exposure to extreme heat in dairy cattle , increased river temperatures in fish , and developmental stress during embryogenesis in birds . While less is known about these relationships in plants, telomere length varies in response to aging and the environment in Arabidopsis , with impacts to fitness . Thus, telomeres may be a valuable biomarker for assessing response to environmental stress in plants. These findings highlight the need to develop new methods to quantifying telomere length for population management in natural and agronomic plant systems.
Telomere length has traditionally been estimated using the terminal restriction fragment (TRF) method . TRF measures the absolute telomere length of an individual by using the length distribution of terminal restriction fragments, which are detected by southern blotting using a probe specific for telomeric DNA . The TRF method uses genomic DNA, digested with restriction endonucleases that cut throughout the chromosome, but not within the telomeric region, assuming restriction sites are absent in the telomere region . Telomere length is then determined by quantifying the signal intensity of the entire TRF smears relative to a DNA ladder with known fragment size . Despite its utility, TRF requires large quantities of DNA (3 µg/individual), and quantification can be sensitive to DNA degradation . An alternate method, quantitative polymerase chain reaction (qPCR) has also been adopted to estimate relative telomere length for diverse taxa . The qPCR approach estimates relative telomere length using the ratio of the threshold cycle (Ct) of the telomeric region (T) to a non-variable copy number reference gene (S) relative to a reference sample . qPCR is widely used in telomere studies as it requires small quantities of DNA (20 ng/individual), provides scalability for high-throughput assessments, and produces results in a shorter amount of time than TRF . However, despite the wide utility of these approaches, recent advances in whole-genome sequencing (WGS) and new bioinformatic tools are enabling the estimation of telomere length using whole-genome sequencing. Rapid advances in high-throughput sequencing coupled with reduced costs have increased the availability of WGS data across model and non-model systems . Thus, given the availability of new bioinformatic resources, comparison of approaches to estimating telomere length is needed, particularly in plants where there is limited evaluation of the accuracy and reliability of different approaches.
Telomere length in humans was initially estimated from WGS data by counting the number of reads containing the human telomere repeat sequence (TTAGGG)4 normalized by genome coverage . To date, several bioinformatic programs, including TelSeq , Telomerecat , Telogator , TelomereHunter , and Computel have been developed to estimate telomere length in humans using WGS data. However, despite the increasing availability of WGS data for plant species, few tools assess telomere length in plants. Only three bioinformatic programs, Computel, K-seek, and Telomeric repeats identification pipeline have been developed that are able to quantify telomere length for diverse organisms more broadly and are appropriate for plants. These bioinformatics tools offer new opportunities for quantifying telomere length variation using WGS across plant systems, essential for advancing the field of telomere ecology.
Despite the biological and ecological value of quantifying telomere length variation in plants, few studies to date have either estimated telomere length or compared telomere length estimates using qPCR and WGS. In this study, we aim to fill this gap, comparing telomere length estimates using multiple bioinformatic approaches for one hundredPopulus clones re-sequenced using Illumina short-read sequencing, comparing Computel , K-seek , and TRIP . In addition, we compare telomere length estimates for the same individuals assayed using qPCR and WGS data. This study will test new approaches to estimating telomere length and will extend the use of telomeres as a potential biomarker to assay response to environmental change needed for plant species management and breeding.