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.