Genetic architecture of natural variation of telomere length in Arabidopsis thaliana

Abstract

Telomeres represent the repetitive sequences that cap chromosome ends and are essential for their protection. Telomere length is known to be highly heritable and is derived from a homeostatic balance between telomeric lengthening and shortening activities. Specific loci that form the genetic framework underlying telomere length homeostasis, however, are not well understood. To investigate the extent of natural variation of telomere length in Arabidopsis thaliana, we examined 229 worldwide accessions by terminal restriction fragment analysis. The results showed a wide range of telomere lengths that are specific to individual accessions. To identify loci that are responsible for this variation, we adopted a quantitative trait loci (QTL) mapping approach with multiple recombinant inbred line (RIL) populations. A doubled haploid RIL population was first produced using centromere-mediated genome elimination between accessions with long (Pro-0) and intermediate (Col-0) telomere lengths. Composite interval mapping analysis of this population along with two established RIL populations (Ler-2/Cvi-0 and Est-1/Col-0) revealed a number of shared and unique QTL. QTL detected in the Ler-2/Cvi-0 population were examined using near isogenic lines that confirmed causative regions on chromosomes 1 and 2. In conclusion, this work describes the extent of natural variation of telomere length in A. thaliana, identifies a network of QTL that influence telomere length homeostasis, examines telomere length dynamics in plants with hybrid backgrounds, and shows the effects of two identified regions on telomere length regulation.

Keywords: Arabidopsis; QTL; centromere-mediated genome elimination; haploid; telomere.

Figure 1

Analysis of 229 natural accessions reveals extensive variation of telomere length. (A) Example TRF blot from 12 accessions. (B) Distribution of mean telomere length over 229 accessions. Range varies from ∼1 to 9.3 kb (see Table S1 for full list of accessions and telomere lengths).

Figure 2

Hov1-10 displays extremely short telomeres. (A) TRF analysis of five individual Hov1-10 plants (ID no. 6035). (B) TeloTool analysis of telomeric smears showing fit quality percentage, mean, and standard deviation (σ). 2σ was used to define upper and lower boundaries, and averages were taken from lanes 3, 4, and 5. Fit quality was deemed too low in lanes 1 and 2 so these samples were excluded. (C) Initially, TeloTool data did not appear to match visually with the gel image from lane 3. Further analysis of intensity profiles showed that a small amount of higher-molecular-weight signal (marked with an asterisk) was present in this lane. This could be explained by incomplete digestion within this sample during TRF preparation. Because of the high fit quality in this lane, the extrapolated values were deemed to be correct and were used in the analysis.

Figure 3

Construction of a new mapping population using centromere-mediated genome elimination. (A) Crossing scheme for Col-0/Pro-0 RIL population. F₁’s derived from crosses between Col-0 and Pro-0 were used to pollinate cenh3/GFP-tailswap haploid inducer plants. Haploids segregated from these crosses are expected to be mosaic for both parental genomes. Diploids were recovered from haploid plants and selfed to the F₅ generation. (B) TRF profiles of Col-0, Pro-0, and five individual F₁ plants. (C) TRF analysis of a subset of F₃ diploid RILs showing variation of telomere length. While F₁ plants contained telomeres from both parents, analysis of F₃ RILs shows that telomere length homeostasis adjusts to a newly defined balance when genomes from both parents are mixed.

Figure 4

Composite interval mapping of RIL populations. Distribution of telomere length in RIL populations and results from CIM analysis (A) Pro-0/Col-0. (B) Cvi-0/Ler-2. (C) Est-1/Col-0.

Figure 5

QTL combinations of Cvi-0/Ler-2 population. To examine the effects of QTL, the most significant markers were taken from each of the four major trans QTL positions: CL-1:EG.198C/200L-Col, CL-2:BH.195L-Col, CL-4:CC.400L-Col, and CL-5:CC.262C. Average telomere length was taken from RILs in each combination. Gray bars represent lines where a Cvi-0 insertion at a single locus conferred telomere lengthening or shortening. Error bars represent ±SE.

Figure 6

Analysis of NILs confirms effects of two loci on telomere length. Cvi-0 insertions into the causative region encompassing the QTL showed telomere lengthening in chromosome 1 (A) and shortening in chromosome 2 (B). Figure illustrating known positions of Cvi-0 insertions is adapted from Keurentjes et al. (2007). Black bars represent known positions of Cvi-0 introgressions, and gray bars indicate crossover areas between markers selected for genotyping.

Figure 7

Segregation of Cvi-0 region from NIL 1–22 confirms its effect on telomere elongation. (A) NIL 1–22 was genotyped with CAPS markers and found to comprise a Cvi-0 insertion in chromosome 1 between ∼12 and 24 Mb. (B) Homozygous NIL 1–22 was backcrossed to the parental Ler-2 line, and single plants homozygous for Ler-2 or Cvi-0 at this region were identified in the F₂. Analysis of these plants showed no apparent difference in telomere length. (C) Pooled plants in the second generation were analyzed, and telomere shortening was observed in two Ler-2 samples (marked by asterisks). (D) Third generation and (E) fourth generation show clear change in telomere length, indicating that this region contains the causative locus.

Figure 8

Summary of QTL positions. Approximate QTL positions are indicated by known positions of significant markers. Horizontal marks present on each chromosome represent 1-Mb intervals. Results indicate that there are shared and unique QTL between Cvi-0/Ler-2 (blue), Est-1/Col-0 (orange), and Pro-0/Col-0 (green) RIL populations. Positions of genes with putative or known effects on telomere biology are highlighted (STN1, Song et al. 2008; CTC1, Surovtseva et al. 2009; TEN1, Leehy et al. 2013; KU70, Riha et al. 2002; KU80, Gallego et al. 2003; TRB1/2/3, Kuchar and Fajkus 2004; TER1 and TER2, Cifuentes-Rojas et al. 2012; TERT, Fitzgerald et al. 1999; POT1a/b/c, Shakirov et al. 2005 and Rossignol et al. 2007; RPA70a, Takashi et al. 2009; RAD50, Gallego and White 2001; NBS1, Najdekrova and Siroky 2012; MRE11, Bundock and Hooykaas 2002; NAP57, Kannan et al. 2008; STEP1, Kwon and Chung 2004; WHY1, Yoo et al. 2007; and TAC1, Ren et al. 2004).