Tandem Repeats Contribute to Coding Sequence Variation in Bumblebees (Hymenoptera: Apidae)

Abstract

Tandem repeats (TRs) are highly dynamic regions of the genome. Mutations at these loci represent a significant source of genetic variation and can facilitate rapid adaptation. Bumblebees are important pollinating insects occupying a wide range of habitats. However, to date, molecular mechanisms underlying the potential adaptation of bumblebees to diverse habitats are largely unknown. In the present study, we investigate how TRs contribute to genetic variation in bumblebees, thus potentially facilitating adaptation. We identified 26,595 TRs from the assembled 18 chromosome sequences of the buff-tailed bumblebee (Bombus terrestris), 66.7% of which reside in genic regions. We also compared TRs found in B. terrestris with those present in the assembled genome sequence of a congener, B. impatiens. We found that a total of 1,137 TRs were variable in length between the two sequenced bumblebee species, and further analysis reveals that 101 of them are located within coding regions. These 101 TRs are responsible for coding sequence variation and correspond to protein sequence length variation between the two bumblebee species. The variability of identified TRs in coding regions between bumblebees was confirmed by PCR amplification of a subset of loci. Functional classification of bumblebee genes where coding sequences include variable-length TRs suggests that a majority of genes (87%) that could be assigned to a protein class are related to transcriptional regulation. Our results show that TRs contribute to coding sequence variation in bumblebees, and thus may facilitate the adaptation of bumblebees through diversifying proteins involved in controlling gene expression.

Fig. 1.

—Repeat unit features for identified bumblebee TRs. (A) Repeat unit length distribution of TRs. Only repeat unit lengths, at which there are >100 TR loci, are shown. (B) The top ten most abundant repeat unit sequences.

Fig. 2.

—Distribution features for bumblebee TR loci. (A) TR locus length distribution. (B) The distance between TRs and predicted genes. As shown in the figure, a majority of TRs identified from assembled portion of Bombus terrestris genome reside within genes.

Fig. 3.

—The relationship between repeat unit length and observed mutation probability for bumblebee TRs. The ratio between the number of TRs showing length variability and the number of TRs that do not exhibit variability in length was plotted against the repeat unit length of TRs.

Fig. 4.

—An example of TRs contributing to bumblebee coding sequence variation. (A) Pairwise alignments of TR arrays between Bombus terrestris and B. impatiens. Colored letters indicate TR array sequences, while black letters show their flanking sequences. The TR array has a repeat unit of CAG, and there are five more repeat units in B. terrestris than in B. impatiens. The coordinate for the variable TR is NC_015770.1: 2190704–2190753 in B. terrestris. (B) Pairwise alignments of protein sequences encoded by genes containing the variable TR. Colored letters indicate TR array sequences, while black letters show their flanking sequences. There are five more glutamine residues (Q) in B. terrestris than in B. impatiens. Genes containing this variable TR encode nuclear receptor corepressor (protein IDs are XP_012166765.1 and XP_012249688.1 in B. terrestris and B. impatiens, respectively).

Fig. 5.

—PCR amplification of variable-length TRs residing in coding sequences in specimens of Bombus terrestris and B. impatiens. (A) A schematic showing the principle of primer design and PCR amplification. (B) PCR amplification of the variable-length TRs residing in the gene that encodes FERM, RhoGEF, and pleckstrin domain-containing protein (protein ID: XP_012169724.1 for B. terrestris). This figure indicates that, for the given TR locus, there is fixed length variation between the two species. (C) PCR amplification of the variable-length TRs residing in the gene that encodes hexamerin (protein ID: XP_012169664.1 for B. terrestris). This figure indicates that, for the given TR locus, there is length variation within species.

Fig. 6.

—Comparison of variable-length TRs in coding sequences across species. (A) TR evolved by the loss of one repeat unit in Bombus impatiens. Colored letters indicate the repeat units of focal TR array, while black letters show its flanking sequences. The sequences involved in the multiple alignments are Apis mellifera (Group9: 2931618–2931857), B. terrestris (NC_015770.1: 10495478–10495717), and B. impatiens (NT_176565.1: 370528–370755). The gene containing this variable TR encodes a cyclin-dependent kinase inhibitor (protein ID is XP_012167390.1 in B. terrestris). (B) TR evolved by the gain of two repeat units in B. terrestris. Colored letters indicate the repeat units of focal TR array, while black letters show its flanking sequences. The sequences involved in the multiple alignments are A. mellifera (Group2: 3483900–3484134), B. terrestris (NC_015763.1: 9110312–9110594), and B. impatiens (NT_176945.1: 210140–210374). The gene containing this variable TR encodes an RNA polymerase-associated protein (protein ID is XP_012175579.1 in B. terrestris).

Fig. 7.

—Functional classification of genes that include variable-length TRs. (A) The number of genes classified in each molecular function category. (B) The number of genes classified in each protein class. The gene number shown in the nucleic acid binding category excludes transcription factors. (C) Biological processes that genes including variable-length TRs are involved in.