Bifurcation and enhancement of autonomous-nonautonomous retrotransposon partnership through LTR Swapping in soybean

Abstract

Long terminal repeat (LTR) retrotransposons, the most abundant genomic components in flowering plants, are classifiable into autonomous and nonautonomous elements based on their structural completeness and transposition capacity. It has been proposed that selection is the major force for maintaining sequence (e.g., LTR) conservation between nonautonomous elements and their autonomous counterparts. Here, we report the structural, evolutionary, and expression characterization of a giant retrovirus-like soybean (Glycine max) LTR retrotransposon family, SNARE. This family contains two autonomous subfamilies, SARE(A) and SARE(B), that appear to have evolved independently since the soybean genome tetraploidization event approximately 13 million years ago, and a nonautonomous subfamily, SNRE, that originated from SARE(A). Unexpectedly, a subset of the SNRE elements, which amplified from a single founding SNRE element within the last approximately 3 million years, have been dramatically homogenized with either SARE(A) or SARE(B) primarily in the LTR regions and bifurcated into distinct subgroups corresponding to the two autonomous subfamilies. We uncovered evidence of region-specific swapping of nonautonomous elements with autonomous elements that primarily generated various nonautonomous recombinants with LTR sequences from autonomous elements of different evolutionary lineages, thus revealing a molecular mechanism for the enhancement of preexisting partnership and the establishment of new partnership between autonomous and nonautonomous elements.

Figure 1.

Structure and Sequence Comparison of gypsy-Type SNARE Elements. The protein coding domains gag, pol, and env-like in an autonomous element SARE^A are represented by boxes with solid outlines, while their corresponding homologous sequences, if any, in nonautonomous elements SNRE^S and SNRE^O are represented by blank boxes with dashed outlines. The solid circle represents the copia-type piggybacking Gmr6 solo LTR, and the arrow above the circle indicates the proposed transcriptional orientation of the LTR in an intact Gmr6 element. Three different families of simple tandem repeats, STR100, STR70, and STR24, are indicated. The plots a and b show the relative nucleotide identity between the SARE^A and SNRE^S elements, and the plots c and d show the relative nucleotide identity between the SNRE^S and SNRE^O elements. To reflect the sequence divergence level among three distinct subfamilies/subgroups, the three elements IN593 (SARE^A), IN834 (SNRE^S), and IN9037 (SNRE^O), as indicated by arrows in Figure 3, were randomly chosen from the relatively young elements of the SNARE family. [See online article for color version of this figure.]

Figure 2.

Structural Comparison and Evolutionary Relationship of the SARE^A and SARE^B Subfamilies. (A) Structural and sequence comparison of SARE^A and SARE^B elements. The protein coding domains in SARE^A and their corresponding homologous sequences are represented with boxes. The plots a and b show the relative nucleotide identity between the SARE^A and SARE^B. The two elements IN593 and IN5410 were randomly chosen from relatively young elements in clade 3 (SARE^A) and clade 4 (SARE^B). Different sizes of simple tandem repeat STR100 and STR62 are also indicated. These two kinds of repeats share no sequence similarity, suggesting their independent origins. (B) Evolutionary relationship and sequence divergence between SARE^A and SARE^B using 5 ′ LTR, conserved protein ORF1, gag, and RT, respectively. Clades 1, 3, and 4 were labeled corresponding to the autonomous element clades shown in Figure 3. The level of nucleotide sequence distance is indicated by the scales. [See online article for color version of this figure.]

Figure 3.

Phylogenetic Analysis of LTR Sequences. (A) Neighbor-joining tree of LTR sequences from random samples of Gmr6 solo LTRs harbored in SNRE^S elements (green circles enclosed in the dashed oval) and intact elements (open diamonds) and solo LTRs (filled diamonds) of Gmr6 outside of SNRE^S elements in the soybean genome. The level of nucleotide sequence distance is indicated by the scales. (B) Neighbor-joining tree of LTR sequences from random samples of intact SARE (red rectangles), SNRE^S (green circles), and SNRE^O (blue triangles) elements. SNRE¹ and SNRE² indicate the two subgroups of SNRE elements formed by two independent subfamily-specific interelement recombination machineries. The green circles enclosed in the dashed oval indicate the lineage of ancestral SNRE elements. Representative elements used for comparisons of sequence divergence in Figure 1 are labeled as black arrows. The level of nucleotide sequence distance is indicated by the scales.

Figure 4.

Evolutionary Model and Insertion Times of SNARE Elements. (A) Evolutionary model of SNARE evolution. Letters a, b, and c indicate three evolutionary events that gave rise to the distinct structural features of SNARE elements: the divergence of SARE^A and SARE^B, the formation of SNRE^O, and the integration of Gmr6 solo LTR, respectively. I and II indicate the proposed two machineries for subfamily- and region-specific interelement recombination. Arrows indicates proposed replacement of LTR sequences of nonautonomous elements by two distinct lineages of autonomous partners during SNARE evolution. The numbers of intact elements within each category are indicated. (B) Age distribution of intact elements. Although SNRE elements were derived from SARE elements, the existing oldest SNRE elements were dated to be older than the existing oldest SARE elements on the basis of their LTR sequence divergence. This may reflects different levels of selection for LTR sequence conservation. [See online article for color version of this figure.]

Figure 5.

Evolutionary Relationship and Sequence Divergence between SARE^A and SNRE^S1. The bootstrap neighbor-joining trees were generated using 5 ′ LTR (A), ORF1 (B), gag (C), and env-like (D) homologous domains. Three clades defined in Figure 3B were labeled with each corresponding number. Clades 1 and 3 represent two SARE^A lineages, while clade 2 represents the ancestral SNRE^S lineage. The level of nucleotide sequence distance is indicated by the scales [See online article for color version of this figure.]

Figure 6.

Transcriptional Activity of SNARE Elements in Different Soybean Tissues. The primers (see Supplemental Table 2 online) were designed based on relatively young elements to specifically amplify fragments unique to different subfamilies. RT domain from SARE^A and an internal region between the gag and env-like gene remnants in SNRE^O and an internal region that covers 5 ′ upstream of Gmr6 solo LTR and part of the LTR in SNRE^S were amplified using these primers. RT-PCR reactions were performed parallel with total RNA (RT−) and with reverse transcribed RNA (RT+) into single strand cDNA. Primers amplifying the housekeeping actin gene fragment (spanning intron2), which is spliced, were used as a control.

Figure 7.

Models for Recombination between Autonomous and Nonautonomous Elements. (A) and (B) Intrastrand unequal recombination between two SARE and SNRE elements to form chimeric structures of nonautonomous recombinants. (C) Initiation of reverse transcription (step 1) from an autonomous element, intraelement template switch (step 2), followed by an interelement template switch (step 3) to form a nonautonomous recombinant with LTRs from the autonomous partner. The arrows underneath R, U5, and U3 represent the synthesized DNA fragments based on the SARE template, while the arrows underneath ORFs represent the synthesized DNA fragments based on the SNRE template. [See online article for color version of this figure.]