Investigation of Polymorphisms Induced by the Solo Long Terminal Repeats (Solo-LTRs) in Porcine Endogenous Retroviruses (ERVs)

Abstract

Homologous recombination events take place between the 5' and 3' long terminal repeats (LTRs) of ERVs, resulting in the generation of solo-LTR, which can cause solo-LTR-associated polymorphism across different genomes. In the current study, specific criteria were established for the filtration of solo-LTRs, resulting in an average of 5630 solo-LTRs being identified in 21 genomes. Subsequently, a protocol was developed for detecting solo-LTR polymorphisms in the pig genomes, resulting in the discovery of 927 predicted solo-LTR polymorphic sites. Following verification and filtration processes, 603 highly reliable solo-LTR polymorphic sites were retained, involving 446 solo-LTR presence sites (solo-LTR⁺) and 157 solo-LTR absence sites (solo-LTR^-) relative to the reference genome. Intersection analysis with gene/functional regions revealed that 248 solo-LTR^- sites and 23 solo-LTR⁺ sites overlapped with genes or were in the vicinity of genes or functional regions, impacting a diverse range of gene structures. Moreover, through the utilization of 156 solo-LTR polymorphic sites for population genetic analysis, it was observed that these solo-LTR loci effectively clustered various breeds together, aligning with expectations and underscoring their practical utility. This study successfully established a methodology for detecting solo-LTR polymorphic sites. By applying these methods, a total of 603 high-reliability solo-LTR polymorphic sites were pinpointed, with nearly half of them being linked to genes or functional regions.

Keywords: genomic variation; polymorphic sites; population genetics; solo-LTR.

Figure 1

The length distribution of solo-LTRs in pig reference genome, Bama, Large White, and Jinhua genomes. The x-axis represents length intervals, and the y-axis represents the number of solo-LTRs.

Figure 2

The methodology and PCR verification for the solo-LTR polymorphic sites. (A) A genome-wide methodology for detecting solo-LTR polymorphic sites across 21 assembled pig genomes. (B) Summary of PCR outcomes for two categories of solo-LTR polymorphic sites: blue indicates solo-LTR⁺, while orange indicates solo-LTR⁻. (C) The gel-electrophoresis results for polymorphic sites are presented. Specifically, sLTRDR-30, sLTRDR-78, and sLTRDR-106 were solo-LTR⁺ sites, whereas sLTRREF-19, sLTRREF-213, and sLTRREF-372 were solo-LTR⁻ sites. For each lane, the larger band represents that the solo-LTR is present, while the small band represents that the solo-LTR is absent. Lane order: DL2000 marker, Duroc, Large White, Landrace, Bamei, Ningxiang, Bama, Wuzhishan, Meishan, Mi, Sushan, Tibetan, and Banna pigs.

Figure 3

603 solo-LTR polymorphic sites source analysis and frequency and distribution analysis. (A) The majority of the solo-LTR polymorphic sites originate from 9 ERV families. (B) The divergence of LTR elements derived from 9 prominent ERV sources within the pig genome. The x-axis indicates the K divergence calculated (%) using the calc-DivergenceFromAlign.pl tool within the RepeatMasker program, while the y-axis represents the total number of bases marked as a specific LTR element in the genome, reflecting the content level at the respective divergence. (C) Frequency distribution of 603 solo-LTR polymorphic sites across 21 genomes; the number represents the genomes presenting the specific solo-LTR. (D) The distribution of 603 solo-LTR polymorphic sites on each chromosome of the pig genome. The above part is the distribution of 603 solo-LTR polymorphic sites on each chromosome. The below is the number of solo-LTR polymorphic sites on each chromosome (blue bar and the numbers at the top) and the density of solo-LTR polymorphic sites per 100 M on each chromosome (orange line and the adjacent numbers).

Figure 4

PCA (A) and pheatmap (B) clustering results with common occurrence sites. (REF: Duroc (Sscrofa11.1); WZS: Wuzhishan; EGM: Ellegaard Gottingen minipig; TB: Tibetan; LW: Large_White; RC: Rongchang; HPS: Hampshire; MS1: Meishan; LD: Landrace; BME: Bamei; PT: Pietrain; JH: Jinhua; BKS: Berkshire; CrosB: Cross-bred (Yorkshire_Landrace_Duroc); NS: Nero Siciliano pig; BMA: Bama miniature; DR: Duroc (Ninghe); MS2: Meishan (Beijing); PK15: PK15 cells; NX: Ningxiang; KY: Kenya domestic pig).