Short interspersed elements (SINEs) are a major source of canine genomic diversity

Abstract

SINEs are retrotransposons that have enjoyed remarkable reproductive success during the course of mammalian evolution, and have played a major role in shaping mammalian genomes. Previously, an analysis of survey-sequence data from an individual dog (a poodle) indicated that canine genomes harbor a high frequency of alleles that differ only by the absence or presence of a SINEC_Cf repeat. Comparison of this survey-sequence data with a draft genome sequence of a distinct dog (a boxer) has confirmed this prediction, and revealed the chromosomal coordinates for >10,000 loci that are bimorphic for SINEC_Cf insertions. Analysis of SINE insertion sites from the genomes of nine additional dogs indicates that 3%-5% are absent from either the poodle or boxer genome sequences--suggesting that an additional 10,000 bimorphic loci could be readily identified in the general dog population. We describe a methodology that can be used to identify these loci, and could be adapted to exploit these bimorphic loci for genotyping purposes. Approximately half of all annotated canine genes contain SINEC_Cf repeats, and these elements are occasionally transcribed. When transcribed in the antisense orientation, they provide splice acceptor sites that can result in incorporation of novel exons. The high frequency of bimorphic SINE insertions in the dog population is predicted to provide numerous examples of allele-specific transcription patterns that will be valuable for the study of differential gene expression among multiple dog breeds.

Figure 1.

Construction of libraries that are enriched for SINEC_Cf elements and flanking sequence. (A) Genomic DNA is cleaved with the frequently cutting restriction enzyme, NlaIII. (B) The cleaved fragments are self-ligated. (C) The circularized products are subjected to PCR using SINEC_Cf-specific primers. (D) The linear products are size-selected and cloned in a plasmid vector. (E) Inserts are sequenced with a vector-specific primer.

Figure 2.

Validation of putative bimorphic SINE insertions. Six representative examples (a-f) of PCR products from loci that were predicted to contain bimorphic SINE insertions after analysis of SINEC_Cf libraries (see Fig. 1). Primers were designed to the flanks of the predicted insertion site, and templates were genomic DNA from a combination of mixed-breed dogs (lane 2), three Bernese Mountain dogs (lanes 3-5), three pugs (lanes 6-8), and three dachshunds (lanes 9-11). Product lengths were derived from DNA markers (lanes M), and were consistent with the presence (+) or absence (–) of a SINE insertion.

Figure 3.

Splicing of a SINEC_Cf sequence into the canine mRNA for Tipin. (A) The 5′-end of the canine Tipin gene differs from that of human by inclusion of an additional untranslated exon that is derived from a SINEC_Cf. (B) Alignment of sequences representing the 5′-ends of Tipin mRNA from human (GenBank accession no. BP248314) and dog (BM540124) with genomic sequences from human (NCBI build 34) and dog (CanFam1). Nucleotide identity between the human and dog genomic sequences is shaded, exons are boxed, and intronic sequences are in lower case. Between exons 1 and 2, the dog genome contains a SINEC_Cf sequence that is flanked by a characteristic 15-bp duplication of the insertion site (white lettering on black). This element provides a 3′-splice acceptor site, and causes activation of a cryptic 5′-splice site downstream of the element, resulting in the incorporation of the SINEC_Cf sequence within the dog Tipin mRNA.