MIPs: multi-locus intron polymorphisms in species identification and population genomics

Abstract

The study of species groups in which the presence of interspecific hybridization or introgression phenomena is known or suspected involves analysing shared bi-parentally inherited molecular markers. Current methods are based on different categories of markers among which the classical microsatellites or the more recent genome wide approaches for the analyses of thousands of SNPs or hundreds of microhaplotypes through high throughput sequencing. Our approach utilizes intron-targeted amplicon sequencing to characterise multi-locus intron polymorphisms (MIPs) and assess genetic diversity. These highly variable intron regions, combined with inter-specific transferable loci, serve as powerful multiple-SNP markers potentially suitable for various applications, from species and hybrid identification to population comparisons, without prior species knowledge. We developed the first panel of MIPs highly transferable across fish genomes, effectively distinguishing between species, even those closely related, and populations with different structures. MIPs offer versatile, hypervariable nuclear markers and promise to be especially useful when multiple nuclear loci must be genotyped across different species, such as for the monitoring of interspecific hybridization. Moreover, the relatively long sequences obtained ease the development of single-locus PCR-based diagnostic markers. This method, here demonstrated in teleost fishes, can be readily applied to other taxa, unlocking a new source of genetic variation.

Keywords: Forensic identification; High-throughput DNA sequencing; Molecular markers; Multiple-SNP haplotypes; Non-model organisms; Teleost fishes.

Figure 1

Transferability of loci across tested species and reproducibility of the approach. (a) Modified version of the Ensembl phylogeny and taxon nomenclature reported by Betancur-R et al. and Nelson et al. with the mean number of loci successfully genotyped per taxon. We reported in the phylogeny only the species for which we used the genome in the first part of the isolation of intron loci. (b) Distribution of the percentage of loci successfully amplified and sequenced in 65 species of fishes tested. Species are grouped based on taxa reported in (Fig. 1a). Figure refined with Biorender.

Figure 2

Plot of the principal component analysis of variation based on 54 intron loci genotyped at a total of 81 icefish individuals (including 8 species defined based on morphological features and 11 individuals with unknown species origin marked as Chionodraco spp.). Colours indicate species. The eigenvalues of the first three axes explain 16.14, 10.19 and 8.75% of the variation. The individual genetically assigned to the species Trematomus scotti was originally mis-assigned to C. hamatus due to sample mislabelling and is highlighted by a red circle. (a) PC1 versus PC2. (b) PC1 vs PC3. Figure refined with Biorender.

Figure 3

Distribution pattern of genetic differentiation between species and populations of the Solea genus based on genotype information at 43 intron loci. (a) Structure bar-plot showing the membership probabilities of cluster assignment for each of the 106 individuals of the two species (71 S. solea and 35 S. aegyptiaca). The best number of clusters (K = 2) that overlaps the species subdivision is reported. The black arrow indicates the three individuals morphologically classified as S. solea, which cluster with S. aegyptiaca according to their mitochondrial barcode information. (b) Plot of the principal component analysis of variation (PC1 vs PC2 on the left, PC1 vs PC3 on the right). Eigenvalues of the first three axes explain 42.5, 4.45 and 3.77% of the variation. Shapes indicate species, while colours indicate populations. Below each PCA, the best cluster subdivision obtained by structure analysis performed within species is also reported as bar-graph showing individual membership probabilities. Figure refined with Biorender.

Figure 4

Sampling sites and population details of the Solea solea and S. aegyptiaca species involved in case studies 2 and 3. Almost all samples are legacy FishPopTrace Consortium (Nielsen et al., 2012). The only exception is represented by three individuals of the North Adriatic population of S. solea (marked with * in the table inset) that were sampled at the Chioggia Fish Market and morphologically classified as S. solea. Wikimedia Commons map refined with Biorender (https://www.biorender.com/).