Pig pangenome graph reveals functional features of non-reference sequences

Abstract

Background: The reliance on a solitary linear reference genome has imposed a significant constraint on our comprehensive understanding of genetic variation in animals. This constraint is particularly pronounced for non-reference sequences (NRSs), which have not been extensively studied.

Results: In this study, we constructed a pig pangenome graph using 21 pig assemblies and identified 23,831 NRSs with a total length of 105 Mb. Our findings revealed that NRSs were more prevalent in breeds exhibiting greater genetic divergence from the reference genome. Furthermore, we observed that NRSs were rarely found within coding sequences, while NRS insertions were enriched in immune-related Gene Ontology terms. Notably, our investigation also unveiled a close association between novel genes and the immune capacity of pigs. We observed substantial differences in terms of frequencies of NRSs between Eastern and Western pigs, and the heat-resistant pigs exhibited a substantial number of NRS insertions in an 11.6 Mb interval on chromosome X. Additionally, we discovered a 665 bp insertion in the fourth intron of the TNFRSF19 gene that may be associated with the ability of heat tolerance in Southern Chinese pigs.

Conclusions: Our findings demonstrate the potential of a graph genome approach to reveal important functional features of NRSs in pig populations.

Keywords: Heat tolerance; Immune ability; Non-reference sequences; Pig pangenome.

Fig. 1

Visualization of the genome synteny of Sus scrofa 11.1 (Sscrofa11) assembly with Shawutou (SWT) assembly and Tunchang (TC) assembly

Fig. 2

Distribution of non-reference nodes (NRNs). A and B Total length and number of NRNs in specific assemblies or shared by multiple assemblies. C Heatmap of the presence/absence variation (PAV) of NRNs within 20 assemblies, with colored blocks on the left representing different lengths of NRNs and blocks on top representing different pig populations

Fig. 3

Characterization of non-reference sequences (NRSs). A Length distribution of NRSs. B and C Proportions of cNRSs (B) and pNRSs (C) in 5'UTR, 3'UTR, CDS, introns, and intergenic regions. D and E Bubble plots of QTL enrichment analysis on QTLs affected by cNRSs (D) and pNRSs (E)

Fig. 4

Evaluation of the population pattern of NRSs using 192 WGS data. A Ration of reads with improved mapping quality when NRSs were taken into consideration. B PCA plot using NRSs. C Heatmap showing the PAV of NRSs in a population scale

Fig. 5

Comparison of frequencies of NRSs in cold-resistant pigs and heat-resistant pigs. A Distribution of NRSs with significant high frequencies in cold-resistant pigs and heat-resistant pigs. The horizontal lines in the chromosome represent NRSs, with the red color indicating a higher significant difference of the NRS between cold-resistant pigs and heat-resistant pigs. The blue circles next to the NRS indicate a higher frequency of the NRS in the cold-resistant pigs, while the red triangles indicate a higher frequency of the NRS in the heat-resistant pigs. B Graph structure of a 665 bp NRS in TNFRSF1 that may contribute to the heat tolerance of pigs. C Overexpression of TNFRSF1 in skin in human GTEx data, produced through GTExPortal (https://gtexportal.org/). D Comparison of read coverage on the 665 bp NRS of cold-resistant pigs (Min), heat-resistant pigs (Bamaxiang) and three other pigs (Erhualian, Landrace and Bamei). The sample IDs on the left side of the figure are the BioSample IDs of these sample in NCBI. The sample IDs in red font represent Bamaxiang pigs, while the sample IDs in blue font represent Min pigs. The sample IDs in black font are from other pig breeds