Advancing the Indian cattle pangenome: characterizing non-reference sequences in Bos indicus

Abstract

Background: India harbors the world's largest cattle population, encompassing over 50 distinct Bos indicus breeds. This rich genetic diversity underscores the inadequacy of a single reference genome to fully capture the genomic landscape of Indian cattle. To comprehensively characterize the genomic variation within Bos indicus and, specifically, dairy breeds, we aim to identify non-reference sequences and construct a comprehensive pangenome.

Results: Five representative genomes of prominent dairy breeds, including Gir, Kankrej, Tharparkar, Sahiwal, and Red Sindhi, were sequenced using 10X Genomics 'linked-read' technology. Assemblies generated from these linked-reads ranged from 2.70 Gb to 2.77 Gb, comparable to the Bos indicus Brahman reference genome. A pangenome of Bos indicus cattle was constructed by comparing the newly assembled genomes with the reference using alignment and graph-based methods, revealing 8 Mb and 17.7 Mb of novel sequence respectively. A confident set of 6,844 Non-reference Unique Insertions (NUIs) spanning 7.57 Mb was identified through both methods, representing the pangenome of Indian Bos indicus breeds. Comparative analysis with previously published pangenomes unveiled 2.8 Mb (37%) commonality with the Chinese indicine pangenome and only 1% commonality with the Bos taurus pangenome. Among these, 2,312 NUIs encompassing ~ 2 Mb, were commonly found in 98 samples of the 5 breeds and designated as Bos indicus Common Insertions (BICIs) in the population. Furthermore, 926 BICIs were identified within 682 protein-coding genes, 54 long non-coding RNAs (lncRNA), and 18 pseudogenes. These protein-coding genes were enriched for functions such as chemical synaptic transmission, cell junction organization, cell-cell adhesion, and cell morphogenesis. The protein-coding genes were found in various prominent quantitative trait locus (QTL) regions, suggesting potential roles of BICIs in traits related to milk production, reproduction, exterior, health, meat, and carcass. Notably, 63.21% of the bases within the BICIs call set contained interspersed repeats, predominantly Long Interspersed Nuclear Elements (LINEs). Additionally, 70.28% of BICIs are shared with other domesticated and wild species, highlighting their evolutionary significance.

Conclusions: This is the first report unveiling a robust set of NUIs defining the pangenome of Bos indicus breeds of India. The analyses contribute valuable insights into the genomic landscape of desi cattle breeds.

Keywords: Bos indicus; BICIs; Cattle; Genome assembly; Linked-reads; NUIs; Pangenome.

Fig. 1

Identification of Non-reference Unique Insertions (NUIs) in Bos indicus. The flowchart illustrates the systematic process for identifying the final set of NUIs. The diagram outlines the sequential steps involved in the selection and refinement of NUIs

Fig. 2

Overview of Non-reference Unique Insertion (NUI) final set and their distribution. A Circos plot of Bos indicus pangenome. From the outer to inner track: Chromosome, Gene track, Red Sindhi, Sahiwal, Tharparkar, Kankrej, Gir. B Size distribution of NUIs with bin size of 500 bp and 50 bp in zoomed area

Fig. 3

Overview of Bos indicus Common Insertions (BICIs) and their distribution patterns. A This ideogram depicts BICIs occurrences across Brahman genome chromosomes. Pink histograms above each chromosome illustrate BICI density using a 100 kb window size. The black line within chromosomes represents gene distribution. B The plot illustrates the size distribution of BICIs with a bin size of 500 bp, providing an overview and 50 bp in the zoomed area. C The first two principal components are based on the BICI occurrence matrix

Fig. 4

Distribution of transposable elements on Bos indicus Common Insertions (BICIs). A Stacked bars represent the total number of BICIs split by three different size ranges. The major transposable elements (TEs) are categorized as SINE (Short Interspersed Nuclear Element), LTR (Long Terminal Repeat), LINE (Long Interspersed Nuclear Element), DNA (DNA Transposon), and NONE (No Interspersed Repeat Detected). “Other TEs” encompasses various minor classes. B Bar plot showing the number of TEs flanking and crossing BICIs

Fig. 5

Characterization of Bos indicus Common Insertions (BICIs) in the transcriptome. A Stacked bars depict the distribution of BICIs across genic and non-genic regions of the genome. B Gene Ontology (GO) annotation analysis for the biological processes associated with BICIs

Fig. 6

Trait enrichment analysis of BICIs in known cattle QTL regions. A Percentage of QTL type (pie chart) associated with BICIs. B Top 10 enriched QTL traits (bar plots) associated with BICIs

Fig. 7

Evolutionary analysis of Bos indicus Common Insertions (BICIs). A Venn diagram illustrating the number of BICIs shared within the Bos genus. The overlapping regions reveal the extent of shared BICIs among species within the Bos genus. B Venn diagram showcasing the number of BICIs shared within the Bovidae family. The intersecting areas depict the shared BICIs among species within the broader Bovidae family