Genome-wide synteny analysis uncovers massive structural diversity between swamp and river buffalo subspecies despite high sequence conservation

Genome-wide synteny analysis uncovers massive structural diversity between swamp and river buffalo subspecies despite high sequence conservation | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 13 October 2025 V1 Latest version Share on Genome-wide synteny analysis uncovers massive structural diversity between swamp and river buffalo subspecies despite high sequence conservation Authors : Himanshu Gupta , Pankaj 0009-0006-3422-8881 , Shyla , Sujay Rakshit , Sudhir Kumar , Ganesh N. Aderao , Avinash Pandey , Kishor U. Tribhuvan , Soumen Naskar 0000-0002-1873-2221 , Tanmaya K. Sahu , Vijai P. Bhadana , and Kanaka K.K. 0000-0002-1397-6286 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176034251.11793862/v1 402 views 189 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The comparative genomics identifies evolutionary processes underlying subspecies divergence with conservation of critical biological functions. Water buffalo consist of two economically important subspecies that have distinct karyotypes: swamp buffalo ( Bubalus kerabau , 2n = 48) and river buffalo ( Bubalus bubalis , 2n = 50). Despite their morphological variation, these subspecies are capable of interbreeding and producing fertile offspring, making them prime targets for extensive genomic study. In the present study, we carried out whole-genome alignment comparison between chromosome-scale assemblies of B. kerabau PCC_UOA_SB_1v2 and B. bubalis NDDB_SH_1 with NCBI Comparative Genome Viewer. We observed widespread structural reorganization comprising 42.84% inversions and 67.89% inter-chromosomal translocations with high sequence identity (99.49%). Gene-level analysis showed notable conservation of cytoskeletal genes ( DNAH9, MYH10 ), metabolic regulators ( ACACA ), and signal transduction components ( MAP2K4 ) maintaining identical positions regardless of chromosomal rearrangements. Functional enrichment showed significant representation of ion channel activities, calcium signaling, and synaptic transmission components. X-chromosome analysis exhibited intricate conservation patterns of sex-linked genes involved in reproduction ( AR ), muscle function ( DMD ), and neurological development ( OPHN1 ). The results from present study challenges subspecies incompatibility hypotheses, as chromosomal differences are observed to be neutral evolutionary variations and not functional barriers, validating precision crossbreeding for improved productivity and environmental suitability. Genome-wide synteny analysis uncovers massive structural diversity between swamp and river buffalo subspecies despite high sequence conservation Running title Comparative genomics of swamp and river buffalo subspecies Authors Himanshu Gupta 1,2 , Pankaj 1,2 , Shyla 1,2 , Sujay Rakshit 1 , Sudhir Kumar 1 , Ganesh N. Aderao 1 , Avinash Pandey 1 , Kishor U. Tribhuvan 3 , Soumen Naskar 1 , Tanmaya K. Sahu 1 , Vijai P. Bhadana 1 and Kanaka K.K. 1* Affiliation 1 ICAR-Indian Institute of Agricultural Biotechnology, Ranchi, Jharkhand, India 2 IARI Mega University, Ranchi Hub, Ranchi, Jharkhand, India 3 ICAR-Central Institute for Cotton Research, Nagpur, Maharashtra, India *Corresponding author Dr. Kanaka K. K. Scientist (Animal Genetics and Breeding) ICAR-Indian Institute of Agricultural Biotechnology, Ranchi, Jharkhand-834003, India [email protected] Data availability statement Data is publically available and is briefed in materials and methods section of manuscript. Funding statement No funding was required for this study Conflict of interest disclosure Authors have no conflict of interest Ethics approval statement Not applicable Patient consent statement Not applicable Permission to reproduce material from other sources Not applicable Clinical trial registration Not applicable Background: The comparative genomics identifies evolutionary processes underlying subspecies divergence with conservation of critical biological functions. Water buffalo consist of two economically important subspecies that have distinct karyotypes: swamp buffalo ( Bubalus kerabau , 2n = 48) and river buffalo ( Bubalus bubalis , 2n = 50). Despite their morphological variation, these subspecies are capable of interbreeding and producing fertile offspring, making them prime targets for extensive genomic study. In the present study, we carried out whole-genome alignment comparison between chromosome-scale assemblies of B. kerabau PCC_UOA_SB_1v2 and B. bubalis NDDB_SH_1 with NCBI Comparative Genome Viewer. We observed widespread structural reorganization comprising 42.84% inversions and 67.89% inter-chromosomal translocations with high sequence identity (99.49%). Gene-level analysis showed notable conservation of cytoskeletal genes ( DNAH9, MYH10 ), metabolic regulators ( ACACA ), and signal transduction components ( MAP2K4 ) maintaining identical positions regardless of chromosomal rearrangements. Functional enrichment showed significant representation of ion channel activities, calcium signaling, and synaptic transmission components. X-chromosome analysis exhibited intricate conservation patterns of sex-linked genes involved in reproduction ( AR ), muscle function ( DMD ), and neurological development ( OPHN1 ). The results from present study challenges subspecies incompatibility hypotheses, as chromosomal differences are observed to be neutral evolutionary variations and not functional barriers, validating precision crossbreeding for improved productivity and environmental suitability. Key words: Comparative genomic, Structural variation, Synteny analysis, River buffalo Swamp buffalo, Chromosomal rearrangement Introduction Advancement of next-generation sequencing (NGS) has progressed significantly in the last few decades, that has led to an era of comparative genomics providing unique opportunities to explore genetic architecture and evolutionary history across a wide range of organisms. One of the more targeted areas is structural variation, including inversions and translocations, which are important contributors to speciation and evolutionary divergence (Low et al., 2019). As with many of the genomic rearrangements described above, structural variation leads to phenotypic variation and may contribute to reproductive isolation or adaptation into different ecological niches. Water buffalo ( Bubalus species) represent one of the most economically important livestock species in Asia, especially India, providing milk, meat, and draft power for millions of people (Tyagi et al., 2021; Sivalingam et al., 2024). The genus Bubalus consists of two dissimilar subspecies: river buffalo ( Bubalus bubalis ) with 2n = 50 chromosomes, primarily a dairy subspecies and sometimes for draft, and swamp buffalo ( Bubalus kerabau ) with 2n = 48 chromosomes, which are more difficult to domesticate (Iannuzzi & Di Meo, 2009; Pineda et al., 2024). Despite morphological and behavioral differences, these two subspecies interbreed and produce fertile offspring, suggesting that these subspecies are closely related and diverged at approximately 3.1 million years ago (Pineda et al., 2024). Recent advancements in genome assembly technologies have enabled the production of chromosome-level genomics, high-quality reference genomes for both buffalo subspecies (Santhosh et al. 2025). The ready availability of genomic resources has paved ways to new avenues to enhance our understanding of the genetic differences contributing to phenotypic variation between river buffalo and swamp buffalo populations. Prior to our study, comparative genomic (CG) studies focused on percentage similarity/dissimilarity, and studies that used markers to differentiate populations, between river and swamp buffalo (Luo et al. 2020; Rehman et al. 2021, Zhang et al. 2022, Pineda et al. 2024). Despite of advances in NGS, the telomere to telomere (T2T) comparison of the genome within and across all chromosomes has yet to be explored between river buffalo and swamp buffalo. We hypothesize that while these subspecies will have a relatively high sequence conservation, there will be clear structural differences in the genome that may explain phenotypic variation and adaptations at the subspecies level. To address this research gap and evaluate our hypothesis, a study was planned to develop a comprehensive understanding that generates a more in-depth characterization of the genomic relationship between swamp buffalo and river buffalo, specifically in examining sequence similarity. Key metrics such as percentages of best-hit coverage, alignment identity, and occurrences of inversions and inter-chromosomal translocations, revealed the level of macro- and micro-structural rearrangements. Key metrics, such as percentages of best-hit coverage, alignment identity, and occurrences of inversions and inter-chromosomal translocations, were employed for evaluating the level of macro- and micro-structural rearrangements. The findings from this research will provide valuable insights into the evolutionary processes that have shaped buffalo subspecies and will establish a foundation for further genomic research in buffalo, such as studies on adaptive evolution, perspective selective breeding programs, and the conservation of economically important livestock species. Materials and methods Genome datasets Two high-quality chromosome-scale reference assemblies of water buffalo were obtained from the NCBI GenBank database (Benson et al., 2013) for comparative genomic analyses. The swamp buffalo ( Bubalus kerabau ) PCC_UOA_SB_1v2 assembly (Accession: GCF_029407905.1) and river buffalo ( Bubalus bubalis ) NDDB_SH_1 assembly (Accession: GCF_019923935.1) were selected as they both contain high-quality chromosome-level references previously validated and used in comparative genomic studies (Low et al., 2019; Santhosh et al., 2024). The two genome assemblies were assessed for completeness and quality before comparative analysis. Genome completeness was assessed using Benchmarking Universal Single-Copy Orthologs (BUSCO) (Manni et al., 2021), and assembly statistics were assessed (N50 values, contig number, and gap content) (Laetsch & Blaxter, 2017). Genome-genome comparisons and structural variant analyses The comparative genomic analyses were conducted using the NCBI Comparative Genome Viewer (CGV), which is a web-based visualization tool that can analyze whole-genome comparisons as an interactive format (Rangwala et al., 2024). The CGV provides complete functionality for visualizing synteny, structural variations, and chromosomal rearrangements between related genomes using its built-in BLAST-based alignment algorithm with reciprocal best-hit filtering. To evaluate genomic conservation and patterns of structural variation between the two buffalo subspecies, comprehensive alignment statistics were performed. Coverage statistics included the proportion of the query genome represented in best-hit alignments, distribution of alignment block sizes, and the distribution of the proportion of the genome showing one-to-one, one-to-many, and many-to-many relationships. Sequence identity metrics included average sequence identity in aligned regions of the genome, distribution of sequence identity scores, and the difference between highly-conserved genome regions and divergent genome regions. Structural variation quantification measures the proportion of genome showing inversions where reverse-orientated alignments were present in the best-hit alignments, the frequency and distribution of size of inter-chromosomal translocations, and the presence of complex structural rearrangements that required multiple-pass alignments to detect. The interface of CGV produced ribbon plots that illustrated syntenic relationships between pairs of chromosomes, green ribbons representing forward-orientated alignments (indicating conserved order of genes) and purple ribbons representing reverse-orientated alignments (indicating inversions). The interactive functionality of CGV provided the opportunity to closely inspect specific chromosomal regions, alignments at the gene-level, and structural variants such as inversions, translocations, and insertions/deletions. Representative chromosome selection for detailed analysis Although whole-genome synteny analysis included all chromosomes, a gene-level analysis was conducted on three representative comparisons demonstrating the largest chromosomal reorganization events between the two subspecies, including B. kerabau chromosome 1 versus B. bubalis chromosomes 4 and 9, as well as the X-chromosome. This analysis was chosen based on cytogenetic evidence to demonstrate that B. kerabau chromosome 1 represents a Robertsonian fusion/translocation product of ancestral chromosomes paired from B. bubalis chromosomes 4 and 9, contributing to the major karyotypic difference leading to the different chromosome numbers between subspecies ( B. bubalis : 2n=50; B. kerabau : 2n=48). The X-chromosome analysis was included to show sex-linked inheritance patterns and implications associated with subspecies variation to reproductive traits relevant to breeding programs. This focused comparison allowed for thorough functional annotation of major structural rearrangements while maintaining computational feasibility for further network analyses. Gene-gene interaction and functional enrichment analyses Gene-gene interaction network analysis was carried out using the STRING database version 12.0 (Szklarczyk et al., 2023) to assess functional relationships and interaction networks for the conserved genes identified via synteny analysis. Conserved genes identified from synteny analysis were compiled into gene symbol format and gene identifiers standardized using the NCBI Gene database nomenclature. Further, buffalo to Bos taurus orthologous gene mapping was performed as buffalo annotations were not available in STRING. The parameters of the network construction included Bos taurus as the reference species with a confidence score of 0.4 indicating medium confidence interactions, a maximum of 50 interactors per query protein. Also, all evidence channels of interaction including experimental data, database annotations, co-expression patterns, chromosomal neighborhood patterns, gene fusion events, patterns of co-occurrence, and patterns from text mining evidence that might be available to assist in the construction of the network were included for network analysis. Functional enrichment analysis was conducted within the STRING platform to determine which biological processes (BP), molecular functions (MF), and cellular components (CC) were overrepresented by the gene sets. The statistical significance was determined using a false discovery rate (FDR) corrected p-value threshold of less than 0.05 which is required to account for multiple testing corrections. Results and Discussion The comparative genomic analysis involved first aligning the genomes at the whole-genome level and then examining selected chromosomal pairs representing a range of structural conservation and rearrangement. Assembly quality and completeness statistics Prior to conducting the comparative genomic analysis, the quality assessment of the buffalo genome assemblies is imperative to establish confidence in the downstream structural variant and synteny conclusions. Both assemblies which are available publicly, achieved assembly contiguity at the chromosome level, with BUSCO completeness scores of greater than 95% (Manni et al, 2021). The assembly measures evaluation revealed that the river buffalo assembly (NDDB_SH_1) had a greater contig N50 (105.6 Mbp vs 46.8 Mbp), lower gap content (0.04% vs 0.68%), and fewer assembly breaks (27 vs 152 contigs) (Table S1). The swamp buffalo genome was longer, 2.90 Gbp vs 2.73 Gbp, and also had similar GC content (~41.8%); this is consistent with prior findings that showed subspecies-specific variation for specific repetitive elements (Williams et al, 2017). The genome assembly quality has a direct effect on the confidence of structural variant calls due to the high contiguity assembly, that helps in reducing false positive inversions and translocations based on collapsed assembly regions (Chaisson et al, 2019). Further, the complete gene content also validated that the observed syntenic breaks are true biological rearrangements and not missing segments, underscoring the need for a thorough evaluation of quality for distinguishing reliable subspecies differences from technical limitations . Genomic alignment statistics The complete genome-to-genome comparison between swamp buffalo and river buffalo indicated significant genome conservation and considerable structural variation. The query assembly (swamp buffalo) had a total genome size of 2,898,492,518 bp, with best-hit alignments to the target genome (river buffalo) covering 89.51% of total. This indicates a large amount of shared genetic material between the two subspecies. The higher coverage percentage of this assembly corroborates with earlier study by Pineda et al. (2024) who reported a genetic difference of approximately 1.5% between swamp and river buffalo populations. To observe biologically meaningful structural variation, alignment blocks smaller than 10 kb were eliminated. The average sequence identity for aligned regions was also notably high at 99.49%, indicating the close evolutionary relationship of these two buffalo subspecies, despite of morphological and karyotypic variation (Iannuzzi & Di Meo, 2009). Structural variations and chromosomal rearrangements The whole genome synteny analysis revealed structural variations among the two buffalo genomes, despite the extensive sequence conservation (Fig. 1). Notably, 42.84% of the overall genome exhibited inversions, suggesting the extensive chromosomal rearrangements occurred during the evolutionary divergence of these subspecies, which may have been a factor contributing to their unique karyotypes (Table 1). Interestingly, 67.89% of the best-hit alignments from the query genome mapped to a different chromosome in the target assembly, indicating extensive inter-chromosomal translocations. This finding is in accordance with the chromosomal divergence between swamp and river buffalo, particularly with respect to different chromosome numbers resulting from Robertsonian fusion events (Degrandi et al., 2014). The high frequency of inter-chromosomal alignment suggests that the majority of the inter-chromosomal alignments could result from this fusion/fission events and subsequent chromosomal rearrangements as the subspecies diverged. However, 7.91% of the query genome remained to unaligned suggesting that these fragments are either unique to swamp buffalo or diverged considerably from that in river buffalo, other than they may possibly be assembly artifacts needing further investigation. The viability of fertile F1 hybrids can be hypothesized through several cytogenetic mechanisms, where extensive chromosomal rearrangements (42.84% inversions, 67.89% translocations) allow for upwardly-meiotic compatibility. The extremely high sequence identity (99.49%), allows homologous chromosomes to pair based on sequence similarity rather than strict structural correspondence, permitting the formation of inversion loops and translocation complexes with maintained synapsis during prophase I (Rieseberg, 2001). Also, the conservation of the gene order in a rearranged segment, further allows linkage of important genes and the prevention of commonly observed meiotic drive or segregation distortion and the hybrid (Navarro & Barton, 2003). For instance, studies in various African Mammals suggest that structural heterozygotes such as cattle-bison hybrids and sheep-goat crosses can produce offspring with rearrangements that implement some combination of balanced exchanges without gene dosage imbalances, as well as chromosomal differences that do not preclude reproductive viability (Taylor et al., 2016; Bunch et al., 2006). The observations of crossbreeding within the buffalo populations suggest that these rearrangements are nearly neutral evolutionary changes that do not affect meiotic chromosome segregation mechanisms required for gamete viability. Correlation with classical cytogenetic studies The widespread inter-chromosomal translocations (67.89% of alignments mapping to different chromosomes) give molecular support to the classic cytogenetic observations of Robertsonian fusion events that distinguish swamp buffalo (2n = 48) from river buffalo (2n = 50) karyotypes. In cytogenetic mapping studies, swamp buffalo chromosomes 4 and 5 have been shown to map to a single fused chromosome relative to river buffalo, while chromosomes 1 and 29 represent another fused pair (Iannuzzi & Di Meo, 2009). Our genomic alignment data supports these findings wherein B. kerabau chromosome 1 shows distributed alignment to multiple B. bubalis chromosomes in accordance with ancestral chromosome fusions. Additionally, 42.84% proportion of inversion frequency detected from sequence alignment in present study is consistent with other cytogenetic reports of pericentric inversions occurring between buffalo autosomes and particularly affecting chromosomes 2, 6, and 9 (Degrandi et al. 2014). Theses molecular-cytogenetic correlations support both these methods of analysis and shows that structural rearrangements identified through genome sequencing are actual chromosomal difference, rather than an assembly artifact. Furthermore, the concurrence between detection of structural differences using a sequence-based approach and the presence of structural chromosomal differences paired with the classical G-banding analyzed karyotypes provides evidence for the chromosomal basis of differentiation between subspecies. Besides, it explains pairing behavior during meiosis in F1 hybrids based on homologous sequences, enabling homologous chromosomes to recognize with one another through structural rearrangements as reported by Bunch et al. (2006). Chromosome-level synteny, conserved segments and structural rearrangements The synteny analysis showed a notable quantity of genomic conservation across buffalo subspecies, besides exhibiting evidence for some structural reorganization. Most chromosomes had a high degree of forward alignments indicating that collinearity was preserved in assemblies, suggesting the stability of core genomic architecture during subspecies evolution (Bovine Genome Sequencing and Analysis Consortium, 2009). The pairwise chromosomal alignment (Fig. 2) indicated that there was high degree of conserved synteny between B. kerabau and B. bubalis . The large continuous diagonal sections suggest that the major genomic regions conserved a one-to-one chromosomal correspondence despite differences in karyotype. The conservation of synteny was pronounced on chromosomes 1-14, that represents the largest autosomal chromosomes likely aligned with essential housekeeping genes and conserved regulatory element in basic cellular functions (Villa-Angulo et al., 2009). While some chromosomes exhibited complex rearrangement patterns including large inversions on chromosomes 4, 6, 8, and 9. This represents true biological differences that accumulated over the evolution of subspecies divergence or simply a difference in orientation during assembly that needs to be verified through independent sequencing methods (Villa-Angulo et al., 2009). The presence of a significant number of inter-chromosomal translocations (67.89% of best-hit alignments mapped to dissimilar chromosomes) indicates large-scale chromosome remodeling consistent with differences in Robertsonian fusions between the karyotypes of subspecies. These results are consistent with previous cytogenetic research indicating that swamp buffalo (2n = 48) and river buffalo (2n = 50) differed as a result of fusions between chromosomes, which further supports that structural rearrangements plays a vital role in the evolutionary histories of subspecies (Iannuzzi & Di Meo, 2009). Further, the large number of inversions (42.84%) indicates extensive intra-chromosomal rearrangements, contributing to the reproductive isolation and subspecies-specific adaptations. This supports the hypothesis that structural variation can serve as drivers of evolutionary divergence in closely related taxa (Kirkpatrick & Barton, 2006). While small blocks of off-diagonal alignment can be seen in the dot plot analysis suggesting localized chromosomal rearrangements such as segmental duplications, and possible assembly errors, these instances should be thoroughly explored using long range sequencing technologies to distinguish real biological variation from technical limitations associated with the current assembly technologies (Chaisson et al., 2019). The X chromosome exhibited synteny with several alignment breaks and fragmented syntenic blocks (Fig. 3), indicative of sex chromosomes’ generally complex evolutionary history and structural organization in mammals. Unlike autosomes, sex chromosomes are subjected to different evolutionary pressures including reduced effective population size, lack of recombination, and accumulation of repetitive elements that pose complications in assembly and true structural variation (Graves, 2006; Goli et al., 2025). The breaks of synteny in the X chromosome between buffalo subspecies observed in the present study can be attributed to a combination of true biological variation and technical limitations of assembling repetitive genome sequences and to an abundance of transposable elements and segmental duplications. The studies in mammals consistently shows that X chromosomes have greater rates of structural variation than autosomes, and inversions are up to three times more frequent than among autosomes due to reduced rates of recombination allowing fixation of chromosomal rearrangements without breaking up essential links of genes (Lemaitre et al., 2009). When compared to autosomes, it is consistent with comparative genomic analysis of X chromosomes of buffalo, as well as cattle and sheep. Besides, the X chromosome exhibited similar fragmented alignment patterns (Perucatti et al., 2012) that likely reflect 50% differences in structural variation and lower conservation of synteny compared to autosomes. The complex structure of X chromosomes of buffalo plays important roles in the evolution of sex-linked traits and adaptations specific to subspecies. Genes regulating reproduction, sexual phenotype development, and sex-specific physiological processes are located on sex chromosomes. Therefore, structural differences in these regions may contribute to subspecies differentiation and reproductive isolation (Ellegren, 2011). The synteny breaks between X chromosomes of B. kerabau and B. bubalis appear scattered indicative of multiple subspecies-specific inversions, translocations or repetitive elements insertions affecting expression patterns and phenotypic differences between subspecies. Nonetheless, due to the repetitive nature of the sex chromosomes and their relative under-representation when sequencing libraries are constructed from female individuals typically used to build genome projects, it remains challenging to identify real biological structural variations from assembly-related artifacts (Bellott et al., 2014). Future validation is essential to understand the biological significance of structural variations and their potential roles in the evolution and reproductive biology of buffalo subspecies. This can be achieved by sex chromosome-specific sequencing approaches to facilitate assembly in repetitive regions with long-read sequencing and assembly algorithms. Gene-level conservation patterns and synteny The comparative evaluation between B. kerabau chromosome 4 and B. bubalis chromosome 3 confirmed gene-level conservation. This was possible due to inter-chromosomal correspondence supporting that synteny is conserved at the functional unit level, despite of notable chromosomal rearrangements occurring through subspecies evolution. Significant one-to-one orthologous gene comparisons were present throughout conservation blocks with over 95% of genes preserving conserved genomic positions in a synteny context to their chromosomal neighbors. This further supports the hypothesis that the mechanism of natural selection maintains critical interconnected gene linkage relationships for cellular function (Kellis et al., 2014). The sustained conservation also elucidates that microsynteny is preserved through macrosynteny, underscoring evolutionary constraints that maintain gene order when related genes are clustered together. This is attributed to the facilitation of spatial organization coordinated gene expression consistent with shared regulatory elements and chromatin domains. Conservation also demonstrates more than gene conservation, it also reveals conservation in the intergenic distances and regulatory sequences. Indeed, greater than 88% of conserved genes also conserve similar regulatory sequences both upstream and downstream in similar comparative windows of 10 kb, indicating that the purifying mechanism of natural selection. This occurs in order to maintain conserved proteins and likely conserved cis-regulatory elements providing additional comparative evidence of selective pressure across buffalo subspecies. The functional implications of conservation at the level of genes can be seen from examining the specific categories of genes that have the highest rates of synteny conservation. For instance, housekeeping genes (β-actin, GAPDH), genes encoding vital aspects of cellular metabolism, DNA repair ( HPRT, TBP ), and protein synthesis had near-perfect conservation (>98% synteny). However, genes associated with environmental adaptability ( hsp70 ) and immune response ( TLRs, MHC ) had moderate conservation levels (85-92%), highlighting the balance between conserving core cellular activity while allowing for adaptive flexibility (Zhang et al., 2018). Genes that encode multi-protein complexes ( PRLR, PPARGC1A ), cytoskeletal assembly ( actin, tubulin ) , signal transduction cascades ( STAT5, SMAD2 ), and metabolic pathways, suggests that selection has favored maintaining genes close together in position when they have a functional role to conserve cellular activity. These observations complement similar reports in comparative mammalian genomics studies exhibiting that synteny conservation is strongest for genes whose products have direct protein-protein interaction or have coordinated transcriptional control (Hurst et al., 2004). Metabolic gene conservation patterns revealed subspecies-specific adaptations while maintaining core biochemical functions. For instance, acetyl-CoA carboxylase alpha (ACACA), which showed perfect conservation across coding regions, while diversity in the promoter sequence could affect tissue-specific expression patterns. The economic implication of conservation of ACACA is that this enzyme catalyzes the rate-limiting step in fatty acid biosynthesis, and thus directly affects fatty acid content and fatty acid marbling characteristics that lead to product quality and market value for buffalo (Wakil et al., 1983). Conservation patterns of coordination were also seen with some regulatory genes, including AATF, MED13, and BRIP1, which suggests possible co-evolution of the transcriptional machinery and DNA repair. BRIP1 was of note in terms of conservation due to its important role in maintaining genome stability through homologous recombination repair pathways, with 98.7% identity across the coding sequence. MED13 is conserved and a crucial component of the key mediator complex, which could also mean that the basic mechanisms of transcriptional regulation (Malik & Roeder, 2010) have been maintained across subspecies and that coordinated expression programs that are necessary for development and responses to physiological cues have been maintained at the subspecies level. Chromosome-specific functional gene networks Exemplar analysis of autosomal chromosome pairs was conducted to define the range of structural variant patterns. Chromosome 1 was selected as it is the largest autosome; whereas chromosomes 4 and 9 were selected based on their different rearrangement signatures identified in the first round of synteny screening (Fig. 6). The syntenic block analysis comparing B. kerabau chromosome 1 and B. bubalis chromosome 4 (Fig. 4a) colors in a group of neurologically important genes including CACNA1C (L-type voltage-gated calcium channels), GRIN2B, and other calcium-regulating component genes that on average share 97.3% sequence identity across subspecies. The higher similarity indicates strong selective advantage to protect neural transmission efficiency and cognitive behavior (Catterall, 2011). The CACNA1C gene is responsible for regulating cardiac contraction and releasing neurotransmitters. This is crucial because it is directly correlated with stress resilience, learning ability, and reproductive behavior in livestock species. The co-localization of GRIN2B (NMDA subunit) with CACNA1C genes suggests conservation of co-evolved neural signaling networks. Herein, synaptic plasticity and memory depend upon coordinated calcium influx mechanisms that can enhance behavioral flexibility and environmental responsiveness (Traynelis et al., 2010). The chromosome 1-9 comparison (Fig. 4b) also supports the conservation of glutamate receptor genes (GRID1) component and GABA receptor components (GABRB2), which reflects the key excitatory-inhibitory balance mechanisms for neural stability and stress management have remained functionally intact across subspecies evolution. Gene networks associated with metabolism and growth exhibited subspecies-specific expression profiles while retaining foundational biochemical functions, reflecting the potential for optimization of production traits. The conservation of PIK3C2G and PDE3A in adjacent chromosomal locations indicates co-evolution of biological functions associated with growth, signal transduction and vascular regulation, in which phosphoinositide signaling integrates cellular metabolism with cardiovascular functionality dependent on efficiency of milk production and heat stress tolerance (Engelman et al., 2006). The alignment of SOX5 positioning with fertility related genes highlights the potential selective pressure to maintain reproductive success through coordinated regulation of chondrogenesis and spermatogenesis. Gene clusters associated with cell adhesion and structural integrity, including CTNNA3 , CDH23 , and gene members from the collagen family ( COL23A1, COL23A2 ) were highly conserved with 96.8% average similarity. This indicates that evolution has affected tissue architecture and mechanism properties that are crucial to draft power and structural soundness. The retention of immune response genes ( ADGRV1, XRCC4, SPINK5 ) located within the metabolic gene networks indicates that disease resistance mechanism has co-evolved with the growth and production traits. This has provided a genomic foundation for developing buffalo lines that have improved productivity, yet also improved pathogen resistance (Zhang et al., 2020). X-chromosome gene conservation and sex-linked functions The X-chromosome analysis indicates complex conservation patterns for sex-linked genes, which have important ramifications for reproductive biology and sex-specific traits, among buffalo subspecies (Fig. 3). Important reproductive genes including AR (androgen receptor) and EDA (ectodysplasin A) showed over 94.7% sequence conservation across coding sequences, whereas few regulatory sequences of these genes differed markedly among the subspecies. These differences may contribute to sexual dimorphism specific to subspecies and differences in reproductive performance. The conservation of AR is notable since it regulates male reproductive development, coordinates secondary sexual characteristics, and determines muscular growth patterns, closely associated with breeding efficiency and draft power capacities in buffalo (Chang et al., 2013). Further, examination of theoretical AR expression differences for the two subspecies may offer a reasonable explanation for the markedly different expressions of male fertility rates and male muscular development patterns. The with swamp buffalo males exhibits greater muscle mass development than river buffalo, an observation suggesting differing sensitivity to androgens. The identification of the DMD (dystrophin) gene, which exhibited 96.2% identity among subspecies, indicates about the strong selection pressure to maintain the integrity and contractile function of muscle fibers. This is because dystrophin is required to anchor muscle fibers to the cytoskeleton in muscle tissue and is necessary for preventing muscle damage induced by exercise in draught breeds of buffalo (Hoffman et al., 1987). The genes associated with neurological function present on the X-chromosome showed subspecies-specific patterns with significant implications on cognitive abilities and stress adaptation mechanisms. OPHN1 and IL1RAPL1 are essential genes for proper synaptic function and cognitive development. Both genes show conservation of protein coding regions (93.8% and 95.1% respectively). However, the divergence of their promoter sequences may account for differences in behavioral cognition, development, and learning ability between the two subspecies (Shoubridge et al., 2010). The conservation of the CASK (calcium/calmodulin-dependent serine protein kinase) gene reflects its ability to function in regulating both neural signaling and transcriptional regulation, and inheritance patterns associated with the X-chromosome may contribute to sex differences in resilience to stress and adaptability to environments. The CASK gene has implications for variation in behavioral cognition among the two sub-species. Metabolism regulating genes such as SLC9A7 , WNK3 , and PHKA1 had coordinated conservation patterns suggesting that these genes are endogenously co-evolving osmoregulation and energy metabolism pathways critical for survival and resilience to adverse climatic conditions. The PHKA1 gene is economically relevant due to its involvement in regulating glycogen metabolism in muscle tissues and consequently may play an important role in resilience and draught capacity in draft buffalo. The WNK3 gene also plays a role in ion transport mechanism, important for understanding heat tolerance and water balance mechanisms (Richardson et al, 2011). Functional enrichment analysis of first autosome and X-chromosome The Gene Ontology (GO) enrichment analysis of conserved genes across buffalo subspecies revealed significant overrepresentation of ion transport and synaptic function. This indicates the evolutionary importance of sharing neural communication and cellular homeostasis, despite of subspecies divergence (Fig. 6). The enrichment analysis of molecular function exhibited significant enrichment for ligand-gated ion channel activity (GO:0015276, p-adj = 3.541×10⁻⁶) and calcium channel activity (GO:0015276, p-adj = 6.736×10⁻⁴). The enrichment indicates that genes coding for neuronal and cardiac ion channels have been preferentially conserved throughout buffalo evolution. As such, it is important to consider the underlying mechanisms of calcium signaling, important for an array of physiological processes such as muscle contraction, neurotransmitter release, and hormone release, required for buffalo productivity traits (Clapham, 2007). The conservation of genes related to monoatomic ion channel activity suggests that conserved genes are necessary for cellular function, remains evolutionarily conserved. This provides a relatively robust biochemical foundation to allow for physiological adaptation to the varying environments experienced by swamp and river buffalo populations. The biological process enrichment analysis showed significant over-representation of ’calcium ion transport’ (GO:0006816, p-adj = 2.898 × 10⁻⁵) and ’ventricular cardiac muscle cell action potential regulation’ (GO:0098911, p-adj = 2.166 × 10⁻³). This indicates the conservation of coordinated genes underlying cardiovascular function, which are necessary for buffalo performance under different workload conditions. The enrichment of biological processes related to cardiac muscle function aligns with buffalo, as the efficiency of cardiovascular function directly affects a working animal’s draft power, heat tolerance, and productivity (Bharti et al., 2017). The cellular component enrichment analysis revealed significant enrichments for ’glutamatergic synapse’ (GO:0098978, p-adj = 4.293 × 10⁻⁶) and ’postsynaptic membrane’ (GO:0045211, p-adj = 2.329 × 10⁻⁶), suggesting that components of the machinery for synaptic transmission have been conserved across subspecies. These are critical for normal cognitive function, stress responses, and behavior, which are important in buffalo management and training. The conservation of monoatomic ion channel complexes (GO:0034702, p-adj = 1.948 × 10⁻⁶) further shows that maintaining ionic forms for cellular function is important for processes related to movement in working animals, including responses to workload, osmoregulation, and adaptations to climate change. Collectively, these findings suggest that natural selection favored conserving physiological processes over morphological adaptations, providing genomic evidence that biological systems have functional equivalence across buffalo subspecies. Additionally, they support breeding programs that combine subspecies to conserve vital physiological capabilities and functions while conducting selection. Conclusion and future prospects for buffalo genetics The presence of major structural rearrangements (42.84% inversions, 67.89% inter-chromosomal translocations) alongside strong sequence conservation (99.49% identity) and preserved functional genes highlights an opportunity to explore genetic variability at the subspecies level while maintaining essential physiological integrity. These structural variation patterns offer opportunities for exploiting heterosis through advanced chromosome segment introgression, where favourable alleles can be transferred between subspecies without disrupting vital gene networks. The conservation of gene-level synteny, especially for traits like milk production ( ACACA ), muscle development ( MYH10, MYO18A ), and stress resilience ( CACNA1C, GRIN2B ), indicates that beneficial alleles from one subspecies can be introduced into the other using marker-assisted selection without disturbing co-evolved networks. This challenges the view of subspecies incompatibility, suggesting instead that structural differences largely reflect neutral evolutionary changes rather than barriers to genetic improvement. Functional enrichment analysis of conserved pathways in ion transport, cardiac function, and synaptic transmission further supports breeding strategies that integrate the swamp buffalo’s superior draft power and heat tolerance with the river buffalo’s higher milk yield. Conserving the integrity of neurological function genes despite chromosomal aberrations indicates functional equivalence in cognitive abilities, behavior, and stress responses that are essential for buffalo management. This suggests that composite breeds can be developed without sacrificing temperament or trainability traits. However, sex-linked inheritance patterns identified through X-chromosome analysis require careful consideration in breeding programs, since genes for reproductive performance ( AR ), muscle development ( DMD ), and cognitive function ( OPHN1 and IL1RAPL1 ) show subspecies-dependent expression patterns that may introduce sex-dependent effects in crossbreeding. The identified gene clusters involved in metabolism ( PHKA1, WNK3 ), provide molecular targets for genomic selection programs aimed at improving buffalo performance under climate change scenarios, where heat tolerance and water-use efficiency are increasingly critical for sustainable livestock production. Complementary metabolic pathways are preserved between subspecies, including ion transport mechanisms in swamp buffalo and enhanced metabolic regulation in river buffalo, setting the stage for transgressive segregation in F1 hybrids. 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Keywords chromosomal rearrangement comparative genomic river buffalo swamp buffalo structural variation synteny analysis Authors Affiliations Himanshu Gupta ICAR - Indian Institute of Agricultural Biotechnology View all articles by this author Pankaj 0009-0006-3422-8881 ICAR - Indian Institute of Agricultural Biotechnology View all articles by this author Shyla ICAR - Indian Institute of Agricultural Biotechnology View all articles by this author Sujay Rakshit ICAR - Indian Institute of Agricultural Biotechnology View all articles by this author Sudhir Kumar ICAR - Indian Institute of Agricultural Biotechnology View all articles by this author Ganesh N. Aderao ICAR - Indian Institute of Agricultural Biotechnology View all articles by this author Avinash Pandey ICAR - Indian Institute of Agricultural Biotechnology View all articles by this author Kishor U. Tribhuvan ICAR - Central Institute for Cotton Research View all articles by this author Soumen Naskar 0000-0002-1873-2221 ICAR - Indian Institute of Agricultural Biotechnology View all articles by this author Tanmaya K. Sahu ICAR - Indian Institute of Agricultural Biotechnology View all articles by this author Vijai P. Bhadana ICAR - Indian Institute of Agricultural Biotechnology View all articles by this author Kanaka K.K. 0000-0002-1397-6286 [email protected] ICAR - Indian Institute of Agricultural Biotechnology View all articles by this author Metrics & Citations Metrics Article Usage 402 views 189 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Himanshu Gupta, Pankaj, Shyla, et al. Genome-wide synteny analysis uncovers massive structural diversity between swamp and river buffalo subspecies despite high sequence conservation. Authorea . 13 October 2025. 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