De novo characterization of the genetic polymorphism and transcript abundance of Toll-like receptors (TLRs) in tissues of swamp buffaloes (Bubalus bubalis) from Guangxi, China

Background: Toll-like receptors (TLRs) are innate signaling receptors, which sense invading pathogens and play roles in induction of immune responses to counter infection. In this study, we for the rst time, cloned, sequenced and characterized the coding sequences of TLRs 1-10 in the Chinese “swamp-type” Guangxi (GX) buffalo breed. Results: Protein sequence analysis of the 10 TLRs showed that GX buffalo share signicant sequence similarity with river buffalo (94.7 to 99.9%) and other ruminants (89.6% to 99%), but similarity with non-ruminants was low (48% to 91%). Prediction of functional domains revealed the presence of Toll/IL-1 receptor domains and variable numbers of leucine-rich repeats (LRRs) in all TLRs of GX buffalo. Transmembrane domain and signal peptide were absent in TLRs 7, 9 and TLRs 1, 3, 9, 10, respectively. When comparing GX buffalo and Nili-Ravi (NR) buffalo, the similarity of TIR domains ranged from 95.9% to 100%, while variations were found in the number of LRR motifs between these two breeds. Phylogenetic analysis based on the coding DNA and protein sequences demonstrated that, GX buffalo and NR buffalo were closely related with respect to TLRs 1, 2, 4, 5, 7, 8 and 10. However, regarding TLRs 3, 6, and 9, GX buffalo clustered with genera Bos and Bison, but not NR buffalo. RT-qPCR analysis provided an overview of TLRs transcription in tissues of GX buffalo. Conclusions: Our study obtained and characterized the full sequences of 10 TLR genes of GX buffaloes, and comparatively quantied the mRNA expression of these genes across various tissues of GX buffaloes. These ndings lay the groundwork for discovering breed-specic differences in the occurrence and progression of infections in buffalo.

and Pakistan (26 million). In many rural areas of China, buffalo breeding is based on free-range farming and pasture grazing, and lacks necessary measures for disease prevention. This farming practice, besides being less sustainable, facilitates the animal-to-animal and accidental animal-to-human transmission of diseases to farmers and milk/meat consumers -leading to illnesses and even death (Wanapat and  In Asia, two main breeds of buffaloes exist: the swamp-buffaloes mainly found in China and southeast of Asia, and the river-buffaloes in India, Pakistan and neighboring regions (Shi et al. 2012). In recent decades, river-buffalo breeds, such as Murrah, Nili-Ravi (NR) and Mediterranean, were introduced by the Chinese government to crossbred with swamp buffaloes in Guangxi (GX) region in order to increase genetic diversity and improve the productivity of the native Chinese breed. Genetic differences exist between buffalo breeds (Kumar et al. 2007; Lei et al. 2007;Degrandi 2010;Yindee et al. 2010), and it is reasonable to hypothesize that genetic differences may also involve genetic polymorphisms in Toll-like receptors (TLRs), which can contribute to a varied immune response that may impact the buffalo breed's resistance or susceptibility to infection.
TLRs are the main family of pattern recognition receptors (PRRs), which recognize speci c pathogenassociated molecular patterns (PAMPs), leading to the activation of antimicrobial host immune responses (Iwasaki and Medzhitov 2015). Increasingly, we are learning about the innate immune response and TLRs in animals and humans. There are ~ 13 known mammalian TLRs: TLRs 1-13 in murine animals ( genes of Indian river type NR buffaloes have been cloned and sequenced (Dubey et al. 2013). However, TLR gene sequences of swamp-type buffaloes from GX region in China have not been evaluated.
Therefore, the aim of this study was to characterize, for the rst time, the gene sequences of TLRs 1-10 of GX buffalo breed from GX region in China. We compared the genetic polymorphism and evolutionary relationship of TLRs of GX buffaloes to that of river buffaloes and other animal species. We also determined the relative mRNA expression levels of TLRs in blood leukocytes and different tissues of GX buffaloes. Our results on sequence characteristics and tissue abundance feature of swamp-type buffalo TLRs, may not only provide basic information for understanding the biological and functional similarities of these TLRs to homologous TLRs of other species, but also extend knowledge for future studies about the 'mammal TLR-pathogens' interactionship that bene ts the researches for both veterinary and human medicine.

Con rming the identity of GX buffaloes
The breed of GX buffaloes was con rmed as a swamp-type based on a combination of morphological and karyotyping analysis, and sequencing of the mitochondria D-loop region. The morphological features of GX buffaloes(swamp-type) were compared with Murrah and NR buffaloes (river-type) as shown in Additional le 1: Figure S1a. The native GX buffaloes had phenotypic characteristics typical to the swamp-type; with a at forehead, wide oronasal area, large abdominal perimeter, long sides, and backwardly swinging horns. The growth direction of the horns is horizontal to the forehead. The spine of GX buffaloes suddenly becomes at after the last thoracic region. The Murrah and NR buffaloes exhibited typical river-type features; with a well-developed udder, and taller and wider body, wider pelvis, upwardly-curled horns, deeply pigmented skin and hair, and spine that extends further into the hindquarters (Cockrill 1976;Zhang et al. 2000;Presicce 2017).
Karyotyping analysis showed that GX buffaloes possess 2n = 48 chromosomes, of which 10 are metaand submetacentric, 36 acrocentric somatic chromosomes, and 2 acrocentric sex chromosomes (see Additional le 1: Figure S1b and c) -features that match the karyotype characteristic of swamp-type buffaloes (Degrandi 2010). The nucleotide sequence of the mitochondria D-loop region of GX buffaloes was ampli ed and sequenced (NCBI accession number: MG491488). NJ tree showed a separation between swamp-type buffaloes and river-type buffaloes, but GX buffaloes were more closely related to swamp-buffaloes, further supporting the breed identity of GX buffaloes as a swamp-type. The similarity of D-loop nucleotide sequences was highest between GX, and WZ and XY buffaloes (99.8%). High similarity was also found with DC (99.7%), XL (99.7%) and FA (95.2%). There was less similarity with river-type buffaloes: Mediterranean (93.3%), Jaffarabadi (92.8%), Surti (92.5%), NR (91.5%), and Murrah (89.3%); similarity with the outgroup species of the genus Bison and genus Bos was 80.1-83.2% (see Additional le 1: Figure S1d). These results show that GX buffaloes are of a swamp-type, and are closely related to the Southwest Chinese cluster (DC, XL, WZ, XY) (Lei et al. 2007;Zhang et al. 2007).
TLRs 1-10 sequence analysis We ampli ed and sequenced full-length TLRs 1-10 of GX buffaloes. The primer sequences, annealing temperature and expected PCR product length are shown in Table 1. The electrophoresis result shown in Fig. 1 indicates that all DNA fragments were ampli ed accorded to the expected size of each amplicon.
The full-length sequences of TLRs 1-10 genes of GX buffaloes have been submitted to the NCBI GenBank, and the accession numbers are listed in Table 1. The coding sequences and deduced AA of these TLRs were compared with those of NR buffaloes, cattle, American bison, zebu, yak, wild yak, gayal, goat and sheep as "ingroups" (Table 3); then with other non-ruminant species including horse, pig, mouse, rat, rabbit, monkey and human as "outgroups" (see Additional le 2: Table S1).  The coding sequence, deduced AA sequence and the predicted TIR domain of most TLRs were conserved across the nine ruminant species (especially river-type buffaloes), but were divergent from non-ruminant mammals. There was a high degree of similarity (> 90%) with coding sequences of ruminant (river-type buffalo, cattle, sheep and goat) TLRs, whereas a 67-89% similarity was detected to the sequences of horse, pig, mouse, rat, rabbit, monkey, and human. At the AA level, the similarity of TLRs between GX buffaloes and river-type buffaloes was 94.7-99.9%, whereas a89.6-99% similarity with ruminant species and 48-91% similarity to non-ruminant species were detected. TLR7 of GX buffaloes showed the highest amino acid homology to that of river-type buffaloes (99.9%). Although the coding sequences of TLR 6 of GX buffaloes had 96.6% identity with river-type buffaloes, TLR6 AA sequence of GX buffaloes was more similar to yak, gayal or bison (~ 98%), cattle (96.9%), wild yak (97.8%) and zebu (97.7%); than to NR buffaloes (94.7%). AA sequences of GX buffalo TLR9 and TLR10 were more similar to domestic cattle, zebu or bison (~ 98% and 97%, respectively) compared to NR buffaloes (97.2% and 95.2%, respectively). The lower AA sequence similarities in TLRs 6, 9 and 10 between the swamp-type and river-type buffaloes point out to breed-speci c differences in the biological function of these TLRs. Compared with nonruminant mammals, the similarities between TLRs of GX buffaloes and ruminant species were high, suggesting that TLRs might have evolutionary conserved functions across ruminants.

Prediction of functional domains
A simple modular architecture tool (SMART) was used to predict the functional domains of TLRs, including the signal peptide (SP), transmembrane(TM), TIR and LRR domains (Fig. 2). Similar to river-type buffaloes, TLRs 1-10 of swamp-type buffaloes possessed LRR and TIR domains, however the TM domains varied across different TLRs. No TM domain was found in TLR7 nor TLR9, of both GX buffaloes and NR buffaloes. This was consistent with an earlier report on river-type buffaloes (Dubey et al. 2013). Surprisingly, only one TM domain was found at the C-terminus of TLR5 and TLR6in GX swamp-type buffaloes, rather than the two TM domains found in the homologous TLRs of river-type buffaloes, which was inconsistent with a previous study (Dubey et al. 2013). TMHMM was employed to con rm the nding, and the result completely matched the SMART prediction. Instead of the N-terminus TM domain, a SP domain was found. This feature suggests that TLR5 and TLR6 of swamp-type buffaloes are secretory proteins.
The transducing TIR domains were subjected to multiple alignments across species. TIR domains were found to be conserved across ruminant TLRs, with a similarity that ranged from 86.1-100%. TIR identity of TLR5, 7 and 9 between swamp-type and river-type buffaloes was 100%, whereas that of TLR6 (96.1%) and TLR10 (95.9%) was relatively lower, when compared to other TLRs. For TLR1 and TLR8, the TIR similarities were up to 100% between GX buffaloes and Bos species, but were slightly lower (99.3%)between GX buffaloes and NR buffaloes. Although GX buffaloes are more closely related to NR buffaloes than other ruminant species in the phylogenetic tree, the result indicated that the TIR domain of TLR6 was more conserved among GX buffaloes, wild yak, yak and sheep (99.2%) compared to river-type buffaloes (96.1%). There was also a 98.6% similarity in TIR10 between GX buffaloes and wild yak, yak, gayal or bison; but only a 95.9% similarity between GX buffaloes and NR buffaloes, and a 90.4% similarity between GX buffaloes and domestic cattle. These results indicate that coding sequences, deduced AA sequence and predicted TIR domains of most TLRs, were relatively more conserved among ruminants -but were divergent from human, mouse and chicken, as expected.
The highly-conserved LRR regions, located extracellularly, are associated with pathogen ligand binding ( swamp-type and river-type buffaloes, which might lead to differential recognition of TLR ligands, despite their highly homologous sequences. Table 4 Number of Leucine-rich repeats (LRRs) identi ed based on the amino acid sequences of the 10 Toll-like receptor (TLR) genes in GX buffaloes and nine other ruminant species. Accession numbers of TLRs used for the prediction analysis are provided in Table 3 TLRs SMART analysis showed that TLRs 2, 4, 5, 6, 7 and 8 of GX buffaloes contained a SP domain at their Nterminus, suggesting their role as secretory proteins, however, TLRs 1, 3, 9 and 10 were non-secretory. As for NR buffaloes, the SP domain was absent in TLRs 1, 3, 5, 6 and 10, but present in the rest of the TLRs.
The absence of a SP domain in TLRs 1, 6 and 10 had been noted in other Indian river-type buffalo breeds buffalo types based on our SignalP-4.1 analysis, but this was not revealed by SMART prediction. This result might be attributed to differences in the algorithms used in the two approaches; however, the presence or absence of a signal peptide does not completely control the localization of a protein.
The LRRCT and LRRNT domains (cysteine anking cap regions of LRR at the C-and N-terminus, respectively) were also predicted in the GX buffalo TLRs 1-10. LRRCT was found to be present in all GX swamp-type buffalo TLRs except TLR9, as was predicted in river-type buffalo TLRs; whereas LRRNT was absent in all TLRs, except TLR3 and TLR7, as expected (Dubey et al. 2013). The high similarity in the structural features of LRRCT and LRRNT, suggest a similar function in receptor activation, or ligandbinding events, among TLRs 1-10 in these two buffalo breeds.

Prediction of protein structure
Analysis of the predicted GX buffalo TLRs 1-10 amino acid sequences indicated that the putative AAs  Figure S3).
Combined analysis using the ProtComp, TargetP and PredictProtein programs, indicated that TLRs 1, 2, 6, 10 are more likely to be located in both the cytoplasm and membrane, whereas TLRs 4, 5, 6 are located on the plasma membrane (extracellular), and TLRs 3, 7, 8, 9 are in the cytoplasm or intracellular organelles. Our analysis also showed a high possibility for the existence of a secretory pathwaycharacterized by containing a SP in TLR3 and TLR4. Prediction of transmembrane helices in TLR proteins was performed by TMHMM 2.0 (see Additional le 7: Figure S4); and showed that the classical type I TM domain was present in TLRs 1, 2, 3, 4, 5, 6, 8 and 10, whereas TM did not appear in either TLR7 and TLR9 of GX buffaloes, which was similar to NR river-type buffaloes, and agreed with the prediction result obtained by SMART analysis. The biological relevance of the absence of a TM domain in bubaline TLR7 compared to other ruminant species remains to be elucidated.
Additionally, to further characterize the tertiary structure, protein homology models for these GX buffalo TLRs were generated by applying the SWISS-MODEL Homology Modelling program (see Additional le 8: Figure S5). The 3D graphics may provide information for further predictions on the active domain of proteins to help design appropriate ligands or reagents. The QMEAN4 ranged between − 2.22 and − 7.28, while GMQE score ranged between 0.55 and 0.77 (if excluding TLR10 which found unmatched template of TLR10 from the database), indicating an overall good quality for most models (see Additional le 9: Table S4).Although this is a rough analysis of homologues for 3D structure for these bubaline TLRs, it has however been revealed that all 3D models for these TLR proteins were closely matched to the homologous TLR templates (with the exception of TLR10), what preliminarily suggests their conservation of tertiary structure therefore the possible biology to those of template TLRs that have been commonly studied.

Evolution of ruminant TLR genes, TIR and LRR domains
The phylogenetic tree revealed the relative genetic distance between different ruminant species, especially between the two types of buffaloes examined in our study. The AA sequences of TLRs 1-10 from GX buffaloes, NR buffaloes and the examined ruminant species, were subjected to phylogenetic analysis using the NJ algorithm with 1,000 bootstrap resampling (Fig. 3a). The same analysis was used for TIRs and LRRs ( Fig. 3b and c). It is clear that clustering of major TLR gene subfamilies with high bootstrap values, could be divided into three clades: TLR1 subfamily (TLRs 1, 2, 6 and 10), TLR7 subfamily (TLRs 7, 8 and 9), and the singletons (TLRs 3, 4 and 5) together as a single clade originating from the same branch.
Regarding the singleton TLRs (TLRs 3, 4 and 5), GX buffalo TLR4 and TLR5, but not TLR3, were highly conserved between the two buffalo breeds -compared with species of genus Bos or Bison. There was a higher similarity between TLR2 or TLR8 of goats with GX buffaloes, but not with river-type buffaloes or any Bos species. An additional phylogenetic analysis of TLR AA sequences -comparing the evolutionary relationship of GX buffaloes, major ruminant species and non-ruminant mammals -revealed a closer relationship of GX buffalo TLRs to those of ruminants than to other mammalian TLRs (see Additional le 10: Figure  mRNA expression in selected tissues from GX buffaloes Transcripts of TLRs 1-10 genes and the reference gene GAPDH were successfully quanti ed in all examined tissues and blood leukocytes of GX buffaloes using a more sensitive SYBR Green dye-based RT-qPCR assay. The expected size of the ampli ed products was veri ed by electrophoresis (Fig. 1b). The results of tissue-speci c TLRs 1-10 expression levels are shown in Fig. 4. Our expression analysis of leukocytes showed that relative TLR4 expression was signi cantly higher than any other TLR in leukocytes (p < 0.0001), and then the expression of TLR4 in all other examined tissues (p < 0.0001). Other TLRs in leukocytes showed low expression levels, among which TLRs 5, 6 and 9 had the lowest expression levels.
Among all examined organs and tissues, TLR1 expression was highest in HLNs and lowest in the lungs. The highest expression level of TLR2 was in the heart (p < 0.0001) and the lung (p < 0.01), and lowest expression was in the liver. TLR2 expression in SILN (p < 0.05), heart (p < 0.0001), lung (p < 0.01), and kidney (p < 0.05) was signi cantly higher than any other TLR in the same group, except TLR1 and TLR10 in the kidney. TLR3 had relatively mild or moderate expression levels in all tissues. TLR4 expression in spleen was signi cantly higher than TLR5 (p < 0.01), TLR6 (p < 0.05), TLR9 (p < 0.01), and TLR10 (p < 0.05). In MLN and spleen, higher expression values of TLR4 (p < 0.01) were found, as well as relatively higher expression of TLR7 and TLR8 mRNAs, compared to all other tested tissues.

Discussion
We have characterized, for the rst time, TLRs 1-10 genes of GX buffaloes -the most popular buffalo's breed in a major buffalo-producing country, China. We also compared the characteristic features of TLRs 1-10 in Chinese GX buffaloes to those of other river-type buffalo's breeds, and other ruminant and nonruminant species. The studies described herein demonstrate that the sequence diversity of TLR genesis conserved across ruminant species, and that distinct phylogenetic clusters of TLRs 1-10 genes exist between different breeds of buffaloes. Furthermore, the protein structure of GX buffalo TLRs 1-10 has also been analyzed by using a series of available bioinformatics tools in this study. Predicting the protein structure will provide basic information especially for further molecular docking which predicts the preferred orientation, binding a nities and signaling cascades for buffalo TLRs to ligands, and is meaningful for posing structural hypotheses of how some pathogenic ligands inhibit TLRs, in the aims to Our evolution tree analyses here concur with a similar study about river-type buffaloes and other reported species, supporting the case for conservation of TLRs. Within the TLR1 and TLR7 subfamilies, GX buffaloes and NR buffaloes were found to be closely related, compared to species of the genus Bos or Bison, except TLR6 and TLR9. With respect to TLR 6 and TLR 9, GX buffaloes were more related to genus Bos; while river-type buffaloes were closer to goat and sheep, as shown in a previous study (Dubey et al. 2013 A phylogenetic analysis, based on TIR or LRR alone, also broadly conformed to the nding shown in the AA sequence tree. However, unlike the tree based on complete AA sequences, the TIR domain of GX TLR9 seems separated from TLR7 subfamily as a single clade; whereas the LRR domain of GX TLR4 was also separated from the TLR3 and TLR5 branch. Interestingly, LRR domains of GX buffalo TLRs 3, 6 and 9, showed a closer relationship with species of Bos and Bison in the tree; whereas NR buffalo LRRs 3, 6 and 9 clustered with goat and/or sheep, a result also reported previously (Dubey et al. 2013). Phylogenetic analysis of the TIR or LRR domains provided more insights across ruminant species. These variations suggest possible differences in the mechanism of pathogen recognition between swamp-type buffaloes and river-type buffaloes.
As known, the mRNA expression pro les of TLRs have been successfully determined in human, mice (Rehli 2002 reports about the tissue abundance of TLR mRNA levels in swamp-type buffalo as yet, what may limit many scienti c researches on buffaloes that are based on the normal expression pattern of TLRs in bubaline organs. Understanding the overall tissue-speci c expression patterns of TLRs in buffaloes can be used to generate focused hypotheses on the regulation of the tissue-speci c expression of TLR isoforms and TLR signaling, thereby be helpful for else possible researches and application on water buffaloes. In the river-type buffaloes, expression of TLRs 1-10 (excluding TLR3) was detected in neutrophils, spleen, heart, lung and liver by using conventional PCR followed by agarose gel electrophoresis -which is considered semi-quantitative and less accurate approach. Our study used RT-qPCR as a more accurate tool to quantify the expression pattern of TLRs in buffalo tissue, provides some more useful data for future studies related to TLR signaling pathways.
Of particular interest was the assessment of the TLRs mRNA expression in blood leukocytes, liver and MLN, because these are important immune related tissues, which are more likely to be affected during infection with Fasciola species. Here, we found that TLR4 expression was signi cant in leukocytes, as well as in most examined organs. TLR4 is widely-expressed in leukocytes, mainly macrophages, and induces Th1 cytokine production, which provokes in ammatory response through binding with LPS . Surprisingly, TLR5 mRNA fell to barely detectable in all tissues in our study. According to the literature research, the abundance of transcripts of TLR5 was also very low in river-type buffaloes (Vahanan et al. 2008). Further study aimed to nd answer for the limited TLR5 expression in buffalo tissue, may possibly bring some surprising discovery about the unclear bubaline immunology. Overall, these ndings indicate that tissue-speci c gene expression pro le of TLRs 1-10 in GX buffaloes share similarities as well as differences to those in river-type buffaloes. It is reasonable to assume that the differential expression patterns of TLR genes in buffalo's tissues can impart the different activities of the TLRs and the diverse repertoire of immune response of buffalo's tissues.

Conclusions
We have carried out the rst characterization of the full sequences of 10 TLR genes of the Chinese swamp-type buffaloes (GX breed). Sequence comparison between TLRs of Chinese swamp-type and Indian river-type buffaloes, revealed differences within the intracellular TIR domains and repeat number of different TLRs-which may underpin breed-speci c differences in susceptibility to infection. We extended our analysis to include comparative quanti cation of TLRs 1-10 mRNA expression, across various tissues of swamp-type buffaloes using RT-qPCR, that found the relatively much higher expression of TLR2 in the heart and the lung, and TLR4 in leukocytes and the MLN. These results can facilitate further investigations of the molecular function of TLRs in buffaloes. Future work should involve examination of the correlation between gene expression and protein abundance in the buffaloes' tissues, and testing of the association between genetic polymorphism in TLRs and the pattern of in ammatory cytokines produced during speci c infections. Other works focusing on the tertiary structure of TLR molecules should focus on determining the function of mutant sites, which may further reveal their contribution to immunity speci cally in swamp-type buffaloes.

Experimental animals
Four female, two-year-old, GX buffaloes (352 ± 13.81 kilogram) were purchased from local breeders in Nanning, Guangxi, China. The river-type buffalo's breeds (NR and Murrah; n = 4 animals per breed) from buffalo's herds at Guangxi Buffalo Research Institute were used as a control for morphological identi cation. Commercial feed and clean water were provided ad libitum. Animals were treated with triclabendazole, penicillin/streptomycin and ivermectin, following the manufacturer's standard dosing regimens, in order to exclude the impact of any pre-existing infection on the expression of TLR genes. Treatment was followed by a 3-week withdrawal period, and animal tested major pathogens (parasites and microbes) -free was allowed to be used for the further experiments. All buffaloes survived the treatment and were euthanized at the end of the experiment, following sample collection for the designed purpose. Euthanasia was performed by head stunning using a captive bolt pistol producing immediate unconsciousness, and exsanguination. Experimental procedures were reviewed and approved by the Animal Ethics Committee of Guangxi University. All animals used in the study were handled in accordance with good animal husbandry practices, as per the Animal Ethics Procedures and Guidelines of the People's Republic of China.
Identi cation of buffaloes Identi cation of the phenotypic features of the animals was performed according to reference textbooks (Zhang et al. 2000). Additionally, karyotyping analysis was performed as described previously (Bongso and Hilmi 1982). Brie y, 1 mL of peripheral blood from each GX buffalo was mixed with 10 mL of RPMI 1640 culture medium supplemented with 20% fetal bovine serum (FBS), 0.05 mL penicillin/streptomycin, and 0.1 mL of phytohemagglutinin (PHA) as a mitogen, and incubated at 37 o C with 5% CO 2 . All reagents used for cell culture were purchased from GIBCO (Thermo Fisher Scienti c, US). After 72 h, 0.2 µg/mL of colchicine was added and cells were harvested, followed by addition of a pre-warmed hypotonic solution (75 mM KCl) and incubation for 15-20 min at 37 ºC. The cell suspension was centrifuged at 1,000 × g for 5 min and the cell pellet was xed with methanol: acetic acid in a ratio of 3: 1 (v/v) for 30 min. The cell suspension was dropped onto clean, chilled glass slides and dried for 2 h in an oven; this was followed by Giemsa staining to determine the diploid numbers and morphological characteristics of each karyotype using a Nikon-80i microscope (Nikon, Japan). For further analysis of the chromosomes, metaphase plates were photographed, and images were enlarged, cropped and arranged into four groups according to their size and shape using Genikon karyotype auto-analysis software (Nikon, Japan).

Sample collection
Whole blood from all four GX buffaloes were collected postmortemly and used for extraction of genomic DNA (gDNA) and mtDNA D-loop sequence analysis as described below. Also, blood leukocytes and tissue samples were collected from four GX breed buffaloes and used for RNA isolation and gene expression analysis. Leukocytes were isolated from EDTA-treated blood samples using Ficoll gradient centrifugation-

Declarations
Ethics approval and consent to participate The study design was reviewed and approved by the Animal Ethics Committee of Guangxi University. Animals used in the study were handled in accordance with good animal practices as required by the Animal Ethics Procedures and Guidelines of the People's Republic of China.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no potential con icts of interest.   Predicted functional domains in TLRs 1-10 proteins of GX buffaloes (swamp-type) compared to NR buffaloes (river-type) as revealed by the genomic mode of SMART tool.  The comparative mRNA expression pro les of TLRs 1-10 in blood leukocytes, hepatic lymph node (HLN), mesenteric lymph node (MLN), super cial inguinal lymph node (SILN), heart, liver, spleen, lung, and kidney in GX swamp buffaloes. Data represent the mean ± standard error from at least three independent experiments. Statistical signi cance between levels of expression of TLRs within the same tissue and across all tissues are indicated by * and # symbols, respectively. (#, *, p < 0.05; ##, **, p < 0.01 or ####, ****, p < 0.0001). TLR4 expression in leukocytes (p < 0.0001) and MLN (p < 0.01) was signi cantly higher than any other TLR in the same cells and tissues, respectively. TLR4 expression in spleen was signi cantly higher than TLR5 (p < 0.01), TLR6 (p < 0.05), TLR9 (p < 0.01), and TLR10 (p < 0.05). TLR2 expression in SILN (p < 0.05), heart (p < 0.0001), lung (p < 0.01), and kidney (p < 0.05) was signi cantly higher than any other TLR in the same group, except TLR1 and TLR10 in the kidney. Across all examined samples, the highest expression of TLR4 was observed in leukocytes (p < 0.0001), followed by MLN (p < 0.01) and the highest expression of TLR2 was observed in heart (p < 0.0001) and lung (p < 0.01).

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