Polymorphic edge detection (PED): two efficient methods of polymorphism detection from next-generation sequencing data.

BACKGROUND: Accurate detection of polymorphisms with a next generation sequencer data is an important element of current genetic analysis. However, there is still no detection pipeline that is completely reliable. RESULT: We demonstrate two new detection methods of polymorphisms focusing on the Polymorphic Edge (PED). In the matching between two homologous sequences, the first mismatched base to appear is the SNP, or the edge of the structural variation. The first method is based on k-mers from short reads and detects polymorphic edges with k-mers for which there is no match between target and control, making it possible to detect SNPs by direct comparison of short-reads in two datasets (target and control) without a reference genome sequence. The second method is based on bidirectional alignment to detect polymorphic edges, not only SNPs but also insertions, deletions, inversions and translocations. Using these two methods, we succeed in making a high-quality comparison map between rice cultivars showing good match to the theoretical value of introgression, and in detecting specific large deletions across cultivars. CONCLUSIONS: Using Polymorphic Edge Detection (PED), the k-mer method is able to detect SNPs by direct comparison of short-reads in two datasets without genomic alignment step, and the bidirectional alignment method is able to detect SNPs and structural variations from even single-end short-reads. The PED is an efficient tool to obtain accurate data for both SNPs and structural variations. AVAILABILITY: The PED software is available at: https://github.com/akiomiyao/ped .

Discovery, primary, and crystal structures and capacitation-related properties of a prostate-derived heparin-binding protein WGA16 from boar sperm.

Mammalian sperm acquire fertility through a functional maturation process called capacitation, where sperm membrane molecules are drastically remodeled. In this study, we found that a wheat germ agglutinin (WGA)-reactive protein on lipid rafts, named WGA16, is removed from the sperm surface on capacitation. WGA16 is a prostate-derived seminal plasma protein that has never been reported and is deposited on the sperm surface in the male reproductive tract. Based on protein and cDNA sequences for purified WGA16, it is a homologue of human zymogen granule protein 16 (ZG16) belonging to the Jacalin-related lectin (JRL) family in crystal and primary structures. A glycan array shows that WGA16 binds heparin through a basic patch containing Lys-53/Lys-73 residues but not the conventional lectin domain of the JRL family. WGA16 is glycosylated, contrary to other ZG16 members, and comparative mass spectrometry clearly shows its unique N-glycosylation profile among seminal plasma proteins. It has exposed GlcNAc and GalNAc residues without additional Gal residues. The GlcNAc/GalNAc residues can work as binding ligands for a sperm surface galactosyltransferase, which actually galactosylates WGA16 in situ in the presence of UDP-Gal. Interestingly, surface removal of WGA16 is experimentally induced by either UDP-Gal or heparin. In the crystal structure, N-glycosylated sites and a potential heparin-binding site face opposite sides. This geography of two functional sites suggest that WGA16 is deposited on the sperm surface through interaction between its N-glycans and the surface galactosyltransferase, whereas its heparin-binding domain may be involved in binding to sulfated glycosaminoglycans in the female tract, enabling removal of WGA16 from the sperm surface.

Transposon Insertion Finder (TIF): a novel program for detection of de novo transpositions of transposable elements.

BACKGROUND: Transposition event detection of transposable element (TE) in the genome using short reads from the next-generation sequence (NGS) was difficult, because the nucleotide sequence of TE itself is repetitive, making it difficult to identify locations of its insertions by alignment programs for NGS. We have developed a program with a new algorithm to detect the transpositions from NGS data. RESULTS: In the process of tool development, we used next-generation sequence (NGS) data of derivative lines (ttm2 and ttm5) of japonica rice cv. Nipponbare, regenerated through cell culture. The new program, called a transposon insertion finder (TIF), was applied to detect the de novo transpositions of Tos17 in the regenerated lines. TIF searched 300 million reads of a line within 20 min, identifying 4 and 12 de novo transposition in ttm2 and ttm5 lines, respectively. All of the transpositions were confirmed by PCR/electrophoresis and sequencing. Using the program, we also detected new transposon insertions of P-element from NGS data of Drosophila melanogaster. CONCLUSION: TIF operates to find the transposition of any elements provided that target site duplications (TSDs) are generated by their transpositions.

Mode of swine hepatitis E virus infection and replication in primary human hepatocytes.

The aim of this study was to investigate the infection and replication of swine-derived hepatitis E virus (HEV) in primary cultured human hepatocytes (PHCs). Hepatocytes were cultured from the resected normal livers of patients with metastatic tumours. These cultured hepatocytes were infected with swine-derived genotype 3 or 4 HEV. Viral replication was monitored using reverse transcriptase-quantitative PCR. The amount of HEV RNA increased in the culture media and cells following infection. Immunofluorescence staining implied that the spread of HEV infection in hepatocytes was attributed mainly to cell-to-cell transmission via the cell membrane. The sequences of the inoculated and propagated HEV were determined to examine whether sequence variation occurred during infection. Sequence analysis showed that there were no differences between inoculated and propagated HEV, demonstrating that in vitro infection and replication of swine HEV in PHCs occurred without sequence variation.

Tag/hybridization-based sensitive detection of polymerase chain reaction products.

The polymerase chain reaction (PCR) is an important technology to amplify a single copy or a few copies of DNA segment in genomic DNAs, visualizing the segment as DNA fragment. Thus, PCR is frequently used in various examinations such as detection of bacteria and fungi in the food industry. Here, we report a simple and sensitive method for detection of PCR products using single-strand tag sequence and hybridization of the tag sequence to the complementary tag sequence immobilized on solid material (STH). The detection sensitivity was found to be at least 50 times higher than electrophoresis/ethidium bromide (EtBr) visualization for approximately a 500-bp fragment and higher than the ordinary hybridization, that is, hybridization of denatured PCR product to probe sequence immobilized on solid material.

The synthesis of cytosolic ascorbate peroxidases in germinating seeds and seedlings of soybean and their behavior under flooding stress.

Cytosolic ascorbate peroxidases (cAPXs) of soybean have been found by proteome analysis to be downregulated in submerged seedlings. To elucidate the physiological meaning of this downregulation, soybean cAPXs were characterized in this study. Vigorous synthesis was detected in germinating seeds and seedlings. Expression of the corresponding genes was detected clearly in tissues that actively underwent cell division. The gene expression was suppressed by flooding stress, but not by salinity, cold or drought stress. The expression recovered 1 d after release from flooding stress, accompanied by growth resurgence.

Pig sperm membrane microdomains contain a highly glycosylated 15-25-kDa wheat germ agglutinin-binding protein.

A highly glycosylated protein, which has unique, novel features in localization, structure, and potential function, is found in pig sperm, and named WGA-gp due to its high binding property with wheat germ agglutinin (WGA). WGA-gp is localized mainly in flagella and enriched in membrane microdomains or lipid rafts. It is not detected by ordinary protein staining methods due to a high content of both N- and O-glycans consisting of neutral monosaccharides. Interestingly, WGA-gp may be involved in intracellular Ca(2+) regulation. Treatment of sperm with anti-WGA-gp antibody enhances the amplitude of Ca(2+) oscillation without changing the basal intracellular Ca(2+) concentrations. All these features of WGA-gp, except for different carbohydrate structures occupying most part of the molecules, are similar to those of flagellasialin in sea urchin sperm, which regulates the intracellular Ca(2+) concentration. Presence of carbohydrate-enriched flagellar proteins involved in intracellular Ca(2+) regulation may be a common feature among animal sperm.

ZNF280BY and ZNF280AY: autosome derived Y-chromosome gene families in Bovidae.

BACKGROUND: Recent progress in exploring the Y-chromosome gene content in humans, mice and cats have suggested that "autosome-to-Y" transposition of the male fertility genes is a recurrent theme during the mammalian Y-chromosome evolution. These transpositions are lineage-dependent. The purpose of this study is to investigate the lineage-specific Y-chromosome genes in bovid. RESULTS: We took a direct testis cDNA selection strategy and discovered two novel gene families, ZNF280BY and ZNF280AY, on the bovine (Bos taurus) Y-chromosome (BTAY), which originated from the transposition of a gene block on the bovine chromosome 17 (BTA17) and subsequently amplified. Approximately 130 active ZNF280BY loci (and ~240 pseudogenes) and ~130 pseudogenized ZNF280AY copies are present over the majority of the male-specific region (MSY). Phylogenetic analysis indicated that both gene families fit with the "birth-and-death" model of evolution. The active ZNF280BY loci share high sequence similarity and comprise three major genomic structures, resulted from insertions/deletions (indels). Assembly of a 1.2 Mb BTAY sequence in the MSY ampliconic region demonstrated that ZNF280BY and ZNF280AY, together with HSFY and TSPY families, constitute the major elements within the repeat units. The ZNF280BY gene family was found to express in different developmental stages of testis with sense RNA detected in all cell types of the seminiferous tubules while the antisense RNA detected only in the spermatids. Deep sequencing of the selected cDNAs revealed that different loci of ZNF280BY were differentially expressed up to 60-fold. Interestingly, different copies of the ZNF280AY pseudogenes were also found to differentially express up to 10-fold. However, expression level of the ZNF280AY pseudogenes was almost 6-fold lower than that of the ZNF280BY genes. ZNF280BY and ZNF280AY gene families are present in bovid, but absent in other mammalian lineages. CONCLUSIONS: ZNF280BY and ZNF280AY are lineage-specific, multi-copy Y-gene families specific to Bovidae, and are derived from the transposition of an autosomal gene block. The temporal and spatial expression patterns of ZNF280BYs in testis suggest a role in spermatogenesis. This study offers insights into the genomic organization of the bovine MSY and gene regulation in spermatogenesis, and provides a model for studying evolution of multi-copy gene families in mammals.

Transcriptional responses to flooding stress in roots including hypocotyl of soybean seedlings.

To understand the transcriptional responses to flooding stress in roots including hypocotyl of soybean seedlings, genome-wide changes in gene expression were analyzed using a soybean microarray chip containing 42,034 60-mer oligonucleotide probes. More than 6,000 of flooding-responsive genes in the roots including hypocotyl of soybean seedlings were identified. The transcriptional analysis showed that genes related to photosynthesis, glycolysis, Ser-Gly-Cys group amino acid synthesis, regulation of transcription, ubiquitin-mediated protein degradation and cell death were significantly up-regulated by flooding. Meanwhile, genes related to cell wall synthesis, secondary metabolism, metabolite transport, cell organization, chromatin structure synthesis, and degradation of aspartate family amino acid were significantly down-regulated. Comparison of the responses with other plants showed that genes encoding pyrophosphate dependent phosphofructokinase were down-regulated in flooded soybean seedlings, however, those in tolerant plants were up-regulated. Additionally, genes related to RNA processing and initiation of protein synthesis were not up-regulated in soybean, however, those in tolerant plants were up-regulated. Furthermore, we found that flooding-specific up-regulation of genes encoding small proteins which might have roles in acclimation to flooding. These results suggest that functional disorder of acclimative responses to flooding through transcriptional and post-transcriptional regulations is involved in occurring flooding injury to soybean seedlings.

Characterization of a novel flooding stress-responsive alcohol dehydrogenase expressed in soybean roots.

Alcohol dehydrogenase (Adh) is the key enzyme in alcohol fermentation. We analyzed Adh expression in order to clarify the role of Adh of soybeans (Glycine max) to flooding stress. Proteome analysis confirmed that expression of Adh is significantly upregulated in 4-day-old soybean seedlings subjected to 2 days of flooding. Southern hybridization analysis and soybean genome database search revealed that soybean has at least 6 Adh genes. The GmAdh2 gene that responded to flooding was isolated from soybean cultivar Enrei. Adh2 expression was markedly increased 6 h after flooding and decreased 24 h after floodwater drainage. In situ hybridization and Western blot indicated that flooding strongly induces Adh2 expression in RNA and protein levels in the root apical meristem. Osmotic, cold, or drought stress did not induce expression of Adh2. These results indicate that Adh2 is a flooding-response specific soybean gene expressed in root tissue.

An integrated RH map of porcine chromosome 10.

BACKGROUND: Whole genome radiation hybrid (WG-RH) maps serve as "scaffolds" to significantly improve the orientation of small bacterial artificial chromosome (BAC) contigs, order genes within the contigs and assist assembly of a sequence-ready map for virtually any species. Here, we report the construction of a porcine: human comparative map for pig (Sus scrofa) chromosome 10 (SSC10) using the IMNpRH2(12,000-rad) porcine WG-RH panel, integrated with the IMpRH(7000-rad) WG-RH, genetic and BAC fingerprinted contig (FPC) maps. RESULTS: Map vectors from the IMNpRH2(12,000-rad) and IMpRH(7,000-rad) panels were merged to construct parallel framework (FW) maps, within which FW markers common to both panels have an identical order. This strategy reduced map discrepancies between the two panels and significantly improved map accuracy. A total of 216 markers, including 50 microsatellites (MSs), 97 genes and ESTs, and 69 BAC end sequences (BESs), were ordered within two linkage groups at two point (2 pt) LOD score of 8. One linkage group covers SSC10p with accumulated map distances of 738.2 cR(7,000) and 1814.5 cR(12,000), respectively. The second group covers SSC10q at map distances of 1336.9 cR(7,000) and 3353.6 cR(12,000), yielding an overall average map resolution of 16.4 kb/cR(12,000) or 393.5 kb per marker on SSC10. This represents an approximately 2.5-fold increase in map resolution over the IMpRH(7,000-rad) panel. Based on 127 porcine markers that have homologous sequences in the human genome, a detailed comparative map between SSC10 and human (Homo sapiens) chromosome (HSA) 1, 9 and 10 was built. CONCLUSION: This initial comparative RH map of SSC10 refines the syntenic regions between SSC10 and HSA1, 9 and 10. It integrates the IMNpRH2(12,000-rad) and IMpRH(7,000-rad), genetic and BAC FPC maps and provides a scaffold to close potential gaps between contigs prior to genome sequencing and assembly. This map is also useful in fine mapping of QTLs on SSC10.

Platelets contribute to the reduction of liver fibrosis in mice.

BACKGROUND AND AIM: Several recent studies have reported that liver cirrhosis (LC) can be ameliorated, but few adequate strategies are available against liver fibrosis. Although LC clinically shows thrombocytopenia and hypersplenism, the correlation with liver fibrosis and platelets remains unclear. The aim of the present study was to investigate the effect of platelets on liver fibrosis in mouse models. METHODS: To induce liver fibrosis, C57BL6 female mice were injected i.p. with 1 mL/kg carbon tetrachloride (CCl(4)) twice a week for 8 weeks. Thrombocytosis was achieved by giving thrombopoietin or splenectomy in addition to CCl(4) intoxication. At 8 weeks, whole blood and liver specimens were obtained for studies as follows: peripheral platelet counts, histopathological examination, hydroxyproline assay, immunostaining, quantification of mRNA expression, and microarray analysis. RESULTS: Thrombocytosis significantly reduced liver fibrosis and hydroxyproline content of liver tissues compared to mice with CCl(4) administration alone. Platelets suppressed increments in mRNA expression for transforming growth factor-beta, and increased matrix metalloproteinase-9 expression in the liver. Microarray analysis of the liver revealed that platelets upregulated gene expressions involved in cell proliferation compared to expression in mice with CCl(4) intoxication alone. Platelets also increased liver volume, proliferative cell nuclear antigen labeling index, and mitotic index in fibrotic mice. CONCLUSION: These results clearly show that platelets reduce liver fibrosis and promote liver regeneration, even under cirrhotic conditions. We, therefore, propose that platelets could offer a potent tool in the treatment of liver cirrhosis.

Expression profile and localization of mouse calcitonin (CT) sense/antisense transcripts in pre- and postnatal tissue development.

Calcitonin (CT) has been shown to have various functions including osteoclast activity and calcium and phosphorus metabolism in mammals. In the present study, we measured the amounts of CT mRNA in the mouse brain, liver, kidney, heart and testis at various development stages, 14 days post-coitum (dpc), 17-dpc, newborn, 1 week and 8 weeks (adult), using real-time PCR. In the brain and kidney, the amount of CT mRNA decreased with development. In the testis, elevated amounts were observed at 17-dpc and 8 weeks. In the liver, the amount increased from the 14 dpc embryo to newborn stage and then decreased. In the heart, elevated amounts were observed at 17-dpc. Additionally, the CT antisense transcript was determined using a modified RT-PCR and nucleotide sequencing in the present study. Organs with high mRNA expressions were examined for localization of transcripts using in situ hybridization. The CT sense and antisense transcripts in the 14 dpc brain were mainly localized in the mesencephalon. In the pre- and postnatal stages, sense and antisense transcripts were shown to exist rather uniformly in the kidney, heart, liver and testis. In the 17-dpc rib and thyroid lobe and the adult ovary, the sense and antisense transcripts were found to be densely localized.

Evaluation of the GeneFields® EHEC/SS PCR dipstick DNA chromatography kit for the detection of enteric bacterial pathogens in stool specimens of healthy humans.

We developed a new GeneFields® EHEC/SS PCR dipstick DNA chromatography kit for the simultaneously detection of invA, ipaH, and stx genes in Salmonella enterica (56 strains), Shigella spp. (44), and enterohemorrhagic Escherichia coli (EHEC) (28), respectively, and evaluated the sensitivity and specificity with other bacteria (57) by this kit. The sensitivity and specificity were 100%, respectively. The detection limit of various methods was determined using 5% (w/v) stool suspensions spiked with each bacterium. The detection limit of the GeneFields® EHEC/SS kit ranged from approximately 102-103 CFU/g. Additionally, the relative sensitivities and specificities of the GeneFields® EHEC/SS kit vs two commercially available real-time PCR kits were >85.0% and >90.0%, respectively. These results indicate that the GeneFields® EHEC/SS kit can be used for genetic screening of S. enterica, Shigella spp., and EHEC in human stool specimens with sensitivities and specificities similar to those of the commercially available real-time PCR kits.

Global transcriptome analysis of pig induced pluripotent stem cells derived from six and four reprogramming factors.

Pigs are important, both for agriculture and as animal models for human diseases. However, due to the lack of embryonic stem cells, the possibility of genetic modification is quite limited. To overcome this limitation, induced pluripotent stem (iPS) cells have been derived from pigs. Despite the public availability of a large number of expression datasets from mice, rats, and primates-derived iPS cells, the expression profile of pig-derived iPS cells is quite limited. Furthermore, there is no dataset focused on the profiling of pig-derived iPS cell with six reprogramming factors (Oct3/4, Sox2, Klf4, c-Myc, Lin28, and Nanog). Here, we used Illumina RNA sequencing platform to characterize the mRNA expression of four-factor derived and six-factor derived pig iPS cells. We observed that the expression levels of whole genes in our established six factors derived iPS cells and parent fibroblast, and compared with that of iPS cells with four factors in public database. These data are valuable in understanding species difference in the reprogramming process of stem cells, and could help identify the key regulating genes involved in the process.

The genomic structure and a novel alternatively spliced form of porcine pTalpha chain.

A complete genomic nucleotide sequence for porcine pTalpha gene was obtained from a BAC clone, which revealed a novel exon 2 missing in human and murine counterparts. Cattle and dog genomic sequences showed the counterparts corresponding to porcine exon 2. Using thymocyte RNA and RT-PCR, three types of porcine pTalpha-chain cDNA sequences, pTalpha1, pTalpha2 and pTalpha3, were obtained. These three different cDNA sequences were alternatively spliced products with pTalpha1 consisting of exons 1, 2, 3, 4, and 5, pTalpha2 consisting of exons 1, 2, 4, and 5, and pTalpha3 consisting of exons 1, 2, 3 and the intron down stream of exon 3. pTalpha1 and pTalpha2 correspond to previously reported pTalphaa, and pTalphab, respectively, and pTalpha3 is reported for the first time. Using RT-PCR, pTalpha3 appeared expressed predominantly in the thymocyte RNA. The chromosome location of pTalpha was investigated using Radiation Hybrid Map and FISH, both of which revealed the location at SSC7q11-q12.

Antisense RNA transcripts in the blood may be novel diagnostic markers for colorectal cancer.

Numerous genetic studies have been conducted regarding the occurrence of colorectal cancer (CRC) and the prognosis using microarrays. However, adequate investigations into the diagnostic application of microarrays have yet to be performed. The simplicity and accuracy of diagnosis and prognosis tracking are important requirements for its processes, and the use of blood cells for diagnosis is considered to be suitable to meet these requirements. The patients involved in the study were 28 preoperative patients with CRC and 6 healthy individuals who served as controls. RNA was extracted from the blood cells of the patients and analyzed using a sense/antisense RNA custom microarray. In the patients with CRC, the expression levels of 20 sense RNA and 20 antisense RNA species were identified as being significantly altered compared with that of the healthy volunteers (P<0.05; fold-change, >2.0). Cluster analysis of these RNA species revealed that the top 10 antisense RNAs significantly clustered patients with cancer and healthy individuals separately. Patients with stage I or II CRC exhibited significant changes in the expression levels of 33 sense and 39 antisense RNA species, as compared with healthy volunteers (P<0.01; fold-change >2.0). Cluster analysis demonstrated that patients with stage I or II CRC and healthy volunteers formed separate clusters only among the top 20 antisense RNA species. A tracking study of expression levels of haloacid dehalogenase-like hydrolase domain-containing 1 (HDHD1) antisense RNA was performed and a significant difference was identified between the CRC and healthy groups revealing that the levels at one week and three months following surgical removal of the cancerous tissue, decreased to almost same levels of the healthy individuals. The results of the current study indicate that HDHD1 antisense RNA may serve as a potential biomarker for the prognosis of CRC.

Jalpha-gene segment usage and the CDR3 diversity of porcine TCRalpha-chain cDNA clones from the PBL of a five-month-old pig and the thymus of a one-month-old pig.

Porcine T-cell receptor alpha (TCRalpha)-chain cDNA clones were isolated from libraries made from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes (PBL) of a 5-month-old Clawn strain pig. Among 109 cDNA clones with the Jalpha-gene segment, 44 different Jalpha-gene segments were found out of the 61 Jalpha-gene segments previously identified in the porcine germline sequence. Among the 103 complete TCRalpha-chain cDNA clones with the rearranged Valpha- and Jalpha-gene segments, 33 different Valpha-gene segments were identified, which randomly rearranged to Jalpha-gene segments indicating lack of any specific combinations between Valpha- and Jalpha-gene segments with only one exception of the same set of Jalpha-gene segments in duplicate clones. Among the cDNA clones from PBL of an individual 5-month-old Clawn strain pig, a broad distribution of the Jalpha-gene segment usage was observed over the entire Jalpha-gene cluster. The Jalpha-gene segment usage in an individual 1-month-old thymus from a LW strain pig also gave a pattern consistent with the 5-month-old pig. These distributions of the Jalpha-gene segment usage were similar to the previously reported patterns for human T-cells and those of adult murine T-cells. Among the porcine cDNA clones isolated, TCRalpha-chain CDR3 length ranged from 4 to 14 amino acids with the average being 9.35 amino acids. Present report provides groundwork for further studies on porcine TCRalpha-chain expression.

Porcine TCR CD3zeta-chain and eta-chain.

Complete porcine CD3zeta-chain cDNA sequence was obtained for the first time, and its genomic nucleotide sequence was investigated from exon 2 down to CD3eta-chain exon 8. The sequence of porcine CD3zeta-chain showed homologous amino acid sequence with human and murine counterparts, in contrast to CD3eta-chain exon 8 with diversity among animals previously investigated. CD3eta-chain peptide is an alternative splice form of CD3zeta-chain exon 7 splicing to CD3eta-chain exon 8 instead of CD3zeta-chain exon 8. The genomic sequences revealed that the splice acceptor sequences of CD3eta-chain exon 8 of all animals investigated to be completely uniform. Further, CD3eta-chain exon 8 amino acid sequences retained the unique characters of having high proline (Pro) and positively charged amino acid content except for rats and mice. Although the biological role of CD3eta-chain remains to be enigmatic, these evidences suggests the evolutional pressure to maintain its sequence.

Genetic structure and relationships of 16 Asian and European cattle populations using DigiTag2 assay.

In this study, we genotyped 117 autosomal single nucleotide polymorphisms using a DigiTag2 assay to assess the genetic diversity, structure and relationships of 16 Eurasian cattle populations, including nine cattle breeds and seven native cattle. Phylogenetic and principal component analyses showed that Bos taurus and Bos indicus populations were clearly distinguished, whereas Japanese Shorthorn and Japanese Polled clustered with European populations. Furthermore, STRUCTURE analysis demonstrated the distinct separation between Bos taurus and Bos indicus (K=2), and between European and Asian populations (K=3). In addition, Japanese Holstein exhibited an admixture pattern with Asian and European cattle (K=3-5). Mongolian (K=13-16) and Japanese Black (K=14-16) populations exhibited admixture patterns with different ancestries. Bos indicus populations exhibited a uniform genetic structure at K=2-11, thereby suggesting that there are close genetic relationships among Bos indicus populations. However, the Bhutan and Bangladesh populations formed a cluster distinct from the other Bos indicus populations at K=12-16. In conclusion, our study could sufficiently explain the genetic construction of Asian cattle populations, including: (i) the close genetic relationships among Bos indicus populations; (ii) the genetic influences of European breeds on Japanese breeds; (iii) the genetic admixture in Japanese Holstein, Mongolian and Japanese Black cattle; and (iv) the genetic subpopulations in Southeast Asia.