Digestion of buffalo genomic DNA with RsaI enzyme, besides minor ones, showed four prominent bands ranging from 1331 base pairs, pDp1; 651, pDp2; 603, pDp3; to 339; pDp4 (Figure 1). Approximately, 15-20 recombinant clones subjected to restriction digestion and slot blot hybridization screening yielded ten positive clones for each repeat. Five-six clones from each fragment were then sequenced. The accession numbers of the recombinant clones are given in the Additional File 1. All the four major repeat elements were AT rich but sequence-wise, were different from one another (Additional File 2). No inter-clonal variations were detected in these sequences. However, random repeats were present in the nucleotide sequences (Table 1). Repeat Masker programme revealed presence of LTR LINE, SINE elements within the four fragments (Table 2). Blast search for each clone showed 69-98% homology with genomic DNA/contigs and 68-93% with transcribing genes in the database (Additional File 1) mostly in UTRs (Additional File 3).

Most striking feature of pDp1 sequence was the presence of 489 bp, +1 ORF (nucleotide position, 841-1329) (Figure 2B). BLASTP search with GenBank sequences using conceptual translation of this ORF (162aa) gave matches to putative reverse transcriptase's domain. This region corresponds to central position of the Reverse transcriptase ORF (Figures 2A and 3). Similarly, pDp2, 111 bp, +3 ORF (nucleotide position, 135-245, 65aa) and pDp4, 135 bp, +3 ORF (nucleotide position, 81-215, 44aa) showed homology to endonuclease reverse transcriptase (Figures 2B and 2C). These ORFs corresponded to central endonuclease reverse transcriptase of LINE1 ORF2 (Figure 3). pDp3 sequence,112 bp, +3 ORF (nucleotide position, 492-602, 37 aa) showed no similarity with endonuclease reverse transcriptase.

Independent cross hybridization of pDp1, pDp2, pDp3 and pDp4 with genomic DNA from different species (mentioned in methods section) under high stringent conditions showed signals only in bovids (Additional File 4). PCR conducted using region specific primers (Table 3) amplified bands in buffalo, cattle, goat and sheep genomic DNA (Figures 3A & 3B). Southern hybridization of two representative RsaI sequence pDp1, and pDp2 showed same number of bands within the genomic DNA of bovids (Additional File 5). Prominent bands within the smear reflect identical size of the fragments dispersed throughout the bovid genomes. ClustalW alignment of 489 bp pDp1derived fragment with cattle (AF060172), goat (AF404302) and sheep (AC148038) showed high level of sequence conservation among the bovids (Additional File 6). Phylogenetically, with respect to all the four sequences, cattle and buffalo were found to be closer to each other (Additional File 7), constituting the same monophyletic group.

RT-PCR analysis using internal primers of pDp1, pDp2, pDp3 and pDp4 (see Table 3) and cDNA from somatic tissues and spermatozoa of buffalo showed amplification of band across tissues and sperm confirming their transcriptional potentials (Additional File 8). Quantitative Real Time PCR analysis of these sequences showed differential expression of RsaI related transcripts across somatic tissues and spermatozoa (Figure 4). pDp1 showed highest expression in lung, pDp2 and pDp3 in Kidney and pDp4 in ovary. Summary of the relative expression (in folds) derived from 2^-ΔΔCt values obtained for various transcripts based on Real Time PCR are given in Table 4.

The pair of internal primers deduced from pDp1-4 used for expression study was also employed for copy number analysis of these fragments in cattle buffalo, goat and sheep using Real Time PCR. Standard curve slope value was between 3.2-3.5. Single melting peak on dissociate curve confirmed primer specificity. pDp1 showed ~2 × 10⁴ copies, pDp2 and pDp3, ~ 3000 copies each and pDp4 showed ~ 1000, in these species (Table 5 and Additional File 9).

In silico analysis of pDp1, pDp2, pDp3 and pDp4 sequences using reference cattle genome revealed its multiple locations on the cow chromosomes (Figure 5). Probing of pDp1 to buffalo genomic DNA digested with RsaI enzyme detected a strong hybridization signal (Additional File 10) giving rise to a single isomorphic band in all the samples. FISH mapping of pDp1, 2, 3 spectrum red labeled cloned probes showed ubiquitous discernible signals over buffalo metaphase chromosomes (Figures 6 and 7). All the chromosomes showed dispersed pattern with all the three probes used independently. In several chromosomes, signals in the centromeric regions were absent or reduced giving rise to inconsistent pattern. FISH with pDp1 sequence showed more localized signals on the metaphase chromosomes as compared to that detected by pDp2 and pDp3. FISH with spectrum red labeled clone pDp4 probe showed background signals even after washing the slides under high stringent conditions (60°C in 0.1× SSC). This might be due to the short length (339 bp) of the probe pDp4 used coupled with its dispersed genomic organization.

Blast search using reference mRNA sequence revealed RsaI repeats to be part of Acyl-coenzyme A thioesterase 11(ACOT11), Vacuolar Protein Sorting 24 (VPS24) and Solute carrier organic anion transporter family member 1A2 (SLCO1A2) genes (Additional File 1) Full length Buffalo ACOT11 cDNA was generated using end point PCR and gene specific (B. taurus ACOT11) primers (Table 3). Assembled cDNA sequence of 2592 base pair fragment lacking poly A tail representing six exons (Additional Files 11 and 12) were deposited in the GenBank (HQ848649 and HQ848650).

Approximately, 10 ml blood samples from both the sexes of buffalo, goat and sheep were collected from local slaughterhouse, Delhi following the guidelines of institute's Ethical and Biosafety Committee. Cattle blood sample was procured from the owner of the animal. Blood samples of human, fish, bird, rat, jungle cat, bonnet monkey, were available from other projects in the lab. Tiger, Indian rhinoceros and leopard samples were obtained with due permission from the competent authorities of the state and union government of India following strictly the guidelines of the Institute's Ethical and Biosafety Committee. Genomic DNA was extracted according to standard phenol-chloroform procedure 41 .

Approximately, 5 μg of buffalo genomic DNA from both the sexes was digested with RsaI restriction enzyme following supplier's (NEB) specification. Fragments were separated on 1% agarose gel in 1× TBE. Distinct bands within the smear were sliced from the gel, purified and cloned into dephosphorylated pBluescript II SK+ vector (Stratagene, USA), using standard protocol 42 . Approximately, ten clones, representing each fragment, were screened with restriction enzyme Xho1/Nde1 for the presence of insert. Slot-blot hybridization was conducted using RsaI fragments as probe labeled by random priming (RediprimeTM II kit, Amersham Pharmacia biotech, USA). Finally, positive clones were selected for sequencing.

Multiple sequence alignment and Phylogenetic tree construction were carried out using ClustalW program. Blast search were performed with the sequences in GenBank using BLASTN program (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome version 2.2.18+) with default parameters. Repeats were calculated using Repeat masker http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker. Clustered repeats were found out using http://zlab.bu.edu/repfind/form.html ORFs and amino acid sequence identification was done using http://www.ncbi.nlm.nih.gov/gorf/gorf.html Conserved domain was determined using site http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi Cattle chromosome map was constructed using NCBI Bos taurus genome view.

For cross hybridization studies, approximately 500 ng heat denatured genomic DNA from 14 species each mentioned earlier were slot-blotted onto the nylon membrane (Amersham) along with cloned plasmid as positive control and 2× SSC as negative control following standard protocol 41 . For Southern hybridization, approximately, 4-5 μg of buffalo, cattle, goat and sheep genomic DNA were subjected to restriction digestion using RsaI enzyme following supplier's (NEB) specifications. The digested DNA was resolved on 1.5% agarose gel and transferred onto the nylon membrane (Amersham) following standard protocol 42 . Membranes were rinsed in 2× SSC, dried and UV cross-linked. Blots were hybridized at 60°C overnight with α-32P-dCTP labeled recombinant plasmid (25 ng) using random priming method (rediprimeTM II kit, Amersham Pharmacia biotech, USA). Washing of the membranes was done using standard protocols and signals were recorded by exposure of the blot to X-ray film 41 .

The extent of sequence conservation across bovid genomes was further determined by PCR analysis. Primers were designed from ORF using Primer 3 software (Table 3). 50 ng of genomic DNA from buffalo, cattle, goat and sheep were PCR amplified using reaction conditions 95°C for 5 minutes followed by 35 cycles each consisting 95°C for 1 minute, 60°C for 1 minute and 72°C for 1 minute, and final extension at 72°C for 10 minutes for all the four fragments. Amplified fragments were resolved on 1.5% agarose gel in 1% TAE buffer (Table 3).

Total RNA was extracted from testis, kidney, liver, spleen, lung, heart, ovary, brain and sperm using TRIzol (Molecular Research Center, Inc., Cincinnati, OH) following manufacturer's instructions 43 44 . Tissues were procured from the local slaughterhouse, Delhi. Fresh ejaculated semen samples from buffalo bulls were obtained from the animal farm, Lucknow, U.P., India, strictly following the guidelines of the Institutes Ethical and Biosafety Committee. To check the contamination of mRNA from the cells other than spermatozoa, RNA extractions from the sperms were tested by RT-PCR for both the CDH1 (E-cadherin) GenBank Accession no. AJ400864 and CD45 (tyrosine phosphatase) GenBank Accession no. NM_001002763 45 . Similarly, presence of DNA was ruled out by PCR using β-actin primers, GenBank accession no. DQ661647 (Table 3). Following this, approximately 10 μg of RNA from different tissues and spermatozoa was reverse transcribed into cDNA using commercially available high capacity cDNA RT kit (Applied Biosystems, USA). The success of cDNA synthesis was confirmed by 35 cycles of PCR amplification using buffalo derived β-actin primers.

Expression analysis of pDp1, pDp2, pDp3 and pDp4 transcripts using 50 ng cDNA from different somatic tissues and spermatozoa of buffalo was done with sequence specific internal primers designed by Primer 3 software (Table 3). PCR conditions were same as that mentioned earlier. β-actin was used as positive control. Relative expression analysis for the four transcripts was done using SYBR Green and Real time PCR Sequence Detection System-7500 (ABI, USA). The primers specific to the sequence were designed using Primer Express Software V2.0 (ABI) (Table 3). Primers for the housekeeping gene GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) GenBank Accession no. XR_083674.1 was used to normalize the values for each sample. The specificity of each primer pair and efficiency of the amplification were tested by assaying serial dilutions of the cDNA. Each reaction was performed in triplicates and the mean value was used for the analyses 46 . The cyclic conditions comprise 50°C for 2 min and 95°C for 10 min, followed by 40 cycles each of 95°C for 10 s and 60°C for 1 min. Each experiment was repeated three times to ensure consistency of the results. The expression level of the desired sequence in different tissues and spermatozoa was calculated in the form of 2^-ΔΔCt value http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042176.pdf 46 .

Real-time qPCR assays were performed in a 25 μl reaction volume containing 12.5 μl 2× SYBR Green^® PCR Master Mix (Applied Biosystems, Foster City, CA, USA), genomic DNA (0.1, 1.0 and 5 ng), forward and reverse primers at final concentration of 100 μM. Copy number estimation for four RsaI fragments in buffalo, cattle, goat and sheep genomes were done using 10 fold dilutions series of recombinant plasmids in the range 30,00,00000 to 3 copies (assuming haploid genome of farm animals =3.3 pg, wt per base pair = 1.096 × 10^-21 gm). Reactions were performed in 96-well MicroAmp Optical Reaction Plates (Applied Biosystems) in triplicates using the Real time PCR Sequence Detection System-7000 (ABI, USA) and SYBR Green dye. Primers and assay conditions were similar to those used for Relative expression studies (Table 3). Reaction specificity was confirmed with melting curves analysis. The standard curve was prepared using 10 folds dilution series of the recombinant plasmids and buffalo, cattle, goat and sheep genomic DNA 45 46 .

Approximately, 200 μl of the whole blood from buffalo was cultured for chromosome preparation following standard protocols 46 . FISH was conducted with spectrum red labeled pDp1, pDp2 and pDp3 and pDp4 cloned probes (Abbott Molecular) on the metaphase chromosomes using Nick Translation Kit, Abbott Molecular, (Illinois, USA). Hybridization was carried out in 20 μl volume containing 50% formamide, 10% Dextran sulphate, Cot 1 DNA and 2× SSC, pH 7 for 16 hours at 37°C in a moist chamber. Post hybridization washes were done in 2× SSC at 37°C (low stringent condition) and then at 60°C in 0.1× SSC (under high stringent condition). Slides were counterstained with DAPI, screened under Olympus Fluorescence Microscope (BX51) and images were captured with Olympus U-CMAD-2 CCD camera. Chromosome mapping was done following the International System for Chromosome Nomenclature (ISCND 2000) for Bovids 47 .

Blast search with pDp1 sequences showed 91% homology with B. taurus ACOT11 gene from nucleotide position 730-1331 encompassing 602 bp. Full length buffalo ACOT11 mRNA was generated using primers designed from B. taurus ACOT 11 (Accession No. NM_001103275). Details of Primer sequences and product size are given in Table 3. PCR amplified products were cloned into and pGEMT-easy vector and sequenced. Finally, buffalo ACOT11 gene sequences were assembled and full length sequence was deposited in the GenBank.