DNA polymorphism in SLC11A1 gene and its association with brucellosis resistance in Indian Zebu (Bos indicus) and crossbred (Bos indicusxBos taurus) cattle.
Solute linked carrier 11A1 (SLC11A1), a transmembrane protein (also referred as Natural Resistance Associated Macrophage Protein 1, NRAMP1), is one of the potential candidate genes that confer innate resistance against various intracellular pathogens. The role of SLC11A1 was first demonstrated by the fact that mice carrying a point mutation G169D (Vidal et al., 1996), located within the fourth transmembrane domain (TM4), were susceptible towards a number of antigenically different intracellular pathogens namely Salmonella typhimurium (Plant and Glynn, 1976), Leishmania donovani (Bradley et al., 1979) and Mycobacterium tuberculosis (Gros et al., 1981). The G169D point mutation, observed in mice, could not be observed in any mammalian species. Several studies, however, have been initiated to identify new DNA polymorphisms and to ascertain its association with the host resistance/susceptibility against different intracellular pathogens in various species including human (Liu et al., 1995; Abel et al., 1998; Bellamy et al., 1998; Jin et al., 2009), cattle (Adams and Templeton, 1998; Horin et al., 1999; Kumar et al., 1999; Martinez et al., 2008), buffalo (Ganguly et al., 2008), sheep (Matthews and Crawford, 1998), pig (Zhang et al., 2000) and horse (Horin and Matiasovic, 2000).
Brucellosis is one of the major zoonotic infections worldwide. The causative organism, Brucella abortus is a facultative intracellular bacteria that is capable of surviving and replicating within the phagosome. Macrophages isolated from naturally susceptible and resistant animals exhibited a differential ability in controlling the intracellular replication of Brucella organisms (Crocker et al., 1984; Price et al., 1990; Campbell and Adams, 1992). In cattle, a [(GT).sub.13] microsatellite allele at 3'UTR of SLC11A1 has a significant association with the natural resistance to brucellosis (Adams and Templeton, 1998). In an in vitro macrophage challenge study, Barthel et al. (2001) showed that [(GT).sub.13] allele restricted the intracellular replication of Brucella organisms. On the contrary, our group (Kumar et al., 2005) and more recently Paixao et al. (2007) demonstrated a lack of association of 3' UTR polymorphisms with the resistance against bovine brucellosis. On the other hand, Capparelli et al. (2007a, 2007b) reported a significant association of polymorphisms at 3' UTR of SLC11A1 gene with resistance/susceptibility to brucellosis in buffalo. Nevertheless, polymorphisms in other regions of SLC11A1 gene can serve as a potential marker (Liu et al., 1995; Abel et al., 1998; Bellamy et al., 1998). Further, TM4 is known to play an important role in positioning the NRAMP1 protein within the phagosomal membrane (Malo et al., 1994). The present study was undertaken to identify polymorphism in and around TM4, and to investigate its association with the incidences of brucellosis in Indian zebu (Bos indicus) and Holstein Friesian crossbred (Bos indicus x Bos taurus) cattle.
MATERIALS AND METHODS
Animals belonging to Hariana breed (Bos indicus) and Holstein Friesian crossbred were included in the study. The crossbred cattle comprised of various genetic constitutions i.e., (1/2 F X 1/2 H); (1/2 F X 1/4 B X 1/4 H); (1/2 F X1/4 J X 1/4 H), where F stands for Holstein Friesian, B for Brown Swiss, J for Jersey and H for Hariana. A random sample of 50 animals from each breed was used for the estimation of gene and genotype frequencies. In addition to the animals mentioned above, 20 animals from Hariana breed and 30 from crossbred cattle having additional records pertaining to date of birth, parity, calving, abortion and vaccination were included for association study.
Isolation of genomic DNA
Genomic DNA was isolated from the venous blood using standard phenol chloroform extraction method (Sambrook et al., 1989).
PCR amplification of TM4 of SLC11A1 gene
A pair of primer (NRE5F 5' TCCGACATGCAGG AAGTCAT 3'; NRE7R 5' GCCGAAGGTCAAGGCCAT TATGG 3') were designed on the basis of aligned sequences of SLC11A1 gene of cattle cDNA (GenBank Acc. No. U12862), human cDNA (GenBank Acc. No. D50403) and human genomic sequences (GenBank Acc. No. AF229163) to amplify a fragment comprising intron 5, exon VI, intron 6 and a part of exon V and VII, corresponding to TM4. The PCR reaction was carried out in 50 [micro]l mixture containing 2.0 mM of Mg[Cl.sub.2], 1.0 U of Taq DNA polymerase, 200 [micro]M of dNTPs, 5 pM of each primer and approximately 100 ng of genomic DNA as template. The PCR product was checked by agarose gel (1%) electrophoresis in 1X TAE buffer after staining with ethidium bromide.
Nucleotide sequencing and sequence analysis
In order to confirm the restriction patterns, the amplicons from two random representative animals of Hariana breed of cattle were sequenced using automated DNA sequencer (ABI Prism Ver 2.0) following Sanger's dideoxy chain termination method. The sequences obtained were subjected to BLAST analysis (www.ncbi.nlm.nih.gov/ BLAST). Using MAPDRAW program of Lasergene Software (DNASTAR), the presence of potential recognition sites for restriction enzyme (RE) was analyzed. The nucleotide and deduced amino acid sequences were compared with that of other species available in the GenBank using the Clustal method of MEGALIGN programme of DNASTAR.
PCR-Restriction fragment length polymorphism (PCR-RFLP)
In order to detect the PCR-RFLP, the amplicon was digested independently with 10 units of AluI and TaqI. The digestion was carried out overnight in a water bath at 37[degrees]C and 65[degrees]C for AluI and Taq I, respectively. For restriction fragment analysis, digested product was run on 2.5% and 1.5% agarose gel, respectively in 1X TBE buffer for 2-3 h at 5 V/cm.
For association study, information pertaining to date of birth, parity, calving, abortion and vaccination was collected. To avoid the interference of brucella antibodies due to vaccination, only non-vaccinated animals were chosen (20 animals of Hariana breed and 30 of crossbred cattle). These animals were then screened using three different serological tests viz., Rose Bengal Plate Test (RBPT), Standard Tube Agglutination Test (STAT) and Indirect Enzyme Linked Immunosorbent Assay (Indirect ELISA) as per standard method described by Kumar et al. (2005). On the basis of results from serological tests and history of abortion, the animals were divided into two groups viz., affected and non-affected. The animals that were serologically positive in all the three tests and had a history of abortion were grouped as "affected". Whereas, animals that were serologically negative in all the three tests and had no history of abortion till third lactation or completion of the study, which ever was later, were grouped as "non-affected".
Amplification and characterizations of TM4
Agarose gel electrophoresis of the amplicon as expected revealed an amplification of a fragment approximately 954 bp. The size of the amplicon was further confirmed by nucleotide sequencing (GenBank Acc. No. AY338470 and AY338471). As predicted, the amplicon comprised a part of exon V (<59 bp) and VII (62>), and the entire intron 5 (423 bp), exon VI (71 bp) and intron 6 (339 bp). A comparison of the obtained nucleotide sequences with the corresponding region of the available genomic sequences of different species (viz. taurine cattle, human, pig and dog), revealed a variation in the size of the fragment of 954, 944, 892 and 953 bp, respectively. The size of the corresponding exonic region of 192 bp was conserved across the species and the variation in size of the amplicons was mainly due to difference in the size of the intron (Table 1). While comparing the 192 bp exonic region of Hariana cattle with the corresponding region of Bos taurus cattle and buffalo, only one and two synonymous nucleotide substitutions were found, respectively. However, on comparison with other species homologues, a total of 63 nucleotide substitutions were observed, amongst them 12 were non-synonymous.
The phylogenetic tree (Figure 1) based on the deduced amino acid sequence showed that Bos indicus (Hariana) cattle are closely related to Bos taurus cattle and with buffalo form a single clad. However, sheep are interestingly placed in a separate group with chicken, and pigs formed a distinct clad from the remaining species.
[FIGURE 1 OMITTED]
Restriction fragment length polymorphisms (RFLP) were identified by restriction digestion of the amplicon independently with AluI and TaqI.
Restriction digestion and sequence analysis indicated the presence of five AluI sites in the amplicon of which the RE site at intron 6 was polymorphic (Figure 2a). AluI digestion should generate six restriction fragments (viz. 281, 260, 255, 79, 51 and 28 bp). However, due to inherent limitation of agarose gel electrophoresis the fragment of 28 bp could not be resolved and the fragments of 260 and 255 bp co-migrated as a single band. Consequently, two alleles were distinguishable viz, A (281, 255, 79 and 51 bp) and B (541, 255, 79 and 51 bp). A total of 35 and 31 AA, 10 and 11 AB, and 5 and 8 BB animals were identified in Hariana and crossbred cattle. The frequencies of A allele were estimated as 0.80 and 0.73, respectively (Table 2).
Digestion of PCR product with TaqI revealed two restriction patterns. Further, nucleotide sequence analysis of the amplicon demonstrated the presence of two TaqI sites in which the RE site present at intron 5 was polymorphic (Figure 2b). Accordingly, two alleles were identified: T (552 and 402) and Q (231, 321 and 402). Among Hariana zebu and crossbred cattle, 46 and 47 TT and 4 and 3 TQ animals were found, respectively. However, none of the animals were homozygous QQ. The allelic frequency of T was estimated as 0.96 and 0.97, respectively (Table 3).
[FIGURE 2 OMITTED]
On the basis of serological tests and abortion history, a total of nine and seven crossbred cattle were grouped as "affected" and "non-affected". In Hariana cattle, none of the animals could be grouped as "affected" and only five of them could be grouped as "non-affected". In TaqI PCR-RFLP, all the animals belonging to both affected and non-affected group were of homozygous TT genotype. However, in case of AluI PCR-RFLP all animals except two were homozygous AA genotype. Accordingly, no association could be established between polymorphism in SLC11A1 gene and the incidences of brucellosis.
This is the first study of its kind trying to associate PCR-RFLP (exon V-VII) polymorphism with the incidences of brucellosis in cattle. One polymorphic restriction site for AluI and TaqI has been identified in intron 6 and 5. In both the breeds, homozygous AA genotype was more frequent than AB and BB. In case of TaqI, homozygous QQ genotype was not observed in either breed. However, PCR-RFLP results showed no variation within the breed in any coding region.
In the present study, we could not find any association between the observed PCR-RFLP and the incidence of brucellosis. It could be argued that in grouping animals for the association study we might have included false positive and/or false negative animals. However, three serological tests and history of abortion were used to exclude false positive and false negative. Nonetheless, it is known that none of the serological tests used for detection of brucellosis are full proof. For example, indirect-ELISA in which Brucella LPS was used as an antigen suffers from two limitations. Firstly, this test sometimes could not distinguish between the animals infected and vaccinated with B. abortus strain19 (Nielsen et al., 1992) but in the present study only non-vaccinated animals were included. Secondly, smooth LPS of several bacteria such as Yersinia enterocolitica (Ahvonen et al., 1969), Vibrio cholerae (Sandulache and Marx, 1978) and Salmonella serotypes Kauffmann white group N (Corbell, 1975) are also reported to cross-react with B. abortus. Consequently, the available history of dams, having late gestational abortion (5-8 months) typical to brucellosis was used to exclude such false positive due to cross-reacting bacteria. Further, we had confirmed the presence of brucella organism by culture examination (in crossbred cattle only) of heart blood, spleen, lung, stomach of fetuses (data not shown).
In mice SLC11A1 was not an important determinant in conferring natural resistance against brucellosis as compared to that against Salmonella, Leishmania, and Mycobacterium (Guilloteau et al., 2003). We (Kumar et al., 2005) and others (Paixao et al., 2007) have also demonstrated the lack of association of [(GT).sub.n] 3' UTR microsatellite polymorphisms with the resistance against bovine brucellosis either in experimental or natural infection. Nevertheless, these findings can not exclude the possible role of bovine SLC11A1 gene restricting intracellular replication of Brucella abortus. As on the contrary, several other studies (Borriello et al., 2006; Capparelli et al., 2007a; Capparelli et al., 2007b) revealed a strong association of [(GT).sub.n] microsatellite polymorphisms with the resistance/susceptibility against brucellosis in water buffalo. Recently, we have also demonstrated that macrophages from [(GT).sub.13] buffaloes produced more nitric oxide and [H.sub.2][O.sub.2] when challenged with brucella LPS (Ganguly et al., 2008). It is plausible that more than a single gene is responsible for determining the resistance/ susceptibility to brucellosis. The classical breeding studies conducted by Templeton et al. (1988) showed that two or more genes are involved in controlling Brucella infection. A M17T mutation in bison prion protein in homozygous condition was found to be associated with the seropositivity to Brucella species (Seabury et al., 2005). SLC11A1 may also have pleiotropic effect on other genes. For instance, SLC11A1 is known to involve in the high level of expression of MHC and surface antigen expression in mice (Denis et al., 1988; Lang et al., 1997).
In conclusion, we identified AluI and TaqI polymorphisms in SLC11A1 gene in cattle, but association of the observed allelic variants with the resistance/ susceptibility to brucellosis could not be established. Future studies need to be directed to explore polymorphisms throughout the entire SLC11A1 gene and to ascertain their suitability as potential genetic marker for brucellosis resistance.
Abel, L., F. O. Sanchez, J. Oberti, N. V. Thuc, L. V. Hoa, V. L. Lap, E. Skamene, P. H. Lagrange and E. Schurr. 1998. Susceptibility to leprosy is linked to the human NRAMP1 gene. J. Infect. Dis. 177:133-145.
Adams, L. G. and J. W. Templeton. 1998. Genetic resistance to bacterial diseases of animals. Rev. Sci. Tech. 17:200-219.
Ahvonen, P., E. Jansson and K. Aho. 1969. Marked cross agglutination between Brucellae and a subtype of Yersinia enterocolitica. Acta. Pathol. Microbiol. Scand. 75(2):291-295.
Altet, L., O. Francino, L. Solano-Gallego, C. Renier and A. Sanchez. 2002. Mapping and sequencing of the canine NRAMP1 gene and identification of mutations in leishmaniasis-susceptible dogs. Infect. Immun. 70(6):2763-2771.
Barthel, R., J. Feng, J. A. Piedrathia, D. N. McMurray, J. W. Templeton and L. G. Adams. 2001. Stable transfection of the bovine NRAMP1 gene into murine RAW264.7 cells: Effect on Brucella abortus survival. Infect. Immun. 69:3110-3119.
Bellamy, R., C. Rumende, T. Corrah, K. P. W. J. McAdam, H. C. Whittle and A. V. S. Hill. 1998. Variation in the human NRAMP1 gene and susceptibility to tuberculosis on Africans. New Engl. J. Med. 338:640-644.
Borriello, G., R. Capparelli, M. Bianco, D. Fenizia, F. Alfano, F. Capuano, D. Ercolini, A. Parisi, S. Roperto and D. Iannelli. 2006. Genetic resistance to Brucella abortus in the water buffalo (Bubalus bubalis). Infect. Immun. 74:2115-2120.
Bradley, D. J., B. A. Taylor, J. Blackwell, E. P. Evans and J. Freeman, 1979. Regulation of Leishmania populations within the host III. Mapping of the locus controlling susceptibility to visceral leishmaniasis in the mouse. Clin. Exp. Immunol. 37:714.
Campbell, G. A. and L. G. Adams. 1992. The long-term culture of bovine monocyte-derived macrophages and their use in the study of intracellular proliferation of Brucella abortus. Vet. Immunol. Immunopathol. 34(3-4):291-305.
Capparelli, R., F. Alfano, M. G. Amoroso, G. Borriello, D. Fenizia, A. Bianco, S. Roperto, F. Roperto and D. Iannelli. 2007a. Protective effect of the Nramp1 BB genotype against Brucella abortus in water buffalo (Bubalus bubalis). Infect. Immun. 75: 988-996.
Capparelli, R., G. Borriello, R. Marabelli, S. Roperto, F. Roperto and D. Iannelli. 2007b. The Nramp1 AA genotype confers susceptibility to Brucella abortus in water buffalo. Mamm. Genome. 18:137-143.
Corbell, M. J. 1975. The serological relationship between Brucella spp., Yersinia enterocolitica serotype IX and Salmonella serotypes of Kauffmann-White group N. J Hyg (Lond). 75(1):151-171.
Coussens, P. M., M. J. Coussens, B. C. Tooker and W. P. Nobis. 2004. Structure of the bovine natural resistance associated macrophage protein (NRAMP 1) gene and identification of a novel polymorphism DNA Seq. 15(1):15-25.
Crocker, P. R., J. M. Blackwell and D. J. Bradley. 1984. Expression of the natural resistance gene Lsh in resident liver macrophages. Infect. Immun. 43(3):1033-1040.
Denis, M., A. Forget, M. Pelletier and E. Skamene. 1988. Pleiotropic effects of the Bcg gene: III. Respiratory burst in Bcg-congenic macrophages. Clin. Exp. Immunol. 73:370-375.
Ganguly, I., A. Sharma, A. Mitra, N. Kumar and A. Ganguly. 2011. Analysis of genetic variations of complete TM4 of buffalo (Bubalus bubalis) Slc11A1 gene. J. Appl. Anim. Res. (In Press)
Ganguly, I., A. Sharma, R. Singh, S. M. Deb, D. K. Singh and A. Mitra. 2008. Association of microsatellite [(GT).sub.n] polymorphism at 3'UTR of NRAMP1 with the macrophage function following challenge with Brucella LPS in buffalo (Bubalus bubalis). Vet. Microbiol. 129(1-2):188-196.
Gros, P., E. Skamene and A. Forget. 1981. Genetic control of natural resistance to Mycobacterium bovis (BCG) in mice. J. Immunol. 127(6):2417-2421.
Guilloteau, L. A., J. Dornand, A. Gross, M. Olivier, F. Cortade, Y. L. Vern and D. Kerboeuf. 2003. Nramp1 is not a major determinant in the control of Brucella melitensis infection in mice. Infect. Immun. 71(2):621-628.
Horin, P. and J. Matiasovic. 2000. Two polymorphic markers for the horse SLC11A1 (RNAMP1) gene. Anim. Genet. 31(2):152.
Horin, P., I. Rychlik, J. W. Templeton and L. G. Adams. 1999. A complex pattern of microsatellite polymorphism within the bovine NRAMP1 gene. Eur. J. Immunogenet. 26:311-313.
Jin, J., L. Sun, W. Jiao, S. Zhao, H. Li, X. Guan, A. Jiao, Z. Jiang and A. Shen. 2009. SLC11A1 (Formerly NRAMP1) gene polymorphisms associated with pediatric tuberculosis in China. Clin. Infect. Dis. 48(6):733-738.
Kumar, G. S. N., M. G. Govindiah, C. S. Nagaraj, A. Bindu and T. J. Rasool. 1999. Does the Natural resistance associated macrophage protein (NRAMP1) gene confer resistance/ susceptibility in bovine against mycobacterial infection? Curr. Sci. 77:809-812.
Kumar, N., A. Mitra, I. Ganguly, R. Singh, S. M. Deb, S. K. Srivastava and A. Sharma. 2005. Lack of association of brucellosis resistance with [(GT).sub.13] microsatellite allele at 3'UTR of NRAMP1 gene in Indian zebu (Bos indicus) and crossbred (Bos indicusxBos taurus) cattle. Vet. Microbiol. 111: 139-143.
Lang, T., E. Prina, D. Sibthorpe and J. M. Blackwell. 1997. Nramp1 transfection transfers Ity/Lsh/Bcg-related pleiotropic effects on macrophage activation: influence on antigen processing and presentation. Infect. Immun. 65:380-386.
Liu, J., T. M. Fujiwara, N. T. Buu, F. O. Sanchez, M. Cellier, A. J. Paradis, D. Frappier, E. Skamene, P. Gros, K. Morgan and E. Schurr. 1995. Identification of polymorphisms and sequence variants in the human homologue of the mouse natural resistance associated macrophage protein gene. Am. J. Hum. Genet. 56:845-853.
Malo, D., K. Vogan, S. Vidal, J. Hu, M. Cellier, E. Schurr, A. Fuks, N. Bumstead, K. Morgan and P. Gros. 1994. Haplotype mapping and sequence analysis of the mouse Nramp1 gene predict susceptibility to infection with intracellular parasites. Genomics 23:51-61.
Marquet, S., P. Lepage, T. J. Hudson, J. M. Musser and E. Schurr. 2000. Complete nucleotide sequence and genomic structure of the human NRAMP1 gene region on chromosome region 2q35. Mamm. Genome 11(9):755-762.
Martinez, R., R. Toro. F. Montoya, M. Burbano, J. Tobon, J. Gallego, S. Dunner and J. Canon. 2008. Bovine SLC11A1 3' UTR SSCP genotype evaluated by a macrophage in vitro killing assay employing a Brucella abortus strain. Anim. Breed. Genet. 125(4):271-279.
Matthews, G. D. and A. M. Crawford. 1998. Cloning, sequencing and linkage mapping of the NRAMP1 gene of sheep and deer. Anim. Genet. 29:1-6.
Nielsen, K.H., D. Gall, W. Kelly, D. Henning and M. M. Gracia. 1992. Enzyme Immunoassay: Application to Diagnosis of Bovine Brucellosis. Monograph, Agriculture Canada, Nepean, Ont.
Paixao, T. A., F. P. Poester, N. A. V. Carvalho, A. M. Borges, A. P. Lage and R. L. Santos. 2007. NRAMP1 3'Untranslated region polymorphisms are not associated with natural resistance to Brucella abortus in cattle. Infect. Immun. 75:2493-2499.
Plant, J. and A. A. Glynn. 1976. Genetics of resistance to infection with Salmonella typhimurium in mice. J. Infect. Dis. 133:72-78.
Price, R. E., J. W. Templeton, R. Smith III and L.G. Adams. 1990. Ability of mononuclear phagocytes from cattle naturally resistant or susceptible to brucellosis to control in vitro intracellular survival of Brucella abortus. Infect. Immun. 58: 879-886.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: A laboratory manual. Vol 3. Cold Spring Harbour Library Press. pp. 6.3-6.4.
Sandulache, R. and A. Marx. 1978. Immunochemical studies on a Yersinia enterocolitica O:9 lipopolysaccharide cross-reacting with Brucella abortus and Vibrio cholerae extracts. Ann. Microbiol (Paris). 129 B(3):425-435.
Seabury, C. M., N. D. Halbert, P. J. P. Gogan, J. W. Templeton and J. N. Derr. 2005. Bison PRNP genotyping and potential association with Brucella spp. Seroprevalance. Anim. Genet. 36:104-110.
Templeton, J. W., R. Smith III and L. G. Adams. 1988. Natural disease resistance in domestic animals. J. Am. Vet. Med. Assoc. 192:1306-1315.
Vidal, S. M., E. Pinner, P. Lepage, S. Gauthier and P. Gras. 1996. NRAMP1 encodes a membrane phosphoglycoprotein absent in macrophages from susceptible (NRAMP1D169) mouse strain. J. Immunol. 157:3559-3568.
Wu, Z. F., W. H. Luo, G. F. Yang and X. Q. Zhang. 2007. Genomic organization and polymorphisms detected by denaturing high-performance liquid chromatography of porcine SLC11A1 gene. DNA Seq. 18(5):327-333.
Zhang, G., H. Wu, C. R. Ross, J. E. Minton and F. Blecha. 2000. Cloning of porcine NRAMP1 and its induction by lipopolysaccharide, tumor necrosis factor alpha, and interleukin-1beta: role of CD14 and mitogen-activated protein kinases. Infect. Immun. 68(3):1086-1093.
Nishant Kumar (1,2), Indrajit Ganguly (1,3), Rajendra Singh (4), Sitangsu M. Deb (1,5), Subodh Kumar (1), Arjava Sharma (1) and Abhijit Mitra (1), *
(1) Genome Analysis Laboratory, Animal Genetics Division, Indian Veterinary Research Institute, Indian Council of Agricultural Research, Izatnagar, Bareilly, U.P. 243122, India
* Corresponding Author: Abhijit Mitra. Tel: +91-581-2303382, Fax: +91-581-2303284, E-mail: firstname.lastname@example.org
(2) Division of Animal Genetics & Breeding, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Science & Technology, R.S. PURA, Jammu-181102, (J&K), India.
(3) Project Directorate on Cattle, Indian Council of Agricultural Research, Meerut, U.P.-250001, India.
(4) Centre for Animal Disease Research and Diagnosis, Indian Veterinary Research Institute, Indian Council of Agricultural Research, Izatnagar, Bareilly, U.P. 243122, India.
(5) Central Institute for Research on Buffaloes, Nabha Campus, Punjab-147201, India.
Received August 24, 2010; Accepted January 3, 2011
Table 1. Size differences of intron 5 and 6 in different mammalian species Species Intron 5 Intron 6 GenBank Acc No. and Ref. Cattle (Bos indicus) 423 339 AY338470, AY338471 Cattle (Bos taurus) 423 340 AY398762, AY398763 (Coussens et al., 2004) Buffalo 423 339 AY860618, AY 707989, AY860620 (Ganguly et al., 2011 in press) Human 418 334 AF229163 (Marquet et al., 2000) Pig 367 333 AY368472, AY368473 (Wu et al., 2007) Dog 425 336 AF091049 (Altet et al., 2002) Table 2. Gene and genotype frequencies of Alu-I PCR-RFLP Sl. Breeds Total no. of Genotype frequency No. individuals AA AB BB 1. Crossbred 50 0.62 0.22 0.16 (n = 31) (n = 11) (n = 8) 2. Hariana 50 0.70 0.20 0.10 (n = 35) (n = 10) (n = 5) Sl. Breeds Gene frequency No. A B 1. Crossbred 0.73 [+ or -] 0.04 0.27 [+ or -] 0.04 2. Hariana 0.80 [+ or -] 0.04 0.20 [+ or -] 0.04 Table 3. Gene and genotype frequencies of Taq-I PCR-RFLP Sl. Breeds Total no. of Genotype frequency No. individuals TT TQ QQ 1. Crossbred 50 0.94 0.06 0.00 (n = 47) (n = 3) (n = 0) 2. Hariana 50 0.92 0.08 0.00 (n = 46) (n = 4) (n = 0) Sl. Breeds Gene frequency No. T Q 1. Crossbred 0.97 [+ or -] 0.017 0.03 [+ or -] 0.017 2. Hariana 0.96 [+ or -] 0.0195 0.04 [+ or -] 0.0195
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|Author:||Kumar, Nishant; Ganguly, Indrajit; Singh, Rajendra; Deb, Sitangsu M.; Kumar, Subodh; Sharma, Arjava;|
|Publication:||Asian - Australasian Journal of Animal Sciences|
|Date:||Jul 1, 2011|
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