Small Ruminant Research 82 (2009) 156–160

Contents lists available at ScienceDirect

Small Ruminant Research

journa l homepage: www.e lsev ier .com/ locate /smal l rumres

Short communication

Single nucleotide polymorphism (SNP) in alpha-lactalbumin gene ofIndian Jamunapari breed of Capra hircus

Anubhav Jaina, D.S. Goura, P.S. Bisenb,1, Prashanta, P.P. Dubeya, D.K. Sharmac,B.K. Joshia, Dinesh Kumara,∗

a Genes and Genetic Resources Molecular Analysis Lab, National Bureau of Animal Genetic Resources, Karnal 132001, Haryana, Indiab Department of Biotechnology, Bundelkhand University, Jhansi 284218, U.P., Indiac Government P.G. College, Guna 473001, M.P., India

a r t i c l e i n f o

Article history:Received 9 September 2008Received in revised form 13 February 2009Accepted 19 February 2009Available online 21 April 2009

Keywords:

a b s t r a c t

Alpha-lactalbumin is a major whey protein found in milk. It influences lactose synthesis bymodifying the substrate specificity of galactosyl-transferase, is important to milk synthesissince lactose, an impermeable disaccharide, is the major osmole of milk. The present studywas undertaken to detect polymorphism in the full coding region of alpha-lactalbumin atthe genetic level and to explore allelic variability of this gene. Samples of Jamunapari breedof goat (n = 50) were included under the present study. Jamunapari is the highest milk pro-

Alpha-lactalbumin geneDairy goatsPolymorphismSNPSSCP

ducer among local Indian goat breeds. PCR-SSCP of all four exons of alpha-lactalbumin (ALA)revealed a total of 9 gel phenotypes of Jamunapari breed of goat. These were sequenced,analyzed and deposited in GenBank, NCBI (accession nos. EU573193–EU573195). Nucleotideand amino acid variations were searched within breeds of Indian goats and homologybetween caprine, ovine, bovine, bubaline and human. In the present study we describe

o nove

for the first time tw

1. Introduction

Alpha-lactalbumin, a calcium metalloprotein, is one ofthe major serum-proteins in ruminant milk (Jenness, 1982)and induces lactose synthesis in the mammary gland byinteracting with the enzyme UDP-galactosyl-transferase,giving rise to the heterodimer enzyme lactose synthase

(Ebner and Brodbeck, 1968; Kuhn, 1983). Because ALA isnecessary for the production of lactose, the movementof water into the mammary secretory vesicles, and thenthe movement of water into the alveolar lumen, it is crit-

Abbreviations: ALA, alpha-lactalbumin; SSCP, single stranded con-formational polymorphism; PCR, polymerase chain reaction; SNP, singlenucleotide polymorphism.

∗ Corresponding author. Tel.: +91 9416111753.E-mail address: dinesh@iastate.edu (D. Kumar).

1 Present address: Department of Biotechnology, Madhav Institute ofTechnology and Science, Gwalior 474005, India.

0921-4488/$ – see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.smallrumres.2009.02.013

l gene variants on the goat alpha-lactalbumin gene exon 4.© 2009 Elsevier B.V. All rights reserved.

ical in the secretion of milk in mammals (Hayssen andBlackburn, 1985). The structure of the gene encoding thisCa2+-metalloprotein has been reported in rat (Qasba andSafaya, 1984), human (Hall et al., 1987), bovine (Vilotte etal., 1987), caprine (Vilotte et al., 1991) and guinea pig (Lairdet al., 1988) species, has an organization similar to that ofhen egg-white and human lysozyme-encoding genes (Junget al., 1980; Peters et al., 1989, respectively) suggesting acommon ancestral origin.

The Indian Jamunapari goats are well known for milkproduction, probably highest amongst the Indian goatbreeds producing 201 kg/lactation (Acharya, 1982). Thebreed is a prolific and non-seasonal breeder (Devendra,1985). Jamunapari (Etawah) breed has been used as

improver goats in several countries in South Asia(Devendra, 1985).

The goat alpha-lactalbumin transcription unit (LALBA),located on chromosome 5 (Hayes et al., 1993), is organizedin 4 exons varying in length from 75 nucleotides (3rd exon)

A. Jain et al. / Small Ruminant Research 82 (2009) 156–160 157

Table 1Sequences of primers used for PCR-SSCP of alpha-lactalbumin gene of Capra hircus.

Primer sequences Tm (◦C)

Fragment 1 747–960a 1F 5′-GGAGGTGAGCAGTGTGGTGA-3′

(Exon 1-214 bp) 1R 5′-TGGGACAAAGCGAAATAGCA-3′ 66Fragment 2 1168–1526a 2F 5′-TGGAGAGCCTTTTTCTGTCTGG-3′

(Exon 2-359 bp) 2R 5′-GGAGGCCAAGGCAGTGAAG-3′ 66Fragment 3 1866–2026a 3F 5′-TTTTCCCACCTGTAACTCCTG-3′

( ′ ′

F(

tp

Aavaiui(imt

2

2

dbtw1wm

2

pocuptdXTf7uMgfe

2

waptw

product affected by single base substitution. Fig. 1 shows

Exon 3-161 bp) 3Rragment 4 2444–2840a 4FExon 4-397 bp) 4R

a Position according to accession no. M63868 (Vilotte et al., 1991).

o 329 nucleotides (4th exon) coding for a 123-amino acidolypeptide chain (Vilotte et al., 1991).

Globally, polymorphism is reported only in exon 3 ofLA gene at position 3E+5 and 3E+7 (Cosenza et al., 2003nd Lan et al., 2008). No nucleotide sequence and thus noariation is reported in ALA gene in Indian goat breeds. Theim of this study was to evaluate the genetic variabilityn ALA nucleotide sequence variation and polymorphism,sing non-radioactive SSCP protocol, followed by sequenc-

ng. This will generate the base line data of the gene variantsSNP and INDELS) to be use in future association stud-es in order to establish a breeding programme based on

olecular-assisted selection for improvement in produc-ivity of goat genetic resources of India.

. Materials and methods

.1. Sample collection and DNA extraction

Blood samples of 50 genetically unrelated Jamunapari goats were ran-omly collected from the different pockets of native breeding tracts of thisreed, avoiding narrow genetic relationships among the sampled animals,hus representing gene pool for SNP discovery. Blood samples (5–6 ml)ere obtained by jugular venipuncture, using vacuum tubes treated with

5% ethylene diamine tetra acetic acid (EDTA) as an anticoagulant. DNAas extracted from blood as described by Sambrook et al. (1989) withinor modifications.

.2. DNA amplification

Four fragments, covering all four exon of ALA gene with theart of their flanking region, was amplified by PCR using four pairsf primers designed on published nucleotide sequence of Capra hir-us ALA gene (GenBank accession no. M63868, Vilotte et al., 1991)sing Primer3 (http://www-genome.wi.mit.edu/genome software/other/rimer3.html). The PCR reaction was performed in a 25 �l reaction mix-ure containing 2 �l of DNA solution (50–100 ng), 200 mM each of dATP,CTP, dGTP and dTTP, 50 mM KCl, 10 mM Tris–HCl (pH 9.0), 0.1% Triton-100, 0.6 unit Taq DNA polymerase, 4 ng �l−1 of each primers (IDT, USA).he following conditions were used: an initial denaturation step of 95 ◦Cor 4 min was followed by 30 cycles of 94 ◦C for 1 min, 66 ◦C for 30 s, and2 ◦C for 30 s, and concluded with a final extension step of 72 ◦C for 10 minsing a PTC-0200 DNA Engine thermal cycler (MJ Research Inc., Waltham,A). Amplification was verified by electrophoresis on 2% (w/v) agarose

el in 1× TAE buffer using a 100 bp ladder as a molecular weight markeror confirmation of the length of the PCR products. Gels were stained withthidium bromide (1 mg/ml).

.3. Single strand conformation polymorphism analysis

The PCR products were resolved by SSCP analysis. Several factorsere tested for each fragment in order to optimize factors such as the

mount of PCR product, denaturing solution, acrylamide concentration,ercentage cross-linking, glycerol, voltage, running time and tempera-ure. The SSCP analysis was carried out as follows: each PCR productas diluted in denaturing solution (95% formamide, 10 mM NaOH, 0.05%

5 -GGCGAAAGGACTGAGAAGAA-3 665′-TGCCTTGTCTCCTTCTTCGTGA-3′

5′-TTTCCTCCCTCTCCCACCAG-3′ 66

xylene cyanol and 0.05% bromophenol blue, 20 mM EDTA) denatured at95 ◦C for 5 min, chilled on ice and resolved (10 h, 375 V, 4 ◦C) on 12%acrylamide:bisacrylamide gels (29:1). The electrophoresis was carriedout in a BioRad Protean II xi vertical electrophoreses unit using 1× Tris-borate–EDTA (TBE) buffer. Gels were silver-stained (Sambrook and Russell,2001) and dried on cellophane using a BioRad Model 583 gel dryer. Thegel phenotypes were identified and scored manually to obtain PCR-SSCPallele frequency.

2.4. Cloning and sequencing

The DNA samples showing different patterns on SSCP gels wereselected for sequencing. For sequencing, the PCR products was either useddirectly in the reaction or firstly cloned into the plasmid vector T4 (cloningkit, Banglore Genei). Primers (IDT, USA) used in sequencing are same asfor SSCP shown in Table 1. PCR products were purified from all the SSCPvariants in the duplicate using PCR cleaning kit (Biogene). Sequencing wascarried out using the big dye terminator chemistry version of ABI as perthe manufacturer’s protocol.

2.5. In silico sequence analysis and allele mining

The nucleotide sequences generated after sequencing for each primerpair was edited using Chromas software and in order to check the identityof our sequence, BLAST analysis was also performed. The sequence wasaligned with Clustalw program with other already deposited nucleotidesequences of ALA gene of C. hircus. The nucleotide sequences andthe deduced amino acid sequences were analyzed with Sequin soft-ware and deposited in Gen Bank (NCBI, USA) under accession nos.EU573193–EU573195.

Extensive allele mining of ALA gene was done and nucleotide varia-tions were searched between species and within breeds of Capra usingMegalign (DNA star-Lasergene® software). The sequences of caprine,ovine, bovine, bubaline and human were compared to search for homol-ogy.

3. Results

The PCR products expected from exonic region of ALAgene were confirmed by 100 bp ladder in agarose gel elec-trophoresis. The ALA gene exhibited polymorphisms in thegoat population.

3.1. Single strand conformational polymorphism analysis

In the present study, genetic variability in alpha-lactalbumin genes assessed by SSCP technique, allowsdetection of changes in the nucleotide sequence of a PCR

SSCP banding pattern for alpha-lactalbumin gene. The PCR-SSCP of ALA gene exons exhibited 9 different conformationsor gel phenotypes, identified in 4 fragments represent 4exons. The genotype and allele frequencies of ALA gene arepresented in Table 2.

158 A. Jain et al. / Small Ruminant Research 82 (2009) 156–160

lamide gxon 4), b

Fig. 1. SSCP banding patterns of alpha-lactalbumin gene on 12% polyacryfor exon 1, E2A, E2B, E2C for exon 2, E3A for exon 3 and E4A, E4B, E4C for e

3.2. Sequence analysis and SNP identification

Comparison between nucleotide sequence of goat ALAgene (accession no. M63868) using MEGALIGN revealedcomplete homology except SNPs at specific nucleotides.Fig. 2 shows alignment of the coding region of caprine ALA,as per Clustal method.

Exon 1, 2 and 3 were found to be monomorphic atnucleotide level and showed no alteration within the breedand showed no transition/transversion related to accessionno. M63868 thus no change in amino acid sequence. Theexon 4 showed two alterations, the first one is T → C tran-sition located at 4E+190 position. Second alteration wasT → C transition at 4E+263 position of nucleotide sequence.These nucleotide alterations are on the untranslated regionof exon 4. The genotypes CT and TT were present at frequen-cies of 1.2 and 8.8, respectively.

3.3. New in silico PCR-RFLP test development

Two bioinformatics software were used for this vizNEB Cutter (online) and CLEAVER (off line, freely avail-able http://cleaver.sourceforge.net/). NEB cutter was usedto find out the restriction enzyme for a particular SNP site(T → C at 217) and cleaver was used to estimate the cut sizefor different genotypes. The alteration in the 4E+190 posi-tion of ALA leads to the introduction of a new restrictionsite [/GTNAC] for restriction enzyme Mae III. Variant A withnew recognition site for Mae III will produce two fragmentsof 212 bp and 185 bp while variant B with no variation willgive a single undigested product of 397 bp. Thus an in silicoPCR-RFLP test is developed for ‘Variant A’ of ALA exon 4 ofJamunapari breed (GenBank accession no. EU573194).

4. Discussion

The alpha-lactalbumin encoding gene is essential forthe biosynthesis of lactose in the mammary gland and is

Table 2Genotype frequency of SSCP variants of alpha-lactalbumin gene.

Specified region of the gene Alleles and their frequency

Exon 1 A = 0.98, B = 0.02Exon 2 A = 0.14, B = 0.28, C = 0.58Exon 3 A = 1Exon 4 A = 0.16, B = 0.8, C = 0.76

el. The haplotypes are named alphabetically for each exons (E1A and E1Based on their electrophoretic mobility on the gel.

a potential quantitative trait locus in dairy animals. It istherefore an interesting candidate gene for marker-assistedselection (MAS). The present work includes complete fourexons of ALA gene with part of intronic region. Thenucleotide coding sequence and the predicted proteinsequence amino acids were compared with bovine, ovineand human sequences. This analysis showed that the C. hir-cus ALA coding region has a high level of similarity withthe Ovis aries, Bos indicus, Bubalus arnee bubalis, Bos tau-rus and Human counterparts (96%, 95%, 95%, 93% and 74%,respectively) and that the amino acid level similarity is98%, 96%, 96%, 96% and 75%, respectively. The publishedsequence of the ALA of C. hircus (GenBank accession no.M63868) (Vilotte et al., 1991) differs at two nucleotidepositions in exon 4 with the sequence determined inthe present work. The predicted protein sequence wasfound to be similar from the published one (M63838) dueto change in nucleotides at untranslated region of exon4.

In the present study the sequence analysis of all SSCPpatterns revealed two SNP. Several authors have pointedout that SSCP analysis is a reliable and reproducible tech-nique for the detection of structural gene polymorphismwhich is primarily due to point mutations (Neibergs etal., 1993; Sheffield et al., 1993; Jain et al., 2008). In ourstudy, five gel phenotypes were observed in fragments ofexon 1 and 2, but in sequencing they did not revealed anySNPs. Such observations are not uncommon. In a study byBarracosa (1996), such unusual patterns of gel phenotypeis reported which is because of existence of more than oneconfirmation of even exactly similar DNA sequence (PCR-SSCP products) especially when SSCP gel is more optimizedhaving highest sensitivity. SSCP has several advantages: itdoes not require specific equipment, it is technically sim-pler and faster, it can be used in most laboratories and isnot very expensive (Orita et al., 1989).

In goat, Cosenza et al. (2003) detected a silent allele atthe alpha-lactalbumin exon 3 using Mval PCR-RFLP, becauseof C → T transition at 3E+5 position MvaL endonucleaserestriction site was removed at third exon, as compared tothe their reference template (M63868). In our study, theexon 3 sequence is similar to the nucleotide sequence of

M63868. Even in other species, which we used for phy-logenetic analysis, the position 3E+5 is conserved, evenin human. Allele mining from reported sequences showedonly one SNP (accession no. DQ673921) in ALA of C. hircusi.e. T → C at position 3E+7 (1897 corresponding to M63868)

A. Jain et al. / Small Ruminant Research 82 (2009) 156–160 159

gene of

wdfgMspr

rcaaslree

5

stsvfsioa

Fig. 2. Alignment of region of exon 4 of alpha-lactalbumin

hich leads to change in amino acid from L → P and intro-uction of MspI restriction site (Lan et al., 2007) and thusound a significant correlation between polymorphism ofoat M63868:g.1897 locus and cashmere yield of Innerongolian white cashmere goats (Lan et al., 2008). In our

tudy two SNPs in exon 4 were discovered in 50 sam-les in alpha-lactalbumin gene. These SNPs have not beeneported ever in caprine previously.

Phylogenetic analysis of ALA gene in different speciesevealed the conserved nature of this gene. ALA sequenceomparison has also highlighted the conservation of aminocids to which functional roles were attributed (Shewale etl., 1984). Reason for conserved sequence is very less diver-ity in the gene due to same physiological functions, i.e.actogenesis in the animal, but we have got SNP in exonicegion may be due to adaptation of animal in differentnvironment as per need of animal to survive in differentnvironment or management condition.

. Conclusion

Our data collection intends to be a first step for a deepertudy of SNP in indigenous goat breeds of India in ordero establish a breeding program based on marker-assistedelection. This result provides the base line data of the geneariants. Though ALA is a key protein in lactogenesis, other

unctional properties have been attributed to this proteinuch as a cell lytic activity (McKenzie and White, 1987),nduction of cell growth inhibition (Thompson et al., 1992)r apoptosis (Hakansson et al., 1995). So, even if the associ-tion studies of these SNPs with the milk protein traits has

variant A and B with the template M63868, showing SNPs.

not been done in this breed, it is very important to conserveeach haplotype in a gene pool.

Acknowledgements

The authors would like to thank Director, NationalBureau of Animal Genetic Resources, Karnal for the facil-ities provided for research. The help and contribution ofState Animal Husbandry Department, Government of UttarPradesh in blood sample and data collection from field isgratefully acknowledged. The grant of Fellowship to Anub-hav Jain by Indian Council of Agricultural Research (ICAR),New Delhi, Government of India and work supported bygrants from Indian Council of Agricultural Research (ICAR),New Delhi, Government of India is duly acknowledged.

References

Acharya, R.M., 1982. Sheep and Goat breeds of India. FAO Animal Pro-duction and Health Paper 30, FAO, United Nations, Rome, Italy, pp.1–190.

Barracosa, H., 1996. Estudo de polimorfismos genéticos e da sua associacãocom capacidades de producão leiteira em caprinos de raca Algarviae ovinos de raca Serra da Estrela. Dissertacão de Mestrado, Univ. doAlgarve, Faro, Portugal.

Cosenza, G., Gallo, D., Illario, R., Di Gregorio, P., Senese, C., Ferrara, L.,Ramunno, L., 2003. A Mval PCR-RFLP detecting a silent allele at thegoat alpha-lactalbumin locus. J. Dairy Res. 70 (3), 355–357.

Devendra, C., 1985. Prolific breeds of goat. In: Land, R.B., Robinson, D.W.(Eds.), Genetics of Reproduction in Sheep. Butterworths Publication,

London, pp. 69–80.

Ebner, K., Brodbeck, U., 1968. Biological role of alpha-lactalbumin: areview. J. Dairy Sci. 51 (3), 317–322.

Hakansson, A., Zhivotovsky, B., Orrenius, S., Sabharwal, H., Svanborg, C.,1995. Apoptosis induced by a human milk protein. Proc. Natl. Acad.Sci. U.S.A. 92, 8064–8068.

ant Res

160 A. Jain et al. / Small Rumin

Hall, L., Emery, D.C., Davies, M.S., Parker, D., Craig, R.K., 1987. Organizationand sequence of the human alpha-lactalbumin gene. Biochem. J. 242,735–742.

Hayes, H.C., Popescu, P., Dutrillaux, B., 1993. Comparative gene mappingof lactoperoxidase, retinoblastoma, and alpha-lactalbumin genes incattle, sheep, and goats. Mamm. Genome 4 (10), 593–597.

Hayssen, V., Blackburn, D.G., 1985. Alpha lactalbumin and the origins oflactation. Evolution 39, 1147–1149.

Jain, A., Gour, D.S., Bisen, P.S., Dubey, P.P., Prashant, Sharma, D.K.,Bhargava, A., Joshi, B.K., Kumar, D., 2008. Single strand con-formation polymorphism detection in alpha-lactalbumin gene ofIndian Jakhrana milk goats. Acta Agric. Scand. Section A 58, 205–208.

Jenness, R., 1982. Inter-species comparison of milk proteins. In: Fox, P.F.(Ed.), Developments in Dairy Chemistry Proteins, vol. 1. Applied Sci-ence Publishers, New York, pp. 87–114.

Jung, A., Sippel, A.E., Grez, M., Schtitz, G., 1980. Exons encode functionaland structural units of chicken lysozyme. Proc. Natl. Acad. Sci. U.S.A.71, 5159–5763.

Kuhn, N.J., 1983. In: Metham, T.B. (Ed.), Biochemistry of Lactation. Elsevier,New York, pp. 159–176.

Laird, J.E., Jack, L., Hall, L., Boulton, A.P., Parker, D., Craig, R.K., 1988.Structure and expression of the guinea-pig alpha-lactalbumin gene.Biochem. J. 254, 85–94.

Lan, X.Y., Chen, H., Tian, Z.Q., Liu, S.Q., Zhang, Y.B., Wang, X., Fang, X.T.,2008. Correlations between SNP of LALBA gene and economic traitsin Inner Mongolian white cashmere goat. Yi Chuan. 30 (2), 69–74.

Lan, X.Y., Pan, C.Y., Chen, H., Zhang, C.L., Zhang, A.L., Zhang, L., Li, J.Y., Cei,

C.Z., 2007. An MspI PCR-RFLP detecting a single nucleotide polymor-phism at alpha-lactalbumin gene in goat. Czech J. Anim. Sci. 52 (5),138–142.

McKenzie, H.A., White, F.H., 1987. Studies on a trace cell lytic activ-ity associated with alpha-lactalbumin. Biochem. Int. 14, 347–356.

earch 82 (2009) 156–160

Neibergs, H., Dietz, A., Womack, J., 1993. Single-strand conformation poly-morphisms (SSCPs) detected in five bovine genes. Anim. Genet. 24,81–84.

Orita, M., Suzuki, Y., Sekiya, T., Hayashi, K., 1989. Rapid and sensitive detec-tion of point mutation and DNA polymorphisms using polymerasechain reaction. Genomics 5, 874–879.

Peters, C.W.B., Kruse, U., Pollwein, R., Grzeschik, K.H., Sippel, A.E., 1989.The human lysozyme gene: sequence organization and chromosomallocation. Eur. J. Biochem. 182, 507–516.

Qasba, P.K., Safaya, S.K., 1984. Similarity of the nucleotide sequences of therat x-lactaibumin and chicken lysozyme genes. Nature 308, 377–380.

Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Lab-oratory Manual, 2nd edn. Cold Spring Laboratory Press, Cold springHarbour, NY.

Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual,3rd edn. Cold Spring Laboratory Press, Cold spring Harbour, NY.

Sheffield, V., Beck, J., Kwitek, A., Sandstrom, D., Stone, E., 1993. The sen-sitivity of single-strand conformation polymorphism analysis for thedetection of single base substitutions. Genomics 16, 325–332.

Shewale, J.G., Sinha, S.K., Brew, K., 1984. Evolution of alpha-lactalbumins.The complete amino acid sequence of the alpha-lactalbumin froma marsupial (Macropus rufogriseus) and corrections to regions ofsequence in bovine and goat alpha-lactalbumins. J. Biol. Chem. 259(8), 4947–4952.

Thompson, M.P., Farrell Jr., H.M., Mohanam, S., Liu, S., Kidwell, W.R., Bansal,M.P., Cook, R.G., Medina, D., Kotts, C.E., Bano, M., 1992. Identificationof human milk �-lactalbumin as a cell growth inhibitor. Protoplasma167, 134–144.

Vilotte, J.L., Soulier, S., Mercier, J.C., Gaye, P., Hue-Delahaie, D., Furet,

J.P., 1987. Complete nucleotide sequence of bovine alpha lactalbumingene: comparison with its rat counterpart. Biochimie 69, 609–620.

Vilotte, J.L., Soulier, S., Printz, C., Mercier, J.C., 1991. Sequence of the goatalpha-lactalbumin-encoding gene: comparison with the bovine geneand evidence of related sequences in the goat genome. Gene 98 (2),271–276.