Gene 532 (2013) 140–145

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Short Communication

Polymorphism of BMP4 gene in Indian goat breeds differing in prolificacy

Rekha Sharma a,⁎, Sonika Ahlawat a, A. Maitra a, Manoranjan Roy b, S. Mandakmale c, M.S. Tantia a

a National Bureau of Animal Genetic Resources, Karnal 132001, Indiab West Bengal University of Animal & Fishery Sciences, Kolkata 700037, Indiac Mahatma Phule Krishi Vidyapeeth, Rahuri 413722, Ahmednagar, MS, India

Abbreviations: 3′ UTR, 3′ Un-Translated Region; BLASTool; BMP15, Bone Morphogenetic Protein 15; BMP4, BBMPR1B, Bone Morphogenetic Protein Receptor type 1Proteins; CDS, Coding DNA Sequence; DNA, DeoxyribDiamine Tetra Acetic acid; Fec, Fecundity; FSH, FollicleGrowth Differentiation Factor 9; ICAR, Indian CouncilMarker Assisted Selection; MgCl2, Magnesium ChloridBiotechnology Information; OD, Optical Density; PCR, PoRFLP, Polymerase Chain Reaction-Restriction Fragment LePolymerase Chain Reaction-Single Strand ConformatioNucleotide Polymorphism; TGF-β, Transforming GrowthPair Group Method with Arithmetic Mean.⁎ Corresponding author. Tel.: +91 9255482422; fax: +

E-mail address: rekvik@gmail.com (R. Sharma).

0378-1119/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.gene.2013.08.086

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 26 August 2013Available online 5 September 2013

Keywords:BMP4Indian goatMicrosatellitePolymorphismSNPsVariations

Bonemorphogenetic proteins (BMPs) aremembers of the TGF-β (transforming growth factor-beta) superfamily,of which BMP4 is the most important due to its crucial role in follicular growth and differentiation, cumulus ex-pansion and ovulation. Reproduction is a crucial trait in goat breeding and based on the important role of BMP4gene in reproduction it was considered as a possible candidate gene for the prolificacy of goats. The objective ofthe present study was to detect polymorphism in intronic, exonic and 3′ un-translated regions of BMP4 gene inIndian goats. Nine different goat breeds (Barbari, Beetal, Black Bengal, Malabari, Jakhrana (Twinning N 40%),Osmanabadi, Sangamneri (Twinning 20–30%), Sirohi and Ganjam (Twinning b 10%)) differing in prolificacyand geographic distribution were employed for polymorphism scanning. Cattle sequence (AC_000167.1) wasused to design primers for the amplification of a targeted region followed by direct DNA sequencing to identifythe genetic variations. Single nucleotide polymorphisms (SNPs) were not detected in exon 3, the intronic regionand the 3′ flanking region. A SNP (G1534A) was identified in exon 2. It was a non-synonymous mutationresulting in an arginine to lysine change in a corresponding protein sequence. G to A transition at the 1534locus revealed two genotypes GG and GA in the nine investigated goat breeds. The GG genotype was predomi-nant with a genotype frequency of 0.98. The GA genotype was present in the Black Bengal as well as Jakhranabreed with a genotype frequency of 0.02. A microsatellite was identified in the 3′ flanking region, only 20 nucle-otides downstream from the termination site of the coding region, as a short sequence with more than nineteencontinuous and repeated CA dinucleotides. Since the gene is highly evolutionarily conserved, identification of anon-synonymous SNP (G1534A) in the coding region gains further importance. To our knowledge, this is thefirst report of a mutation in the coding region of the caprine BMP4 gene. But whether the reproduction trait ofgoat is associated with the BMP4 polymorphism, needs to be further defined by association studies inmore pop-ulations so as to delineate an effect on it.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

India ranks second in the goat population of the world with morethan 140 million goats which constitute 25.6% of the country's totallivestock (Livestock census, 2007). Goats have been an integral compo-nent of the farming system and support a large rural population of

T, Basic Local Alignment Searchone Morphogenetic Protein 4;B; BMPs, Bone Morphogeneticonucleic acid; EDTA, EthyleneStimulating Hormone; GDF9,

of Agricultural Research; MAS,e; NCBI, National Centre forlymerase Chain Reaction; PCR-ngth Polymorphism; PCR-SSCP,n Polymorphism; SNP, SingleFactor β; UPGMA, Unweighted

91 184 2267654.

ights reserved.

landless and marginal farmers in India. Kidding/lambing percentage isthe most important factor affecting profitability in small ruminants.Hence a moderate increase in litter size can lead to a larger profit forthe rural poor. Goat (Capra hircus) is a highly diverse species withmore than 23 different domestic breeds in India that vary substantiallyin their physiological characteristics including ovulation rate and fecun-dity. How to fully exploit their multiple birth ability is the major goal ofresearch at this moment. The genetic basis of fecundity in sheep hasbeen extensively studied. Studies have revealed that the ovulation rateand litter size of domesticated sheep (Ovis aries) could be geneticallyregulated by a set of different genes, collectively named as Fec genes.Several genes affecting ovulation rate in sheep have been discoveredsince the first major gene FecB (Fecundity Booroola) had been detectedin 1980 (Davis, 2004). In recent years, many studies on the genetics ofprolificacy in sheep lead to highlight the importance of three majorgenes: BMPR1B, BMP15 andGDF9, which have been shown to affect ovu-lation rate and litter size through different mechanisms (Davis, 2004).However, mutations in the candidate genes associated with fecundityin sheep were not detected in Indian goats (Ahlawat et al., 2013).

141R. Sharma et al. / Gene 532 (2013) 140–145

None of these genes were associated with the fecundity of goats (Heet al., 2010; Hua et al., 2008). Thus, the genetic basis of caprine prolifica-cy remains to be explored. Improvement of reproduction by traditionalselective breedingmethods has proved to be difficult due to low herita-bility (Morris, 1990) and a long reproductive cycle. Thus there is a needto find out key mutations in the candidate genes, how these mutationsaffect reproduction and how to increase reproductive abilities such aslitter size, which will be a rapid and economic method to improve thespeed of goat breeding. The incorporation of amajor gene for prolificacyinto a flock using marker assisted selection (MAS) can allow increasedselection pressure on the traits leading to increased genetic gain(Davis, 2005). The major gene has the advantage that it can be intro-duced into any new breed while retaining the new breed's characteris-tics (Davis, 2005). Effective breeding programs exist in a number ofcountries, the largest one in Australia and New Zealand aiming for ge-netic improvement of fecundity trait (Van der Werf, 2006).

The process of ovarian folliculogenesis is composed of proliferationand differentiation of the constitutive cells in developing follicles(Silva et al., 2005). The number of mature oocytes released during onereproductive cycle and estrous is determined by a complex exchangeof endocrine signals between the pituitary gland and the ovary as wellas the paracrine and autocrine signals within ovarian follicles involvingthe oocytes and its adjacent somatic cells (Knight andGlister, 2003). Lit-tle information is available on the local factors that regulate this processin goats. Bonemorphogenetic proteins (BMPs) aremembers of the TGF-β (transforming growth factor-beta) superfamily, which is a multifunc-tional cytokine and is expressed in a variety of cells (Wozney et al.,1988). More than thirty members have been identified in the BMP fam-ily of which BMP4 is themost important. The crucial role of BMP4 in fol-licular growth and differentiation, cumulus expansion and ovulation hasbeen established. BMP4 expression is very high in healthy follicles butbarely detectable in follicles undergoing atresia, which can act directlyon granulosa cells and cause important changes in FSH (follicle stimulat-ing hormone) action (Shimasaki et al., 1999). BMP4 can inhibit proges-terone production by granulosa cells and decrease basal granulosa cellprogesterone secretion and totally abolish FSH-stimulating action bothin cattle and sheep (Juengel et al., 2006). So, BMP4 could have implica-tions for reproductive functions in mammals.

Based on the important role of BMP4 gene in reproduction, BMP4gene was considered a possible candidate gene for the prolificacy ofgoats. The objective of the present study was to detect polymor-phism in coding regions, intronic (partial intron 2) and partial 3′untranslated region (3′ UTR) of BMP4 in a panel of Indian goatbreeds for future association with prolificacy of goats. Nine indige-nous goat breeds (Barbari, Beetal, Black Bengal, Malabari, Jakhrana(Twinning N 40%), Osmanabadi, Sangamneri (Twinning 20–30%),Sirohi and Ganjam (Twinning b 10%)) differing in prolificacy andgeographic distribution were employed for polymorphism scanning.

Table 1Characteristics of Indian goat breeds investigated in the study.

Phenotype Breed Geographicaldistribution

Twinningpercentage

High prolificacy Beetal Punjab N40Barbari Uttar PradeshBlack-Bengal West Bengal, Bihar, JharkhandMalabari KeralaJakhrana Rajasthan

Medium prolificacy Osmanabadi Maharashtra 20–30Sangamneri Maharashtra

Low prolificacy Ganjam Orissa b10Sirohi Rajasthan

2. Materials and methods

2.1. Sample collection

To explore the genetic variations within BMP4 gene, blood samplesfrom a panel of Indian goat breeds were used. The panel included ninebreeds (Table 1) based on phenotype (prolificacy), geographical distri-bution (Fig. 1) and genetic diversity. The blood samples (100) were col-lected from the jugular vein into EDTA containing vacutainer tubes from12 animals of Black Begal goat breed and eleven animals each of Barbari,Beetal, Malabari, Jakhrana, Osmanabadi, Sangamneri, Sirohi and Ganjamgoat breeds. GenomicDNAwas isolated andpurified from theblood cellsusing standard phenol–chloroform–isoamyl alcohol extraction followedby ethanol precipitation (Sambrook and Russell, 2001). The quality andquantity of isolated DNA was determined using agarose gel electropho-resis (0.8%) and Nanodrop spectrophotometer (GE Healthcare). A single

band on agarose gel and 1.8–2 OD 260/280 ratio for all the samples indi-cated the recommended quality of purified DNA.

2.2. Primer designing

Three pairs of primers were designed to amplify the goat BMP4gene based on the gene sequence of cattle (GenBank accession no.AC_000167.1). These included one pair of primer designed forexon 2 and two overlapping oligonucleotide primers designed toamplify exon 3 along with upstream and downstream 3′ UTRs,using PRIMERSELECT program of LASERGENE software (DNASTARInc., Madison, WI, USA). Primer sequence, amplified region andproduct size were listed in Table 2.

2.3. PCR amplification

Polymerase chain reaction (PCR) was carried out in a final reactionvolume of 25 μl on i-cycler (BIO-RAD, USA). PCR cocktail consisted of50 to 100 ng of genomic DNA, 200 μM of each dNTP, 50 pM of eachprimer, 0.5 units of high fidelity Taq DNA polymerase and Taq bufferhaving 1.5 mMMgCl2 for each reaction. The PCR reaction cycle was ac-complished by denaturation for 1 min at 94 °C; 30 cycles of denatur-ation at 94 °C for 45 s, annealing at specific temperature for 45 s,extension step at 72 °C for 45 s with a final extension at 72 °C for5 min. The PCR products were visualized following electrophoresisthrough a 1.8% agarose gel.

2.4. Sequencing and analysis

The amplified region was sequenced using ABI 3100 (AppliedBiosystems, USA) Automated DNA Sequencer. Prior to sequencing, PCRproducts were purified by enzymatic method using Exonuclease I andAntarctic Phosphatase (New England Biolab, USA). Raw sequence datawere edited using Chromas (Ver. 1.45, http://www.technelysium.com.au = chromas.html). Multiple sequence alignments were performedwithMegAlign programof LASERGENE software version 5.07 (DNASTARInc., Madison, WI) to identify polymorphisms (mutations or Single Nu-cleotide Polymorphisms, SNPs) in different regions of the BMP4 genein indigenous goats. Samples with variants were also sequenced withthe reverse primer to confirm these findings. Polymorphic sites identi-fied by sequence comparisonwere further confirmed bymanual inspec-tion of chromatograms. The coding DNA sequences of different exonicregions were conceptually translated to amino acid sequences usingChromaspro software. The BLAST algorithm was used to search theNCBI Genbank (http://www.ncbi.nlm.nih.gov) databases for homolo-gous sequences. Phylogenetic analysis was performed with CLC MainWorkbench version 5.0.2.

3. Results and discussion

The size of a goat BMP4 gene is 3283 bp (EU104684.1) ofwhich exon2 (1085…1464) and exon 3 (2427…N3283) are translated. DNA of nine

Fig. 1. Geographical distribution of selected goat breeds.

142 R. Sharma et al. / Gene 532 (2013) 140–145

goat breeds was successfully amplified using three pairs of primers forBMP4 gene. Sequenced regions of the gene included exonic, intronicand 3′ UTR. The results showed that the amplification fragment sizeswere consistent with the target ones. Sequences have been entered inthe NCBI GenBank database (http://www.ncbi.nlm.nih.gov/genbank/)under the following accession numbers: exon2 (KC571785.1) andexon3 (KC571786.1). The polymorphic sites were explored in the ninedifferent goat breeds including the high fecundity and low fecunditygoat breeds such as Black Bengal, a meat-type Indian goat breed, whichis famous for its quality lean meat production and well known for givingmultiple births with a prolificacy of above 180% (Zeshmarani et al., 2007)and Ganjam goat a low prolificacy, late maturing, annual single kiddingand slow growing goat (Acharya, 1982).

3.1. Sequence analysis of exonic regions

Sequences of Indian goat exons 2 and 3 were compared with goat(EU104684.1), sheep (EU183348.1), as well as cattle (AC_000167.1).No variations were recorded in exon 2 of Indian goat BMP4 gene(Table 3). Three variations in exon 3 were observed in Indian goat as

Table 2Oligonucleotide primers for the caprine BMP4 gene.

Primer Oligonucleotide sequence (5′ to 3′) Product size (bp)

BMP2 F-ATTTATTCTTTACCCTTCCACCTCR-AACTCCTCGCCTTCCCACAG

482

BMP3.1 F-TTCACTACTCATCACCCAACTCTTR-GTCATTCCAGCCCACATCACT

715

BMP3.2 F-GTAGCCCCAAGCATCACCCACAGR-CCCCAACCCCACCTTAACAGAAAA

724

a Corresponding to cattle sequence (accession no. AC_000167.1).

compared to Huanghuai goat of China. Whereas, five variations wererecorded in Indian goat BMP4 gene as compared to sheep which in-cluded deletions of two consecutive nucleotides. The maximumnumber of nucleotide changes (12) was recorded in comparison tocattle (AC_000167.1) of which three variations were in intron 2, 6were in exon 3 and 3 were in 3′ UTR of the gene (Table 3).

BLAST analysis was carried out to find the percentage homology inthe coding region of Indian goat BMP4 gene at the nucleotide levelwith other species. Exon 2 of Indian goat exhibited 100% similaritywith the corresponding sequences of exotic goat (Huanghuai goat)(EU104684.1), sheep (EU183348.1) as well as cattle (AC_000167.1).Exon 3 DNA sequences of BMP4 gene revealed a homology of 95% ofIndian goat sequence with Bos taurus (AC_000167.1) and 99% with O.aries (EU183348.1) and C. hircus (EU104684.1). Homology of BMP4 se-quences among sheep and goat are especially higher, which coincideswith the fact that these are evolutionarilymore related. A similar picturewas depicted on phylogenetic analysis of BMP4 gene following aUPGMA algorithm. Phylogenetic tree (Fig. 2) revealed that Indian goatand exotic goat were closer to each other and formed a distinct cluster.Sheep being a small ruminant was closer to goats than cattle (large

Amplified regiona (location) Annealing temperature (°C)

Exon2–partial intron2(1817–2298)

57

Partial intron2–partial Exon3(3082–3796)

57

Partial exon3–3′ UTR(3699–4422)

56.8

Table 3Variations in nucleotide of Indian goat BMP4 gene as compared to goat, sheep and cattle.

Region Position as per goat(EU104684.1)

Goat(EU104684.1)

Indian goat(KC571785, KC571786)

Sheep(EU183348.1)

Cattle(AC_000167.1)

Exon 2 1354 G G/A G GIntron 2 1474 – – – T

2373 G G G T2384 T T T –

2406–07 – – TT –

Exon 3 2506 T T T C2572 G G G T2686 A A A G2802 A G G G2887 C C T T2918 C C C A2980 C C T C3058 G G G A3262 A G G G3274 T G G G

3′ UTR 3462⁎ NA C C T3472⁎ NA T NA C3509⁎ NA A NA C

⁎ Corresponding to cattle sequence (accession no. AC_000167.1).

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ruminant) whereas, ruminants (Bovidae family) were distant fromhuman (Hominidae family) and rat (Muridae family) were most distantas expected.

3.2. SNP identification in exonic regions

SNPs are widely used in linkage analysis and for evaluation of vari-ability in natural populations, due to their robustness in laboratory han-dling and data interpretation. Comparison of BMP4 gene sequencesfrom 100 samples of nine Indian goat breeds resulted in identificationof a SNP (G1354A) in a coding region (exon 2) of the gene (Fig. 3).The numbering of SNP is with respect to Huanghuai goat sequence(EU104684.1). This is the first report of polymorphism in the exonic re-gion of BMP4 gene in goats. It is a non-synonymous mutation (AGA toAAA) resulting in a change of amino acid from arginine to lysine at the87th position of a corresponding 124 amino acid protein sequencefrom exon 2. Exon 3, intron 2 and 3′ UTR did not display polymorphismin the investigated Indian goats. These results are in concordance withthe fact that BMP4 is one of the best evolutionary conserved growth fac-tors (Winnier et al., 1995). Hence there are few reports of SNPs in BMP4gene.

The first polymorphic site (147ANV) of BMP4 gene was found byHphI enzyme digestion which presented with similar frequency inCaucasian, Hispanic, and African populations (Mangino et al., 1999).Ramesh Babu et al. (2005) detected a major haplotype defined by G-C-T allele in SNPs −5826GNA, 3564CNT and 6007CNT, respectively ofBMP4 gene in postmenopausal women. Information regarding goatare scanty. Fang et al. (2010) for the first time described polymorphismin the BMP4 gene of goats. They could not detect polymorphismwithinthe coding region of three different breeds (Boer, Xuhuaiwhite goat andHaimen goat) reared in China whereas, two new SNPs (EU104684:g.1986ANG, 2203GNA) were identified in the intronic region. Chu et al.(2010) executed experiments to detect SNPs of exon 2 and intron 2 ofBMP4 gene in both high fecundity (Jining Grey goat) and low fecunditygoat breeds (Boer, Angora and Inner Mongolia Cashmere goats) by sin-gle strand conformation polymorphism. Polymorphismwas not detect-ed for exon 2 (primer P1) of BMP4 gene in four goat breeds. For intron 2,three genotypes (AA, AB and BB)were detected in JiningGrey and Inner

Fig. 2. Phylogenetic analysis of BMP4 coding DNA sequence of different species followin

Mongolia Cashmere goats, two genotypes (AB and BB) in Angora goats,and only one genotype (AA) in Boer goats. Sequencing revealed onemutation (2203GNA) of BMP4 gene in the genotype BB in comparisonto the genotype AA. The differences of litter size between AA, AB andBB genotypes were not significant (P N 0.05) in Jining Grey goats.

Chu et al. (2008) found a SNP (305CNA) in exon 3 of BMP4 gene ofSmall Tail Han, Chinese Merino, Corriedale and South African MuttonMerino sheep, which resulted in an amino acid change of alanine toaspartic acid. The Small Tail Han ewes with genotype BB had0.61(P b 0.05) or 1.01 (P b 0.05) lambs more than thosewith genotypeAB or AA. This mutation had an additive effect for litter size in Small TailHan sheep. Polymorphism (G1354A) identified in the present study ofIndian goats is the first report of a SNP in exon 2 of caprine BMP4gene. Identification of novel SNP in Indian goats may be attributed tothe fact that goat population presents a different molecular spectrumdue to biodiversity. Diversity and presence of unique SNPs reflect theantiquity of the Indian goat population. G to A transition at 1354 locusrevealed two genotypes GG and GA in nine investigated Indian goatbreeds (Fig. 3). GG genotype was predominant with a genotype fre-quency of 0.98. GA genotype was present in one animal each of BlackBengal as well as Jakhrana breed with genotype frequency of 0.02. NoAA genotype was detected among the hundred samples of differentgoat breeds. It has been reported that this gene is highly evolutionarilyconserved, so identification of non-synonymous SNP (G1534A) in acoding region gains further importance. The presence of A allele in thehigh prolific groups only, such as the Black-Bengal, the most prolificgoat breed of India (Zeshmarani et al., 2007),makes thismutation an in-teresting site for further exploration with reference to prolificacy asso-ciated markers. In the present study no AA was identified across thepanel of goat breeds. The reasonsmay be: i) A allele is rare, ii) AA geno-type frequency is so low that homozygous AA could not be detected in100 goats, and iii) homozygous AA suffered from developmental orreproductive anomalies. It is notable that non-synonymous SNPs,which have lower frequency, are more likely to be population-specific(Halushka et al., 1999). Our results have two major implications forSNP discovery and applications. First, polymorphism detection in di-verse samples can increase the yield of SNPs; consequently, a mixedsample is ideal for SNP discovery. Secondly, conserved genes like

g UPGMA algorithm (branch number represent bootstrap value in 1000 replicates).

Fig. 3. Genotype of novel SNP (G1354A) in exon 2 of BMP4 gene in Indian goats (arrowpointed to the mutation site).

144 R. Sharma et al. / Gene 532 (2013) 140–145

BMP4 are under strong selective pressure and hence are expected to de-pict a lower frequency of nucleotide sequence polymorphism.

Due to the small size of the samples analyzed in this study, there is aneed to undertake further research on a substantially larger number ofthe population to reach some logical conclusion. An enzyme restrictionsite which recognizes the novel SNP in exon 2 has been identified usingNEB cutter V2.0 (Vincze et al., 2003). The polymorphism (G1354A) ofBMP4 gene can be detected by restriction fragment length polymor-phism (RFLP) as G to A transversion changes the recognition site of re-striction endonuclease PshAI. Since PCR-RFLP method is efficient, lowcost, reproducible and convenient for laboratories with a limited levelof technology worldwide, it should be useful for researchers willing towork with genetic markers in goats, genotyping in case–control associ-ation or population genetic studies.

3.3. Microsatellite in 3′ UTR

In the present study, a microsatellite was found. Sequencing analysissuggested that there existed a short sequence with nineteen or morethan nineteen continuous CA dinucleotide repeats starting at 20 bpdownstream from the termination site of the coding region (Fig. 4).

Fig. 4. Chromatogram depicting microsatellite (C

Fang et al. (2010) and Chu et al. (2010) have also described polymor-phism in the 3′ flanking region of goat BMP4 gene that contained a dinu-cleotide repeated sequence (CA). The lengths of repeated sequence (CA)were 24, 17 and 14, respectively and microsatellite locus had rich poly-morphism (Fang et al., 2010). Chu et al. (2010) described polymorphismin the four goat breeds by microsatellite analysis too. For primer P3,three genotypes (CC, CD and DD) were detected in four goat breeds. Se-quencing revealed onemore CA dinucleotide in genotype DD than in ge-notype CC. The CA repeats were 19 and 20 in CC and DD genotypes. TheJining Greywith genotype CC had 0.55 (P b 0.05) or 0.72 (P b 0.05) kidsmore than thosewith genotype CD or DD. These results preliminarily in-dicated that allele C of BMP4 gene is a potential DNAmarker for improv-ing litter size in goats. It is important tomention that allele C (CA19) wasidentified in the Indian goat breeds too. Moreover, the functional signif-icance of microsatellites cannot be ignored as numerous microsatelliteshave been proposed as hotspots for recombinations. Somemicrosatellitesequences may influence recombination directly by their effects on DNAstructure. It has been proposed that GT, CA, GT, GA, GC or AT repeat-binding proteins could participate in the recombination process by in-ducing Z-conformation or another alternative secondary structure(Biet et al., 1999; Karlin et al., 1998). Additionally the microsatellitemay be in linkage disequilibrium with variations in other regions of thegene with functional or structural significance (Karlin et al., 1998). Asthe identified microsatellite is located in the 3′ flanking region of exon3, it may also have the ability to affect the structure of mRNA and trans-lation efficiency (De Smit and van Duin, 1994). In humans (GenBank ac-cession no. NM_001202.3), mice (GenBank accession no. NM_007554.2),horse (GenBank accession no. XM_001494966.2), sheep (GenBank ac-cession no. EE851370.1) and goat,without exception, there is amicrosat-ellite of the dinucleotide-repeated (CA) sequence in the 3′ flankingregion of the BMP4 gene, andmaybe this can imply the functional signif-icance of the microsatellite. Thus identification of a similar CA repeat inIndian goat becomes very important and further investigation shouldbe directed towards detecting polymorphism of this microsatellitelocus of BMP4 gene in random animals of a larger number of differentgoat breeds.

4. Conclusions

The polymorphic site in goat BMP4 gene has been described so far inintronic (intron 2 of exotic goat) and 3′ flanking regions of BMP4 gene.To our knowledge, this is the first report of mutation (G1354A) in thecoding region of caprine BMP4 gene. Amicrosatellite has been identifiedin the 3′ flanking region of the BMP4 gene. Due to the crucial role ofBMP4 in follicular growth and differentiation, cumulus expansionand ovulation, further studies should be performed in order to vali-date the association and/or physiological significance of novel SNPsand microsatellites identified in the present study with more numberof breeds aswell as individuals of goats. The present studywill be signif-icant in applying this in future directions and augment production ofthe goat-based industry.

A)n site in 3′ flanking region of BMP4 gene.

145R. Sharma et al. / Gene 532 (2013) 140–145

Conflict of interest

There is no conflict of interests.

Acknowledgments

This work was financially supported by the Network Project on An-imal Genetic Resources (ICAR).

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