Indian Journal of Biotechnology

Vol 11, July 2012, pp. 274-279

ARMS-PCR as an alternative, cost effective method for detection of

FecB genotype in sheep

S R Saste1*, P M Ghalsasi

1, R S Kataria

2, B K Joshi

2, B P Mishra

2 and C Nimbkar

1

1Animal Husbandry Division, Nimbkar Agricultural Research Institute, Phaltan 415 523, India 2National Bureau of Animal Genetic Resources, Karnal 132 001, India

Received 4 January 2011; revised 21 July 2011; accepted 12 September 2011

Authors have standardized and report here a simplified amplification refractory mutation system (ARMS)-PCR test as

an alternative technique to PCR-RFLP for detection of FecB genotype in sheep. An ARMS-PCR test to detect the FecB

mutation in Black Bengal goats has recently been described in the literature. A number of modifications to this technique

has been made to simplify and standardize the protocol to use it for sheep DNA, isolated from blood samples on FTA paper.

The modifications included reducing the PCR reaction volume to half of original protocol, using a single touchdown

annealing temperature and combining two PCR amplicons amplified with different primer pairs (one for each allele of

FecB) for each sample prior to loading on the gel. These modifications can make the technique cheaper and more suitable

for large scale, rapid FecB genotyping in sheep for use in FecB introgression breeding programmes.

Keywords: ARMS-PCR, FecB, genotyping, PCR-RFLP, sheep

Introduction The FecB (BMPR1B) mutation increases ovulation

rate in sheep (Ovis aries)1-3

. BMPR1B is expressed in

oocytes and granulosa cells. It is a point mutation

A746G (Q249R), which occurs on sheep chromosome

number 6, and constitutes a change from the normal

or wild type allele encoding glutamine (Q) to that

encoding arginine (R) in FecB carriers1-3

. The

Nimbkar Agricultural Research Institute (NARI) at

Phaltan, Maharashtra, has introgressed the FecB gene

from the Garole breed of Sunderban, West Bengal,

into the local Deccani breed to increase ewe

productivity and incomes of smallholder shepherds.

Garole sheep, the probable original source of the

FecB gene, are small-sized (average adult live weight

15 kg) and have a reported mean litter size of 2.27 in

the Sundarban region but have a lower mean litter size

of 1.74 in the semiarid environment of the Deccan

plateau of Maharashtra4. The Deccani are the native

sheep of the semi-arid Deccan plateau and adult ewes

weigh about 27 kg. However, the reproductive

performance of Deccani is low, with an average litter

size of 1.044. The improved Awassi dairy sheep strain

from Israel, which was available at NARI was also

used for crossbreeding to improve milk yield of

prolific ewes, lamb weight and growth rate5. Two

fecund strains were developed at NARI—the NARI

Suwarna with contributions from only Garole and

Deccani breeds, and the NARI Composite with

additional contribution from Awassi breed. Garole

breed percentage was kept below 25% so that the

FecB gene carrier ewe would have a phenotype

similar to the Deccani. One copy of FecB led to an

increase in ovulation rate from 1.1 to 2.1 eggs and an

increase in live litter size at birth from 1.0 to 1.6 in

the NARI flock and from 1 to 1.4 in smallholder

flocks6.

The test usually used to detect the genotype of

sheep at the FecB locus is a modified form of the

PCR-RFLP test described by Wilson et al3. In

PCR-RFLP, incomplete restriction enzyme digestion

may lead to errors in genotyping. An ARMS-PCR test

has been described by Polley et al7 to detect the FecB

genotype of Black Bengal goats. They used specific

primers designed around the mutation locus (A746G)

according to the principle of tetra-primer ARMS-PCR

to test genomic DNA of 88 Black Bengal goats. This

paper describes the modifications made to the Polley

et al7 ARMS-PCR protocol to detect the FecB

genotype of sheep and make it more suitable and

cheaper for large-scale genotyping in a breeding

programme.

_______________ *Author for correspondence:

Tel: + 91-2166-262106/200783; Fax: + 91-2166-262106

Email: sonali_gene@yahoo.com

SASTE et al: DETECTION OF FecB GENOTYPE IN SHEEP BY ARMS-PCR

275

Materials and Methods

Experimental Animals

A total of 35 ewes of 3 breeds and their crosses

were genotyped at the FecB locus in this study. They

were Garole (n=5), Deccani (n=5), Awassi (n=5) and

crosses (n=20) of Garole, Deccani and Awassi with

varying proportions of the three breeds. Crossbred

ewes comprised of two fecund strains, viz., NARI

Suwarna (n=10) and NARI Composite (n=10) ewes.

They had 12.5-25% contribution of Garole breed,

0.0-26.6% contribution of Awassi breed and

75-87.5% contribution of Deccani breed in their

breed composition. The mean litter size of Garole

ewes was 1.79±0.15, while the mean litter sizes for

Deccani, Awassi ewes and Crossbred ewes were

1.03±0.0, 1.12±0.13 and 1.69±0.10, respectively.

The number of ewes of different breeds, their

average number of lambings, and range and average

of litter size are given in Table 1. These ewes were

not randomly selected from the population. Instead,

they were from the breeding flocks maintained at

NARI. In these flocks, ewes of known FecB

genotypes were mated to sires of known FecB

genotypes. Genotyping using a DNA test is

necessary to ascertain the FecB genotype of

crossbred ewes to be used for further breeding.

DNA Isolation and PCR Amplification

Blood samples from the 35 ewes described above

were collected on Whatman FTATM

Classic Cards and

DNA was extracted using the alkaline lysis method8.

The PCR reaction volume for the ARMS-PCR test

was reduced to half of that used by Polley et al7 and

PCR amplification (12.5 µL final volume) was

performed using 1.25 µL thermophilic DNA

polymerase 10× buffer, 200 mM dNTPs, 25 mM

MgCl2, 10 pmol of each primer, 0.5 units of Taq DNA

polymerase (GeneiTM

) and 5 µL of the DNA extracted

from FTA punches. Two separate PCR reactions, one

with mutant allele primers and one with wild type

allele primers, were carried out in a single PCR run

for each DNA sample.

Primer Sequences

The sequences of primers7 used in the present study

are given below:

Wild type allele primers

Forward 82-GCTGGTTCCGAGAGACAGAAATA

TATCA-109

Reverse 1178-CCCCGTCCCTTTGATATCTGCAG

CAATG-1151

Mutant allele primers

Forward 1-GTCGCTATGGGGAAGTTTGGAT

GGGAA-27

Reverse 136-ATGTTTTCATGCCTCATCAACA

CCGTCC-109

A single touchdown PCR procedure was used in

contrast to the two different touchdown temperatures

for mutant and wild type allele primers as described

by Polley et al7. PCR amplification was carried out

at 94ºC for 4 min, followed by 30 cycles of

denaturation at 94ºC for 30 sec, annealing

touchdown from 60°C to 52°C (8 cycles) with

remaining cycles at 52°C for 45 sec, and extension at

72ºC for 30sec, followed by a final extension at 72ºC

for 6 min. The PCR products amplified with wild

type and mutant allele primers for each sample were

then pooled in a single tube, mixed together and

loaded in a single well on the gel. The amplified

products were electrophoresed for 90 min on a 1.5%

agarose (Ameresco) gel at 150 V. The gel was

stained with ethidium bromide solution (0.5 µg/mL)

and photographed using a molecular Gel imager

(Gel Doc, Star Micronics).

Validation of Genotypes Obtained with ARMS-PCR

To validate the accuracy of genotyping by

ARMS-PCR, conventional PCR was carried out,

followed by a forced RFLP test3. This method uses a

primer that introduces a mismatch so that amplified

PCR product of a mutant allele will contain an AvaII

restriction site. The FecB (BMPR-1B) allele will

produce a fragment of a different length from a wild

type allele, which can be detected by sizing the

fragments on an agarose gel. Each PCR reaction was

Table 1—Ewes genotyped at the FecB locus

Ewe breed/

cross

No. of

ewes

(n)

Av. no. of

lambings1

Av. litter

size1

Range

of litter

size

Garole 5 3.80±0.58 1.79±0.15a* 1.0-3.0

Deccani 5 6.00±0.32 1.03±0.00b 1.0-2.0

Awassi 5 2.00±0.71 1.12±0.13b 1.0-2.0

Crossbred2 20 4.15±0.33 1.69±0.10a 1.0-3.0

1Values are expressed as mean±standard error of the mean 2Crossbred ewes had 12.5-25% contribution of Garole breed,

0.00-26.6% contribution of Awassi breed and 75-87.5%

contribution of Deccani breed in their breed composition *Values with different superscripts differed significantly

(P<0.05)

INDIAN J BIOTECHNOL, JULY 2012

276

carried out in a total volume of 8 µL, containing

0.8 µL True Hot Start Taq DNA polymerase PCR

buffer, 200 mM dNTPs, 25 mM MgCl2, and

7 pmol each of the following primers: 5′-

GTCGCTATGGGGAAGTTTGGATG-3′ and 5′-

CAAGATGTTTTCATGCCTCATCAACACGGTC-3′;

and 0.5 units of True Hot Start Taq DNA

polymerase (Fermentas). The fragments were

amplified using 34 cycles at 94ºC for 15 sec, 60ºC

for 30 sec, and 72ºC for 30 sec, followed by 72ºC

for 5 min. The 8 µL PCR product was digested with

AvaII enzyme (Fermentas) at 37ºC for 3 h and

electrophoresed on a 3% high resolution blend

agarose (Ameresco) gel. The gel was stained with

ethidium bromide solution (0.5 µg/mL) and

photographed using a molecular Gel imager

(Gel Doc, Star Micronics).

Results and Discussion The ARMS-PCR amplifications yielded two

bands of mol wt 1100 bp (wild type allele product)

and 136 bp (mutant allele product). Concordant

results were obtained with PCR-RFLP test and two

bands of mol wt 140 bp (wild type allele product)

and 110 bp (mutant allele product) were seen on the

gel. FecB genotypes of all 35 tested ewes are given

in Table 2. Genotyping revealed that out of five

Garole ewes, four were homozygous and one ewe

was heterozygous for the FecB (BMPR1B)

mutation. The FecB mutation was absent in the

5 Deccani and 5 Awassi ewes. Out of 20 crossbred

ewes 11 were heterozygous and 9 were

homozygous for the FecB (BMPR1B) mutation.

The electrophoresis pattern showed that both the

ARMS-PCR and PCR-RFLP assays gave the same

genotyping results (Figs 1 & 2). The genotypic and

allelic frequencies at FecB locus in 35 tested ewes

were calculated and are presented in Table 3. The

homozygous mutant genotype (FecBB/FecB

B) was

found to be the predominant genotype in Garole

sheep. While in Deccani and Awassi sheep, the

homozygous wild type genotype (FecB+/FecB

+)

was found to be the predominant genotype. In

Crossbred sheep both homozygous mutant

(FecBB/FecB

B) and heterozygous (FecB

B/FecB

+)

genotypes were found.

Table 2—FecB genotypes of ewes genotyped using an

ARMS-PCR test and validated using a PCR-RFLP test

Ewe breeds/

crosses

FecBB/

FecBB

FecBB/

FecB+

FecB+/

FecB+

Total

Garole 4 1 0 5

Deccani 0 0 5 5

Awassi 0 0 5 5

Crossbred 11 9 0 20

Total 15 10 10 35

Table 3—Allele and genotype frequency of the 35 ewes genotyped at the FecB locus

Allele frequency Genotype frequency Gene

mutation

Sheep breed/

crosses

No. of

animals FecB+ FecBB FecBB/FecBB FecBB/FecB+ FecB+/FecB+

Garole 5 0.10 0.90 0.80 0.20 0.00

Deccani 5 1.00 0.00 0.00 0.00 1.00 BMPR1B

(FecBB)

Awassi 5 1.00 0.00 0.00 0.00 1.00

Crossbred 20 0.23 0.77 0.55 0.45 0.00

Fig. 1—ARMS-PCR genotyping of three different breeds and their crosses in 1% agrose at FecB locus [Lanes 1-10: Amplification of

only wild type allele (W=1100 bp product; lanes 1-5, Deccani ewes; Lane 6-10, Awassi ewes); lanes 11-13, 15-20 & 33: Amplification of

both W and mutant alleles (M=136 bp product; lanes 11-13 & 15-20, Crossbred ewes; lane 33, Garole ewe); lanes 14, 21-27, 28-32 & 34-

35: Amplification of only mutant allele (lanes 14, 21-27 & 28-30, Crossbred ewes; lanes 31-32 & 34-35, Garole ewes); lanes FC: Positive

controls with FTA paper extracted DNA of mutant homozygous, heterozygous and wild type homozygous sheep, respectively; lanes GC:

Positive controls with genomic DNA of homozygous mutant, heterozygous and homozygous wild-type sheep, respectively; lanes N: PCR

cocktail without genomic DNA (template negative control); & lanes M: DNA mol wt marker (GeneiTM Low Range DNA Ruler)].

SASTE et al: DETECTION OF FecB GENOTYPE IN SHEEP BY ARMS-PCR

277

Most Indian sheep produce a single offspring in a

lambing except Garole sheep from West Bengal and

Kendrapada (Kuzi) sheep from Orissa9,10

. In the

present study, genotyping of 35 ewes at the FecB

locus revealed that FecB mutation was present in

the tested Garole and crossbred animals and it was

absent in Deccani and Awassi animals. Davis et al9

used the conventional PCR-RFLP method for FecB

genotyping developed by Wilson et al3 and reported

the presence of FecB mutation in Garole ewes.

Deccani is a non-prolific sheep with average litter

size 1.04; hence, it is not expected that the mutation

would exist in Deccani ewes. Pardeshi et al11

genotyped four Indian breeds of sheep, viz., Garole,

Deccani, Bannur and Madras Red, using the

PCR-RFLP technique and reported that FecB gene

was absent in Deccani, Bannur and Madras Red

sheep, while it was present in Garole sheep. The

average litter size of Awassi ewes in Bedouin and

Fellahin Awassi flocks was not more than 1.03,

with less than 5% of the adult ewes giving birth to

twins12

. Gootwine et al

13 reported the average litter

size of 1.28 in FecB non-carrier (FecB++

) Awassi

ewes from Israel. The absence of FecB mutation in

Deccani and Awassi breeds, thus, agreed with

earlier published results.

Though the PCR-RFLP method is the common

test for genotyping of single nucleotide

polymorphisms, tests like ARMS-PCR can be

developed and used as an alternative for validation

of results obtained by conventional genotyping

methods. In principle, ARMS tests can be developed

for any mutation and the technique has already been

applied to the detection of several genetic

polymorphisms. These include bovine acyl-

CoA:diacylglycerol acyltransferase 1 K232A

polymorphism in cattle14

, alpha-lactalbumin

polymorphism in bovine milk protein15

, tumor

necrosis factor (TNF) gene, interleukin 6 (IL 6),

ApaI and TaqI polymorphisms of the vitamin D

receptor gene (VDR) in humans16

and angiotensin

receptor 1 (AGTR1) gene polymorphisms in

humans17

. Duta-Cornescu et al16

concluded that

ARMS-PCR method is the most adequate for

detecting the alternative genotypes determined by

single base mutations.

Use of DNA isolated from blood samples on

FTA paper instead of genomic DNA and use of the

ARMS-PCR technique both reduced the time and

cost of the FecB genotyping test (FTA paper DNA

extraction: Rs. 62 per sample; Genomic DNA

extraction : Rs. 125 per sample). The use of FTA

paper for sample collection made the genotyping

simpler and faster than whole blood collection as

DNA could be extracted from 96 blood samples in

2 h. In authors’ laboratory, the modified ARMS-

PCR test (modifications listed in Table 4) resulted

in a 30% cost reduction as compared to the PCR-

RFLP test (ARMS-PCR: Rs. 141 per sample; PCR-

RFLP: Rs. 200 per sample) because of fewer

chemicals needed to be used and less time required

(Table 5). The ARMS-PCR test is also more

suitable as it reduces false negative results due to

incompletely digested PCR products, which can be

a problem with the PCR-RFLP test. The PCR-RFLP

method is more laborious, needing more

Fig. 2—PCR-RFLP genotyping of three different breeds and their crosses in 3% agarose at FecB locus [Lanes 1-10: Amplification of

wild type 140 bp product (lanes 1-5, Deccani ewes; Lane 6-10, Awassi ewes); lanes 11-13, 15-20 & 33: Amplification of heterozygous

genotype having both 140 bp and 110 bp products (M=110 bp product; lanes 11-13 & 15-20, Crossbred ewes; lane 33, Garole ewe); lanes

14, 21-27, 28-32 & 34-35: Amplification of mutant type 110 bp product (lanes 14, 21-27 & 28-30, Crossbred ewes; lanes 31-32 & 34-35,

Garole ewes); Lanes FC: Positive controls with FTA paper extracted DNA of homozygous mutant, heterozygous and homozygous

wild-type sheep, respectively; lanes GC: Positive controls with genomic DNA of mutant homozygous, heterozygous and wild type

homozygous sheep, respectively; lanes N: PCR cocktail without genomic DNA (template negative control); & lanes M: DNA mol wt

marker (GeNeiTM PhiX 174 DNA/HaeIII Digest).

INDIAN J BIOTECHNOL, JULY 2012

278

optimization steps, reagents and human handling,

which increases the cost.

Acknowledgement The present work was carried out under the project

‘Increasing profitability of sheep production by

genetic improvement using the FecB (Booroola)

mutation and improved management’ funded by the

Department of Biotechnology, Government of India,

New Delhi. Authors acknowledge Dr Sachinandan De

for his valuable suggestions during standardization of

FecB ARMS-PCR technique.

References 1 Mulsant P, Lecerf F, Fabre S, Schibler L, Monget P et al,

Mutation in bone morphogenetic protein receptor-IB is

associated with increased ovulation rate in Booroola

Merino ewes, Proc Natl Acad Sci USA, 98 (2001)

5104-5109.

2 Souza C J H, MacDougall C, Campbell B K, McNeilly A S,

Baird D T et al, The Booroola (FecB) phenotype is associated

with a mutation in the bone morphogenetic receptor type 1 B

(BMPR1B) gene, J Endocrinol, 169 (2001) R1-R6.

3 Wilson T, Wu X Y, Juengel J L, Ross I K, Lumsden J M

et al, Highly prolific Booroola sheep have a mutation in

the intracellular kinase domain of bone morphogenetic

protein IB receptor (ALK-6) that is expressed in both

oocytes and granulosa cells, Biol Reprod, 64 (2001)

1225-1235.

4 Nimbkar C, Ghalsasi P M, Ghatge R R & Gray G D,

Establishment of prolific Garole sheep from

West Bengal in the semi-arid Deccan Plateau of

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25 (1998) 257-260.

Table 4—Modifications made over Polley et al7 protocol

Parameter ARMS-PCR test

(Polley et al7)

ARMS-PCR test

(Present protocol)

DNA extraction method Genomic DNA extraction FTA paper DNA extraction

Animals tested Black Bengal goats Sheep (3 breeds and their crosses)

Animals selected Randomly With known lambing and litter size records

PCR reaction conditions and

touchdown protocol

Two PCR protocols

(two touchdown annealing temperatures)

Single PCR protocol

(Single touchdown annealing temperature)

Loading in gel Two wells in agarose gel for each sample Single well in agarose gel for each sample

Validation with standard PCR-RFLP test Not done Done and validated

PCR reaction volume 25 µL 12.5 µL

Table 5—Benefits and cost details in use of ARMS-PCR and PCR RFLP tests at NARI

Parameter Forced PCR-RFLP ARMS-PCR

Time More

• 1.5 h for PCR

• 3 h for RFLP digestion

Less

• 2.5 h for PCR

Enzymes and buffers in PCR More

• Used doubly expensive True Hot Start Taq

DNA polymerase (Fermentas) to get clean

single band of 140 bp PCR products. Additional

cost of restriction enzymes and buffers.

Less

• Used cheaper thermophilic Taq DNA

polymerase (Sigma); large PCR products, so True

Hot Start Taq DNA polymerase not needed. Cost

of only PCR enzymes and buffers.

Electricity Extra for RFLP digestion, 3 h Only for PCR

Gel electrophoresis • 3% agarose gel (high resolution blend, more

expensive)

• Smaller digested PCR products

• 30 bp resolution on agarose, more time for

gel electrophoresis.

• 1% agarose gel (simple and cheaper agarose)

• Large PCR products

• Less time for gel electrophoresis.

Merits/demerits Lengthy, incomplete digestion problems Fast, no digestion problems

Cost Rs. 200/- per sample

True start Hot start Taq DNA polymerase

[Fermentas]

High Resolution blend Agarose [Ameresco]

AvaII restriction enzyme[Fermentas]

Digestion for 3 h

Electricity consumption

Cost Rs. 141/- per sample

Thermophilic Taq DNA polymerase

[Sigma]

Simple agarose [Sisco-SRL]

No restriction enzyme

No digestion

No digestion hence less electricity is consumed

SASTE et al: DETECTION OF FecB GENOTYPE IN SHEEP BY ARMS-PCR

279

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acyltransferase 1 K232A polymorphism in cattle,

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