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 Ghalsasi1, R S Kataria2, B K Joshi2, B P Mishra2 and C Nimbkar1
1

Animal Husbandry Division, Nimbkar Agricultural Research Institute, Phaltan 415 523, India
2
National 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
_______________
*Author for correspondence:
Tel: + 91-2166-262106/200783; Fax: + 91-2166-262106
Email: sonali_gene@yahoo.com

used for crossbreeding to improve milk yield of

prolific ewes, lamb weight and growth rate5. Two
fecund strains were developed at NARIthe 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.

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

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 1.790.15, while the mean litter sizes for
Deccani, Awassi ewes and Crossbred ewes were
1.030.0, 1.120.13 and 1.690.10, 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.

1.790.15a*

1.0-3.0

Validation of Genotypes Obtained with ARMS-PCR

1.0-2.0

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 1Ewes genotyped at the FecB locus

No. of
ewes
(n)

Av. no. of
lambings1

Av. litter
size1

3.800.58

Deccani

6.000.32

1.030.00

Awassi

2.000.71

1.120.13b

1.0-2.0

4.150.33

1.0-3.0

Crossbred
1

20

1.690.10

Values are expressed as meanstandard error of the mean

Crossbred 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)
2

Primer Sequences

Range
of litter
size

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 L final volume) was
performed using 1.25 L thermophilic DNA

Garole

polymerase 10 buffer, 200 mM dNTPs, 25 mM

MgCl2, 10 pmol of each primer, 0.5 units of Taq DNA
polymerase (GeneiTM) and 5 L 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.
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 94C for 4 min, followed by 30 cycles of
denaturation at 94C for 30 sec, annealing
touchdown from 60C to 52C (8 cycles) with
remaining cycles at 52C for 45 sec, and extension at
72C for 30sec, followed by a final extension at 72C
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 g/mL)
and photographed using a molecular Gel imager
(Gel Doc, Star Micronics).

DNA Isolation and PCR Amplification

Ewe breed/
cross

275

INDIAN J BIOTECHNOL, JULY 2012

276

carried out in a total volume of 8 L, containing

0.8 L True Hot Start Taq DNA polymerase PCR
buffer, 200 mM dNTPs, 25 mM MgCl2, and
7 pmol each of the following primers: 5GTCGCTATGGGGAAGTTTGGATG-3
and
5CAAGATGTTTTCATGCCTCATCAACACGGTC-3;
and 0.5 units of True Hot Start Taq DNA
polymerase (Fermentas). The fragments were
amplified using 34 cycles at 94C for 15 sec, 60C
for 30 sec, and 72C for 30 sec, followed by 72C
for 5 min. The 8 L PCR product was digested with
AvaII enzyme (Fermentas) at 37C for 3 h and
electrophoresed on a 3% high resolution blend
agarose (Ameresco) gel. The gel was stained with
ethidium bromide solution (0.5 g/mL) and
photographed using a molecular Gel imager
(Gel Doc, Star Micronics).
Table 2FecB genotypes of ewes genotyped using an
ARMS-PCR test and validated using a PCR-RFLP test
FecBB/
FecBB

FecBB/
FecB+

FecB+/
FecB+

Total

Garole
Deccani
Awassi
Crossbred

4
0
0
11

1
0
0
9

0
5
5
0

5
5
5
20

Total

15

10

10

35

Ewe breeds/
crosses

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/FecBB) 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/FecBB) and heterozygous (FecBB/FecB+)
genotypes were found.

Table 3Allele and genotype frequency of the 35 ewes genotyped at the FecB locus
Gene
mutation
BMPR1B
(FecBB)

Sheep breed/
crosses

No. of
animals

Garole
Deccani
Awassi
Crossbred

5
5
5
20

Allele frequency
FecB+
FecBB
0.10
1.00
1.00
0.23

0.90
0.00
0.00
0.77

FecBB/FecBB
0.80
0.00
0.00
0.55

Genotype frequency
FecBB/FecB+
0.20
0.00
0.00
0.45

FecB+/FecB+
0.00
1.00
1.00
0.00

Fig. 1ARMS-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 & 3435: 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

Fig. 2PCR-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).

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 al13 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 acylCoA: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 ARMSPCR test (modifications listed in Table 4) resulted
in a 30% cost reduction as compared to the PCRRFLP test (ARMS-PCR: Rs. 141 per sample; PCRRFLP: 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

INDIAN J BIOTECHNOL, JULY 2012

278

Table 4Modifications made over Polley et al7 protocol

Parameter

ARMS-PCR test
(Polley et al7)

ARMS-PCR test
(Present protocol)

DNA extraction method

Animals tested
Animals selected
PCR reaction conditions and
touchdown protocol

Genomic DNA extraction

Black Bengal goats
Randomly
Two PCR protocols
(two touchdown annealing temperatures)

FTA paper DNA extraction

Sheep (3 breeds and their crosses)
With known lambing and litter size records
Single PCR protocol
(Single touchdown annealing temperature)

Loading in gel
Validation with standard PCR-RFLP test
PCR reaction volume

Two wells in agarose gel for each sample

Not done
25 L

Single well in agarose gel for each sample

Done and validated
12.5 L

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

Enzymes and buffers in PCR

Electricity
Gel electrophoresis

Merits/demerits

Forced PCR-RFLP
More

1.5 h for PCR

3 h for RFLP digestion

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.
Extra for RFLP digestion, 3 h
3% agarose gel (high resolution blend, more
expensive)
Smaller digested PCR products
30 bp resolution on agarose, more time for
gel electrophoresis.

ARMS-PCR
Less
2.5 h for PCR
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.
Only for PCR
1% agarose gel (simple and cheaper agarose)
Large PCR products
Less time for gel electrophoresis.

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

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.
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