Improved RAPD-PCR for discriminating breeds of water buffalo

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Biochemical Genetics
ISSN 0006-2928
Biochem Genet
DOI 10.1007/s10528-012-9502-8
Improved RAPD-PCR for Discriminating
Breeds of Water Buffalo
Rubigilda C.Paraguison, Maria Pia
P.Faylon, Ester B.Flores & Libertado
C.Cruz

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NOTE
Improved RAPD-PCR for Discriminating Breeds
of Water Buffalo
Rubigilda C. Paraguison •Maria Pia P. Faylon •
Ester B. Flores •Libertado C. Cruz
Received: 29 June 2011 / Accepted: 4 January 2012
ÓSpringer Science+Business Media, LLC 2012
Introduction
The classification of the water buffalo is uncertain. Some authorities record the species
Bubalus bubalis with three subspecies, the river buffalo (B. bubalis bubalis) of South
Asia, the carabao or swamp buffalo (B. bubalis carabanesis) of the Philippines and
Southeast Asia, and the arni, or wild water buffalo (B. bubalis arnee). Others regard
these as closely related, but separate, species (Roth and Myers 2004).
The adult size range for domestic breeds of water buffalo is 400–900 kg
(880–2,000 lb). Wild animals are nearly 3 m (9.8 ft) long and 2 m (6.6 ft) tall, weighing
up to 1,200 kg (2,600 lb); females are about two-thirds this size (Encyclopaedia
Britannica Online 2009). River buffaloes are usually black, have curled horns, and are
native to the western half of Asia, whereas swamp buffalo can be black, white, or both
colors, with long, gently curved,swept-backhorns;theyarenativetotheeasternhalfof
Asia from India to Taiwan (United Nations Food and Agriculture Organization 2000). The
swamp buffalo has 48 chromosomes; the river buffalo has 50 chromosomes. These two
types do not readily interbreed, but fertile offspring can occur.
Carabao is a tamed type of water buffalo (B. bubalis or sometimes B. car-
abanesis). In the Philippines, it is the farm animal of choice for draft purposes
(pulling the plow to prepare the soil for farming and pulling carts used to carry loads
to market). Recent developments in Asia include transforming the swamp buffalo to
producers of milk and meat by organized crossing and backcrossing with the
riverine type, as is now aggressively pursued in the Philippines and China (Cruz
2010).
R. C. Paraguison (&)M. P. P. Faylon E. B. Flores L. C. Cruz
Molecular Genetics Laboratory, Genetic Improvement Program Unit, Philippine Carabao Center,
Science City of Mun
˜oz 3119, Nueva Ecija, Philippines
e-mail: rubigee@gmail.com; pcc-oed@mozcom.com
E. B. Flores
e-mail: esterflrs@yahoo.com
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DOI 10.1007/s10528-012-9502-8
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Part of the genetic improvement program for water buffalo in the Philippines is
the genetic characterization of the native water buffalo. This may be considered an
important step in the management and conservation of the species. Also, this may be
necessary in developing new breeding strategies to enhance the genetic potential of
the Philippine carabao. As a consequence of the rapid changes in production
systems that lead to either extinction or replacement of breeds, documenting the
diversity of livestock genetic resources and designing strategies for their conser-
vation should be important.
Conservation of genetic resources involves several activities, such as applying
molecular marker technology. Random amplification of polymorphic DNA (RAPD)
is known to be a simple yet powerful genotyping method for many organisms
(Wang et al.1993). Scientists performing conventional RAPD generally create
several arbitrary, short primers (8–12 nucleotides) and proceed with the PCR using a
template of genomic DNA, hoping that fragments will amplify. By resolving the
resulting patterns, they can glean a semi-unique profile from a RAPD reaction
(http://en.wikipedia.org/wiki/RAPD). A study was published on the genetic simi-
larity and polymorphisms among the three Egyptian water buffalo populations using
the conventional RAPD technique (Abdel-Rahman and Hafez 2007). Other reports
use conventional RAPD for farm animals (Rao et al. 1996). Our study employs a
modified RAPD method wherein DNA markers were designed of 20-mer oligo-
nucleotides and the PCR annealing temperature was more stringent, ramping to
55–60°C (Lim et al. 2006). Conventional RAPD uses a very low annealing tem-
perature to amplify PCR products (usually 28–40°C). The use of stringent condi-
tions for the modified RAPD could therefore improve the specificity of the primers
and the reproducibility of the products (Kang et al. 2002). Some advantages of
RAPD are its speed and simplicity and its ability to generate many loci with variable
levels of polymorphism. The disadvantages include a lack of information about the
underlying sequences, its sensitivity to reaction conditions (requiring the mainte-
nance of highly consistent experimental conditions), an inability to interpret band
profiles in terms of loci and alleles, and low reproducibility. The use of modified
RAPD, however, improved the specificity and reproducibility of the PCR products.
Most of the time, it is rather difficult to distinguish crossbred from purebred animals
when it comes to their physical features. This study suggests that the modified RAPD-
PCR method can distinguish between the riverine and swamp buffaloes genetically
and possibly confirm the genetic identity and breed purity of crossbred individuals.
The multilocus character of RAPD markers allows complex DNA profiling using only
one PCR and primer, which makes this method effective for routine purposes such as
the breed verification of calves generated by embryo transfer.
Materials and Methods
Sample Collection
Representative blood samples were collected from each of the Philippine carabao,
Bulgarian murrah and their crosses, Indian murrah, American, and Cambodian
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buffaloes. Blood samples were collected in vacutainers and kept cold until
processed in the laboratory.
DNA Extraction
Genomic DNA was isolated from whole blood samples following a high salt
concentration extraction procedure to facilitate lysis of erythrocytes and to remove
unwanted debris. White blood cells were concentrated by adding an approximate
volume of ammonium chloride solution (about 2–3 times the volume of sample) to
the whole blood samples in microcentrifuge tubes. The solution was mixed for
15–20 min and centrifuged at 2,500 rpm for 5 min. Samples were washed
successively with ammonium chloride solution until a white pellet was obtained.
Genomic DNA was extracted using a standard DNA extraction kit (Wizard
Promega) as recommended by the manufacturer. DNA samples were stored at 4°C
for the succeeding analysis.
Modified RAPD-PCR Analysis
Modified RAPD reactions were performed as described by Lim et al. (2006)on
Philippine rice using the 12 primers of the SRILS (Seoulin Research Institute of Life
Science) UniPrimer kit (Seoulin Scientific Co., Korea; Kang et al. 2002).
PCR amplifications were performed in a final reaction volume of 10 lL,
containing the following reaction buffer: 19PCR buffer, 1.5–2 mM MgCl
2
,1mM
dNTPs, 10–50 ng/lL DNA template, 1.5 lM UBC or SRILS primer, and 0.05 U
Taq DNA polymerase (Intron/Go-Taq, Invitrogen). Thermal cycling temperature
settings were 94°C for predenaturation for 4 min; then 35 cycles of 94°C for 1 min,
annealing temperature of 53–60°C (depending on the melting temperature of each
primer) for 1 min, and elongation at 72°C for 2 min; and a final elongation for
7 min. All RAPD reaction products were electrophoresed in 1.5–3% (w/v) agarose
gels in 19TAE (Tris, acetic acid, EDTA) buffer. Gels were run for 45 min at 100 V
in the Mupid gel electrophoresis system, stained, viewed under UV light, and
photographed with a UVtech gel documentation system.
SRILS 2, 5, and 6 were also tested for breed identification of embryo transfer
calves to determine whether the calves produced were purebred or crossbred.
Samples were taken from the embryo transfer calf, dam, and the suspected sires.
Results and Discussion
To enhance the probability of detecting differences between breeds of buffaloes, all
SRILS random markers were prescreened. These primers were evaluated by
analyzing their polymorphic band products. Each primer generated a unique PCR
amplification profile, and each had to be optimized with a certain annealing
temperature and/or different MgCl
2
concentration in a PCR mixture. Apparently,
SRILS 2, SRILS 5, and SRILS 6 are potential markers that can differentiate swamp
buffaloes such as the Philippine carabao and the Cambodian buffaloes among the
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riverine buffaloes (Fig. 1). SRILS 2 is capable of distinguishing the Philippine
carabao from all the riverine and crossbred buffaloes by the absence of the *850-bp
band from the carabao, but it cannot differentiate crossbred from riverine animals.
In the same way, SRILS 5 can distinguish swamp from riverine buffaloes by the
absence of the *350-bp band in the swamp buffalo banding profile, but it could not
distinguish the purebred Philippine carabao from crossbred animals. With SRILS 6,
we can distinguish swamp from riverine buffaloes by the presence of the *175-bp
band in Philippine carabao. On the other hand, it cannot distinguish the pure
Philippine carabao from crossbred buffaloes.
One of the disadvantages of the RAPD method is the inconsistent reproducibility
of the banding pattern. To confirm the reliability of the amplified products using
SRILS 2 and SRILS 5 primers, we analyzed 342 DNA isolates from stock using
SRILS 2
SRILS 5
m PC BMB CB m IMB
m PC BMB CB IMB
SRILS 6
m PC BMB CB IMB C AMB
~175bp
~850bp
~350bp
Fig. 1 RAPD band profiles amplified using SRILS 2, SRILS 5, and SRILS 6 primers. In the SRILS 2
profile, the absence of an 850 bp band distinguishes the Philippine carabao from both riverine and
crossbred buffaloes. In the SRILS 5 profile, the absence of a 350 bp band differentiates the Philippine
carabao from riverine buffaloes but not from crossbred animals. In the SRILS 6 profile, the presence of a
175 bp band differentiates the Philippine carabao from riverine buffaloes but not from crossbred animals.
Lanes:PC Philippine carabao, a swamp type. BMB Bulgarian murrah, a riverine type. CB crossbred
buffalo. IMB Indian murrah, a riverine type. CCambodian buffalo, a swamp type. AMB American murrah
buffalo. Lane m 1 kb DNA ladder
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these primers. The acquired results further validated the reliability and repeatability
of the amplified products.
Breed Verification of Calf Produced by Embryo Transfer Using SRILS
The results analyzed with SRILS 2, 5, and 6 show that a calf produced by embryo
transfer is crossbred (Fig. 2). The presence of the *850-bp band in SRILS 2
eliminates the possibility that the calf is a pure Philippine carabao; it must be either
a riverine or a crossbred buffalo. The absence of the *350-bp band in SRILS 5
eliminates the possibility that it is a pure riverine buffalo and the presence of a faint
band in SRILS 6 shows that it could be crossbred or a carabao. Since the SRILS 2
test has eliminated the pure carabao option, the calf must be considered a crossbred
buffalo.
The use of the modified RAPD improved the specificity and reproducibility of the
PCR products. This study presents potential markers for the modified RAPD-PCR
method, which can verify genetic identities among riverine and swamp buffaloes
and confirm breed purity. This modified RAPD technique can also be very useful in
breed identification among purebred and crossbred buffaloes, but the analyses may
need more than one random primer in order to establish the identity of a crossbred
animal.
Acknowledgments This study was supported by the Philippine Department of Agriculture Biotech-
nology Project Implementation Unit. The Philippine Carabao Center is gratefully acknowledged for
hosting this research project in the institute, and thanks are extended to the staff of the Genetic
Improvement Program unit for their assistance and support.
References
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using RAPD-PCR and PCR-RFLP techniques. Res J Agric Biol Sci 3(5):351–355
SRILS 2 SRILS 5 SRILS 6
M ET D S1 S2 (-) ET D S1 S2 (-) ET D S1 S2 (-)
~850bp
~350bp
~175bp
Fig. 2 Breed identification of an embryo transfer calf using three SRILS markers. Lanes:ET calf
produced by embryo transfer. Ddam. S1 and S2 suspected sires. (-), negative control. Mmolecular
weight marker. The DNA band profiles identify this as a crossbred calf
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