SPECIES AUTHENTICATION OF GOAT (Capra hircus) MEAT

USING SPECIES-SPECIFIC PCR PRIMERS FROM

MITOCHONDRIAL CYTOCHROME B GENE1

MARY RANZELLE ASIN PASANG

Bachelor of Science in Agricultural Biotechnology

Thesis outline is submitted for the fulfilment of the requirements for graduation
with the degree of Bachelor of Science in Agricultural Biotechnology major in Animal
Biotechnology, Animal and Dairy Science Cluster, College of Agriculture, University of the
Philippines Los Baos, College, Laguna. Prepared under the supervision of Mr. Medino Gedeun
N. Yebron Jr.
INTRODUCTION

Goats (Capra hircus) are one of the oldest domesticated livestock (Spencer, 2008) and are
useful to humans as a renewable provider of milk, manure, and fiber when they are living and as
meat and hide when slaughtered (Mahmoud Abdel Aziz, M. A., 2010). They are also used for
driving and packing purposes, and are easier and cheaper to manage compared to other
ruminants thus providing relative income to impoverished people in some poor countries (Hirst,
2008).
Goat and sheep meat is the fourth most consumed meat, following pork, poultry, and beef
(The Pennsylvania State University, 2012).The goat meat is one of the choicest edible
commodities and carries premium value in the market (Agarwal, 2013) due to its unique flavor
and palatability. Although it has distinct taste as compared to other red meats, it is still liable to
frauds. For instance, goat meat (chevon) is being substituted with that of dog meat and cat meat,
and is also used as an adulterant of mutton (sheep meat) as reported by Kangehte et al. in 1986.
Despite of these fraudulent practices, the demand for meat products continues to escalate in
almost all regions of the globe, especially in developing countries, as the world population rises
(Delgado, 2003). This high demand for meat invites special attention to the import and export
regulations regarding legitimacy of meat. There are various religious communities worldwide,
who have strict preferences regarding their meat for consumption. Moreover, it is difficult to
differentiate meat source by look or taste especially for processed meat. The public can rely only
on the authenticity statement made on the menus in restaurants and labels in the markets.
The ability to detect less desirable or objectionable species in meat products is important not
only for economic, health, religious and ethical reasons, but also to ensure fair trade and
compliance with legislation (Ballin et al., 2009; Nakyinsige et al., 2012; Spink & Moyer, 2011).
Most analytical methods utilized to date for meat authentication have relied on the detection of
2

species-specific proteins or DNA (Ballin et al., 2009; Leighton Jones, 1991; Meyer & Candrian,
1996). Today, however, DNA is considered to be the most appropriate molecule for species
detection and identification in foods (Singh & Neelam, 2011). Unlike proteins, DNA is relatively
stable at high temperatures, meaning that it can be analysed not only in fresh and frozen food
products, but also in processed, degraded and mixed commodities (Lenstra, 2003). In addition,
while the presence and characteristics of proteins depend on the tissue type being analysed, DNA
exists and is identical in almost all cells, and the diversity afforded by the genetic code permits
the discrimination of even closely-related species (Ballin, 2010; Lockley & Bardsley, 2000).
Therefore, molecular traceability based on DNA analysis is recognized as the most appropriate
means of identifying and authenticating the origin of species of meat samples to detect various
types of fraudulent practices.
Most of literature refers to the use of mitochondrial DNA (mtDNA) rather than nuclear
DNA for the identification of the origin of meat products. This is largely because processed
meats are likely to contain degraded DNA and in this matter, mitochondrial DNA is more
suitable than nuclear DNA due to the high copy number of mtDNA per cell, which thereby
increases the chance of getting good DNA from samples (Hsieh et al., 2003). Furthermore, the
mutation rate on mtDNA is higher than nuclear DNA and this gives a greater chance to
accumulate several point mutations, which allows the differentiation of even closely-related
species (Tamimi & Ashhab, n.d.). Some of the molecular methods wherein mitochondrial DNA
is usually utilized for meat species identification are DNA hybridization, PCR or polymerase
chain reaction based methods such as sequencing of PCR products, RFLP analysis, RAPDPCR,
PCR-SSCP, and Multiplex PCR (Matsunaga et al., 1998; Lockely & Bardsley, 2000).

Mitochondrial cytochrome b (cyt b) has been considered one of the most useful genes for
phylogenetic work, and is probably the best-known mitochondrial gene with respect to structure
and function of its protein product (Esposti et al., 1993). Cyt b gene contains both slowly and
rapidly evolving codon positions, as well as more conservative and more variable regions or
domains overall. Therefore, this gene has been widely used for the identification of various
species (e.g., Satish et al., 2009; Jain et al, 2007; Matsunaga et al, 1998; and Zarringhabaie et al.,
2011) and is essential for the traceability of the origin of meat species. Hence, the aim of this
study is to design a primer from mitochondrial cytochrome b gene using PCR-based methods,
specific only for goat (Capra hircus) which could be used for species identification and
authentication of fresh (raw), cooked, processed and putrefied meat products.

REVIEW OF RELATED LITERATURE

Goat
Goats (Capra hircus) are one of the earliest domesticated animals, providing humankind
with milk, meat, hides, and fiber. They include several species of small, cloven-hoofed ruminants
constituting the genus Capra. Similar to other ruminants, including cows and sheep, goats
process plant roughage through a fermentation process within their compartmentalized stomachs,
4

and they chew regurgitated, partially digested food known as cud. Unlike other ruminants, goats
are agile browsers, preferring to reach upwards for foods such as the leaves, fruit, and bark of
small trees rather than grazing on grasses. When the desired foods are unavailable, however,
goats will consume any plant material accessible. It is this foraging ability and flexibility of diet
that has secured the importance of goats as a food source in the world's subsistence economies
(Kittler, 2003).
Goat Meat
The United States Department of Agriculture describes quality goat meat as firm and finely
grained. The color can vary between females and males, from light pink to bright red. Kids
defined as less than one year old are often slaughtered at three to five months of age. Their meat
is less flavorful and juicy, but more tender than the meat of older goats (Kittler, 2003).
Goat meat has a taste similar to mutton, with a slightly gamy flavor. It is lower in fat than
either beef or mutton (due to a fat layer exterior to the muscle rather than marbled through it),
and can be drier.
The domestic goat (Capra aegagrushircus) is a member of the family Bovidae and is closely
related to the sheep as both are in the goat-antelope subfamily Caprinae (Hirst, 2008). For this
reason, goat is often being associated with that of sheep. This property and the fact that goat and
sheep meats have similar tastes contribute to fraudulent substitutions and adulterations of sheep
meat (mutton) with that of goat meat (chevon). In addition, cat and dog meats are used as
adulterants and/or alternatives for goat meat as reported by Kangehte et al. in 1986.
Meat Adulteration

Food authenticity issues in the form of adulteration and improper description have been
around for a long time and probably for as long as food has been offered for sale (Ioannis et al.,
2005). The adulterated food often enters the supply chain and jeopardizes the sentiments as well
as health of the people. The driving force behind any adulteration is the revenue maximization,
either by using a low cost ingredient to (partially or totally) substitute a more expensive one, or
to (partially) remove the valued component in the hope that the adulterated product neither will
be perceived nor detected by the authorities and the consumer (Ioannis & Nikolaos, 2005).
Substantial proportion of population has religious considerations towards the consumption of
meat of a particular animal species. Hence, the meat adulteration has got social, religious,
economic, and public health concerns (Girish et al., 2013).
Molecular Traceability for Meat Species Identification
Identification of the species of origin in meat samples is relevant to consumers for the
possible economic loss from fraudulent adulterations, medical requirements of individuals that
might have specific allergies, and for religious reasons (Asensio et al., 2008a). For some
consumer groups, such as vegetarians, the contamination of food with meat residue is strictly
prohibited. This scenario is similar with that of the Halal food for the Muslim consumers, who
are prohibited from consuming pork (Unajak et al., 2011). Another example is the adulteration of
buffalo meat with that of beef is a common fraudulent practice in India because of the
prohibition on cow slaughter in most states of India (Girish et al., 2013). As for goat meat
adulteration, it is commonly substituted for mutton while cat and dog meats are its usual
adulterants (Kangehte et al., 1986). Hence, extensive development of identification techniques
had been a great challenge over the past decades and recent studies had proven that molecular

techniques are the most reliable method available for speciation (Edris et al, 2012; Girish et al.,
2013).
Molecular Traceability Using PCR Based Methods
The polymerase chain reaction (PCR) is a scientific technique in molecular biology to
amplify a single or a few copies of a piece of DNA across several orders of magnitude,
generating thousands to millions of copies of a particular DNA sequence. PCR is now a common
and often indispensable technique used in medical and biological research laboratories for a
variety of applications. There are three major steps involved in the PCR technique: denaturation,
annealing, and extension (Joshi & Deshpande, n.d.).Among DNA-based methods, polymerase
chain reaction (PCR) is the most well developed molecular technique up to now and provides a
simple, rapid, highly sensitive and specific tool for detecting constituents of animal origin in
foods (Mafra et al., 2008; Tobe& Linacre, 2008).Some of the PCR-based methods being used for
species identification includes sequencing of DNA amplicons (PCR-sequencing), analysis of
PCR-single strand conformation polymorphism (PCR-SSCP), simultaneous amplification of two
or more fragments with different primer pairs (multiplex PCR), analysis of PCR-restriction
fragment length polymorphism (PCR-RFLP), analysis of random amplified polymorphic DNA
(PCR-RAPD), and real-time fluorescence PCR assays (Fajardo et al., 2010).
The use of specifically designed oligonucleotides under restrictive PCR conditions has made
possible the direct and specific identification of defined DNA fragments and its application to
control food authenticity. By means of PCR with species-specific primers, a target sequence can
be amplified very sensitively from a food matrix containing a pool of sequences, avoiding
subsequent sequencing or restriction fragment length polymorphism (RFLP). PCR using species-

specific primers has the preferences of being useful for routine analysis of large numbers of
samples, even when aggressive processing treatments have been applied to the food (Rojas et al.,
2009; Mafra et al., 2008).
Mitochondrial DNA
According to a study made by the Polish Academy of Sciences, Institute of Genetics and
Animal Breeding in Jastrzbiec, Poland, mitochondrial genome is evolutionary the oldest
attribute of living organisms. The human mitochondrial DNA or mtDNA genome is
approximately 16, 569 bases in length and has two general regions: the control region, so called
D-loop; and the coding region containing genes for 13 polypeptides utilized in metabolic
respiration and 24 RNA molecules. It is often chosen as a target in identifying investigations
also due to faster evolution rate than nuclear DNA. As a consequence, the mtDNA contains more
variable positions in sequence that is very useful in identification of closely related species
(Vawter and Brown, 1986). The copy number of the mitochondrial genome exceeds that of the
nuclear genome by a factor up to 10, 000 (Alberts et al., 1990). The most often used targets in
mtDNA for species identification is the variable control region and cytochrome b gene (Barallon,
1989).
Mitochondrial Cytochrome B Gene
The mitochondrial cytochrome b (cyt b) gene is approximately 1141 bp and is widely used
in systematic studies to resolve divergences at many taxonomic levels (Farias et al., 2001). The
amino acid sequence of cytochrome b gene is highly conservative, but because of degeneracy of
the genetic code, the genes for cytochrome b differ at least in a few nucleotides even in closely
related species. Previous research (Hsieh et al., 2001; Hsieh et al., 2003) showed that the
8

fragment size of cytochrome b gene (about 300 bp) is sufficient for discriminating between close
species. Variation within species is less than between species. This enables to work out methods
allowing differentiation between many species in a single test. Since all mitochondrial genes
behave as a haploid locus therefore the problem of heterozygosity can be avoided. Another
advantage of the cytochrome b gene is the possibility of amplifying the certain segment of the
gene from any vertebrate species using only one pair of primers (Kocher et al, 1989).
Cytochrome b gene has wide representation in nucleotide databases. It is therefore a high
likelihood of finding a sequence entry of unknown sample or at least of a related species. It is
also used in studies of molecular evolution (Kocher et al., 1989, Montgelard et al., 1997, Prusak
et al., 2004) and forensic medicine (Barlett and Dawidson, 1992).
Mitochondrial Cytochrome B Gene
as a Tool for Meat Traceability
In 2009, Murugaiah, et al. developed a method utilizing PCR-restriction fragment length
polymorphism (RFLP) in the mitochondrial genes for beef (Bos taurus), pork (Sus scrofa),
buffalo (Bubalus bubalis), quail (Coturnix coturnix), chicken (Gallus gallus), goat (Capra
hircus), rabbit (Oryctolagus cuniculus) species identification and Halal authentication. The
genetic differences within the cytochrome b gene among the meat were successfully confirmed
by PCR-RFLP; thus developing a reliable typing scheme of species which revealed the genetic
differences among the species.
Zarringhabaie, et al. reported in 2011 that an accurate analytical technique for sheep, goat,
beef, and buffalo meat identification, based on PCR analysis of the cytochrome b gene of
mitochondrial DNA for enforcement of labelling regulations, is useful and feasible to trace meat
adulteration and differentiate species present in mixed meat. Therefore, it can be suggested as a
9

useful laboratory tool for species identification, especially for meat traceability and Halal
authentication.
Edris et al. presented a study in 2012 that suggests an accurate analytical technique for
detecting meat adulteration by conventional multiplex PCR analysis of the cyt b gene of animal
mtDNA. This technique was used to detect and trace meat adulteration and to differentiate
species present in meat mixture. The test could also be used and applied by researchers and
quality control laboratories for verification and control of industrial meat products, such as Halal
authentication and raw material origin certification.
Goat-Specific PCR Primers from
Mitochondrial Cytochrome B Gene
PCR primers are strands of nucleic acid that serve as starting point for DNA synthesis.
These primers are required for DNA replication because the enzymes that catalyze this process,
DNA polymerases, can only add new nucleotides to an existing strand of DNA (Rossi, 2009).
Designing primers specific for goat species requires numerous considerations to amplify the
target regions from a DNA template. Some of the published available primers specific for goat
species are shown in Table 1.
Table 1.Some of the published goat-specific PCR primers.
Author
Matsunaga
al. (1998)

PCR product
size
et 5-GAC CTC CCA GCT CCA 5-CTC GAC AAA TGT
157 bp
TCA AAC ATC TCA TCT TGA GAG TTA CAG AGG GATGA AA-3
3

Zarringhabaie
et al. (2011)

Forward

Reverse

5-CGC CAT GCT ACT AAT 5-TGT CCT CCA ATT

TCT TGT T-3
CAT GTG AGT GT-3

330 bp

10

The significant association of goat meat with that of sheep meat has shown great
contributions to fraudulent practices in the industry thus, having been able to design sheepspecific primers are also important for this study. Although the two animals are closely related,
they are two different species; goats having 60 chromosomes while sheep having only 54
chromosomes. This difference can already be a useful property to design and create goat-specific
and sheep-specific primers that would distinguish one species from another. Some of the
published sheep-specific primers are shown in Table 2.
Table 2.Some of the published sheep-specific PCR primers.
Author
Matsunaga
al. (1998)

Forward

Reverse

et 5-GAC CTC CCA GCT CCA 5-CTA TGA ATG CTG

TCA AAC ATC TCA TCT TGA TGG CTA TTG TCG CA-3
TGA AA-3

Zarringhabaie
et al. (2011)

PCR product
size
331bp

5-TAC CAA CCT CCT TTC 5-TGT CCT CCA ATT

AGC AAT T-3
CAT GTG AGT GT-3

585bp

Dooley et al. 5-GAG TAA TCC TCC TAT 5-AGG TTT GTG CCA
(2004)
TTT GCG ACA-3
ATA TAT GGA ATT-3

133 bp

11

METHODOLOGY
Sample Collection
Meat samples of goat species will be collected from a local slaughter house and meat
markets. The samples will be transferred to the laboratory in a chilled condition using an ice
container and will be stored at -20C until processing.
The meat samples will be subjected to various experimental procedures to obtain fresh,
cooked, putrefied and processed meat samples. The tissue will be cleaned of extraneous fat,
connective tissue and blood vessels to obtain fresh meat samples while processed meat samples
will be bought from meat markets.
12

As for cooked meat samples, meat will be wrapped in aluminium foil and will be cooked at
100C and 120C in a dry hot air oven and in moist heat (water bath and autoclave) for 45
minutes to simulate various methods of cooking. For putrefied meat samples, on the other hand,
the meat will be allowed to putrefy in a natural (unpreserved) condition at room temperature for
about 3 months to stimulate the autolysis in meat. In this treatment, 5 samples will be collected
from certain incubation periods and will be stored at -20C until processing.
100 mg of meat samples from each treatment will be collected for DNA extraction.
DNA Extraction
Mitochondrial DNA, along with genomic DNA will be extracted using the method described
by Lenstra et al. (2001). This extraction protocol will provide a rapid procedure for DNA
extraction by using an alkaline extraction buffer (0.5 M NaOH, 10 mM EDTA).It will yield
single-stranded DNA (6-15 g DNA/g meat), which can be spotted directly onto a positivelycharged nylon membrane for subsequent hybridization.
100mg of the meat samples will be weighed and placed in a 1.5mL Eppendorf tube. 0.2mL
of the extraction buffer, which will include a mixture of 0.5 M NaOH and 10 mM EDTA, will be
added into the tube. This will then be mixed using vortex mixer and will be incubated at 100C
for 7 minutes in a hot water bath. The tube will be spun using a centrifuge (room temperature)
for 2 minutes at 12,000 rpm and the supernatant will be transferred into a new 1.5mL Eppendorf
tube, avoiding floating fat. The latter step will be repeated where in the transferred supernatant
will be spun in a centrifuge for 2 minutes at 12,000 rpm and the supernatant will be transferred
into a new 1.5mL Eppendorf tube, avoiding floating fat. The second extracted supernatant will

13

contain the isolated genomic DNA from the meat samples and will be stored at -20C until
processing.
Moreover, approximate quantification of the DNA isolate will be determined using
spectrophotometry.
Neutralization Protocol
The alkaline extracted DNA samples will be neutralized by adding one volume of 1M TrisHCl, pH 7.75, into the isolated DNA. This neutralized DNA samples will then be stored at -20C
until processing.
PCR Amplification
In 2012, Naidu et al. developed novel primers for complete mitochondrial cytochrome b
gene sequencing of mammals. These novel oligonucleotide primers will be used for PCR
amplification

of

the

cytochrome

CCHCCATAAATAGGNGAAGG-3;

b
and

gene:
the

the

forward

reverse

primer

primer

will
will

be
be

55-

WAGAAYTTCAGCTTTGGG-3. PCR amplification will be conducted in 50 ml of 10 mM TrisHCl, (pH 8.3) containing 50 mM KCl, 1.5 mM MgCl 2, 200 mM dNTP mix, primer mix (4-60
pmol each), 250 ng template DNA and 1.25 unit Taq DNA polymerase (Perkin-Elmer).
Oligonucleotide primers from mitochondrial cytochrome b gene will be amplified and will have
a product size of about 1,424 bp. Thirty-five cycles of amplification will be run using G-Storm
Premium PCR Thermal Cycler as follows: denaturation at 94C for 30 seconds, annealing at
60C for 30 seconds, and extension at 72C for 30 seconds. Following amplification, 7 ml PCR
solution will be electrophoresed on 2% agarose gel for 30 minutes at 100 V in TAE buffer (40

14

mM Tris-acetate, 1 mM EDTA, pH 8.0) and will be stained with ethidium bromide (0.5 mg
ml1) and will be destained for 30 minutes.
Molecular Cloning
PCR products will be cloned using the pGEM-T Easy Vector System and the Rapid DNA
Ligation System. This will involve a DNA ligation step in which the PCR product, pGEM-T
vector, ligase buffer and T4 DNA ligase enzyme will be incubated. Reactions using this buffer
may be incubated for 1 hour at room temperature. The incubation period may be extended to
increase the number of colonies after transformation. Generally, an overnight incubation at 4C
will produce the maximum number of transformants. Competent cells will be transformed with
the pGEM-T vectors using standard transformation protocols and colonies carrying the clones
are identified by blue and white screening using X-GAL/IPTG LB agar plates.

Plasmid Isolation
In 1979, Birnboim and Doly described an alkaline lysis method of plasmid isolation which
will be used to isolate plasmid DNA or other cell components such as proteins by breaking the
cells open. In this procedure, bacteria containing the desired plasmid will be harvested from
culture medium. Suspension of bacteria will be prepared in isotonic solution which will be
subsequently subjected to lysis by an alkaline solution containing a detergent sodium dodecyl
sulphate (SDS) and NaOH. While detergent will serve to lyse cells and denature proteins,
alkaline condition will denature genomic DNA, plasmid DNA as well as proteins. This mixture
15

in subsequent step will be neutralized by potassium acetate (pH 5.2). Neutralization will result in
renaturation of plasmid and genomic DNA. Since plasmid DNA is covalently closed, it will
reanneal properly and will remain in solution in soluble form while genomic DNA will reanneal
randomly, which will result in the formation of precipitate. Precipitate will then be separated by
high speed centrifugation. Plasmid from the supernatant will be recovered by precipitation using
isopropanol or ethanol.
Sequencing
PCR products will be cycle sequenced using the PCR primers in a cycle sequencing reaction
that will be done by the 1st BASE DNA Sequencing Services of Asia Gel Corporation in
Malaysia.
Sequence Analysis
After sequencing, the obtained sequences will be confirmed and aligned manually to
identify species, using the online BLAST search engine of the National Center for Biotechnology
Information (NCBI) and Vector NTI. Sequences where a similarity of the query is found will be
displayed according to the degree of sequence match. The cleaning of the sequences will be done
to ensure its purity, following DNA sequence alignment for primer designing.
Figure 1 shows the alignment of the available sequences of goat and sheep species from
NCBI (see page 18).
Designing of PCR Primer Specific for Goat Species
For primer designing, the mitochondrial Cytb gene sequences (accession numbers) of goat
species will be taken from GenBank: Capra hircus (AB110597.1). The sequences will then be
16

aligned using BLAST, Vector NTI and NCBI online multiple alignment tools (www.ebi.ac.uk).
Primers will be designed by evaluating various parameters to create ideal species-specific
primers, which include: primer length (optimum: 18-22bp); product size (optimum for
traceability: 150bp); GC content (at least 50 % GC); annealing and melting temperature (Tm) of
the forward and reverse primers (at most 5C difference); 3-end stability; primer amplification
or extension capability; primer specificity and homology or complementary primer sequence;
and absence of repeats, dimerization, hairpin loops, duplex, palindrome, and other primer
secondary structure conformations. Vector NTI as well as various online software applications
such as Primer3Plus, GenScript Primer Design and Primer-Blast, will be used in this parameter
evaluation.
In addition, some of the published goat-specific and sheep-specific primers will also be used
to serve as basis of designing.

Goat
Sheep
Consensus
Goat
Sheep
Consensus
Goat
Sheep
Consensus
Goat
Sheep
Consensus
Goat
Sheep
Consensus
Goat
Sheep

1
75
(1) ATGACCAACATCCGAAAGACCCACCCATTAATAAAAATTGTAAACAACGCATTTATTGACCTCCCAACCCCATCA
(1) ATGATCAACATCCGAAAAACCCACCCACTAATAAAAATTGTAAACAACGCATTCATTGATCTCCCAGCTCCATCA
(1) ATGANCAACATCCGAAANACCCACCCANTAATAAAAATTGTAAACAACGCATTNATTGANCTCCCANCNCCATCA
76
150
(76) AACATCTCATCATGATGAAACTTTGGATCCCTCCTAGGAATTTGCCTAATCTTACAAATCCTGACAGGCCTATTC
(76) AATATTTCATCATGATGAAACTTTGGCTCTCTCCTAGGCATTTGCTTAATTTTACAGATTCTAACAGGCCTATTC
(76) AANATNTCATCATGATGAAACTTTGGNTCNCTCCTAGGNATTTGCNTAATNTTACANATNCTNACAGGCCTATTC
151
225
(151) CTAGCAATACACTATACATCCGACACAATAACAGCATTTTCCTCTGTAACTCACATTTGTCGAGATGTAAATTAT
(151) CTAGCAATACACTATACACCTGACACAACAACAGCATTCTCCTCTGTAACCCACATTTGCCGAGACGTAAACTAT
(151) CTAGCAATACACTATACANCNGACACAANAACAGCATTNTCCTCTGTAACNCACATTTGNCGAGANGTAAANTAT
226
300
(226) GGCTGAATCATCCGATACATACACGCAAACGGAGCATCAATATTCTTTATCTGCCTATTCATACATATCGGACGA
(226) GGCTGAATTATCCGATATATACACGCAAACGGGGCATCAATATTTTTTATCTGCCTATTTATGCATGTAGGACGA
(226) GGCTGAATNATCCGATANATACACGCAAACGGNGCATCAATATTNTTTATCTGCCTATTNATNCATNTNGGACGA
301
375
(301) GGTCTATATTATGGATCATATACCTTTCTAGAAACATGAAACATTGGAGTAATCCTCCTGCTCGCGACAATGGCC
(301) GGCCTATACTATGGATCATATACCTTCCTAGAAACATGAAACATCGGAGTAATCCTCCTATTTGCGACAATAGCC
(301) GGNCTATANTATGGATCATATACCTTNCTAGAAACATGAAACATNGGAGTAATCCTCCTNNTNGCGACAATNGCC
376
450
(376) ACAGCATTCATAGGCTATGTTTTACCATGAGGACAAATATCATTTTGAGGGGCAACAGTCATCACTAATCTTCTT
(376) ACAGCATTCATAGGCTATGTTTTACCATGAGGACAAATATCATTCTGAGGAGCAACAGTTATTACCAACCTCCTT

17

Consensus
Goat
Sheep
Consensus
Goat
Sheep
Consensus
Goat
Sheep
Consensus
Goat
Sheep
Consensus
Goat
Sheep
Consensus
Goat
Sheep
Consensus
Goat
Sheep
Consensus
Goat
Sheep
Consensus
Goat
Sheep
Consensus
Goat
Sheep
Consensus

(376) ACAGCATTCATAGGCTATGTTTTACCATGAGGACAAATATCATTNTGAGGNGCAACAGTNATNACNAANCTNCTT
451
525
(451) TCAGCAATCCCATATATTGGCACAAACCTAGTCGAATGAATCTGAGGGGGATTCTCAGTAGACAAAGCCACTCTC
(451) TCAGCAATTCCATATATTGGCACAAACCTAGTCGAATGAATCTGGGGAGGATTCTCAGTAGACAAAGCTACCCTC
(451) TCAGCAATNCCATATATTGGCACAAACCTAGTCGAATGAATCTGNGGNGGATTCTCAGTAGACAAAGCNACNCTC
526
600
(526) ACCCGATTCTTCGCCTTCCACTTTATCCTCCCATTCATCATCACAGCCCTTGCCATAGTCCACCTGCTTTTCCTC
(526) ACCCGATTTTTCGCCTTTCACTTTATTTTCCCATTCATCATCGCAGCCCTCGCCATAGTTCACCTACTCTTCCTC
(526) ACCCGATTNTTCGCCTTNCACTTTATNNTCCCATTCATCATCNCAGCCCTNGCCATAGTNCACCTNCTNTTCCTC
601
675
(601) CACGAAACAGGATCGAACAACCCCACAGGAATTCCATCAGACACAGATAAAATCCCATTTCACCCTTACTACACC
(601) CACGAAACAGGATCCAACAACCCCACAGGAATTCCATCGGACACAGATAAAATTCCCTTCCACCCTTATTACACC
(601) CACGAAACAGGATCNAACAACCCCACAGGAATTCCATCNGACACAGATAAAATNCCNTTNCACCCTTANTACACC
676
750
(676) ATTAAAGATATCTTAGGCGCCATGCTACTAATTCTTGTTCTAATACTACTAGTACTATTCACACCCGACCTACTC
(676) ATTAAAGACATCCTAGGTGCTATCCTACTAATCCTCATCCTCATGCTACTAGTACTATTCACGCCTGACTTACTC
(676) ATTAAAGANATCNTAGGNGCNATNCTACTAATNCTNNTNCTNATNCTACTAGTACTATTCACNCCNGACNTACTC
751
825
(751) GGAGACCCAGACAACTATATCCCAGCAAATCCACTCAATACACCCCCTCACATTAAACCTGAGTGGTATTTCCTA
(751) GGAGACCCAGACAACTACACCCCAGCAAACCCACTTAACACTCCCCCTCACATCAAACCTGAATGATACTTCCTA
(751) GGAGACCCAGACAACTANANCCCAGCAAANCCACTNAANACNCCCCCTCACATNAAACCTGANTGNTANTTCCTA
826
900
(826) TTTGCATACGCAATCCTACGATCAATTCCCAACAAACTAGGAGGAGTCCTAGCCCTAGTCCTCTCAATCCTAATC
(826) TTTGCGTACGCAATCTTACGATCAATCCCTAATAAACTAGGAGGAGTCCTCGCCCTAATCCTCTCAATCCTAGTC
(826) TTTGCNTACGCAATCNTACGATCAATNCCNAANAAACTAGGAGGAGTCCTNGCCCTANTCCTCTCAATCCTANTC
901
975
(901) TTAGTACTTGTACCCTTCCTCCACACATCTAAACAACGAAGCATAATATTCCGCCCAATCAGCCAATGCATATTC
(901) CTAGTAATTATACCCCTCCTCCATACATCAAAGCAACGGAGCATAATATTCCGACCAATCAGTCAATGTATATTC
(901) NTAGTANTTNTACCCNTCCTCCANACATCNAANCAACGNAGCATAATATTCCGNCCAATCAGNCAATGNATATTC
976
1050
(976) TGAATCCTGGTAGCAGATCTATTAACACTCACATGAATTGGAGGACAGCCAGTCGAACATCCCTACATTATTATT
(976) TGAATCCTAGTAGCCGACCTATTAACACTCACATGAATTGGAGGCCAGCCAGTTGAACACCCCTACATCATTATT
(976) TGAATCCTNGTAGCNGANCTATTAACACTCACATGAATTGGAGGNCAGCCAGTNGAACANCCCTACATNATTATT
1051
1125
(1051) GGACAACTAGCATCTATCATATATTTCCTCATCATTCTAGTAATAATACCAGCAGCTAGCACCATTGAAAACAAC
(1051) GGACAACTAGCATCTATTATATATTTCCTTATCATTCTAGTCATAATACCAGTAGCTAGCATCATCGAAAACAAC
(1051) GGACAACTAGCATCTATNATATATTTCCTNATCATTCTAGTNATAATACCAGNAGCTAGCANCATNGAAAACAAC
1126
1140
(1126) CTTCTAAAATGAAGA
(1126) CTCCTAAAATGAAGA
(1126) CTNCTAAAATGAAGA

Figure 1. The alignment of the available sequences of goat and sheep from NCBI, via Vector
NTI.

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