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DNA BARCODING OF
LIVESTOCK AND POULTRY
BREEDS/STRAINS
IN THE PHILIPPINES
Bangko Sentral ng Pilipinas Professorial Chair Lecture
Dr. Orville L. Bondoc, Ph.D.
Professor of Animal Breeding/Genetics
Animal and Dairy Sciences Cluster
College of Agriculture
University of the Philippines Los Baños
olbondoc.DNAbarcoding.2011
ACKNOWLEDGEMENTS
Department of Agriculture Philippine Agriculture and Fisheries
Biotechnology Program
Bangko Sentral ng Pilipinas
… for the opportunity in using the science
approach to appreciate and be truly grateful for
just a few of God’s many amazing creations!
Andy Abella, Romy Almeda, Modesto Alonzo, Artemio Almoroto, Mr. Am’mak, Amado Angeles, Bert Anido, Amando Apoderado, Ruben Arsinas, Edwin Atabay, Fileteo Atendido, Joel Bagarinao, Philbert Baguilat, Elmer Bandian, Elmer Baure,
Elizabeth Beltran, Jose Bigcas, Josenieto Bihis, Peale Jon Bondoc ,
olbondoc.DNAbarcoding.2011
Antonio Rayos, Marichele Rebenque, Reli Ann Rogado, George Roxas, Agapita Salces, Arturo Samodio, Edwin Sanchez, Mario Sandoval, Rene Santiago, Julius Santos, Eduardo Soriano, Pablo Subong, Fidel Talay, Antonio Tamayo, Rogelio Tamayo,
Wedamar Tanaman, Tomas Tello, Loida Valenzuela, Anthony Yap
Sabin Larrizabal, Raymund Ledesma, Roger Lopez, Archie Lluz, Anastacia Lucas, Jessie Malab, Bernard Malavi, Arnulfo Monleon, Rustico Morales,
Renelyn Morcoso, Gime Mortalla, Manuel Nalzaro, Gideo Napecole, Angela Oliva, Jade Pabico, Bernie Patricio, Francisco Peñalba, Maying Pilapil, Mercy Porsuelo,
Nestor Ebuenga, Audi Edralin, Joselito Elizaga, Herminio Esguerra, Celso Evangelista, Edward Foronda, Beatriz Garcia, Noel Genturo,
Francisco Geromo, Karlo Gicana, Rommel Herrera, Undin Hurtada, Peter Icalia, Walter Israel, Tata Jimenez, Pedro Kauntaw, Reymundo Lagazo, Nancy Lapid
Eduardo Briones, Jaime Cabarles, Rodrigo Cachuela, Flomella Caguicla, Jeric Paul Casilag, Dennis Castasus, John Paul Caunan, Josie Centeno, Wilson Cerbito,
Cenon Cerezo, Guada dela Cerna, Arnel del Barrio, Tintin Defensor, Adena Detera, Dominador Discion, Jorge Dominguez, Adelino Dotaro, Willie Eda,
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
TOPIC OUTLINE
I. Introduction
A. Historical milestones in DNA barcoding
B. Effectiveness of DNA barcoding as an identification tool
C. Benefits and limitations of DNA barcoding
D. Practical applications of DNA barcoding
II. Generating DNA barcodes for livestock and poultry
breeds/strains
A. Field sampling and blood collection
B. Laboratory analysis (DNA extraction, purification, elution,
amplification, and sequencing)
C. DNA barcodes generated for common livestock and
poultry breeds/ strains in the Philippines
D. COI sequence analysis
olbondoc.DNAbarcoding.2012
III. Evolutionary analysis of livestock and poultry
breeds/strains using DNA barcodes
A. Among livestock (mammals) families/species
1. Among ruminant breeds/strains
2. Among pig breeds/strains
B. Among poultry (avian) families/species
1. Among chicken breed/strains
2. Among duck breeds/strains
IV. Future outlook
A. Local DNA barcode library (database), laboratory
equipment and standard protocols
B. Applications of DNA barcodes in the local livestock and
poultry industry
C. Establishment of a national DNA barcode library for
agriculture
Inspired by barcode on products in supermarkets
(i.e. Uniform Product Code barcodes on
manufactured goods)
olbondoc.DNAbarcoding.2012
I. Introduction
DNA barcoding
Use of DNA sequence analysis of a uniform target
gene to enable species identification
>HOL - Holstein Friesian GGTATAGTAGTGAACAGCTAATAAGCCTTCTAATTCGCGCTGAA
TTAGGCCAACCCGGAACTCTGCTCGGAGACGACCAAATCTACA
ACGTAGTTGTAACCGCACACGCATTTGTAATAATCTTCTTTATAG
TAATACCAATCATAATTGGAGGGTTCGGTAACTGACTTGTTCCC
CTAATAATTGGTGCTCCCGATATAGCATTTCCCCGAATAAATAAT
ATAAGCTTCTGACTTCTCCCTCCCTCATTCCTACTACTCCTCGCA
TCCTCTATAGTTGAAGCTGGGGCAGGAACAGGCTGAACCGTGT
ACCCTCCCTTAGCAGGCAACCTAGCCCATGCAGGAGCTTCAGT
TGATCTAACCATTTTCTCTTTACACTTAGCAGGAGTTTCCTCAAT
TTTAGGAGCCATCAACTTCATTACAACAATTATCAACATAAAGCC
CCCCGCAATGTCACAATACCAAACCCCTCTATTCGTATGATCCG
TAATAATTACCGCCGTACTACTACTACTCTCGCTCCCTGTATTAG
CAGCCGGCATCACAATGCTATTAACAGACCGGAACCTAAATACA
ACTTTCTTCGACCCGGCAGGAGGAGGAGATCCTATTCTATACCA
ACACTTATTCTGATTTTTTGGTCACCTGGGAAAGTTATAAAA
Used as standard barcode marker
for animals especially birds, fishes,
amphibians, and lepidopterans,
with a species discrimination rate
of more than 95%.
New method for identifying and classifying species of
organisms using the cytochrome c oxidase subunit I
(COI) in the mitochondrial genome
DNA
barcode
DNA barcode
olbondoc.DNAbarcoding.2012
Historical milestones in DNA barcoding
Species identification using PCR-based approaches (bacterial studies, microbial biodiversity surveys, pathogenic
diagnoses, taxonomy, food and forensic molecular ID) - Teletchea et al. (2008)
Comparison of genetic polymorphisms and
distances in native farm animals in the Philippines ( blood typing, karyotyping and electrophoretic methods in
the 1970s ; polymorphic DNA in the 1990s; DNA finger
printing in verifying parentage in farm animals) - Bondoc (2000)
Measurement of genetic diversity using anonymous
markers (microsatellites, minisatellites, amplified fragment
length polymorphisms, gene markers, single nucleotide
polymorphisms, large scale or directed sequencing,
mitochondrial genotyping, y chromosome genotyping, etc.)
olbondoc.DNAbarcoding.2012
Mitochondrial DNA (mtDNA)
Approx. 16-17 kbp in size in most animals; encodes 22
tRNAs, 2 rRNAs, 13 polypeptides (Chinnery and Schon, 2003)
Used in studies of phylogenetic relationships among
animal populations, species, and subspecies, and
evaluation of maternal genetic constitution for a specific
population
Evolves much more rapidly than nuclear DNA, resulting in
accumulation of differences between closely related
species (Brown et al. 1979; Moore 1995; Mindell et al. 1997)
More abundant (also has greater sequence differences among
species) than nuclear DNA and therefore easier to recover,
esp. from small or partially degraded samples (Stoeckle and
Hebert, 2008)
Maternally inherited, does not undergo recombination
olbondoc.DNAbarcoding.2012
DNA Barcodes
1970s Carl Woese (University of Illinois) was the first to
show that DNA sequences could be used to
reconstruct the Tree of Life.
Hebert et al. (2003a) first provided proof that DNA
barcoding can distinguish at least some species
through an analysis of relatively short sequences of
DNA (i.e. cytochrome c oxidase subunit I or COI)
among closely related species across diverse phyla
in the animal kingdom.
2003 Establishment of an identification system for all
plant and animal life using genetic sequences
from a uniform locus was proposed.
olbondoc.DNAbarcoding.2012
COI barcode sequences
Easily recovered and provides good resolution, as
concluded from deep sequence divergences between
13,000 closely related pairs of animal species across
diverse phyla in the animal kingdom (Hebert et al. 2003b)
Gene for barcoding animals however, is not practical for
plants; Chloroplast genes, i.e. RuBisCO large subunit
(rbcL) and maturase K (matK) are used instead as standard
barcode for plants (CBOL Plant Working Group, 2009)
Complement the inherent limitations of morphology-based
systems of taxonomic classification
Allows rapid automated identifications by shifting the
process of species recognition from traditional
morphological approaches to one based on discrete
genetic codes
olbondoc.DNAbarcoding.2012
Consortium for the Barcode of Life (CBOL)
Established in 2004 including 150 institutions
from 45 countries through a grant from the Alfred
P. Sloan Foundation, CBOL plans to process five
million specimens from 500,000 species by 2014 (Stoeckle and Hebert, 2008)
Mission is to
rapidly accelerate compiling of DNA barcodes of
known and newly discovered plant and animal
species
establish a public library of sequences linked to
named specimens
promote development of portable devices for DNA
barcoding
olbondoc.DNAbarcoding.2012
International Barcode of Life (iBOL) Project
Systematic recording of DNA barcodes
for the identification of all animal and plant life
Based at the Biodiversity Institute of Ontario (BIO),
University of Guelph, Canada, BOLD since 2008 has
over 460,000 records from more than 46,000 animal
species.
Established a public and global online database called
the Barcode of Life Data systems or BOLD (see
www.barcodinglife.org) - an informatics workbench aiding
the acquisition, storage, analysis and publication of DNA
barcode records (Ratnasingham and Hebert, 2007)
Uses a standardized, rapid and inexpensive species
identification method (i.e. accurate, reliable, practical,
and cost-effective) accessible to non-specialists
olbondoc.DNAbarcoding.2012
Effectiveness of DNA barcoding as
an identification tool
DNA barcoding gene should be sufficiently conserved
to be amplified with broad range primers, yet divergent
enough to resolve closely related species.
Barcode region for animals:
650-base fragment of the 5’- end of the mitochondrial gene
cytochrome c oxidase I (COI, cox1)
Effective DNA barcoding:
More than 95% of species possess unique COI
barcode sequences
Higher inter-species than intra-species variability
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
Useful properties of the COI gene in
animals:
1. Present in all eukaryotes
2. Contains enough sequence
diversity to differentiate most animal
species (with the exception of
Cnidaria)
3. Short enough to be readily amplified
and sequenced
4. Can be amplified from diverse phyla
with broad-range primers
5. Relatively abundant in each cell (as
a mitochondrial gene), facilitating
recovery from suboptimal
specimens (i.e. from museum
collections preserved in formalin)
olbondoc.DNAbarcoding.2012
Effective DNA barcoding was reported in:
Other groups of organisms
Plants (Kress et al, 2005; Chase et al., 2007)
Macroalgae (Saunders, 2005)
Fungi (Summerbell et al. 2005)
Protists (Scicluna et al., 2006)
Bacteria (Sogin et al., 2006)
Lepidopterans (Hebert et al., 2004b; Janzen et al., 2005;
Hajibabaei et al., 2006)
Spiders (Barret and Hebert, 2005)
Fish (Ward et al., 2005)
Birds (Hebert et al., 2004a; Yoo et al., 2006; Kerr et al., 2007
and 2009)
olbondoc.DNAbarcoding.2012
More recent publications by the author:
Poultry (Bondoc, 2013 - ALS)
Phil. Red jungle fowls (Bondoc, 2012 - JESAM)
Ducks (Bondoc and Santiago, 2012 - PJVM)
Mammalian livestock (Bondoc, submitted ALS)
Goats and sheep (Bondoc and Cerbito, submitted PJVM)
Chickens (Bondoc and Santiago, submitted PAS)
Cattle and buffaloes (Bondoc, submitted PAS)
Swine (Bondoc, Dominguez and Peñalba, submitted PJVAS)
Dogs (Bondoc, Gicana and Hurtada, submitted PJVAS)
olbondoc.DNAbarcoding.2012
Benefits and limitations of DNA barcoding
Benefits: (Hebert et al., 2003a)
Contributes to conservation biology
Amplification and sequencing are inexpensive
per specimen with sequences made publicly
available through the Internet.
Provides insight into the evolutionary history
of life
Facilitates species identification and flagging
specimens from new species
Provides efficient method for mapping the
extent of species
Enables identifications where traditional
methods are unrevealing
olbondoc.DNAbarcoding.2012
Limitations:
There is no universal DNA barcode gene for all species
Hybridization (crossbreeding) will lead to shared or very
similar sequences at COI and other gene loci. Because
mitochondrial DNA is maternally inherited, a COI
barcode will assign F1 hybrids to the species of their
female parent (Hebert et al., 2004a).
DNA barcoding focuses on delineation between
species rather than their relationships (Rubinoff, 2006)
DNA barcoding is not optimal for the study of deep
evolutionary relationships especially involving recently
diverged species (Funk and Omland, 2003)
More data is needed to distinguish intra-specific from
inter-specific genetic variation - which are unknown and
may differ between groups (Santamaria et al., 2007)
olbondoc.DNAbarcoding.2012
Practical applications of DNA barcoding
DNA barcoding complements
Taxonomy
Molecular phylogenetics (evolutionary relationships
among deeper clades)
Population genetics (variation within and among
populations of a single species)
DNA barcoding is used as the primary source of information
in medicine, agriculture, health, fraud, smuggling, exploring
our planet's prehistoric life
Paleo-ecological/ ancient DNA studies (Willerslev et al., 2007)
Forensics (Dawnay et al., 2007)
Conservation genetics and molecular
ecology (Rubinoff, 2006; Ward et al., 2008)
olbondoc.DNAbarcoding.2012
II. Generating DNA barcodes for livestock and poultry breeds/strains
Field sampling and blood collection
olbondoc.DNAbarcoding.2012
Used stratified sampling design
Blood samples are placed in NucleoSave
storage cards
Demographic and morphological data, pictures
and video clips (i.e. more than 7,500 digital
images ~ 81 gigabytes) were taken per sample
Two or more breeds/strains of each species were used
to provide a general sense of intra-specific sequence
divergences, as well as a preliminary indication of
variation in each species
At least one individual from each farm species was
examined to ascertain COI sequence divergences
among species
olbondoc.DNAbarcoding.2012
Laboratory analysis
Laboratory protocols were developed separately for
livestock (mammals) and poultry (birds) specimens at
the Animal Biotechnology Laboratory, Animal and
Dairy Sciences Cluster, College of Agriculture,
University of the Philippines Los Baños.
Laboratory analysis
olbondoc.DNAbarcoding.2012
Laboratory
Protocol Livestock (mammals) Poultry (birds)
DNA
extraction
Using a Harris 1.2 mm micropunch, at least 30 discs from each
dried NucleoSave card or sample were collected and placed in
labeled microcentrifuge tubes.
In case of PCR failure due to low DNA
yield, rapid extraction of high quality
DNA from whole blood stored at 4oC
for long period was used using the
methods described by Iranpur and
Esmailizadeh (2010) such as for
buffalo and goats.
Laboratory
Protocol Livestock (mammals) Poultry (birds)
DNA
purification
Sample discs were washed with 200 μl of FTA Purification Reagent
(Whatman Inc., USA) for 4-5 times and rinsed with 200 μl sterile
molecular biology grade water. Sample discs were then dried in a
laminar hood overnight.
olbondoc.DNAbarcoding.2012
Laboratory
Protocol Livestock (mammals) Poultry (birds)
DNA elution
Six dried sample discs were transferred in a sterile PCR tube and
added with 55 μl sterile molecular biology grade water. DNA was
eluted using Veriti 96 Well Thermal Cycler (Applied Biosystems) at
90oC for 10 minutes. Eluted DNA was stored at -20oC for further
use.
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
Laboratory
Protocol
Livestock
(mammals) Poultry (birds)
DNA
amplification
(I)
The COI gene was
amplified using
primers LCO1490
(5’
GGTCAACAAATCAT
AAAGATATTGG 3’)
and HCO2198 (5’
TAAACTTCAGGGTG
ACCAAAAAATCA 3’).
The COI gene was amplified using primers
BirdF1 (5’
TTCTCCAACCACAAAGACATTGGCAC 3’and
BirdR1 (5’
ACGTGGGAGATAATTCCAAATCCTG 3’).
In cases where this primer pair failed, an
alternate reverse primer (BirdR2 - 5’
ACTACATGTGAGATGATTCCGAATCCAG 3’)
was used such as for Coturnix (quail) and
Struthio (ostrich).
The 20-μl PCR reaction mix included 13.44 μl sterile ultrapure water,
2.0 μl of 10X buffer, 1.0 μl of MgCl2, 0.8 units of Taq polymerase, 0.4
μl (0.2 mM) of each forward and reverse primer and 2.0 μl of DNA
template.
olbondoc.DNAbarcoding.2012
Laboratory
Protocol Livestock (mammals) Poultry (birds)
DNA
amplification
(II)
The optimized PCR
amplification program was
composed of 3 min at 94°C
followed by five cycles of 40 sec
at 94°C, 30 sec at 52°C and 45
sec at 72°C, followed by another
30 cycles of 40 sec at 94°C, 30
sec at 54°C, and 45 sec at 72°C,
and finally 7 min at 72°C.
The optimized PCR
amplification program was
composed of 3 min at 94°C
followed by five cycles of 40 sec at
94°C, 30 sec at 56°C and 45 sec
at 72°C, followed by another 30
cycles of 40 sec at 94°C, 30 sec at
58°C, and 45 sec at 72°C, and
finally 7 min at 72°C.
Laboratory
Protocol Livestock (mammals) Poultry (birds)
DNA
amplification
(III)
PCR products were visualized in a 1.0% agarose gel with ethidium
bromide. Post stained gels are viewed using Molecular Imager® Gel
DocTM XR System (Bio-Rad, USA).
PCR products were purified using GF-1 PCR Clean Up Kit (Vivantis,
Malaysia). In cases where multiple bands occur (e.g., pseudogenes
or short DNA sequences less than 200bp), gels were excised and
purified using GF-1 Gel DNA Recovery Kit (Vivantis, Malaysia).
The DNA amplification regime was repeated four (4) times for each
sample specimen.
The final PCR product for each sample specimen (about 30 to 50 μl
final volume) was obtained from pooled amplicons of all 4 PCR
reactions (replicates).
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
Laboratory
Protocol Livestock (mammals) Poultry (birds)
DNA
sequencing
PCR products were sent to Macrogen Inc., Seoul, Korea for
unidirectional sequencing using appropriate forward primer
and analyzed using 3730L DNA analyzer (AB, USA) and
BigDye (AB, USA).
DNA barcodes generated for common livestock
and poultry breeds/strains in the Philippines
olbondoc.DNAbarcoding.2012
60 livestock (Class Mammalia) specimens
- 3 orders, 4 families, 7 genera and 8 species, excluding horses (Equidae) and cats (Felidae)
world total: 5,702 species in 1,229 genera, 153 families,
and 29 orders (Wilson and Reeder,2005)
Each of the farm animal species had a different COI
sequence.
82 poultry (Class Aves) specimens
- 4 orders, 6 families, 9 genera, and 9 species
world total: 10,000 species
COI barcodes in a breed/strain represented by two or
more individuals were either identical or most similar
to other sequences of the same breed/strain.
GenBank Accession Numbers: (National Center for
Biotechnology Information or NCBI- http://www.ncbi.nlm.nih.gov.)
olbondoc.DNAbarcoding.2012
Buffaloes: JX218048 - JX218054, JX280474 - JX280478
Cattle: JX218055 - JX218067, JX280479 - JX280482
Goats: X218068 - JX218073
Sheep: JX218074 - JX218081
Pigs: JX218082 - JX218087, JX280483
Dogs: JX280484 - JX280490
Chickens: JX177989 - JX178001, JX178014 - JX178043,
JX280461 - JX280473
Quails: JX178002 - JX178003, JX178044 - JX178045
Turkey: JX178003 - JX178004
Guinea fowl: JX178046
Ducks: JX178006-JX178011, JX178047-JX178054
Geese: JX178012, JX178055
Pigeons: JX178013, JX178056
Ostrich: JX178057
Figure 1. Taxonomic arrangement of common livestock
(Class Mammalia) in the Philippines
olbondoc.DNAbarcoding.2012
Figure 2. Taxonomic arrangement of common poultry
animals (Class Aves) in the Philippines
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
UPLB-DA DNA Barcoding Project: an online library information
system for the Philippine livestock and poultry sector (Oliva and Rogado, 2011) - in collaboration with ICS, CAS, UPLB
Encourages the acquisition, storage, analysis and
publication of DNA barcode records for domestic
animal genetic resources in the Philippines
Web-based online repository that runs in parallel with
the Barcode of Life Data systems or BOLD (Ratnasingham and Hebert, 2007)
Accessible at: http://www.ics.uplb.edu.ph/UPLB-DA-
DNA-barcoding-project
Converts text-based COI sequences to illustrative DNA
barcodes, in addition to digital images and videos
Allows viewers of the public information library and
contributors of new DNA barcode information
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
COI sequence analysis
olbondoc.DNAbarcoding.2012
Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011):
diversity analysis
distance analysis
phylogeny analysis
Diversity analysis:
Calculate sequence divergence using Kimura 2-parameter
or K2P model (Kimura, 1980) and standard errors of
estimates using bootstrap method (Nei and Kumar, 2000)
Genetic diversity within taxa (intra-specific divergence)
of 2% may justify effectiveness of COI barcodes as an
identification tool to discriminate among species of
mammals and birds (Hebert et. al., 2003a).
Distance analysis:
Compute evolutionary distances using Kimura 2-
parameter method (Kimura, 1980) with their variances
estimated by a bootstrap approach
Test whether genetic distances within species
(or group) are less than those between
species (or group)
Average distance between sequence pairs are in the
units of the number of base substitutions per site
(i.e. d units) = number of transition and/or transversion
or differences occurring between them.
olbondoc.DNAbarcoding.2012
Phylogeny analysis:
Examine the nearest-neighbour distance, the minimum
genetic distance between a species and its closest
congeneric relative, using the Neighbour-Joining (NJ)
method
High bootstrap support for species nodes suggests
neighbour-joining analysis of COI barcode sequences will
be widely effective (e.g., Ward et al. 2005; Hajibabaei et al.
2006).
Create a NJ tree of K2P distances to provide a graphic
representation of the pattern of divergences among taxa
or animal breeds/strains (Saitou and Nei, 1987)
Another way of identifying species in need of taxonomic
scrutiny involves the search for taxa whose specimens
from two or more distinct clusters with high bootstrap
support (i.e. 98%) in a NJ tree. olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
III. Evolutionary analysis of livestock and poultry breeds/strains using DNA barcodes
Livestock (mammals) families/subfamilies/species
Among large ruminant breeds/strains
Among small ruminant breeds/ strains
Among pig breeds/strains
Among dog breeds
Poultry (avian) families/genera/species
Among chicken breed/strains
Among red jungle fowls from different
mountain areas
Among duck breeds/strains
Mean diversity (overall mean distance)
Among livestock families/subfamilies/species
olbondoc.DNAbarcoding.2012
Farm animals No. of
nucleotide
sequences
N
positions
Diversity (%)
Mean Standard
Error
Livestock (mammals) 58 513 57.82 2.41 Family Bovidae 43 516 56.33 3.13
Subfamily Bovinae 29 622 10.66 0.91
Buffalo 12 637 6.41 0.57
Cattle 17 631 1.94 0.91 Subfamily Caprinae 14 568 67.33 2.91
Goats 6 589 67.33 2.91
Sheep 8 674 32.91 1.44
Family Suidae (pigs) 8 619 29.98 1.72
Family Canidae (dogs) 7 671 2.98 1.72
Family Leporidae (rabbits) 2 616 70.66 5.12
olbondoc.DNAbarcoding.2012
Neighbour-Joining tree with
bootstrap support showing the
evolutionary relationships among
LARGE RUMINANTS (Subfamily Bovinae)
Korean cattle
Longhorn cattle
Japanese Black cattle
Marinduque native cattle
F3 62.5 Holstein x 37.5 Sahiwal cattle
Jersey cattle
Australian Friesian Sahiwal - black cattle
American Brahman cattle
F1 50 Jersey x 50 Australian Friesian Sahiwal cattle
Siquijor native cattle
Guimaras native cattle
F2 75 Brown Swiss x 25 Brahman cattle
F1 50 Brown Swiss x 50 Brahman cattle
F3 62.5 Brown Swiss x 37.5 Brahman cattle
F1 50 Holstein x 50 Sahiwal cattle
Australian Friesian Sahiwal - brown cattle
Holstein Friesian cattle
Batangas native cattle
Ilocos Norte native cattle
Iloilo native cattle
Brazilian Murrah buffalo
Bulgarian Murrah buffalo (BMU)
Indian Murrah buffalo
F3 87.5 BMU x 12.5 Carabao
Batangas carabao
Cagayan carabao
Ilocos Norte carabao
Marinduque carabao
Nueva Ecija carabao
F4 93.5 BMU x 6.25 Carabao
F1 50 BMU x 50 Carabao
Leyte carabao
Haikou (Chinese) buffalo
7693
8399
6546267
51
9746
51
63
100
88
92
38
42
0.02 divergence
olbondoc.DNAbarcoding.2012
Estimates of evolutionary divergence between mtDNA COI
sequences (d units)
- Water buffaloes
Diagonals = w/ in group mean distance;
Lower off-diagonal = b/w group difference;
Upper off-diagonal = net b/w group mean distances
Buffalo
groups
River-
type
Swamp
type
Cross-
breds
River-type 0.104 ±
0.011
0.028 ±
0.005
0.014 ±
0.001
Swamp
type
0.088 ±
0.009
0.017 ±
0.003
-0.001 ±
0.001
Cross-
breds
0.106 ±
0.009
0.048 ±
0.005
0.082 ±
0.009
olbondoc.DNAbarcoding.2012
Estimates of evolutionary divergence between mtDNA COI
sequences (d units)
- Cattle
Diagonals = w/ in group mean distance;
Lower off-diagonal = b/w group difference;
Upper off-diagonal = net b/w group mean distances
Cattle
groups Purebred
Native
strains
Cross-
breds
Purebred 0.022 ±
0.004
-0.001 ±
0.000
0.000 ±
0.001
Native
strains
0.016 ±
0.003
0.011 ±
0.003
0.000 ±
0.001
Cross-
breds
0.025 ±
0.003
0.019 ±
0.003
0.026 ±
0.004
olbondoc.DNAbarcoding.2012
Estimates of evolutionary divergence between mtDNA COI
sequences (d units)
- Goats
Diagonals = w/ in group mean distance;
Lower off-diagonal = b/w group difference;
Upper off-diagonal = net b/w group mean distances
Goat
groups Purebred Native strains
Purebred 0.111 ±
0.012
0.712 ±
0.059
Native
strains
0.911 ±
0.062
0.286 ±
0.020
olbondoc.DNAbarcoding.2012
Neighbour-Joining tree with
bootstrap support showing the
evolutionary relationships among
GOATS
Inner Mongolia White Cashmere goat
De Co (Vietnamese) goat
Polish White Improved goat
Leyte native goat
Bohol native goat
Cagayan native goat
Boer
Anglo Nubian
Saanen100
100
61
100
80
85
0.02 divergence
olbondoc.DNAbarcoding.2012
Estimates of evolutionary divergence between mtDNA COI
sequences (d units)
- Sheep
Diagonals = w/ in group mean distance;
Lower off-diagonal = b/w group difference;
Upper off-diagonal = net b/w group mean distances
Sheep
groups Hair-type Wool-type
Hair-type 0.250 ±
0.016
-0.002 ±
0.006
Wool-type 0.312 ±
0.013
0.378 ±
0.019
olbondoc.DNAbarcoding.2012
Neighbour-Joining tree with
bootstrap support showing the
evolutionary relationships among
SHEEP
Dorset
St. Croix
Suffolk
Kathadin
Damara
Dorper
Merino
Philippine sheep
Merino Landschaf sheep
Afec-Assaf sheep
Black Welsh Mountain sheep44100
99
100
100
99100
99
0.02 divergence
olbondoc.DNAbarcoding.2012
Neighbour-Joining tree with
bootstrap support showing the
evolutionary relationships among
PIGS Chinese Meishan AF304200
Berkshire AY574045
Large White AF486874
Landrace AF304202
Duroc AF486858
F1 50 LDR x 50 LWH crossbred pig
Pietrain (PTR)
F2 DUR-PTR-LDR-LWH crossbred pig
Duroc (DUR)
Quezon native pig
Kalinga native pig
Landrace (LDR)
Large White (LWH)73
62
66
100
100
100
100
90
37
68
0.02 divergence
olbondoc.DNAbarcoding.2012
Estimates of evolutionary divergence between mtDNA COI
sequences (d units) in pigs
Diagonals = w/ in group mean distance;
Lower off-diagonal = b/w group difference;
Upper off-diagonal = net b/w group mean distances
Pig
groups Purebred
Native
strains
Cross-
breds
Purebred 0.273 ±
0.017
0.002 ±
0.007
0.026 ±
0.012
Native
strains
0.234 ±
0.015
0.190 ±
0.019
0.119 ±
0.019
Cross-
breds
0.344 ±
0.021
0.395 ±
0.026
0.363 ±
0.028
olbondoc.DNAbarcoding.2012
Companion animals
olbondoc.DNAbarcoding.2012
Neighbour-Joining tree with
bootstrap support showing the
evolutionary relationships among
DOGS Chihuahua EU408262
Pit Bull Terrier EU408293
French Bulldog EU408275
Great Dane EU408276
Toy Poodle EU408302
German Shepherd EU408277
Pug EU408294
Dachshund EU408272
Shih Tzu
Rough Collie
Dachshund
Chihuahua
French Bulldog
Dalmatian
Toy Poodle
80
51
43
94
92
64
100
19
16
0.02 divergence
olbondoc.DNAbarcoding.2012
Mean diversity (overall mean distance)
Among poultry families/species
olbondoc.DNAbarcoding.2012
Farm animals No. of
nucleotide
sequences
N
positions
Diversity (%)
Mean Standard
Error
Poultry (birds) 82 521 61.67 3.76 Family Phasianidae 60 565 57.88 4.44
Gallus (chickens) 56 590 58.37 4.73 Coturnix (quails) 4 656 22.82 1.50
Family Meleagridae (turkey) 2 863 14.53 1.39
Family Anatidae 16 617 43.44 2.52
Anas/Cairina (ducks) 14 631 19.52 1.25 Anser (geese) 2 667 - -
Family Columbidae (pigeons) 2 664 16.63 1.68
Mean diversity (overall mean distance)
Among chicken groups
olbondoc.DNAbarcoding.2012
Chicken Taxa No. of
nucleotide
sequences
N
positions
Diversity (%)
Mean Standar
d Error
Standard breeds 11 633 71.41 5.31
Commercial hybrids 3 666 2.50 0.51
Phil. native chickens 7 646 51.05 3.13
Phil. red jungle fowls 25 627 62.05 5.25
Fighting cocks 10 639 38.29 2.45
All Chickens 56 590 58.37 4.73
olbondoc.DNAbarcoding.2012
Neighbour-Joining tree
with bootstrap support
showing the evolutionary
relationships among
CHICKENS
Palawan Lasak native chicken Mt Taal Batangas RJF
Mt Agustin Camarines Sur RJF Mt Sierra Madre Quirino RJF Mt Sierra Madre Cagayan RJF Mt Bulusan Sorsogon RJF Mt Mayon Albay RJF
Mt Guinatungan Camarines Norte RJF Mt Pandan Masbate RJF Mt Silungan Catanduanes RJF
Mt Supu Capiz RJF Paraoakan native chicken Mt Palomok Zamboanga Sibugay RJF
Mt Makiling Laguna RJF Black Jersey SASSO range chicken Mapolo Hill Batangas RJF Mt Daraitan Rizal RJF Cobb broiler Babcock layer Frost Grey gamefowl Kelso Jimmy East gamefowl Hatch Gilmore gamefowl Hatch Yellow Legged gamefowl Radio gamefowl Blue Cochin bantam Taiwan Yellow Joloano native chicken
Australorp Darag native chicken
Igon native chicken Mt Naujan Mindoro Oriental RJF
Barred Plymouth Rock White Leghorn
Banaba native chicken Hatch Mel Sims gamefowl
Kelso Johnny Jumper gamefowl Black Giant
Sweater McGinnis gamefowl Silky
Hatch Leaper gamefowl Mt Sierra Madre Isabela RJF
New Hampshire Mt Bayugon Palawan RJF
Mt Kamandingan Ilocos Norte RJF Mt Rizal Bohol RJF
Camarines native chicken Mt Tumatangis Sulu RJF Mt Cambandilaan Siquijor RJF Mt Pangasugan Leyte RJF Silver Seabright bantam Mt Natib Bataan RJF Mt Kanlaon Negros Occ Mt Castilla Sorsogon RJF Golden Seabright bantam Malaysian Asil gamefowl
804547282401
1010
1
3663
7499
4449
7979
69
73
99
4563
6635
5391
82857287
7576
36
587336
72
64
5081
83
78
6145
54
40
51
35
45
41
26
47
0.02 divergence
olbondoc.DNAbarcoding.2012
Estimates of evolutionary divergence between mtDNA COI
sequences (d units) in standard breeds of chickens
Diagonals = w/ in group mean distance;
Lower off-diagonal = b/w group difference;
Upper off-diagonal = net b/w group mean distances
Standard breeds Meat and/or
egg type Fancy type
Meat and/or egg type 0.543 ± 0.042 0.034 ± 0.014
Fancy type 0.788 ± 0.059 0.966 ± 0.077
Black Jersey
Taiwan Yellow
Blue Cochin
Australorp
Barred Plymouth Rock
White Leghorn
Black Giant
Silky
Silver Seabright bantam
New Hampshire
Golden Seabright bantam63
100
52
73
50
97
88
90
0.02 divergence
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
Cobb broiler chickens
Babcock layer chickens
SASSO range chickens
0.02 divergence
olbondoc.DNAbarcoding.2012
Palawan Lasak
Paraoakan
Joloano
Banaba
Darag
Igon
Camarines
88
92
80
80
0.02 divergence
olbondoc.DNAbarcoding.2012
Estimates of evolutionary divergence between mtDNA COI
sequences (d units) in Philippine red jungle fowls
Mt Kanlaon Negros Occidental Chocolate Hills Bohol
Mt Castilla Sorsogon Mt Natib Bataan
Mt Camandingan Ilocos Norte Mt Tumatangis Sulu Mt Cambandilaan Siquijor Mt Pangasugan Leyte
Mt Bayugon Palawan Mt Sierra Madre Isabela
Gallus gallus gallus AP003322 Gallus gallus bankiva AP003323 Gallus sonneratii AP006746
Gallus gallus spadiceus AP003321 Gallus lafayettei AP003325
Gallus varius AP003324 Mt Halcon Oriental Mindoro
Mt Palomok Zamboanga Sibugay Mt Makiling Laguna Mapolo Hill Batangas Mt Supu Capiz Mt Daraitan Rizal Mt Guinatungan Camarines Norte Mt Silungan Catanduanes Mt Pandan Masbate Mt Mayon Albay Mt Bulusan Sorsogon Mt Sierra Madre Cagayan Mt Sierra Madre Quirino
Mt Taal Batangas Mt Agustin Camarines Sur
6360
4252
100
23262743
5266
50
64
100
100
9087
8580
76
86
62
6660
42
33
24
19
0.02 divergence
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
Hatch Gilmore
Hatch Yellow Legged
Radio
Frost Grey
Kelso Jimmy East
Hatch Mel Sims
Kelso Johnny Jumper
Hatch Leaper
Sweater McGinnis
Malaysian Asil
99
99
99
77
77
90
71
0.02 divergence
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
Neighbour-Joining tree
with bootstrap support
showing the evolutionary
relationships among
DUCKS
Phil. Mallard Quezon duck
F3 87.5 Pekin x 12.5 Pateros duck
F1 50 Pateros x 50 Pekin duck
Tsaiya duck
Pekin Czech. duck
Phil. Mallard Batangas duck
Pekin duck
Phil. Mallard Pampanga duck
BAI Pateros White duck
Commercial layer duck
Laguna mule duck
Khaki Campbell duck
BAI Pateros Black duck
Muscovy duck
84
67
71
90
99
93
99
79
29
21
62
0.02 divergence
olbondoc.DNAbarcoding.2012
Estimates of evolutionary divergence between mtDNA COI
sequences (d units) in ducks
Diagonals = w/ in group mean distance;
Lower off-diagonal = b/w group difference;
Upper off-diagonal = net b/w group mean distances
Duck
groups
Common
mallard
breeds
Phil.
mallard
strains
Hybrid
ducks
Common
mallard
breeds
0.176
± 0.015
-0.025
± 0.003
-0.023
± 0.004
Phil. mallard
strains
0.140
± 0.011
0.154
± 0.012
-0.021
± 0.002
Hybrid ducks 0.146
± 0.011
0.137
± 0.010
0.162
± 0.013
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
1. Identification of breeds in local genetic
improvement and conservation programs
Applications in local selection and
crossbreeding programs:
Establishment of breed standards
Breed certification and registry work
Local accreditation programs for nucleus
and multiplier breeding farms, stock farms,
breeding centers/stations
IV. Future outlook
Selection program in elite/nucleus
purebred herds/flocks
Commercial crossbreeding programs
in multiplier and base herds
- to ascertain pedigrees, reconstruct
phylogenies, identify phylogeographic
patterns, and estimate patterns of
gene flow
- to resolve disputes about
purity/ homozygosity of
breeding populations and
differences between
breeds, lines, or strains
olbondoc.DNAbarcoding.2012
Applications in genetic conservation programs:
Determine species groups needing more
detailed analysis
Monitor, catalogue, and image biodiversity
of livestock and poultry breeds/strains (e.g.,
UPLB-CA Agripark, per province or per region)
Pre-screening important breeds/strains
that are considered for conservation
and/or cryopreservation in their pure form
olbondoc.DNAbarcoding.2012
olbondoc.DNAbarcoding.2012
2. Tracking of invasive species
Applications of DNA barcoding:
May be used by regulatory agencies in
testing and tracking of invasive pests
in smuggled animals and imported feeds
with forbidden items likely to spread
illnesses
May be used in introgression
studies to assess the effective-
ness of local animal dispersal
programs
May aid public health authorities to
identify carrier of infectious diseases
and other disease vectors
olbondoc.DNAbarcoding.2012
3. Product labeling in the marketing and trade
of animal products
Applications of DNA barcoding:
Promote proper labeling of breeding animals
including breed certificates required for
imported and threatened breeds/strains of
livestock and poultry animals
Prevent and control smuggling, illegal
trade, and poaching of threatened breeds/
strains and their wild relatives
Test for adulteration and contamination of
new and traditional animal food and non-food
products
olbondoc.DNAbarcoding.2012
Thank you. GenBank Accesion No. JX218091
Dr. Orville L. Bondoc’s - mtDNA COI, 640 bp
CTCTGTATCCTTCTTCTTCAAGCTGAGCTGGGCCAGCCTCGCAACCTTCTATGTAACGACCACATCTACAACGTAATCGTCACAGCCCATGCATTTGTAATAATCTTCTTCATAGTAATACCCATCATAATCGGAGGCTTTGGCAACTGACTAGTTCCCCT
AATAATCGGTGCCCCCGATATGGCGTTTCCCCGCATAAACAACATAAGCTTCTGACTCTTACCTCCCTCTCTCCTACTCCTGCTCGCATCTGCTATAGTGGAGGCCGGAGCAGGAACAGGTTGAACAGTCTACCCTCCCTTAGCAGGGAACTACTCCCA
CCCTGGAGCCTCCGTAGACCTAACCATCTTCTCCTTACACCTAGCAGGTGTCTCCTCTATCTTAGGGGCCATCAATTTCATCACAACAATTATCAATATAAAACCCCCTGCCATAACCCAATACCAAACGCCCCTCTTCGTCTGATCCGTCCTAATCACA
GCAGTCCTACTTCTCCTATCTCTCCCAGTCCTAGCTGCTGGCATCACTATACTACTAACAGACCGCAACCTCAACACCACCTTCTTCGACCCCGCCGGAGGAGGAGACCCCATTCTATACCAACACCTATTCTGATTTTTTGGTCACCCTGGAAGTTTAA
olbondoc.DNAbarcoding.2011
Recommendations:
Update information on morphology, ecology,
adaptive differences, and genetic data from the
mitochondrial and nuclear genomes of livestock and
poultry breeds/strains
Expand the DNA barcoding project to include:
Endemic pest and disease organisms (vectors)
Forage pastures
Important milk and meat pathogens in food
safety and food processing technologies
Farm by-products and waste microorganisms
Increased intra- and interspecies sampling
Compare with published barcode sequences found
in the GenBank (NCBI) - http://www.ncbi.nlm.nih.gov
Local biotechnology laboratories should be equipped
to allow extraction of DNA from tissue, hair, feathers,
semen, milk and other animal parts apart from the
usual blood samples
olbondoc.DNAbarcoding.2011
Researchers and graduate students in
collaboration with DA’s regulatory agencies and
bureaus involved in data collection and laboratory
analysis through the assistance of various LGUs
Optimized DNA barcoding protocols (i.e. DNA
extraction, purification, elution, and amplification)
should be made available to local scientific community
New research projects can be developed through
inter-laboratory and institutional collaborations