professor of animal breeding/genetics animal and dairy ... abella, romy almeda, modesto alonzo,...

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 ,


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,


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


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)


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


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.)


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


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.


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


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


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

http://www.barcodinglife.org/


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


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)


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)


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)


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


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)


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)


II. Generating DNA barcodes for livestock and poultry breeds/strains

Field sampling and blood collection


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


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


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


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.


Laboratory


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.



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.


Laboratory


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


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).



Laboratory


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


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.)


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


Figure 2. Taxonomic arrangement of common poultry

animals (Class Aves) in the Philippines



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


http://www.ics.uplb.edu.ph/UPLB-DA-DNA-barcoding-project









COI sequence analysis


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.


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


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


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


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


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



sequences (d units)

- Cattle




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



sequences (d units)

- Goats




Goat

groups Purebred Native strains

Purebred 0.111 ±

0.012

0.712 ±

0.059

Native

strains

0.911 ±

0.062

0.286 ±

0.020





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



sequences (d units)

- Sheep




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





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





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



sequences (d units) in pigs




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


Companion animals





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


Among poultry families/species


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


Among chicken groups


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


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



sequences (d units) in standard breeds of chickens




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


Cobb broiler chickens

Babcock layer chickens

SASSO range chickens

0.02 divergence


Palawan Lasak

Paraoakan

Joloano

Banaba

Darag

Igon

Camarines

88

92

80

80

0.02 divergence



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


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


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



sequences (d units) in ducks




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


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


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



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


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


Thank you. GenBank Accesion No. JX218091

Dr. Orville L. Bondoc’s - mtDNA COI, 640 bp

CTCTGTATCCTTCTTCTTCAAGCTGAGCTGGGCCAGCCTCGCAACCTTCTATGTAACGACCACATCTACAACGTAATCGTCACAGCCCATGCATTTGTAATAATCTTCTTCATAGTAATACCCATCATAATCGGAGGCTTTGGCAACTGACTAGTTCCCCT

AATAATCGGTGCCCCCGATATGGCGTTTCCCCGCATAAACAACATAAGCTTCTGACTCTTACCTCCCTCTCTCCTACTCCTGCTCGCATCTGCTATAGTGGAGGCCGGAGCAGGAACAGGTTGAACAGTCTACCCTCCCTTAGCAGGGAACTACTCCCA

CCCTGGAGCCTCCGTAGACCTAACCATCTTCTCCTTACACCTAGCAGGTGTCTCCTCTATCTTAGGGGCCATCAATTTCATCACAACAATTATCAATATAAAACCCCCTGCCATAACCCAATACCAAACGCCCCTCTTCGTCTGATCCGTCCTAATCACA

GCAGTCCTACTTCTCCTATCTCTCCCAGTCCTAGCTGCTGGCATCACTATACTACTAACAGACCGCAACCTCAACACCACCTTCTTCGACCCCGCCGGAGGAGGAGACCCCATTCTATACCAACACCTATTCTGATTTTTTGGTCACCCTGGAAGTTTAA


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

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


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

professor of animal breeding/genetics animal and dairy ... abella, romy almeda, modesto alonzo,...

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