Conservation of Red Junglefowl Gallus Gallus in India IJGC

8/12/2019 Conservation of Red Junglefowl Gallus Gallus in India IJGC

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2009 World Pheasant Association.

International Journal of Galliformes Conservation, 1, 94101

Conservation of red junglefowl Gallus gallusin India

MERWYN FERNANDES1,MUKESH1,S.SATHYAKUMAR1*,RAHUL KAUL2,RAJIV S.KALSI3andDEEPAKSHARMA41Wildlife Institute of India, P.O. Box 18, Chandrabani, Dehradun 248001, Uttarkhand, India.2Wildlife Trust of India, A-220, New Friends Colony, New Delhi 110 065, India.3

M.L.N. College, Yamuna Nagar 135 001, Haryana, India.4Central Avian Research Institute, Bareilly, Uttar Pradesh, India.*Correspondence author - [email protected]

Paper presented at the 4thInternational Galliformes Symposium, 2007, Chengdu, China.

AbstractThe red junglefowl (RJF) is one of the most important species for mankind, due to economicand cultural reasons. Recently, fears have been expressed that the wild RJF may be geneticallycontaminated, leading to an inference that there may not be any pure RJF left in the wild. In order toassess the distribution of RJF in India, field surveys were carried out and secondary information wascollated. Historically, RJF occurred in 270 districts in 21 states across India, but now it is found in 205districts in the 21 states. Of the 255 Protected Areas (PAs) that occur within the RJFs distributionalrange in India, 190 PAs (31 National Parks [NPs] and 159 Wildlife Sanctuaries [WSs]) have reported its

presence. A composite set of trait characters that are presumed to be indicators of wild RJF was usedfor characterising RJF in the field. A total of 563 (293 males and 270 females) RJF were characterisedof which 7% of birds in the central region had reports of white ear patch. Eclipse plumage wasobserved in wild and captive birds. Ninety-two RJF samples and twenty five domestic chicken sampleswere collected and processed for DNA extraction. Thirty highly polymorphic microsatellite markerswere utilised for RJF and domestic chicken genotyping. Preliminary studies showed polymorphismwithin RJF at these microsatellite loci.

Keywords DNA extraction, hybridisation, microsatellite markers, PCR, red junglefowl, traits.

Introduction

The red junglefowl Gallus gallus (RJF), theancestor of the domestic chicken, is one of themost important species for mankind, due to itseconomic and cultural significance. Liu et al.,(2006) suggested that there are distinctdistribution patterns and expansion signaturessuggesting that different clades may originatefrom different regions, which support the theoryof multiple origins in South and Southeast Asia.The present day, multi-billion dollar poultryindustry is based on the RJF and may have todepend on it in the future. Andersson et al.(1994) stated that populations of domestic

animals and their wild ancestors provide avaluable source of genetic diversity that may beexploited to develop animal models forquantitative traits of biological and medicalinterest. Hence conservation of genetically purewild forms or their representatives have greatpotential to make a significant contribution tothe study of some economically importantgenetic traits (Brisbin et al., 2002).

The RJF is widely distributed and its five sub-species are spread from the Indian sub-continent eastwards across Myanmar, SouthChina, Indonesia to Java (Johnsgard, 1986). InIndia, two sub-species occur, the typespecimen, Gallus gallus murghii and Gallusgallus spadiceus (Ali & Ripley, 1983). While theformer is found in the north and central part ofIndia, extending eastwards to Orissa and WestBengal, the latter is confined to the northeastern parts of India. Recently, fears havebeen expressed that the wild RJF populationsmay be genetically contaminated withdomesticated chickens, leading to an inferencethat there may not be any pure RJF populations

in the wild (Peterson & Brisbin, 1998), causingintrogression of domestic genes into wild birds.An analysis of skins by Peterson & Brisbin(1998) showed a lack of phenotypic traits,which characterise true wild RJF, as describedby Morejohn (1968).

This study investigated the status of RJF inIndia and aimed to identify ways to safeguardremaining pure wild birds. In 2006, acollaborative research project on the


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Conservation of red junglefowl 95

2009 World Pheasant Association.International Journal of Galliformes Conservation, 1, 94101

conservation of RJF was initiated focussing on,1) an assessment of the current status anddistribution of RJF in India; 2) the identificationof pure RJF populations by molecular studies;3) investigations into social interactionsbetween wild RJF and domestic or feral chickenand 4) the development of a conservationaction plan for RJF.

This paper presents the preliminary findings onthe current status and distribution of RJF inIndia, standardisation of DNA extractionprotocols for various sample types, optimisationof PCR condition and amplification of sevenmicrosatellite loci in extracted DNA samples.The proposed plan of work is also presentedand discussed.

Methods

Assessing the status and distribution of

RJF in IndiaIn order to assess the distribution of RJF inIndia, primary and secondary data was collated.Secondary data was gathered throughliterature, questionnaire surveys and reliablepersonal communication. Ad libitum surveysusing the encounter rate method were carriedout to obtain abundance estimates using direct(sightings) or indirect (calls) methods (Bibby etal., 1992). The data (primary and secondary)were digitalized in a Geographical InformationSystem (GIS) using ARCinfo software. A rule-based model was created using forest coverand elevation below 2500 m. A minimum of

two transects were selected within representedhabitat types, which were traversed a minimumof three times. The encounter rates werepooled for the different forest types.

Identification of pure RJF by moleculargenetic studiesA composite set of traits were complied toidentify pure wild RJF from introgressed hybrids(Morejohn, 1968; Johnsgard, 1986; Peterson &Brisbin, 1998; Kaul et al., 2004). The traitsused were yellow-coloured hackles, slendersmooth thin black legs, a white patch on the

upper tail coverts, elongated sickle shapecentral tail feathers and horizontal tail carriagein males. Males moult during the post breedingseason and have an eclipse plumage.

Sampling for genetic analysis

Wild RJF were live-trapped using modified foot-hold snares (Ramesh & Kalsi, 2007) with thehelp of local bird-trappers in different RJF rangestates in India. The birds were carefullyhandled during sample collection and released.

Samples were also collected from the captiveRJF. Blood was collected from the brachialveinof live trapped individuals and stored in DNA zolBD as well as on FTA cards (Mackey et al.,1997). Freshly pulled primary feathers werestored in 70% alcohol, while moulted feathersand hatched egg shells were collected andstored in a zip lock bag for dry preservation.

DNA Extraction from whole blood, tissue,feather and egg shell membrane

Genomic DNA was extracted from bloodfollowing DNA zol BD based protocol Mackey etal., 1996. For tissue, feathers and egg shellmembrane DNA was extracted using QiagenDNeasy tissue kit (Qiagen, Germany) accordingto the manufacturers protocol with thefollowing alterations: addition of 100 mg/mlDTT solution in the lysis buffer, digestion wasperformed overnight at 550C in a shaking waterbath and addition of ice chilled ethanol forbetter precipitation and DNA was finallyrecovered in 80-100 l of elution buffer. DNAwas quantified with UV spectrophotometer andconcentration was adjusted to approximately50-80 ng/l with TE buffer.

Microsatellite markersA set of thirty highly polymorphic microsatellitemarkers were utilised for genotyping of RJF anddomestic chicken (TABLE 3). (http://dad.fao.org/en/refer/library/guidelin/marker.pdf)

Optimisation of cycling condition and

amplification of microsatellite lociFor initial standardisation, reported conditionswere tested first and modified to obtain optimalresults. Annealing temperature for each locuswas optimised in a gradient PCR and sevenmicrosatellite loci were amplified in extractedDNA samples. PCR amplification was performedin a 10 l reaction volume. Each reactionconsists 1X PCR Buffer (50 mM Kcl, 10 mM Tris-Hcl), 1.5 mM Mgcl2, 200 M of each d-NTPs,25X BSA, 10 p-mole of each primer (forwardand reverse), 0.5 unit of Taq DNA polymerase,50 to 80 ng of genomic DNA. Protocol for PCR

reaction was comprised of an initialdenaturation at 940C for 2 minute, followed by35 cycles of denaturation at 940C for 45seconds, primer annealing for 45 seconds at550C, primer extension for 1 minute at 720Cand a final extension of 10 minutes at 720C.About 5 l of PCR product was resolved on 2.0% Agarose, 100 bp ladder was used asmolecular size markers.


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TABLE 1 Collection of RJF and domestic chickensamples in different states

Results

Distribution of RJF Gallus gallus in IndiaThe current distribution of RJF based on therule model is c652,000 km2 (FIG. 1). Thespecies is present in 205 Districts in 21 rangestates in India. Of the 255 PAs that occur withinthe RJF distribution range in India, 190 PAs (31NPs and 159 WSs) have reported presence intheir areas. During March 2007, there were 209

individuals (74 males: 80 females: 55 chicks)RJF in captivity in the various zoos andpheasantries in the states of Andhra Pradesh,Delhi, Haryana, Himachal Pradesh and UttarPradesh.

As there are no population estimates for RJF inthe Pas, an encounter rate method was used. Atotal of 38 trails covering 358 km weretraversed taking 546 man hours. Theencounters rate was pooled together for similarhabitats (TABLE 2). The encounter rate was thehighest for the summer months in the Shivalik

Region. This area also had the highest variancearound the estimate, suggesting that moresampling is needed for the area.

Morphological traitsIn total, 563 RJF (293 males and 270 females)were sighted during the field surveys. All maleshad the presence of yellowish hackles, slateyblack thin tarsus and white ear lobes in 7% (n=5) of the central region, while other areas hadpinkish ear lobes. The tail carriage was difficult

to characterise, but the presence of the sicklefeather was prominent in all cases. In 30% offemales, the comb was either rudimentary orabsent, while in the rest, classification was notpossible. This was mainly due to crypticcolouration and behaviour. Eclipse plumage wasalso observed in RJFs (n = 70), howevercaptive populations that were examined at the

Morni Hill Pheasantry during the period July September and these also had eclipse plumage.

FIG.1 Current distribution of Red Junglefowl inIndia.

Isolation of genomic DNA from different

tissuesA total of 92 samples of RJF and 25 samples ofdomestic chicken were collected from variousparts of India (TABLE 1). Of these 50 sampleswere from wild and 42 samples from captivity.The genomic DNA was extracted from blood,tissue, feather follicles and egg shellmembrane. A good quality and quantity of

genomic DNA was obtained from whole bloodfollowing DNA zol BD protocol (FIG.2). DNA wasalso extractable from tissue, feather follicle andegg shell membrane, but the quantity as well asquality of genomic DNA extracted from featherfollicle and egg membrane was much lower incomparison to the DNA extracted from tissue(FIG.3).

RJF SamplesState

Wild Captivity

DomesticChicken

Samples

Jammu andKashmir

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HimachalPradesh 2 25 1Uttarakhand 9 2 4Haryana - 9 2Uttar Pradesh - 16 -Chhatisgarh 2 - 1Bihar 8 2 5West Bengal - - 1Sikkim 1 - -Orissa 4 - 2AndhraPradesh

- 4 -

Assam 2 - -Nagaland 1 - -Mizoram 1 - -Meghalaya 2 - -


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TABLE 2 Encounter rate (No/km) for Red Junglefowl in various habitat types in India.

Habitat types Area Months Transects(no, km)

Birds (mean noper km s.e)

Mangroves Bhittarkanika Nov 4, 9.8 1.36 0.29Moist mixed forest parts of Orissa & Udanti

WSNov-Dec 10, 46 0.17 0.09

Dry deciduous parts of Andhra Pradesh Dec-Jan 16, 103 0

Moist mixed forest Meghalaya & Assam Feb-Mar 18, 72 0.47 0.26Grassland &Woodland

Assam floodplains Mar-Apr 18, 40 1.43 0.21

Shivaliks Uttarakhand May 12,19.2 5.06 1.26Himalayan foothills Himachal Pradesh Jun-July 8, 18 0.69 0.30

FIG. 2 Gel electrophoresis (0.8% agarose) ofextracted DNA, L - 1Kb ladder, Lane 1 to 12 RJFsamples, Lane 13 to 24 domestic chickens.

L 1 2 3 4 5 6 L

FIG 3 Gel electrophoresis (0.8% agarose) ofextracted DNA, L - 1Kb ladder, lanes 1 = blood,2 & 3 = tissue, 4 = pulled feathers, 5 = fallenfeathers and 6 = egg shell.

Optimisation of PCR assay for

microsatellite genotypingPCR cycling condition was optimised for allmicrosatellite loci and the optimised annealingtemperature are given in TABLE 3. Annealingtemperature for each locus was optimised in agradient PCR (FIG. 4) and seven microsatelliteloci were amplified in extracted DNA samples.The amplification success rate showed adiscrepancy with sample types as the blood andtissue sample showed a higher success rate,followed by feathers while with egg shell, the

success rate was least (FIG. 5). The averageallele sizes for microsatellite loci was similar tothose reported by Weigend (pers. comm.).

TABLE 3 Microsatellite markers used.

Marker Chrom

osome

Allele Range TA

ADL0268 1 102-116 600CMCW0206 2 221-249 600 CLEI0166 3 354-370 600 CMCW0020 1 179-185 600 CMCW0037 3 154-160 640 CLEI0192 6 244-370 600 CADL0112 10 120-134 580 CMCW0295 4 88-106 600 CMCW0067 8 176-186 600 CMCW0104 13 190-234 600 CMCW0111 1 96-120 600 CMCW0034 2 212-246 600 C

MCW0222 3 220-226 60

0

CLEI0094 4 247-287 600 CMCW0216 13 139-149 600 CMCW0081 5 112-135 600 CMCW0330 17 256-300 600 CLEI0234 2 216-364 600 CMCW0103 3 266-270 640 CMCW0098 4 261-265 600 CMCW0284 4 235-243 600 C

MCW0069E60C04W23

158-176 600 C

MCW0016 3 162-206 600 CMCW0078 5 135-147 600 CMCW0014 6 164-182 580 C

MCW0183 7 296-326 580 CMCW0123 14 76-100 600 CMCW0165 23 114-118 600 CMCW0248 W29 205-225 600 CADL0278 8 114-126 600 C

L 1 2 3 4 5 6 7 8 9 10 11 12 L

L 13 14 15 16 17 18 19 20 21 22 23 24 L


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0

20

40

60

80

100

SSK 1 SSK 2 SSK 3 SSK 4 SSK 5 SSK 6 SSK 7 SSK 8

Microsatellite loci ID

Amplificationsuccessrate(%)

Blood (n=86) Tissue (n=5) Pulled feathers (n=6)

Fal len feathers (n=17) Egg shells (n -=3)

FIG.5 Amplification success rate in reference ofsample types

Due to the varied habitat in the distributionrange there were constraints in transectrepeatability in different seasons hencedetection probabilities were not included in theabundance estimates. However, we propose tocorrect for detection probabilities in thedifferent habitats and seasons for the differentzones in India by repeating surveys withinthese same areas, which will give an abundanceestimate.

In this study different samples were used forDNA extraction. The blood and tissue proved tobe very good samples types for DNA extraction.Avian blood, having nucleated red bloodcorpuscles (RBCs), is always the sample ofchoice for DNA extraction. Earlier studies alsoshowed that genomic DNA of good quality, aswell as quantity, can be extracted from frozen

and stored chicken blood (Sharma & Appa Rao,2000). Extraction of genomic DNA from featherfollicle and egg shell membrane is the non-invasive method. The quantity of genomic DNAextracted was quite low with these samples butwas enough for PCR amplification.

PCR amplification is influenced by number offactors such as quality and quantity of genomicDNA, concentration of magnesium ions anddNTPs and annealing temperature (Williams etal. 1993) hence, the initial standardisation ofPCR amplification conditions are often

necessary to get optimum amplification. Theseamplified products were resolved on 3%agarose gel and the average allele sizeobserved for these microsatellite markers werein accordance with that reported by Weigend(pers. comm.). Since the microsatellite allelesdiffer in few base pairs with each other at agiven locus, the ordinary agarose gel is notsufficient to differentiate the alleles. Hence forbetter allele differentiation, the amplifiedproduct will need to be resolved on metaphoragarose or 4 - 5% denaturing polyacrylamide

gels or on automated DNA sequencer. Sharma(2006) reported polymorphism not only withinRJF population, but also between RJF andchicken breeds on 3.5 % metaphor agarose.

Though these preliminary studies showed thepresence of polymorphism within RJFpopulation at these seven microsatellite loci,

these microsatellite markers, as well as another23 markers, will be used to develop themicrosatellite profile of RJF population usingautomated DNA sequencer. These microsatelliteallelic profiles will be utilised for estimating thegenetic diversity present within the RJFpopulation. Such estimates will be suggestive ofexisting genetic variability between the RJFpopulations from different regions of thecountry. Further genetic distance analysis willbe undertaken using the microsatellite allelicprofile of RJF with that of chicken and used intesting the purity of RJF. Since theintrogression of domesticated chicken genes inwild RJF might affect the phenotypic expressionof physical traits such as eclipse plumage, henscomb, leg colour, horizontal body posture andtail carriage, simpler and a shorter call, it maybe necessary to correlate the genetic purity andthe physical traits in order to identify pure RJF.

A few sites within the distribution range such asSariyanj (Himachal Pradesh) will be taken upfor intensive studies. These sites will try toaddress issues with respect to the ecology andbehaviour (interactions) of wild RJF with thedomestic fowl.

Acknowledgements

We thank the Director, Dean and FacultyMembers Wildlife Institute of India, ExecutiveDirector and staff of Wildlife Trust of India,Principal and staff of MLN College, for theirencouragement and support. We thank theState Forest/Wildlife Departments of the RJFrange states for granting us permission toconduct field work and sample collections. Wethank the PCCFs , CWLWs, Park Managers, ZooCurators and field staff in the RJF Range States

for their help and cooperation during field work.Our thanks are due to Shri Qamar Qureshi andDr K. Ramesh for reviews and comments on themanuscript; and to Dr. S.P.Goyal, Dr K. Sankar,Shri Pratap Singh, Shri Dhananjay Mohan, Dr.Manoj Aggarwal and Dr. R. Jayapal for theirinputs. We acknowledge the help provided bythe researchers of the WIIs All India TigerMonitoring Project, field assistants and birdtrappers.


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Biographical sketches

MERWYN FERNANDES has a Masters in WildlifeBiology, currently working as a Research Fellowwith the Wildlife Institute of India. His areas ofinterest include wildlife law and policies, urbanlandscapes and behaviour. MUKESH has a

Masters in Biotechnology, currently working asa Research Fellow with the Wildlife Institute ofIndia. His areas of interests include populationgenetics, conservation biology and wildlifeforensics. S. SATHYAKUMAR has a Masters andD.Phil Degree in Wildlife Science and iscurrently a Scientist at the Wildlife Institute ofIndia, Dehradun. He is interested in theecology and conservation of high altitudewildlife, particularly mammals and pheasantsand has been carrying out research on theHimalayan fauna since 1989. His subjectinterests include Habitat Ecology, Population

Ecology and Quantitative Ecology. RAHUL KAULhas a Masters and D.Phil Degree in Zoology andis currently Director of ConservationProgrammes at the Wildlife Trust of India, NewDelhi. He is interested in the conservation ofpheasants and other wildlife, particularlymammals in India. RAJIV S.KALSIhas a Mastersand D.Phil Degree in Zoology and is currently aFaculty in the Department of Zoology, M. L. N.College, Yamuna Nagar. He is interested in theconservation of birds and conservation


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2009 World Pheasant Association.Inte national Jo nal of Gallifo mes Conse ation 1 94 101

genetics. DEEPAK SHARMA has a Masters andD.Phil degree in Zoology and is a seniorScientist at the Central Avian ResearchInstitute, Bareilly, U.P., and has been

conducting studies on the comparative genomeof RJF and domestic chicken.

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Conservation of Red Junglefowl Gallus Gallus in India IJGC