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SHORT COMMUNICATION A panel of polymorphic microsatellite markers in Himalayanmonal Lophophorus impejanus developed by cross-speciesamplification and their applicability in other Galliformes Thakur Mukesh & Ishwari D. Rai & Rishi P. Mandhan & Sambandam Sathyakumar Received: 30 March 2010 /Revised: 30 December 2010 /Accepted: 6 January 2011 # Springer-Verlag 2011 Abstract Isolation and development of new microsatellitemarkers for any species is still labour-intensive and requiressubstantial inputs of time, money and expertise. Therefore,cross-species microsatellite amplification can be an effec-tive way in obtaining microsatellite loci for closely relatedtaxa in bird species. We have reported microsatellite loci for Himalayan monal for the first time. Fifteen microsatellitemarkers developed for chicken were cross-amplified inHimalayan monal. All the tested 15 microsatellite markerswere polymorphic, with mean (± s.e.) allelic number of 4±1.51, ranging 2 – 7 per locus. The observed heterozygosityin the population ranged between 0.285 and 0.714, withmean (± s.e.) of 0.499±0.125, indicating considerablegenetic variation in this population. While 12 loci conformedto Hardy – Weinberg equilibrium ( P >0.05), 3 loci, i.e.MCW0295, MCW0081, MCW0330 deviated from it ( P <0.05). No evidence for linkage disequilibrium was observedamong pair of loci. Our study show that these 15 micro-satellites loci could be employed in population geneticstudies for Himalayan monal and their applicability in JungleBush Quail, Grey francolin and Kalij pheasant. Keywords Chicken microsatellites.Cross-speciesamplification.Himalayan monal. Lophophorus impejanus .Galliformes Introduction The Himalayan monal, Lophophorus impejanus Latham1790, also known as the Impeyan Pheasant, is a bird of genus Lophophorus of the family, Phasianidae (Ali andRipley1983). It is a large, brightly coloured bird that isstrongly associated with the forested and alpine habitats inthe Himalaya and in the folklore of local communities. It isthe national bird of Nepal and the state bird of Uttarakhand,India. It is distributed in the northwestern, western, centraland eastern Himalayas (Jammu & Kashmir, HimachalPradesh, Uttarakhand, Sikkim & Arunachal Pradesh) inIndia between 2,300 – 4,875 m and down to 2,000 m duringwinter in western Himalaya, and between 2,800 – 4,575 mand down to 2,500 m during winter in eastern Himalaya (Sathyakumar and Kaul2007). Although it is listed as a ‘ least concern ’ species by the IUCN red list, the species isthreatened by poaching and habitat fragmentation within itsdistribution. It is listed in schedule I of the Indian Wildlife(Protection) Act and listed in appendix I of the CITES(Sathyakumar and Kaul2007). However, the current data are not enough to assess the present status of Himalayanmonal because only few biological and ecological studiesare available on the Himalayan monal (Ramesh2003;Suresh Kumar et al.2006), and no detailed genetic studyhas been carried out so far.Microsatellite DNA markers are simple sequence repeats(Tautz1989) distributed along the genome (Litt and Luty Communicated by C. Gortázar T. Mukesh : I. D. Rai : S. Sathyakumar ( * )Department of Endangered Species Management,Wildlife Institute of India,P.O. Box No. 18, Chandrabani,Dehradun 248001 Uttarakhand, India e-mail:
[email protected]. Mukesh : R. P. MandhanDepartment of Biotechnology, Kurukshetra University,Kurukshetra 136119 Haryana, India Eur J Wildl ResDOI 10.1007/s10344-011-0494-1 1989). By virtue of this feature, they have become powerfultools used in forensic studies, kinship investigation, genemapping, conservation biology and population genetics(Jarne and Lagoda 1996; Zane et al.2002; Gibson et al. 2005). Despite their usefulness, isolation and development of microsatellite markers is most time-consuming, labour-intensive, expensive and requires a skilled molecular biologist. Thus, using loci, already developed in a relativespecies, may provide a cost-effective alternative to micro-satellite isolation and development in a species of interest.Cross-species amplification is only effective if the primer sequences are conserved between species. Generally, thenumber of loci amplifying tends to decrease with increasingdivergence between species (Moore et al.1991; Peakall et al.1998). Besides, the relationship between amplificationsuccess and the evolutionary divergence of source-target species has also been widely studied in birds (Primmer et al.1996; Dallimer 1999; Dawson et al.2000; Galbusera et al.2000). Although the conclusions of these studies wereinconsistent, they can still be used as valuable referenceswhen conducting studies on cross-species microsatelliteamplification (Primmer et al.2005). Several early studieshave shown the applicability of microsatellite loci amongclosely related species by means of cross-species amplifi-cation (Moore et al.1991; Primmer et al.1996; Pang et al. 1999; Baratti et al.2001; Wilson et al.2004; Huang et al. 2005; Kupper et al.2007; Sruoga et al.2008; Zhou et al. 2009). In this paper, we present the information on theestablishment of a set of 15 polymorphic microsatellites for Himalayan monal by cross-species amplification and their use in other related birds. Materials and methods Sample collection and DNA extractionShed feathers of Himalayan monal ( n =18) and KalijPheasant ( Lophura leucomelanos ) ( n =2) were collectedfrom different locations in Kedarnath Wildlife Sanctuary,Uttarakhand, India (30°30 ′ N, 79°15 ′ E) (Fig.1), whilefeathers from Jungle Bush Quail ( Perdicula asiatica )( n =2) and Grey francolin ( Francolinus pondicerianus )( n =2) were collected from different reserved forests of Uttarakhand. Genomic DNAwas then extracted from thesefeather samples using Qiagen DNeasy tissue kit (Qiagen,Germany) following the manufacturer's protocol with thefollowing alterations: (1) addition of 100 mg/ml DTTsolution in the lysis buffer; (2) digestion was performedovernight at 55°C in a shaking water bath and addition of ice-chilled ethanol for better precipitation; and (3) DNAwas finally recovered in 30 – 40 μ l of elution buffer andstored at − 20°C.Microsatellite genotypingA total number of 15 microsatellite loci, which weredeveloped for chicken and earlier used in the (AVIANDIV,Weigend S Coordinator et al.1998) project, were selectedfor this study. Polymerase chain reactions (PCR) were performed on an Applied Biosystems thermal cycler (2700and 2720) in a 10- μ l reaction mixture containing 1× PCR Buffer (50 mM KCl, 10 mM tris – HCl), 1.5 mM MgCl 2 ,200 μ M of each d-NTP, 1.25 μ g BSA, 4 p-mole of each primer, 0.5 unit of Taq DNA polymerase (MBI, Fermentas)and approximately 25 ng genomic DNA. The amplificationconditions were 2-min initial denaturation at 94°C, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at specific temperature (Table1) for 45 s and extension at 72°Cfor 2 min with a final extension at 72°C for 15 min.Annealing temperature for each microsatellite loci was first tested as defined in the target species and then modifiedaccordingly to get the optimum amplification. Approximately,5 μ l of PCR products were mixed with 1 μ l of loading buffer and then loaded onto a 2% agarose gel along with the sizestandard and visualised over UV light to detect amplification.Primers producing visible and expected bands were labelledwith fluorescent dyes (FAM, VIC, PET, NED) at the 5 ′ end.PCR products were pooled and denatured at 95°C for 5 minand electrophoresed using ABI 3130 Genetic Analyser (Applied Biosystem) with GeneScan 500 (-250) LIZ as theinternal lane size standard. Data were collected and analysedusingGeneMapperSoftware(Version3.7,AppliedBiosystem).There was less samples of Jungle Bush quail, Grey francolinand Kalij pheasant ( n =2), so we excluded these species for further analysis, while all the Himalayan monal samples( n =18) were amplified with 15 microsatellite loci, therefore,subjected to further statistical analysis.Statistical analysisMicrosatellite data quality was statistically assessed for genotyping errors due to non-amplified alleles (null alleles),short allele dominance (large allele dropout) and the scoringof stutter peaks using the computer programme MICRO-CHECKER 2.2 (Van Oosterhout 2004). The observed andeffective number of alleles, percentage of polymorphic lociobserved and expected heterozygosity estimates werecomputed after Nei (1973), as executed in POPGENEsoftware (Yeh et al.1999). Using allelic frequencies, polymorphic information content (PIC), a measure of marker's informativeness, was calculated with the Cervus(ver. 3.0) computer programme (Kalinowski et al.2007).Hardy – Weinberg equilibrium (HWE) estimations wereconducted using the exact test of POPGENE (Yeh et al.1999). Linkage disequilibrium (LD) test between pairs of loci was performed in FSTAT (Goudet 1995). Eur J Wildl Res Results All the 15 microsatellites developed for chicken wereamplified successfully in the four gallinaceous bird speciesthrough cross-species amplification. Jungle Bush quail,Grey francolin and Kalij pheasant could not be analysedfurther for diversity estimates due to low sample size, andtheir genotype is presented in Table1. PCR amplificationcould not be tested because of depletion of DNA in fewsamples of these four species, and therefore indicated asunknown (UK) in Table1. All the 18 samples of Himalayanmonal were amplified, and a total number of 64 distinct alleles were distinguished over the 15 microsatellite loci.Genotype biases were evaluated with Micro-Checker (Van Oosterhout et al.2004), and null alleles were not found with any of the 15 loci used. The genotype data revealed a reasonable amount of polymorphism (Table2).The numberofobservedallelesrangedfromtwo(MCW0222,MCW0016, MCW0078) to seven (MCW0081), with overallmean number of alleles per locus of 4.0 (±1.51 s.e. [standarderror]). The observed number of alleles for all the 15 lociexceeded the effective number of alleles, which varied from1.8 (MCW0016, MCW0078) to 4.23 (MCW0330) with mean(± s.e.) of 2.62±0.091 (Table2). Eight microsatellite markersshow PIC values higher than 0.5, which is normallyconsidered as informative in population-genetic analyses(Botstein et al.1980). These loci were informative in the present study; while PIC value was lower than 0.5 for sevenloci (ADL0268, ADL0112, MCW0222, LEI0234,MCW0016, MCW0078 and MCW0165). The mean PIC inour samples was 0.5249. Mean (± s.e.) observed heterozy-gosity over the 15 loci was 0.499±0.125, which was lower than the expected heterozygosity 0.632±0.124. Expectedheterozygosity in Himalayan monal samples ranged from0.484 (MCW0016, MCW0098) to 0.857 (MCW0081). Theaverage observed heterozygosity estimation in this study was0.391±0.134. All the 15 loci were tested for any deviation toHWE and 12 loci conformed to HWE ( P >0.05) while three Fig. 1 Map of Kedarnath Wildlife Sanctuary showing the sampling location of Himalayan monalEur J Wildl Res T a b l e 1 C h a r a c t e r i s a t i o n o f 1 5 c h i c k e n m i c r o s a t e l l i t e m a r k e r s i n J u n g l e B u s h q u a i l , G r e y f r a n c o l i n a n d K a l i j p h e a s a n t L o c u s A l l e l e r a n g e s G e n b a n k ( a c c e s s i o n n o . ) P r i m e r s e q u e n c e ( 5 ′ - > 3 ′ ) ( f w d & r e v e r s e ) A n n e a l i n g t e m p e r a t u r e T A ( ° C ) J u n g l e B u s h q u a i l ( J B Q ) G r e y f r a n c o l i n ( G F ) K a l i j p h e a s a n t ( K J P ) J B Q 1 J B Q 2 G F 1 G F 2 K J P 1 K J P 2 A D L 0 2 6 8 1 0 2 – 1 1 6 G 0 1 6 8 8 C T C C A C C C C T C T C A G A A C T A C A A C T T C C C A T C T A C C T A C T 6 0 U K U K 1 0 2 / 1 0 2 1 0 4 / 1 0 6 U K 1 0 4 / 1 0 4 L E I 0 1 6 6 3 5 4 – 3 7 0 X 8 5 5 3 1 C T C C T G C C C T T A G C T A C G C A T A T C C C C T G G C T G G G A G T T T 6 0 U K U K 3 4 6 / 3 4 6 3 4 8 / 3 5 6 U K 3 5 4 / 3 5 4 A D L 0 1 1 2 1 2 0 – 1 3 4 G 0 1 7 2 5 G G C T T A A G C T G A C C C A T T A T A T C T C A A A T G T A A T G C G T G C 5 8 U K U K U K U K U K U K M C W 0 2 9 5 8 8 – 1 0 6 G 3 2 0 5 2 A T C A C T A C A G A A C A C C C T C T C T A T G T A T G C A C G C A G A T A T C C 6 0 8 4 / 9 0 8 6 / 9 4 9 0 / 9 0 8 6 / 9 4 7 2 / 8 8 8 8 / 9 0 M C W 0 0 6 7 1 7 6 – 1 8 6 G 3 1 9 4 5 G C A C T A C T G T G T G C T G C A G T T T G A G A T G T A G T T G C C A C A T T C C G A C 5 8 1 8 2 / 1 8 2 1 8 2 / 1 8 4 1 9 4 / 1 9 6 1 9 2 / 1 9 2 1 8 4 / 1 8 4 1 8 4 / 1 8 6 M C W 0 1 1 1 9 6 – 1 2 0 L 4 8 9 0 9 G C T C C A T G T G A A G T G G T T T A A T G T C C A C T T G T C A A T G A T G 6 0 1 0 0 / 1 0 0 1 0 2 / 1 0 4 U K U K U K U K M C W 0 2 2 2 2 2 0 – 2 2 6 G 3 1 9 9 7 G C A G T T A C A T T G A A A T G A T T C C T T C T C A A A A C A C C T A G A A G A C 6 0 2 2 0 / 2 2 4 2 2 0 / 2 2 0 U K U K 2 1 8 / 2 1 8 2 1 8 / 2 2 0 L E I 0 0 9 4 2 4 7 – 2 8 7 X 8 3 2 4 6 G A T C T C A C C A G T A T G A G C T G C T C T C A C A C T G T A A C A C A G T G C 6 0 2 2 7 / 2 4 7 2 2 7 / 2 4 7 2 2 1 / 2 2 1 2 2 1 / 2 2 3 2 3 9 / 2 4 3 2 3 9 / 2 3 9 M C W 0 0 8 1 1 1 2 – 1 3 6 n o n e G T T G C T G A G A G C C T G G T G C A G C C T G T A T G T G G A A T T A C T T C T C 5 8 1 3 0 / 1 3 2 1 3 0 / 1 3 2 1 1 2 / 1 1 2 1 1 2 / 1 2 8 1 0 8 / 1 1 8 1 1 2 / 1 1 2 M C W 0 3 3 0 2 5 6 – 3 0 0 G 3 2 0 8 5 T G G A C C T C A T C A G T C T G A C A G A A T G T T C T C A T A G A G T T C C T G C 6 0 2 5 6 / 2 5 6 2 5 6 / 2 6 2 2 5 0 / 2 5 0 2 5 0 / 2 5 2 2 4 8 / 2 5 8 2 4 8 / 2 4 8 L E I 0 2 3 4 2 1 6 – 3 6 4 Z 9 4 8 3 7 A T G C A T C A G A T T G G T A T T C A A C G T G G C T G T G A A C A A A T A T G 6 4 2 2 0 / 2 2 0 2 1 4 / 2 2 0 2 4 6 / 2 4 6 2 4 6 / 2 5 0 2 2 0 / 2 5 2 2 4 6 / 2 4 6 M C W 0 0 1 6 1 6 2 – 2 0 6 n o n e A T G G C G C A G A A G G C A A A G C G A T A T T G G C T T C T G A A G C A G T T G C T A T G G 6 0 2 5 8 / 2 7 2 2 6 0 / / 2 6 7 2 5 0 / 2 6 2 2 5 0 / 2 5 0 2 4 6 / 2 5 8 2 5 0 / 2 5 4 M C W 0 0 7 8 1 3 5 – 1 4 7 n o n e C C A C A C G G A G A G G A G A A G G T C T T A G C A T A T G A G T G T A C T G A G C T T C 6 0 1 5 1 / 1 7 7 1 5 7 / 1 7 7 U K U K 1 5 3 / 1 5 3 1 5 3 / 1 5 7 M C W 0 1 8 3 2 9 6 – 3 2 6 G 3 1 9 7 4 A T C C C A G T G T C G A G T A T C C G A T G A G A T T T A C T G G A G C C T G C C 5 8 U K U K 2 9 4 / 2 9 4 2 9 8 / 3 1 0 U K U K M C W 0 1 6 5 1 1 4 – 1 1 8 n o n e C A G A C A T G C A T G C C C A G A T G A G A T C C A G T C C T G C A G G C T G C 6 0 1 2 0 / 1 2 0 1 1 8 / 1 2 0 1 2 0 / 1 2 4 1 2 0 / 1 2 0 1 1 6 / 1 1 8 1 1 6 / 1 1 6 U K u n k n o w n ( P C R a m p l i f i c a t i o n c o u l d n o t b e t e s t e d f o r t h e s e s a m p l e s b e c a u s e o f t h e d e p l e t i o n o f D N A e x t r a c t e d f r o m s h e d f e a t h e r s ) Eur J Wildl Res loci, i.e. MCW0295, MCW0081, MCW0330 deviate toHWE. No significant linkage disequilibrium was observedamong the tests for each pair of loci (all adjusted P values>0.0007). Discussion A total of 15 polymorphic microsatellite loci wereestablished in Himalayan monal by means of cross-speciesamplification, and all of them showed ample polymorphismfor evaluating genetic variation. However, most loci investi-gated here show small number of alleles when compared withother studies carried out on domestic chicken and red junglefowl populations (Romanov and Weigend2001; Cucet al.2006; Mukesh et al.2010). In the present study, eight loci were found informative (PIC>0.50) while PIC value for seven microsatellite loci was lower than 0.50. The lowaverage number of alleles per locus and lower PIC value(PIC<0.50) may result from the ascertainment bias (Ellegrenet al.1995,1997). This hypothesis could be tested by the combination of nuclear and mtDNA data and an increasedsample size. Deviations of three loci (MCW0295,MCW0081 and MCW0330) from HWE could have beencaused due to several reasons, such as Wahlund effect,inbreeding due to consanguineous mating and assortativemating (Hedrick 2005). Since, we examined our data set through MICRO CHECKER 2.2 and did not find presenceof null alleles, large allele drop-out (Wattier et al.1998) andscoring errors, associated with peak stuttering (Ewen et al.2000). Therefore, none of these is the cause of existing HWdisequilibrium. Though all the feather samples used in thestudy were collected from the Tungnath region of KedarnathWildlife Sanctuary, they were collected over a period of 1 year and in area of about 3 km 2 . The subalpine and alpinehabitats of Tungnath region have a monal density of 20 birds/km 2 (Sathyakumar et al.1992). Inbreeding might be present locally or analysed samples might have belonged toclosely related individuals. This might be the possible causeof departure from HWE.The present study is the first exhaustive attempt to test and to ensure the applicability of 15 chicken microsatelliteloci in Himalayan monal. These loci could be employed for further studies on ecological traits such as kin selection andmating systems in Himalayan monal, and the study wouldsuggest measures for better conservation of species.Increased samples may also provide an insight of theexisting genetic diversity in the population and inbreedinglevel estimates. We conclude that 15 cross-species micro-satellite markers were successfully transferred to Himalayanmonal. In addition, these loci were also genotyped in JungleBush quail, Grey francolin and Kalij pheasant. Theapplicability of these microsatellites would allow inevaluating genetic characteristics of many Galliformes inorder to understand different aspects of their conservationin relation with dispersal and colonisation, genetic aspectsof mating and breeding performance (Aparicio et al.2007).These results suggest that time, effort and funds spent ininvestigating the cross-species amplification of these micro-satellite loci in non-target species is being well justified,and the results in getting heterologous markers will be of use to other researchers to work on Galliformes. Locus n a * n e * H o * H e * PIC* HWE P ValueADL0268 4 1.8491 0.4286 0.4945 0.427 0.4151LEI0166 3 2.5128 0.2857 0.6484 0.523 0.111610ADL0112 3 2.1778 0.4286 0.5824 0.453 0.497636MCW0295 6 2.7222 0.5714 0.6813 0.605 0.033752MCW0067 5 2.5789 0.5714 0.6593 0.571 0.081786MCW0111 5 3.1613 0.7143 0.7363 0.647 0.170831MCW0222 2 1.9231 0.4 0.5333 0.365 0.527089LEI0094 4 2.6667 0.6667 0.6818 0.559 0.913466MCW0081 7 4.9 0.5714 0.8571 0.772 0.007864MCW0330 5 4.2353 0.5 0.8333 0.726 0.032568LEI0234 3 2.0851 0.4286 0.5604 0.464 0.266507MCW0016 2 1.8 0.6667 0.4848 0.346 0.300623MCW0078 2 1.8 0.3333 0.4848 0.346 0.388367MCW0183 4 3.1304 0.5 0.7424 0.622 0.171712MCW0165 5 1.8846 0.4286 0.5055 0.448 0.211511Mean 4 2.6285 0.4997 0.6324 0.5249Standard error 1.5119 0.9194 0.1256 0.1241 Table 2 Genetic polymorphismof 15 cross-species microsatel-lite markers in Himalayan monal* na – number of allele,ne – effective number of allele,Ho – observed heterozygosity,He – expected heterozygosity,PIC – polymorphism informa-tion content, HWE- Hardy-Weinberg equilibriumAmplification success was 100%with all the 15 microsatelliteloci used.Microsatellite loci, developedfor chicken and earlier used inthe (AVIANDIV, Weigend SCoordinator et al.1998) project,were used in the present studyand detail information is avail-able onwww.aviandiv.tzv.fal.de/ primer_table.htmlEur J Wildl Res
