Genetic characterization of indigenous duck of Tripura state in India using microsatellite markers

DOI: https://doi.org/10.21203/rs.3.rs-1932589/v1

Abstract

Exploring genetic variability by microsatellite markers is essential for genetic improvement, preservation of indigenous breeds and production of high quality offspring. Lack of information on microsatellite profiling of indigenous duck of Tripura has endorsed interest for this present study. Genomic DNA from randomly selected thirty six (36) native ducks was analysed at twenty five duck specific microsatellite loci, alleles were separated on 3.4% MetaPhore™ agarose and their sizes were determined by Image Lab software. Allelic data were analyzed by POPGENE version 1.31. All the studied loci demonstrated polymorphic patterns resolving 112 alleles. Allele number varied from 2 to 15 and average number of allele (Na) was 4.480 ± 0.659. Allele sizes and allele frequency ranged from 96 to 357 bp and 0.014 to 0.819, respectively. Average Nei’s heterozygosity, effective number (Ne) of alleles and Shannon’s Information index (I) were 0.617 ± 0.036, 3.538 ± 0.527 and 1.184 ± 0.112, respectively. The estimated Botstein’s polymorphic information content (PIC) ranged from 0.252 (CAUD020) to 0.911 (CAUD019) with an average of 0.562 ± 0.040 and sixteen loci were moderate to highly polymorphic and informative (PIC˃0.5). Ne was less than Na at all the loci, indicating prevalence of heterozygosity. Chi-square and G-square statistics revealed Hardy-Weinberg disequilibrium at all the loci. Moderate to high level of polymorphism at all analyzed microsatellite loci indicated that these markers might be helpful for genetic characterization and adoption of appropriate conservation strategies to exploit optimum genetic potentiality of indigenous duck of Tripura.

Introduction

Duck plays a substantial role after chicken to meet the demand of egg and meat in India. In North-Eastern India, indigenous ducks are mostly reared by local farmers under a traditional system and play a major role in development of socio-economic values of the small and marginal farmers. Indigenous ducks are hardy, acclimatize rapidly to various adverse environments and are resistant to many general poultry diseases. In rural areas, ducks are mainly maintained on free-range system as they are good foragers and have ability to find their own feed in a proportion of considerable amount. India has a total 33.51 million duck population distributed mainly in West Bengal, Assam, Kerala, Bihar, Andra Pradesh, Orissa and Tamil Nadu states of the country (Das et al., 2020). Tripura, one of the states in the North-East, has 8.5 lacs ducks (Livestock Census, 2019) and ranks in 3rd position after Assam and Manipur in the North-Eastern duck population (Das et al., 2020). The indigenous duck of Tripura is more or less distributed throughout the state but mostly seen in Sepahijala, Gomati and South district of Tripura. It has mixed colored feather, dark brown colour plumage, head colour varied from green, to black, white, brown, grey and yellowish brown where as bill, shank and feet is orange or yellow in colour (Phookan et al., 2018). At present, India has only two defined breeds of duck (ICAR-NBAGR, 2021) and various non-descript duck populations. These indigenous non-descript ducks have inherent potential to produce eggs and meat at substantial quantity with minimum input and the preference of eggs and meat of local duck over chicken is considerably high in this region (Phookan et al., 2018). Characterization and conservation efforts of native ducks are essential in alleviation of poverty and supply of sustainable contribution of animal protein in food stock. Scanty knowledge on genetic characterization of indigenous ducks for breeding program will interrupt the introduction of exotic breeds germplasm (Susanti et al., 2021). Presently genetic literature on duck for heritability and genetic relationship of some traits and epidemic disease cases of duck are only available to study (Susanti et al., 2021). Reports on genetic polymorphism of native duck of India using microsatellite markers are very few (Alyethodi et al., 2010, Susanti et al., 2021).

Microsatellite or simple sequences repeats are short repetitive elements of 1 to 6 bases which are by virtue polymorphic, copious, reproducible and inherited co-dominantly (Susanti et al., 2021) and used efficiently for diversity and relationship analysis of poultry and other livestock species, particularly for indigenous species and to characterise genetic diversity in various farm animals than other molecular markers like RAPD, RFLP and ISSRs (Vijh et al., 2008, Chatterjee et al., 2008, Alyethodi & Kumar, 2010, Debnath et al., 2017). Hence, DNA based neutral and highly polymorphic markers, particularly microsatellites are currently the marker of choice for determination of genetic variation and breed differentiation as recommended by FAO (FAO, 2004). This methodology is also helpful to provide necessary genetic information for determining preservation priorities for livestock breeds (Veeramani et al., 2015).

In order to conserve and to exploit the genetic potentiality of indigenous duck population, study on genetic characterization using microsatellite markers is pre-requisite. Therefore, the present study was under taken to characterise the indigenous duck of Tripura using microsatellite markers.

Materials And Methods

Experimental birds and sampling procedures

The present investigation used the indigenous ducks of Tripura which is more or less distributed throughout the state and generally reared by small or marginal farmers. Approximately, 0.5 ml venous blood was collected (during 2019–2020) at random from 36 (Thirty Six) numbers of indigenous ducks distributed in various areas of the main breeding tract and genomic DNA was extracted by following Phenol-Chloroform-Isoamyl alcohol method (Kagami et al., 1990). Quality of the isolated DNA was checked on 0.8% horizontal submarine agarose gel electrophoresis followed by its purity and quantity determination by using Eppendrof Bio-spectrophotometer. DNA samples showing intact band and optical density ratio (260 nm: 280 nm) between 1.7 and 1.9 were used in subsequent experiments. Final concentration of PCR ready DNA samples was optimized at 50 ng/µl for subsequent experiment.

Microsatellite markers and primers

A panel of twenty five (25) duck specific microsatellite markers reported by Huang et al. (2005) was screened for use in this study and their primers were got synthesized from GCC Biotech, Joychandipur, Bakrahat, West Bengal (India). PCR conditions were optimized for each of the primer as per Alyethodi & Kumar (2010) and Huang et al. (2005). The primer sequences and optimized annealing temperatures (Ta) are presented in Table 1.

Table 1

A panel of duck specific microsatellite markers along with their nucleotide sequences and optimized annealing temperatures ((Ta)

Markers

Accession Number

Nucleotide Sequence of Left (L) & Right (R) Primers

Ta (oC)

Optimized Ta

Ta as per Huang et al.(2015)

CAUD001

AY493246

L- ACAGCTTCAGCAGACTTAGA

54

55.5

R- GCAGAAAGTGTATTAAGGAAG

CAUD002

AY493247

L- CTTCGGTGCCTGTCTTAGC

60

60.8

R- AGCTGCCTGGAGAAGGTCT

CAUD003

AY493248

L- CCTGGCATTCTGCTAAGTTC

51.4

51.4

R- TGGGTTTGAACAGTGTAGCC

CAUD004

AY493249

L- TCCACTTGGTAGACCTTGAG

60.8

60.8

R- TGGGATTCAGTGAGAAGCCT

CAUD005

AY493250

L-CTGGGTTTGGTGGAGCATAA

60

60.8

R- TACTGGCTGCTTCATTGCTG

CAUD006

AY493251

L- ATGGTTCTCTGTAGGCAATC

56

63.5

R- TTCTGCTTGGGCTCTTGGA

CAUD007

AY493252

L- ACTTCTCTTGTAGGCATGTCA

60

60.8

R- CACCTGTTGCTCCTGCTGT

CAUD009

AY93253

L- AGGGATTTTGGAGCGGAGC

63

60.8

R- TGTGCGGCGTTTTCCTCTG

CAUD010

AY493254

L- GGATGTGTTTTTCATTATTGAT

50

50.3

R- AGAGGCATAAATACTCAGTG

CAUD011

AY493256

L-TGCTATCCACCCAATAAGTG

50.3

50.3

R-CAAAGTTAGCTGGTATCTGC

CAUD013

AY493258

L-ACAATAGATTCCAGATGCTGAA

58

58.1

R- ATGTCTGAGTCCTCGGAGC

CAUD016

AY493261

L-ITT AGG TAA AAC TGT GAA

56

51.4

R-ATC AAA GCA GGG AGC TAA G

CAUD017

AY493262

L-AGA AAT ACA CTT ACA GCA CT

52

58.1

R-TGTCATAAA ATG G'IT AAT TGC

CAUD018

AY493263

L-TTA GAC AAA TGA GGA AAT AGT A

54

50.3

R-GTC CAA ACT AAA TGC AGG C

CAUD019

AY493264

L-CTTAGCCCAGTGAAGCATG

58.1

58.1

R-GCAGACTTTTACTTATGACTC

CAUD020

AY493265

L-TAGGGTCAATAG TAA GAAACA

56

53.2

R-TAA CTG TGT GAT AAG GGA

CAUD023

AY493268

L-CACATTAACTACATTTCGGTCT

52

51.4

R-CAGCCAAAGAGTTCAACAGG

CAUD024

AY493269

L-TCGCATTAAGCTCTGATCT

55.5

55.5

R-ATCAACAGAATCCAAAATATG

CAUD026

AY493271

L-ACGTCACATCACCCCACAG

61

60.8

R-CTTTGCCTCTGGTGAGGTTC

CAUD027

AY493272

L-AGAAGGCAGGCAAATCAGAG

64

66

R-TCCACTCATAAAAACACCCACA

CAUD028

AY493273

L-TACACCCAAGTTTATTCTGAG

56

55.5

R-ACTCTCCAGGGCACTAGG

CAUD029

AY493274

L-GACCTCAAGAATTTACCAC

54

55.5

R-ATTATTTTCTTCTGGCAGCA

CAUD030

AY493275

L-ATTATTCCTGATGGCGTGGT

53

50.3

R-TCATGCTGAATTTGGCTGTT

CAUD032

AY493277

L-GAAACCAACTGAAAACGGGC

58.1

58.1

R-CCTCCTGCGTCCCAATAAG

CAUD035

AY493280

L-GTGCCTAACCCTGATGGATG

R-CTTATCAGATGGGGCTCGGA

64

63.5

PCR reaction mix and amplification program

Each PCR reaction was performed in a volume of 25 µl reaction mixture containing 50 ng of template DNA, 10 pM of each primer, 0.5 µl 10mM dNTPs, 2.5 µl 10X PCR buffer, 2.5 µl MgCl2, and 1U Taq DNA polymerase into nuclease free water in 0.2 ml nuclease free maxiamp flat cap PCR tubes (Tarsons Products Pvt. Ltd., India). PCR amplification was carried out in programmable thermal cycler (Eppendrof nexus gradient master cycler) using PCR program as initial heat activation at 94°C for 5 min, followed by 30 cycles of (i) denaturation at 94°C for 1 min, (ii) optimised annealing temperature (Ta, °C) for each microsatellite primer pair for 1 min presented in Table 1, and (iii) extension at 72°C for 1 min followed by final extension at 72°C for 5 min and 4°C forever.

Gel electrophoresis

The amplified PCR products were checked on 2% horizontal submarine agarose gel electrophoresis for 60 min at 2–5 volts/cm and subsequently the developed bands were examined under UV light in Gel Doc™ EZ System (Biorad Laboratories, U.S.A). Molecular sizes of amplified products were adjudged by loading approximate 10 µl of PCR product in agarose gel along with 3 µl of 50 bp DNA ladder (molecular marker) for detection of desired bands. The confirmed amplified products were run onto horizontal 3.4% Metaphore™ Agarose Gel Electrophoresis (MAGE) along with 50 bp DNA ladder in parallel @ 6–8 V/cm for 2 h 30 min for identification of microsatellite alleles and visualized in Gel Documentation system (Biorad Laboratories, U.S.A). Allelic patterns of microsatellites were documented for further genotyping.

Determination of allele size, data handling and analysis

The molecular sizes (bp) of microsatellite alleles were estimated by comparing of standard ladder DNA marker with Image Lab 6 software of Gel Doc™ EZ System (Biorad Laboratories, U.S.A). The number of observed alleles with probable genotype in each sample at each microsatellite loci was recorded.

Data on genotype at twenty five microsatellite loci in all the samples were compiled and analysed for population genetic parameters viz., allele frequencies, observed and the effective number of alleles, Shannon’s information index as well as goodness of fit (Chi-square) and likelihood ratio (G-square) through POPGENE® 1.31 software as described by Yeh et al. (1999).

Average heterozygosity at each microsatellite locus was calculated according to Nei (1978) by using following formula:

$${H}_{i}= \frac{2\text{N}}{2\text{N}-1}\left(1-{\sum }_{j=1}^{k}{P}_{j}^{2}\right)$$

Polymorphic Information Content (PIC) at each microsatellite locus was calculated as per Botstein et al. (1980) with the help of following formula:

$$PIC=1-{\sum }_{i=1}^{k}{p}_{i}^{2}-{\sum }_{i=1}^{k-1}{\sum }_{j=i+1}^{k}{2p}_{i}^{2}{p}_{j}^{2}$$

Results And Discussion

The present investigation documented a profile of microsatellite alleles in native ducks of Tripura (India) presented in Table 2, and resolved 112 distinct alleles at 25 microsatellite loci. Allele number varied from 2 to 15 and the lowest number of alleles was observed in microsatellite CAUD003, CAUD006, CAUD007, CAUD009, CAUD018, CAUD020, CAUD028 and CAUD029 locus, whereas the abundant number of alleles in CAUD019 locus. The molecular sizes of the alleles (Fig. 1 for representative samples) ranged from 96 bp (CAUD013) to 357 bp (CAUD024). The allele frequencies at various microsatellite loci ranged from 0.014 (CAUD035) to 0.819 (CAUD020). Out of total 112 alleles resolved, 75 alleles (66.96%) had high frequencies with a level of more than 10%. It is well known fact that different breeds represent different number of microsatellite alleles, for example, 139 and 126 number of alleles in Keeri and Sanysi ducks, respectively, at 23 microsatellite loci (Veeramani et al., 2015), 281 alleles at 20 loci in six Chinese duck population (Wu et al., 2008), 236 alleles at 24 loci in six Chinese duck breeds (Li et al., 2006), and 117 alleles at 35 microsatellite loci in Peking ducks (Huang et al., 2005). Earlier literatures also witnessed varied number of observed alleles reported as 2 to 14 for Peking ducks (Huang et al.,2005), 2 to 5 for Moti native ducks (Aleythodi & Kumar, 2010), 3 to 15 for Keeri as well as 3 to 19 for Sanyasi ducks (Veeramani et al., 2015), and 2 to 10 for Central Javanese duck (Susanti et al., 2021). The present investigation reported various locus specific alleles whose sizes were quite comparable with earlier findings. Huang et al. (2005) reported some quite similar alleles at CAUD001, CAUD003, CAUD004, CAUD010, CAUD0013, CAUD016, CAUD018, CAUD019, CAUD020, CAUD027, CAUD029, CAUD030, CAUD032 and CAUD035 in Peking ducks. Alyethodi & Kumar (2010) documented a number of quite comparable alleles at CAUD001, CAUD002, CAUD003, CAUD004, CAUD007, CAUD013, CAUD 016, CAUD018, CAUD023, CAUD027 and CAUD035 in Moti native ducks. Goel et al. (2016) resolved few quite similar alleles at CAUD004, CAUD010, CAUD016 and CAUD027 in Indian Muscovy duck. Sultana et al. (2017) reported some quite similar alleles at CAUD035 in Asian ducks. Susanti et al. (2021) also reported some comparable alleles at CAUD001, CAUD005, CAUD016 and CAUD032 in Central Javanese duck. In present study, molecular sizes of alleles at CAUD026 microsatellite locus greatly differed from the estimation of Huang et al. (2005) and Alyethodi & Kumar (2010). However, the differences in the allele sizes might be due to the methodological differences adopted for resolution and size assessment, besides the breed differences. In context to the frequency distribution of alleles, it could be referred the reports of Susanti et al. (2021) and Rajkumar et al. (2008) that alleles with frequency about 10% levels might be more appropriate to tag as a specific population. The present study revealed that these alleles could be used as characterization markers for indigenous duck of Tripura as a distinct breed with other Indian native ducks.

Table 2

Microsatellite allele profile of indigenous duck of Tripura state of India

MS Loci

No. of alleles

Allele sizes (bp) and respective allele frequencies in the parenthesis

CAUD001

5

310(0.069), 316(0.125), 330(0.306), 338(0.250), 344(0.250)

CAUD002

4

177(0.222), 190(0.306), 204(0.278), 220(0.194)

CAUD003

2

116(0.444), 124(0.556),

CAUD004

5

191(0.097), 202(0.222), 213(0.319), 221(0.139), 228(0.222)

CAUD005

6

245(0.111), 257(0.208), 265(0.111), 276(0.306), 287(0.097), 297(0.167)

CAUD006

2

215(0.722), 222(0.278)

CAUD007

2

113(0.722), 118(0.278)

CAUD009

2

131(0.333), 137(0.667)

CAUD010

3

109(0.222), 117(0.278), 121(0.500)

CAUD011

5

133(0.528), 138(0.097), 144(0.250), 147(0.083), 161(0.042)

CAUD013

4

96(0.167), 103(0.208), 108(0.444), 114(0.181)

CAUD016

5

189(0.042), 195(0.139), 208(0.306), 213(0.444), 216(0.069)

CAUD017

5

210(0.111), 225(0.278), 234(0.431), 245(0.097), 264(0.083)

CAUD018

2

99(0.250), 104(0.750)

CAUD019

15

132(0.028), 137(0.056), 141(0.056), 145(0.083), 152(0.042), 162(0.028), 168(0.042), 184(0.125), 189(0.111), 192(0.056), 196(0.125), 200(0.069), 206(0.056), 218(0.097), 224(0.028)

CAUD020

2

115(0.181), 125(0.819)

CAUD023

5

166(0.139), 173(0.236), 179(0.194), 186(0.181), 195(0.250)

CAUD024

14

273(0.028), 280(0.069), 284(0.083), 288(0.041), 291(0.097), 295(0.153), 301(0.069), 304(0.028), 307(0.125), 315(0.069), 326(0.111), 336(0.042), 343(0.042), 357(0.042)

CAUD026

3

258(0.139), 271(0.194), 276(0.667)

CAUD027

4

102(0.125), 113(0.333), 118(0.306), 123(0.236)

CAUD028

2

145(0.278), 153(0.722)

CAUD029

2

114(0.250), 122(0.750)

CAUD030

4

246(0.111), 257(0.278), 265(0.528), 272(0.083)

CAUD032

5

115(0.472), 118(0.153), 121(0.069), 127(0.236), 132(0.069)

CAUD035

4

217(0.264), 223(0.458), 229(0.264), 252(0.014)

Total

112

 

In the present investigation, the estimated means (± S.E.) of the observed (Na) and effective (Ne) number of alleles per locus were 4.480 ± 0.659 and 3.538 ± 0.527, respectively (Table 3). The effective (Ne) number of alleles ranged from 1.420 (CAUD020) to 12.056 (CAUD019). The lower effective number of alleles than the observed number of alleles across the loci indicated that allele frequencies were widely distributed. To discuss, Aleythodi & Kumar (2010) reported an average number of allele per locus as 3.1 across 21 microsatellite loci in Moti native ducks which was quite lesser than the present findings and Veeramani et al. (2015) estimated higher mean number of alleles i.e. 5.61 ± 0.66 and 5.91 ± 0.76 in Keeri and Sanyasi ducks, respectively, across 23 microsatellite loci. Our estimate was in accordance with the report of Huang et al. (2005) who documented the average number of alleles per locus as 4.18 in Peking ducks across 35 microsatellite loci. In contrast to the present investigation, lesser effective (Ne) number of alleles was reported by Veeramani et al. (2015) as 2.43 ± 0.22 in Keeri ducks and 2.89 ± 0.41 in Sanyasi ducks, whereas higher number of alleles was reported as 4.8 by Li et al. (2006). Kumar et al. (2011) reported 3.6 numbers of effective alleles in Moti and Indian runner ducks, which was quite comparable with the present findings. The reason of variation in the findings might be due to the differences in the genetic architecture of the duck population, various number and type of microsatellite primers used for study, breed differences and methodological differences under the present investigation.

Table 3

The measures of heterozygosity and diversity statistics, Chi-square (ꭓ2) and likelihood ratio (G-square) test for Hardy-Weinberg equilibrium at duck specific microsatellite loci in indigenous duck of Tripura

Primer Name

df

Nei’H (%)

PIC

Na

Ne

I

Chi-square

G-square

CAUD001

10

0.761

0.721

5.000

4.187

1.501

137.900***

106.308***

CAUD002

6

0.742

0.695

4.000

3.880

1.371

111.656***

101.806***

CAUD003

1

0.494

0.372

2.000

1.976

0.687

37.055***

50.482***

CAUD004

10

0.770

0.734

5.000

4.356

1.534

39.040***

38.481***

CAUD005

15

0.801

0.774

6.000

5.033

1.703

48.751***

42.195***

CAUD006

1

0.401

0.321

2.000

1.670

0.591

37.564***

43.569***

CAUD007

1

0.401

0.321

2.000

1.670

0.591

37.564***

43.569***

CAUD009

1

0.444

0.346

2.000

1.800

0.637

37.298***

46.854***

CAUD010

3

0.624

0.553

3.000

2.656

1.037

75.749***

76.702***

CAUD011

10

0.641

0.593

5.000

2.784

1.250

59.014***

36.765***

CAUD013

6

0.699

0.651

4.000

3.319

1.295

72.897***

62.056***

CAUD016

10

0.683

0.631

5.000

3.157

1.315

50.959***

55.279***

CAUD017

10

0.709

0.664

5.000

3.433

1.396

92.670***

64.422***

CAUD018

1

0.375

0.305

2.000

1.600

0.562

37.758***

41.519***

CAUD019

105

0.917

0.911

15.000

12.056

2.589

193.482***

115.355***

CAUD020

1

0.296

0.252

2.000

1.420

0.472

31.760***

26.351***

CAUD023

10

0.792

0.759

5.000

4.809

1.589

29.817***

33.400***

CAUD024

91

0.909

0.902

14.000

11.030

2.509

169.997***

109.209***

CAUD026

3

0.499

0.446

3.000

1.994

0.863

77.930***

64.224***

CAUD027

6

0.724

0.673

4.000

3.625

1.329

36.179***

26.991***

CAUD028

1

0.401

0.321

2.000

1.670

0.591

37.564***

43.569***

CAUD029

1

0.375

0.305

2.000

1.600

0.562

37.758***

41.519***

CAUD030

6

0.625

0.568

4.000

2.667

1.144

120.999***

85.585***

CAUD032

10

0.688

0.645

5.000

3.208

1.353

43.932***

38.005***

CAUD035

6

0.651

0.582

4.000

2.861

1.120

68.035***

70.254***

Mean ± SE

 

0.617

± 0.036

0.562±

0.040

4.480 ± 0.659

3.538

± 0.527

1.184

± 0.112

 
df = Degree of freedom; H = Nei’s heterozygosity; PIC = Polymorphic information content; Na = Observed number of alleles; Ne = Effective number of alleles; and I = Shannon’s information index; ***P ≤ 0.001

Shannon et al. (1949) reported that estimates Shannon’s information index as a measure of gene diversity. The present estimate of Shannon’s information index (I) was 1.184 ± 0.112 indicating the prevalence of gene diversity in the population investigated. No earlier reports were available on Shannon’s diversity estimates for duck to compare or contrast with the present findings. The estimates of observed and effective numbers of alleles and Shannon’s information index indicated the prevalence of heterozygosity in the studied population of the native ducks of Tripura constituting a more diverse population, which could be due to the reason that this population was not subjected to selection.

In the present study, all the microsatellite loci (100%) demonstrated polymorphic patterns and CAUD019 was the most polymorphic marker recorded with fifteen alleles whose molecular sizes ranged from 132 bp to 224 bp. The mean polymorphic information content (PIC) values for the markers varied from 0.252 (CAUD020) to 0.911(CAUD019) with a mean of 0.562 ± 0.040 (Table 3). As of indicative of examining genetic variation in the population (Rahim et al., 2017), the PIC value is a good index for genetic diversity evaluation and symbolizes the degree of informativeness of a marker (Parmar et al., 2007). According to Botstein et al. (1980), Vanhala et al. (1998) and Susanti et al. (2021), a PIC score higher than 0.50 indicates high gene diversity, a score of lesser than 0.25 indicate low diversity and a score between 0.25 and 0.50 is suggestive of a moderate degree of polymorphism at a particular locus. In the present investigation, sixteen loci (CAUD001, CAUD002, CAUD004, CAUD005, CAUD010, CAUD011, CAUD013, CAUD016, CAUD017, CAUD019, CAUD023, CAUD024, CAUD027, CAUD030, CAUD032 and CAUD035) out of twenty five loci showed higher degree of polymorphism and nine loci revealed moderate polymorphism. CAUD019 and CAUD024 microsatellite loci revealed more high PIC values (0.911 and 0.902, respectively) as compared to other analyzed microsatellite loci, which was in accordance with the report of Huang et al. (2005). Alyethodi & Kumar (2010) also documented quite similar PIC value at CAUD001, CAUD013 and CAUD035 microsatellite loci in Moti native duck. In comparison to the present finding of average PIC value (0.562 ± 0.040), it could be referred that Huang et al. (2005) and Alyethodi & Kumar (2010) estimated overall PIC value as 0.41 ± 0.01 and 0.45 ± 0.01 in Peking and Moti native ducks, respectively. Higher overall PIC value in contrast to the present finding was estimated by Veeramani et al. (2015) as 0.5985 ± 0.06 for Keri ducks of Tamil Nadu. Also in the present study, the microsatellite loci CAUD004, CAUD003, CAUD006, CAUD007, CAUD009 and CAUD028 were found polymorphic in native duck of Tripura, whereas, were monomorphic in Peking ducks (Huang et al., 2005). The differences in PIC value at various microsatellite loci might be due to genetic differences of studied population as well as differences in methodology adopted. The present investigation revealed that selected set of microsatellite loci provide enough information for estimation of genetic diversity in population.

Genetic variation of each population could be measured by estimation of heterozygosity. In the present study, the Nei’s heterozygosity at all studied polymorphic microsatellite loci ranged from 0.296 (CAUD020) to 0.917(CAUD019) with an average of 0.617 ± 0.036 (Table 3). Huang et al. (2005) observed the highest heterozygosity at CAUD019 (0.97) microsatellite loci which was quite similar to present estimation. In contrast to the present findings, lower average heterozygosity was reported as 0.52 ± 0.02 in Moti ducks (Alyethodi & Kumar, 2010), 0.47 ± 0.01 in Peking ducks (Huang et al., 2005) and 0.56 ± 0.02 in Indian Runner native duck (Sankhyan, 2007) for the same set of primers. Wu et al. (2008) and Su et al. (2009) reported mean heterozygosity values of more than 0.6 in different Chinese duck population which were quite comparable with the present estimation. The variation in the findings might be due to genetic architecture differences of studied population and differences in adopted methodology. Heterozygosity value of 0.3 to 0.8 at microsatellite marker could be useful for measuring estimation of genetic variation as reported by Veeramani et al. (2015) and heterozygosity value of 0.10 at polymorphic microsatellite loci should be considered useful for genetic diversity estimation as reported by Rahim et al. (2017). In the present study, the values of heterozygosity were found within the specified range for all microsatellite markers. Hence, the markers used in the present investigation might be quite suitable for estimation of genetic diversity in duck population.

In the present study, the results of Chi-square and G-square estimates were found significant at all the studied microsatellite markers and revealed that all the loci were maintaining Hardy-Weingberg disequilibrium in native duck population of Tripura in consistence with the earlier reports in Moti native duck population (Alyethodi et al., 2010) for the same set of primers as used in the present investigation and in six Chinese duck populations (Yeh et al., 1999). Veeramani et al. (2015) also reported the same for few markers in Keeri and Sanyasi ducks of Tamil Nadu which might be in agreement with the present findings, whereas, Kumar et al. (2011) observed Hardy-Weingberg equilibrium for few microsatellite loci (CAUD001, CAUD005, CAUD016 and CAUD035) in Indian runner duck. The Hardy-Weinberg equilibrium test was performed to validate whether the genotypes were conserved in equilibrium or deviated from equilibrium. In the present study, all the loci were not found in Hardy-Weinberg equilibrium for the population of native duck of Tripura. This result revealed that the studied population structures have become unbalanced, which might be due to influence of some forces like selection, non-random mating, inbreeding, mutation, genetic drift etc.

Conclusion

It could be concluded that the studied 25 microsatellite markers used in the present investigation have high discrimination power for genetic description and breeds identification, and could also be used for investigation of population structure and genetic diversity estimation in various duck population because of most of markers showing highly polymorphic alleles and PIC values of more than 0.5 in native duck of Tripura. Furthermore, this present study could help for genetic characterization of indigenous duck of Tripura in tagging as a distinct breed of Indian native ducks.

Declarations

Acknowledgements

The authors would like to convey sincere thanks to the Department of Biotechnology, Government of India, New Delhi, for the research funding support through NER research grant fiscal year 2018. The facilities provided by the Director, Animal Resources Development Department and the Principal, College of Veterinary Sciences & A.H., R.K. Nagar, Government of Tripura are also acknowledged. 

Funding: 

The funding for the work was provided by the Department of Biotechnology, Government of India, New Delhi through NER research grant fiscal year 2018, vide No. BT/PR24311/NER/95/645/2017, 25th Sept, 2018. 

Conflict of interest/Competing interest

Authors of this manuscript declare that there is no conflict of interest for this research work. 

Ethics approval 

All the experimental procedures on animals were carried out according to the recommendations and approval of the Institute Animal Ethics Committee (IAEC) as per the guidelines set forth by the Institutional Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). 

Consent to participate 

Not applicable. 

Consent for publication 

All authors are agreed to let their consent for publication of the research. 

Availability of data and material

All the data that supports the findings of this study are available with the data repository of the College of Veterinary Sciences and Animal Husbandry, R.K. Nagar, Tripura-799008, India. 

Code availability

Not applicable 

Authors contribution 

JD: Project administration, Supervision, Conceptualization, Methodology, Formal analysis, Writing, SD & DS: Methodology, Lab work, Data recording, Data curation, Original Draft, BD & Mk: Review/Editing, AKD: Writing, Review/Editing. 

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