Microsatellites analysis in Tripureswari duck of Tripura

Introduction

Ducks are considered as viable livelihood option for farmers community and the second most important poultry species just after chicken in rural India for its high resistance ability to disease, more production year length, easy to train, large sized egg, early morning egg laying character and natural foraging inclination (Padhi and Giri 2024) [15] . In addition, people in North-East India favour native duck meat and eggs over chicken. In 2020, India ranked fifth amongst the world with the duck population of 33.51 million and the states of Assam, West Bengal, Andra Pradesh, Orissa, Bihar, Tamil Nadu, and Kerala were home to majority of duck population of the country (Debnath et al., 2023; Padhi and Giri 2024) [7,15] . As per 20 th Livestock census (2019) [1] , about 97% of the total duck population of the country is reared under backyard poultry and Assam, Manipur and Tripura are the leading states in duck population in the North-Eastern region of India. Tripura ranks in 3 rd position amongst North-Eastern states with a population of 8.5 lacs duck (Debnath et al., 2023) [7] . In India, the production of egg in different desi ducks under backyard and commercial farms vary highly indicating huge scope of duck population improvement in the country (Padhi and Giri 2024) [15] . Native ducks are raised by farmers using a traditional free-range system, and they produce a significant amount of meat and eggs with relatively little outside assistance (Phookan et al., 2018) [18] . In addition of native ducks, other duck genetic groups, such as Pati Hans, Khaki Campbell, Vigova Super M, Nageswari, etc. are commonly seen in Tripura (Debnath, 2022) [8] . Most of the Indian duck populations are of indigenous origin as reported by Padhi (2014). Native duck population of Tripura constitutes 81% of all the ducks in the state and raised by majority of local farmers of Tripura (Das and Rahman 2019) [6] . Indigenous duck of Tripura has been recognized as the Tripureswari duck breed of India (Kumar and Dwivedi, 2025) [12] . Research on native ducks is still in its infancy, despite the species' significant population and ability to improve the socioeconomic status of small and [11] . [3,9] and inherited co-dominantly (Susanti et al., 2021) [20] . Therefore, the present studies were undertaken on estimating genetic diversity of the local duck population of Tripura using microsatellite markers.

Materials and Methods Experimental birds and sampling procedures

The Tripureswari ducks are mostly found in West Tripura, South Tripura, Gomati, Sepahijala, Kowai and Dhalai district of Tripura and traditionally raised by small and marginal farmers, were utilized for this present study. Approximately, 0.5 ml venous blood samples were randomly collected from thirty-six numbers of the Tripureswari ducks distributed in different areas of the breeding tract and genomic DNA was extracted from each blood sample by Phenol-Chloroform-Isoamyl alcohol method (Kagami et al., 1990) [10] . Quality of the DNA was assessed on 0.8% horizontal submarine agarose gel electrophoresis, followed by its purity checking and quantity determination by Eppendorf Bio-spectrophotometer. DNA samples with intact bands and OD (260/280 nm) between 1.7 and 1.9 were used in subsequent experiments. PCRready DNA samples were prepared in a concentration of 50 ng/µl.

Scanning birds for microsatellite genotypes

The present investigation employed a panel of twelve duck-specific microsatellite markers that were described by Magpantay et al. (2019) [13] and primers for these markers were synthesised from GCC Biotech, Joychandipur, West Bengal (India). PCR conditions were optimized for each of the primer pairs as per Magpantay et al. (2019) [13] . Table 1 shows the primer sequences and optimal annealing temperatures (T a ). PCR reaction mixture was prepared for each primer pair in 0.2 ml nuclease-free maxiamp flat cap PCR tubes (Tarsons Products Pvt. Ltd., India) in a volume of 25 µl into nuclease-free water containing 50 ng of template DNA, 2.5 µl 10X PCR buffer, 2.5 µl MgCl 2, 0.5 µl 10 mM dNTPs, 10 pM of each primer, and 1U Taq DNA polymerase. The PCR amplification was performed in a programmable thermal cycler (Eppendorf nexus gradient master cycler). The PCR program included initial denaturation at 94 °C for 5 min, thereafter 30 cycles of (i) denaturation at 94 °C for 1 min, (ii) optimised annealing temperature (°C) for each primer pair for 1 min, and (iii) extension at 72 °C for 1 min, followed by final extension at 72 °C for 5 min and 4 °C forever. The amplified PCR products were run on 2% horizontal submarine agarose gel electrophoresis for 60 min at 2-5 volts/cm, and subsequently, checked for the DNA bands developed onto the gel by viewing under UV light in GelDoc TM EZ System (Biorad Laboratories Inc., USA). The molecular sizes of the amplified products were determined by loading around 10 µl of the PCR product and 3 µl of a 50 bp DNA ladder onto an agarose gel so as to identify the appropriate bands. The verified amplified products were run onto horizontal 3.4% Metaphore TM agarose gel electrophoresis (MAGE) along with 50 bp DNA ladder in parallel @ 6-8 V/cm for 2 h 30 min. Thereafter, the gel was visualized in the GelDoc system and the allelic patterns of the microsatellites were documented for further genotyping. The molecular sizes (bp) of the alleles were estimated by comparing with the standard ladder DNA marker under Image Lab 6 software of the GelDoc System. At each microsatellite locus, the number of detected alleles with a likely genotype was noted for each sample.

Table 1: A panel of duck-specific microsatellite markers along with their primer sequences and optimized annealing temperature (Ta.).

MS Loci Accession Number

Forward (F) and reverse (R) primer sequences

Reported Ta (°C) (Magpantay et al., 2019)

International Journal of Advanced Biochemistry Research https://www.biochemjournal.com R 5′-GGGCTTGAGGCATACACTCCCTA-3′

Statistical analysis

All DNA samples' genotypic data at twelve microsatellite loci were compiled and analysed using POPGENE® 1.31 software, following Yeh et al. (1999) [22] instructions for population genetic parameters such as observed and effective numbers of alleles, allele frequencies, Shannon's information index, goodness of fit (Chi-square), and likelihood ratio (G-square) statistics.

Assuming that the population was under Hardy-Weinberg equilibrium, average heterozygosity at each microsatellite locus was computed as per Nei (1978) [14] using the following formula:

Where, P j is the frequency of the j th allele at i th locus with k number of alleles in a population and N is the number of individuals. Polymorphic information content (PIC) at each microsatellite locus was calculated as per Botstein et al.

(1980) [4] with the help of following formula:

Where, P i and P j are the frequencies of i th and j th alleles, respectively at a locus with k number of alleles in the population.

Results and Discussion

The present investigation documented a profile of microsatellite alleles (Figure 1) and resolved overall 46 distinct alleles at 12 microsatellite loci in Tripureswari duck of Tripura and the results are presented in Table 2. The observed number of alleles ranged from 2 (SMO010 locus) to 7 (APL080 locus) at different loci with an average number of 3.83±0.46 per locus. Molecular size of the alleles ranged from 105 bp (SMO010) to 263 bp (APL079). The overall allelic frequencies ranged from 0.01 (APL079) to 0.83 (SMO010). Out of total 46 (73.91%) alleles, 34 had a level of more than 10% frequencies (Table 2). The corresponding means (±S.E) of the observed (N a ) and effective (N e ) number of alleles and Shannon's information index (I) were 3.83±0.46, 2.89±0.34 and 1.09±0.11. The effective number of alleles at each locus was less than the observed number of alleles and ranged from 1.38 (SMO010) to 5.31 (APL080) (Table 3). Polymorphic patterns were observed in all the loci (100%).

The highest polymorphism was recorded at APL080 locus with seven alleles and their molecular size ranged from 107 to 137 bp. Polymorphic information content (PIC) values indicated that all the loci were moderate to highly polymorphic. The PIC values varied from 0.24 (SMO010) to 0.79 (APL080) with a mean of 0.54±0.05. APL002, APL011, APL012, APL079, APL080, SMO07 and SMO013 had the PIC value more than 0.50 (Table 3). Average heterozygosity was approximated as 0.60±0.04 with a range from 0.28 (SMO010) to 0.81 (APL080). The results of Chisquare and G-square statistics were found significant at all the microsatellite loci and revealed that all the loci were maintaining Hardy-Weinberg disequilibrium in the duck population (Table 3). The present microsatellite allelic profile was developed exploring APL002, APL011, APL012, APL077, APL078, APL079, APL080, APL081, APL083, SMO07, SMO010 and SMO013 loci in an indigenous duck population native to Tripura and such type of reports were not available in Indian native ducks to compare or contrast with the present findings. However, outside India, Philippine mallard duck was investigated (Magpantay et al., 2019) [13] exploring 22 microsatellite loci including the present panel of markers using Fragment Analyzer. SMO013 locus had quite same number of observed alleles while other loci contained variable sizes with number of observed alleles. Likewise, Shaoxing duck population was also reported (Chepiha et al., 2018) [5] to contain quite similar number of observed alleles at SMO013 locus and dissimilar sizes with observed allelic numbers at APL077, APL078, APL079, SMO07 and SMO010 loci. Agatep et al. (2016) [2] reported quite comparable number of observed alleles at APL011, APL083 and SMO07 but variable sizes with number of observed alleles at APL02, APL012, APL077, APL078, APL079 and APL081 in three genetic groups of Philippine Mallard, Khaki Campbell and Pekin ducks. The inscribed differences in allele sizes and numbers in different studies might be due to the attributed variation in different duck population or breeds along with adopted methodologies for resolution and molecular size estimation. In context to the distribution of allelic frequency, Debnath et al. (2023) [7] and Susanti et al. (2021) [20] reported that the alleles with frequency about 10% levels might be more suitable to tag as specific population. The present study revealed that alleles with more than 10% level of frequency could be used as marker of choice for genetic characterization of indigenous duck of Tripura. Debnath et al. (2023) [7] reported that heterozygosity and polymorphic information content could be the indicative of genetic variation measures in each population. No reports were available on heterozygosity and polymorphic information content (PIC) in Indian indigenous ducks to compare or contrast with the present investigation. However, lower observed heterozygosity was reported by Magpantay et al. (2019) [13] in Philippine mallard duck at APL02, APL011, APL012, APL077, APL078, APL079, APL081, APL083 and SMO07 loci. In contrast to the present findings, Agatep et al. (2016) [2] reported quite comparable heterozygosity estimates at APL02 (Khaki Campbell duck), higher estimates at APL02 (Pekin), APL078 (Philippine Mallard) and APL083 (Khaki Campbell and Pekin duck) and lower estimates at APL02, APL011, APL12, APL077, APL083 and SMO07 (Philippine Mallard duck). The variation in the findings might be due to the attributed differences in the different duck population and adopted methodology. Debnath et al. (2023) [7] and Veeramani et al. (2015) [21] reported that those microsatellite markers with heterozygosity estimates ranged from 0.3 to 0.8 could be suggestive for usefulness of genetic variation and diversity estimation. In the present investigation, the heterozygosity estimates were found within the specified range for all the studied microsatellite markers. Hence, all the markers used in the present study might be quite suitable for assessment of genetic variation in duck population. Polymorphic information content (PIC) symbolises the degree of informativeness of a marker (Parmar et al. 2007) International Journal of Advanced Biochemistry Research https://www.biochemjournal.com [17] and the PIC value is a good index for genetic diversity evaluation in the population (Rahim et al. 2017) [19] . According to Susanti et al. (2021) [20] and Debnath et al.

(2023) [7] , a PIC score more than 0.50 specifies high genetic variation, lesser than 0.25 specifies low diversity and between 0.25 and 0.5 indicates moderate degree of polymorphism at a particular microsatellite locus. In present study, seven loci (APL02, APL011, APL012, APL079, APL080, SMO07 and SMO013) showed higher degree of polymorphism, four loci revealed moderate and one marker revealed lower polymorphism. No information was available on the PIC values of Indian indigenous duck at the present panel of microsatellites to compare or contrast with the present findings. However, Magpantay et al. (2019) [13] reported quite comparable PIC values at APL011, APL012, APL078 and SMO07, higher PIC values at APL077 and lower value at APL02, APL079, APL080, APL081, APL083, SMO07 and SMO013 in Philippine mallard duck.

In contrast to the present findings, Chepiha et al. (2018) [5] reported the SMO010 as monomorphic locus, higher PIC values at APL077 and lower values at APL078, APL079, SMO07 and SMO013 loci in Shaoxing duck population. The differences in polymorphic information content of various microsatellite loci might be due to the differences in genetic architecture of the population analysed or probably due to the differences in adopted methodology.

In the present study, there was prevalence of heterozygosity in the studied population as the average effective numbers of alleles (2.89±0.34) were lesser than an average observed number of alleles (3.83±0.46) which was in agreement with the earlier report in Tripura-native duck for different microsatellites (Debnath et al. 2023) [7] . No other reports were available to compare or contrast with the present findings. Debnath et al. (2023) [7] reported Shannon's information index as a measure of gene diversity. In the present investigation, the estimate of the index was 1.09±0.11 indicating prevalence of heterozygosity in the studied population. The present estimates of observed and effective numbers of alleles and Shannon's information index indicated the prevalence of heterozygosity in the Tripura native ducks constituting a more diverse population, which might be due to the reason that this population was not subjected to selection other than natural. The Hardy-Weinberg equilibrium test was performed to validate whether the genotypes were conserved in equilibrium or deviated from equilibrium (Debnath et al.

2023) [7] . In the present study, the results of chi-square and G-square statistics were found significant at all the studied microsatellite markers and revealed that all the loci were maintaining Hardy-Weinberg disequilibrium. This result revealed that studied population structures have become unbalanced, which could be due to probable influence of some forces like selection, non-random mating, inbreeding, mutation and genetic drift.

Conclusion

In view of the findings that the 12 studied duck specific microsatellite loci revealed polymorphism in Tripureswari duck and most of the microsatellite loci showed high PIC values (>0.50), it may be concluded that the usefulness of studied microsatellite as genetic markers to disentangle the genetic variability in various duck population. Further study of these microsatellites may unravel more vital information for their application in marker assisted selection programme in duck and may also be employed as marker for genetic characterization study of other indigenous duck populations in India.

Acknowledgement

AcknowledgementsThe authors express their profound gratitude to the Department of Biotechnology, Government of India, New Delhi, for the financial assistance provided under the NER Research Grant Fiscal Year 2018 to the first author vide No. BT/PR24311/NER/95/645/2017, September 25, 2018. They also acknowledge the facilities provided by the Principal of the College of Veterinary Sciences and Animal Husbandry, R.K. Nagar (West Tripura), and the Director of the Animal Resources Development Department, Government of Tripura.

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