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.