Genetic Characterization of Indigenous Duck of North-East Region using Microsatellite Markers

North-East India is rich repository of watershed and potential place for duck farming. Duck farming is a major component in integrated farming system and plays a significant role in women empowerment and uplift ment of socio-economic status of the farming community of North-East India. Due to hot - humid climatic condition of the region along with extensive availability of resources like ponds, river, marshy wet lands etc. provides suitable natural habitats for duck rearing in rural areas of the states. Duck eggs and meat are widely accepted by the different sections of the society and provide nutritional security. 

To exploit the genetic potential of native duck breeds genetic characterization is a pre-requisite. DNA based markers, particularly microsatellites are the marker of choice for such studies and alsowell recommended by FAO (Fao, 2007). The hyper variability, co-dominant nature, uniform and pervasive nature in genome, easiness of detection and reliability and possibility of analysis of a number of microsatellites simultaneously make it more popular than minisatellites which are otherwise difficult to detect and not distributed uniformly. Reports on the genetic characterization of ducks using microsatellite markers are slowly accumulating across the globe in recent years but are scanty in the Indian native duck breeds (Maak et al., 2000; Huang et al., 2005; Wu et al., 2008). Pati duck is native breed of Assam, squat in posture with solid brown plumage in ducks and dark brown plumage and greyish black head in drakes. In the neck white ring may or may not be seen in both sexes. The beak, shank and feet are predominantly yellow in colour. The age at sexual maturity is between 220-235 days with a mean body weight of 1.56kg. The annual egg production ranges from 75 to 90 with the average weight of 60.5g (Kalita et al., 2009; Mahanta et al., 1993). Another duck variety Chara- Chambeli originated in Kerela and is commonly reared in North-East India. Reports on the genetic characterization of ducks using microsatellite markers are slowly accumulating across the globe in recent years but are scanty in the Indian native duck breeds (Maak et al., 2000; Huang et al., 2005; Wu et al., 2008). Therefore, the present investigation was carried out to characterize these two duck breeds using microsatellite markers to evaluate the genetic diversity in these two duck population.
 

A total of 100 (50 nos. of Patiducks and 50 nos. of Chara-Chambeli) ducks from different parts of North –East India region were selected for the present study. Approximately 1 ml of venous blood was collected aseptically from the wing vein of the birds in vaccutainer tube containing EDTA (2.7%). The samples were properly labelled and transported immediately in ice box to the Department of ILFC, C.V.Sc, A.A.U, Khanapara and later stored in deep freezer at -20°C temperature till the isolation of genomic DNA. Genomic DNA was isolated from 100ìl of whole blood. The concentration of genomic DNA was estimated by Picodrop spectrophotometer (Model no. PICOPET01). The concentration of the genomic DNA was estimated at UV-VIS Spectrophotometer at 260 nm Optical Density (OD). The ratio of OD values at 260 nm and 280 nm was used as a criterion to check the purity of the extracted genomic DNA.The present study was conducted using sixteen duck microsatellite specific primers for the PCR amplification. The gel preparation was done using 3% Metaphor agarose was slowly added to the chilled 1x TBE buffer with continuous swirling. It was soaked in that cold buffer for about 10-15 minutes before heating in the microwave (to prevent it from foaming and not to over boil the gel). Just prior to pouring the gel, Ethidium bromide (0.3 µg/ml) was mixed to the dissolved agarose. Once the molten gel was solidified after being poured into a cast, it was kept at 4°C for 20 minutes before use to obtain a better resolution and for easier handling of the gel (Anonymous 2004).

After adding 3 µl 6x loading dye to the 8 µl PCR product, 10 µl of it was loaded on the Metaphor agarose gel submerged in chilled 1x TBE buffer and electrophoresed for about 1-2 hours at 6 Volt/cm using horizontal electrophoresis system. After electrophoresis, the finely resolved products were visualized under UV transilluminator. The data were analysed for probable genotype of each sample at each microsatellite loci and population genetics (Shanan index, effective nos. of alleles, observed and expected heterozygosity, Hardy-Weinberg equilibrium, numbers of alleles per microsatellite marker, allele frequency) analysis were done by POPGENE software v 1.32 (Table 1).

Analysis generated a total of 41 microsatellite alleles across all studied loci by using 16 microsattelites. The study utilized the same set of primers as reported by Alyethodi et al., (2010). Highest numbers of alleles 5 (A/B/C/D/E) were recorded at locus CAUD 04 and CAUD 05. The number of alleles at various polymorphic microsatellite loci ranged from 1 to 5 and frequency ranges from 0.04 to 1. The polymorphic patterns were observed in 12 out of 17 microsatellite loci studied in the present study. The average number of alleles per locus is 2.41. Similar studies was reported by Sankhyan, (2007) on Indian Runner and Moti native ducks, where Moti native duck had higher number of alleles (5) than Indian Runner, whereas Huang et al., (2005) recorded the highest numbers of alleles (3) at locus CAUD 011. The number of alleles at various polymorphic microsatellite loci ranged from 1 to 3. They used automated DNA sequencer/genotyper and reported higher average allele number with same primer pairs which may be attributed to the sensitivity of instrument/techniques employed in their studies, besides the genetic differences. In the present study, the frequencies of most of alleles were high and 36 out of 43 (83. 72%) polymorphic alleles had a frequency of more than 10 per cent. Another study indicating that Moti native duck can be tagged as a distinct population from other native Indian ducks based on the microsatellites studies (Rajkumar et al., 2008). Polymorphic patterns were observed in 14 out of 16 microsatellite loci studied. The average number of alleles per locus (2) was in accordance with the report of Maak et al., 2000. They estimated the same as 2.9 and 3.5 in Muscovy and Pekin ducks, respectively. Sankhyan (2007) reported an average number of allele as 3.0 in Indian Runner duck. All the loci investigated in the present study were polymorphic in nature. The number of observed alleles (Na) with an over all mean of 1.93± 0.258. However, the effective number of alleles (Ne) with a mean of 1.6933± 0.2712.Shannon’s information index with a mean value of 0.5685±0.1693. The overall means for observed (HO) and expected (He) heterozygosities were 0.2889±0.2477 and 0.5289 ± 0.0853 respectively. Similar findings were reported by Sankhyan, 2007 with an average heterozygosity of 0.56±0.02 in Indian Runner native duck. Rajkumar et al., (2008) also reported an average heterozygosity at polymorphic microsatellite loci as 0.52±0.02 in Moti ducks. Whereas, Huang et al., (2005) recorded average heterozygosity of 0.47±0.01 for the same set of primers. The chi-square (c2) test for Hardy-Weinberg equilibrium revealed that all the loci is in within Hardy-Weinberg equilibrium. The Nei’s genetic identity and genetic distance has been depicted in Fig 1.

The present study may help to explore the genetic diversity of native duck breed namely Pati duck in Assam and it will work as baseline information for these unique duck breed.

The authors are thankful to the Department of Biotechnology, Govt. of India, New Delhi (Project ID 102/IFD/SAN/1335/2018-2019) for financial assistance to carry out the research work.

There is no conflict of interest.