Characterization of Microsatellite Markers in Crossbred Ducks

Some important traits are quantitative traits which is controlled by polygene. The molecular genetics mapping tools enables the identification of quantitative trait loci in the genome. Application of marker assisted selection for QTL has the potential to enhance the accuracy in animal breeding program, particularly for the traits that are difficult to improve through traditional selection methods. As we know that microsatellites or short tandem repeats (STR), are tandem repeated motifs of 1-6 bases. They were found abundantly and at random throughout most eukaryotic genomes (Stallings et al.,1991). A large number of microsatellites have been isolated and widely used for these purposes.  In case of duck populations less genetic markers have been established. Thus, we attempted to isolate microsatellite markers for cross bred duck and to investigate their polymorphism. Exploring genetic variability by microsatellite markers is essential for genetic improvement, preservation of indigenous germplasm and production of high-quality offspring (Jowel et al., 2023).
       
Chicken represents a valuable genetic resource and protein source, it remains a potential threat to human health as they serve as a reservoir for diseases and food borne pathogens. Diversification of the poultry production is one of the viable options to enhance the food production with less susceptibility to the threats from the emerging diseases and changing climatic conditions. Ducks are promising species suitable for the diversification and will be complimentary with an adaption potential from small to commercial farming. Majority of ducks in the country are desi type with a meagre proportion of improved duck strains. Recent livestock census indicated that the duck population has been drastic decreasing over years from 27.6 million in 2007 to 23.5 million in 2012. Therefore, there is an urgent need to augment the duck production through improved breeding, feeding, disease control and other managemental strategies. Further, fine-tuning of production performance and effective health care management can be achieved with the help of newer technologies. Converged use of various conventional molecular and health care technologies will augment duck egg and meat production. Since majority of ducks are desi or non-descript type having low production potential therefore there is an ample scope of duck improvement. Besides, Limited research and scientific intervention has been paid to characterize them, to improve their productivity and to exploit their unique characteristics. Hence, any attempt to improve the duck farming will have direct bearing on the economically weaker section of the society. Improvement of productivity of ducks through identification of molecular markers could be a method of choice. Therefore, genetic characterization by developing duck specific microsatellite markers and designing suitable improvement program are required to be undertaken at the earliest. The microsatellite markers are extensively used for assessing genetic structure, diversity and relationships. Information about genetic diversity of indigenous duck breeds is important to design effective improvement and conservation strategies. Therefore, the current studies aimed at genetic characterization and evaluation of growth, production and reproduction traits of the crosses of local duck population besides undertaking a suitable duck improvement program.

DNA isolation protocol       
 
About 1.5 mL of distilled water was added to the duck blood (35-50 µL) that was placed in the anticoagulation tube. The content was mixed well and transferred into a 1.5 mL micro centrifuge tube and centrifuged for 2 min at 13500 rpm in a micro centrifuge. The supernatant was discarded and the repeated the steps. The pellet was resuspended in a a 55°C pre warmed 1 mL of WBC lysis buffer (10 mM Tris-Cl pH 7.7, 1.5 M NaCl, 2 mM EDTA, 0.5% SDS) and then the whole suspension was mixed well and micro centrifuged at 13500 rpm for 4 min. The supernatant containing DNA thread mass was picked up with a micropipette equipped with a wide orifice and placed in a new tube. To the supernatant, 1 ml of absolute ethanol was added and the tube was inverted several times. The DNA threads were picked up and placed in a new micro centrifuge tube containing 1 mL of ice cold 70% ethanol and mixed well. Micro centrifugation was performed at 13500 rpm for 4 min. The supernatant was discarded.  The DNA was resuspended in 0.5 mL of TE buffer.
  
Spectrophotometric evaluation of DNA extraction
 
The spectrophotometric evaluation of the concentration and and purity of DNA was carried out by spectrophotometer. DNA concentration was evaluated. DNA purity with regards to protein and salt contaminants was based on the A 260/280 and A 260/230 absorbance ratios respectively.
 
The electrophoretic evaluation of DNA extraction
 
The genomic DNA integrity was checked electrophoretically in agarose gel.
 
Microsatellite primers
 
Presently, a panel of 16 informative duck microsatellite markers (Table 1) were identified from database as reported by Alyethodi and Kumar (2010) and Huang et al., (2006) and used for the studies. The synthesized primer pairs were obtained in lyophilized form and were reconstituted with nuclease-free water as per manufacturer’s instructions. A stock of 100 μM was prepared and from this working primer solution of 10 pM was prepared and used in PCR.
 

 
Polymerase chain reaction (PCR)
 
Each PCR assay was carried out in a total of 25 µL containing 12.5 µL PCR master mix(2X), 5.5 µL nuclease free water, 5 µL template DNA, 1 µL each of reverse and forward primer. Initial denaturation was done at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 min, primer annealing at optimized temperature for 1 min and extension at 72°C for 1 min and then final extension at 72°C for 5 min. The PCR products were analyzed by Agarose gel electrophoresis.
 
Following PCR cycling conditions were optimized for 16 microsatellite loci
 
Heat inactivation at 95^°C for 5 minutes 30 cycles of
a.  Denaturation at 94^°C for 1 minute.
b.  Annealing  at T_a^°C (T_a=optimized annealing temperature for each Microsatellite primer pair)for 45 seconds.
c.  Extension at 72^°C for 45 Seconds.
►  Final extension at72^°C for 5minutes.
►  4°C forever.
 
The PCR products were kept a- 20°C until further analysis
 
Documentation of PCR products by Agarose Gel Electrophoresis
 
Approximately, 10 µl of PCR product was added with 2 µl Bromophenol blue dye (6X loading dye, GCC Biotech, India Pvt. Ltd.) for loading in the gel. Samples were loaded into wells of 2% agarose gel containing ethidium bromide (5 µl per 100 ml of 1X TBE buffer) along with 5 µl of 100 bp DNA ladder (GCC Biotech, India Pvt. Ltd.) as molecular size marker for identification of the desired product. The electrophoresis was done at 2-5 volts/cm. The products were examined under UV light in Gel Documentation system (Bio rad Laboratories,USA) and documented.
 
Metaphor agarose gel electrophoresis (MAGE)of microsatellites alleles
 
The confirmed amplification of all the samples, the amplicons were run on 3% metaphor agarose gel electrophoresis (MAGE) to resolve microsatellite alleles for further genotyping.
 
Determination of molecular size of microsatellite alleles and genotypes
 
The molecular sizes (in bp) of all the alleles at sixteen studied microsatellites were determined with the help of Image Lab software (Bio-Rad Laboratories Inc., U.S.A.) through Gel Doc system. Genotypes of all the birds were determined on the basis of presence of microsatellite alleles.
 
Statistical analysis of population genetics data
 
Data on genotype of all experimental birds at sixteen microsatellites were compiled and analyzed using POPGENE® 3.1 software (Yeh et al., 1999) for their population genetics parameters. The primary data on genotype was subjected to co-dominant marker diploid data analysis to estimate observed and expected genotypic frequencies, Hardy-Weinberg (HW) equilibrium status, allele frequency, observed and effective number of alleles, percentage of polymorphic loci, observed and expected homozygosity and heterozygosity and Shannon index.
 
Genetic variability analysis
 
Average heterozygosity per microsatellite marker was calculated according to Nei (1978).
  Where
Pj 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, assuming that the population was under Hardy-Weinberg equilibrium.
       
Polymorphic information Content (PIC) at each microsatellite locus was calculated using the following formula (Botstein et al., 1980) :
   
Where,
P_i and P_j are the frequencies of i^th and j^th  alleles, respectively at a locus with k numbers of alleles in the population.

The primers along with their nucleotide sequences were summarised in Table 1. the characteristics of the 16 microsatellite loci with allele frequencies were summarized as Table 2. Table 3 depicts Polymorphic Information Content (PIC), Shannon’s index, Number of observed and expected alleles, Nei’s heterozygosity and Wright’s fixation index, observed and expected heterozygosity, chi-square and G-square value at duck specific microsatellite loci in crossbred  duck (F1) generation. A total of 16  microsatellites were used to detect polymorphisms in 50 cross bred ducks. A total of 34 nos of alleles were observed and all loci were polymorphic. The number of alleles ranged from 1 to 4 with an average of 2.125±0.07 per microsatellite locus. The observed and expected heterozygosity of these polymorphic makers ranged from 0.00 to 0.42  and 0.41 to 0.84 with an average number of 0.068±0.04 and 0.616±0.02 respectively. Among the polymorphic markers, the observed heterozygosities of loci were less than 0.50. The polymorphism information content (PIC) of 21 loci ranged from 0.32 to 0.78 with an average of 0.52±0.03.
 

 

       
Based on the classification of Hamilton et al., (1999), Botstein et al., (1980), ten (50%) polymorphic markers were highly informative (PIC>0.50) and rest six  (50%) were reasonably informative (0.50>PIC>0.25) which can be comparable to Hsu et al., (2003) and Maak et al., (2003), Dwi Nur Happy Hariyono et al., (2018) and Jowel et al., (2023).

In conclusion, the identified appropriate microsatellite marker systems for crossbred  ducks will  provide a good choice for genetic monitoring of the quality and the population genetic diversity of poultry stocks.

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.

All the Authors have equally contributed for the research article.

The authors declare that there is no Conflict of Interests regarding the publication of this article.