Full Length Research Article
Assessment of genetic diversity and genetic characterization of Nili Ravi buffalo breed utilizing microsatellite markers
Atia Bukhari1, Naveed Ahmed1*, Fizza Khan1, Muhammad Shafique2, Asif Suleman Sahi1
Adv. life sci., vol. 7, no. 4, pp. 277-280, August 2020
*- Corresponding Author: Naveed Ahmed (Email: naveed753@outlook.com)
Authors' Affiliations
2. National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore – Pakistan
Abstract
Introduction
Methods
Results
Discussion
References
Abstract
Background: Livestock contribution to Pakistan’s GDP is 11.2% and it engages 8 million of rural families for their livelihood. Estimated population of buffalo is 40.00 million heads in Pakistan mostly consisting of low genetic worth population resulting in low productivity and pressure on natural resources. Assessment of genetic diversity and genetic characterization of indigenous livestock resources is an essential step towards conservation and to gauge effects of various breed improvement efforts on population genetics.
Methods: In present study genetic diversity of Nili Ravi buffalo population was assessed. A total of 196 unrelated Nili Ravi buffaloes from Punjab, Pakistan were sampled in this study. Genomic DNA was extracted and subjected to amplification using FAO recommended 12 Short Tandem Repeats (STRs) microsatellite markers. Among 12 microsatellite loci, 11 were successfully amplified (TGLA227, BM2113, ETH10, SPS115, TGLA126, TGLA122, INRA23, BM1818, ETH3, ETH225 and BM1824) whereas microsatellite locus TGLA53 was not amplified. Amplicons were resolved by genetic analyzer instrument and gene mapper software. Allele count, frequencies, gene diversity, heterozygosity, polymorphic information content (PIC) and linkage disequilibrium values were calculated by using Microsatellite toolkit v3 and Power Marker version 3.25.
Results: A total of 96 alleles were detected in 196 samples with average of 8.73 alleles per locus and range of 5 alleles (ETH 3) to 18 alleles (ETH 225) per locus. Gene diversity ranged from 0.198 (BM1824) to 0.841 (ETH225), observed heterozygosity values ranged from 0.081 (ETH10) to 0.831 (BM2113) and PIC values ranged from 0.191 (BM1824) to 0.825 (ETH225).
Conclusion: This study which will serve as a baseline to understand genetic dynamics of Nili Ravi buffalo breed. Highly polymorphic nature of STR markers will help in understanding effects of various breed improvements efforts on genetic diversity of Nili Ravi breed in future studies.
Keywords: Microsatellite; STR; Nili Ravi Buffalo; Genetic Characterization; Punjab
Introduction
Hearing impairment (HI) is a heterogeneous infirmity The livestock genetic resources are integral part of agricultural ecosystem. They contribute to nutritional requirements of population by providing meat and milk. Other products and by-products from livestock resources such as hide, fiber, manure and fuel provide raw material for other industries. This contribution constitutes significant share of GDP of under develop and developing countries. Livestock sector accounts for 11.2% of total GDP of Pakistan. As most of livestock in Pakistan is raised in rural areas in small holder setups, it engages about 8 million families of rural community providing more than 35-40% of their income [1].
The water buffalo (Bubalus bubalis) population in Pakistan is estimated at 40.00 Million with estimated milk production of 36.18 Million tons [1]. Buffalo population in Pakistan consists of five breeds’ i.e. Nili, Ravi, Nili Ravi, Azakheli and Kundi. Nili and Ravi are morphologically distinct breeds but due to intensive interbreeding among population, it is becoming increasingly difficult to find purebred Nili or Ravi specimen in field. Breed characterization based on morphological features is insufficient to understand breed’s genetic dynamics, improvement and conservation. Hence molecular studies of these breeds are warranted to underscore genetic variations and to gauge effect of various breed improvement efforts in buffalo breeds of Pakistan [2]. Selection of high producing animals based on performance of their progeny and disseminating their germplasm through artificial insemination (AI) is being carried out successfully in Punjab toimprove genetic potential of native buffalo breeds. On the other hand such efforts create pressure on genetic diversity of population due to availability and utilization of few high genetic worth males in AI programs resulting in low genetic diversity in population. Food and Agricultural Organization (FAO) in collaboration with International Society of Animal Geneticists (ISAG) addressed this issue by publishing guidelines on Measurement of Domestic Animal Diversity (MoDAD) [3].
Breed characterization exploiting Short Tandem Repeats (STRs) markers is recommended method by FAO. Briefly, STRs are tandem repeats of 2 to 7 nucleotide innon coding regions of genome. STRs are excellent source of measuring quantitative genetic variation in population owing to their polymorphism. STRs are non-coding regions hence they are not prone to selection pressure. Moreover, they are randomly distributed in genome thus providing reliable genetic dynamics of population. Using STRs panels in genetic characterization is also helpful in interpretation and utilization of data at global scale as well as its comparison with other genetically characterized breeds. Using similar panel of STRs markers in future will also show clearer picture of genetic trends in that breed. Therefore, the present study was designed to genetically characterized and to assess its genetic diversity in Nili Ravi buffalo population of Punjab province of Pakistan utilizing STRs markers [4].
Methods
A total of 196 blood samples were taken from genetically unrelated animals across Punjab. Samples were taken from adult female animals present at Livestock experiment station (LES) Bhunikey district Kasur, LES Rakh Ghulaman district Bhakkar, LES Khushab, LES ChakKatora district Hasilpur, LES Haroonabad district Bahawalnagar and from field area in Punjab. All sampled animals were phenotypically true to Nili Ravi breed. Venous blood samples of 5 to 10 ml were taken from each animal in vials containing EDTA as anticoagulant. Samples were stored at -20 °C prior to DNA extraction. DNA was extracted using Favor Prep ® DNA isolation kit (cat No. FABGK 001-1) following kit manual. DNA was quantified by Nano Drop Onec (Thermo Scientific®) spectrophotometer and diluted appropriately for PCR reaction. Samples were amplified in thermal cycler (Bio Rad® C-1000 Touch) by using Bovine Genotyping Panel 1.2 (Thermo Scientific® cat No. F904S). Samples were initially denatured at 98 °C for 1 min following by 30 cycle of amplification. Thermal profile for amplification was: 98 °C for 20 sec for denaturation, 60 °C for 75 sec for annealing and 72 °C for 30 sec for extension. 5 minutes at 72 °C were provided for final extension. Every batch included standard DNA specimen provided with Bovine genotyping 1.2panel for accurate genotyping and allele calling. Samples were genotyped by using Genetic Analyzer 3130 (Applied Biosytems®) with GeneScan™ 500 LIZ® as size standard. GeneMapper® Software version 5 was used for analysis of genotyping data and allele calling. Microsatellite loci TGLA227, BM2113, ETH10, SPS115, TGLA126, TGLA122, INRA23, BM1818, ETH3, ETH225 and BM1824 were amplified successfully, whereas Microsatellite locus TGLA53 was not amplified. Resulting data were analyzed for number and frequency of alleles, expected heterozygosity and polymorphism information contents (PIC), Linkage disequilibrium (LD) Matrix by using Microsatellite toolkit v3 and Power Marker version 3.25 tool.
Results
A total of 96 alleles were detected in 196 samples with average of 8.73 alleles per locus and range of 5 alleles (ETH 3) to 18 alleles (ETH 225) per locus. Details of locus wise number of alleles are shown in table 1. Overall population statistics calculated by using Microsatellite toolkit v3 are described in table 2. Gene diversity, Heterozygosity and Polymorphic information content values were calculated by Power Marker version 3.25 tool [5]. Heterozygosity values indicate genetic variability and population bottlenecks. Gene diversity or expected Heterozygosity is probability of finding heterozygous alleles in a random sample from population under Hardy-Weinberg Equilibrium[6].It ranged from 0.198 (BM1824) to 0.841 (ETH225). Observed Heterozygosity values were highest0.831 for locus BM2113 and lowest 0.081 for locus ETH10. Polymorphic information content (PIC) values ranged from 0.191 (BM1824) to 0.825 (ETH225). The Expected and Observed heterozygosity as well as PIC values for each locus are shown in Table 3. Details of polymorphic allele size, count and frequency at each locus is shown in Table 4. Linkage disequilibrium (LD) is association of alleles at two different loci and shows dependence of gene frequencies. LD Matrix for Nili Ravi Buffalo is shown in Table 5.
Figures & Tables
Discussion
Genetic diversity of Nili Ravi Buffalo breed based on FAO approved STR markers was reported in this study which will serve as a baseline to understand genetic dynamics of breed. Highly polymorphic nature of STR markers will help in understanding effects of various breed improvements efforts on genetic diversity of Nili Ravi breed in future studies.The use of FAO approved markers will be helpful for future studies on same breed as well as comparative studies on other breeds.
Authors' Contribution
AB designed and conducted the study. NA and FK helped in lab work. NA conducted field work and write up. FK, NA and MS helped in data compilation and analysis. AB and ASS reviewed and approved the draft.
The authors declare that there is no conflict of interest regarding the publication of this paper.
References
- Pakistan Go (2019) Economic Survey of Pakistan. Islamabad.
- Hussain T, Ellahi Babar M, Ali A, Nadeem A, Rehman ZU, et al. Microsatellite based Genetic Variation among the Buffalo Breed Populations in Pakistan. Journal of veterinary research, (2017); 61(4): 535-542.
- FAO (2011) Molecular genetic characterization of animal genetic resources.
- Hoffman I. New MoDAD marker sets to be used in diversity studies for the major farm animal species : recommendations of a joint ISAG/FAO working group. Proceedings of the 29th International Conference on Animal Genetics, September 11-16th 2004, Meiji University, Tokyo, Japan, (2004).
- Liu K, Muse SV. PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics, (2005); 21(9): 2128-2129.
- Harris AM, DeGiorgio M. An Unbiased Estimator of Gene Diversity with Improved Variance for Samples Containing Related and Inbred Individuals of any Ploidy. G3: Genes|Genomes|Genetics, (2017); 7(2): 671-691.
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