Bottleneck Effect Analysis in Indigenous Poonchi Chicken Located Near International Border of India and Pakistan

Livestock production constitutes a very important component of the agricultural economy of developing countries, with multipurpose uses, such as skins, meat, egg, fibre and fertilizer (George et al., 2024). Poonch and Rajouri (Fig 1) are remote districts of Jammu located near line of control and share international borders with Pakistan. Backyard poultry farming with local native indigenous chicken is common practice in these hilly areas. These native indigenous chickens play an important role in socioeconomic upliftment of hilly tribal farmers. The present study was carried out with the main aim to evaluate the presence or absence of bottleneck effect in the Poonchi chicken as previously no study has been done in Poonchi chicken using these recommended microsatellite markers. Earlier studies have documented that local indigenous breeds /strains exhibit more diversity as compared to exotic commercial breeds (Lee et al., 2000; Okumura et al., 2006). It is very imperative to chracterize these native breeds and register them with national breed registration organization for conservation and research purposes. Breed registration involves genotypic chracterization using different molecular markers for estimation of genetic variabilty. Microsatellite markers have been reported by many research workers as one of the most accurate and easy ways to estimate genetic diversity estimation and its relationships among populations (Takezaki and Nei, 1996). Microsatellite markers are highly polymorphic, randomly repeated motifs or simple sequence nucleotide repeats (Tautz, 1989).
 

       
The term ²bottleneck² refers to narrow neck of a bottle which decreases the flow of any substance into the bottle. In conservation studies population bottlenecks attributes to the loss of genetic variation in the population in due course of time due to drastic climate change, predation, limited resources that lead to random fluctuation in the size of the existing population. This reduction in size of the populations results in development of smaller gene pool, this decrease in gene pool is not contemplative of the diversity in the original gene pool. Probable reasons for genetic bottle neck in Poonchi chicken population is loss of genetic diversity due to natural disaster like flash floods in hilly areas of Jammu and Kashmir, inbreeding and small population size of Poonchi chicken flocks. Founder effect is another reason for reduction in diversity as a large habitat of the Poonchi chicken have gone to Pakistan after independence. Microsatellite markers have been used for bottleneck analysis in various livestock species like cattle (Ganapathi et al., 2012; Thiagarajan, 2012), sheep (Girish et al., 2007; Radha et al., 2011) goat (Thiruvenkadan et al., 2014) and poultry (Vij et al., 2006; Pandey et al., 2005, Sharma et al., 2017. Microsatellite markers are useful in assessing genetic diversity, population structure and gene flow analysis and identifying inbreeding and out breeding levels. Microsatellite profiling helps assess the genetic variability within and between population, helps to identify the low genetically diverse population and simultaneously helps to conservation actions. Microsatellite markers can detect levels of inbreeding within a population, guiding breeding plans to introduce new genetic material or promote outbreeding to enhance heterozygosity.

Blood samples were collected aseptically from wing vein of Poonchi chicken from remote villages of Poonch (Gursai, Ari, Harni, Digvaar) and Rajouri district (Androoth, Hanjana, Guni, Rajpur Bhatta) Rajouri. Genomic DNA isolation was carried out by both phenol-chloroform method and by sigma AldrichGenelute blood genomic DNA kit.50 best DNA samples with spectrophotometer values ranging from 1.7 to 1.9 were selected for further PCR amplification with FAO recommended microsatellite markers namely ADL0268, MCW0206, MCW0081, ADL0278, MCW0069, MCW0248, MCW0111, MCW0222, MCW0016 and LEI0094 (Table 1). PCR was standardized for all 10 primers and positively amplified samples were then resolved at 10% urea polyacrylamide gel electrophoresis for a period of 4hrs at 100 -120 volts. The gels were then stained with silver staining to visualize and analyse the number of alleles in each sample.The bands obtained after staining were further genotyped, scored and different alleles were identified as different bands. Alleles were marked as A,B,C….. in ascending order of size. Popgen 32 software was used to analyze the genetic diversity. Three tests Infinite allele model (IAM), two phase model (TPM) and step wise mutation model (SMM) were performed to evaluate the Poonchi chicken for mutation drift equilibrium. Three tests namely sign rank, standardized differences and wilcoxon test were used to investigate the presence of bottleneck effect in Poonchi chicken breed. The method for detecting bottleneck effect involves comparison of expected heterozygosity (H_e) with heterozygosity expected at mutation disequilibrium (H_eq). If H_e is higher than H_eq then there is heterozygosity excess whichmeans a recent bottleneck effect (Cornuetand Luikart 1996). Heterozygosity excess was tested with one tailed wilcoxon sign rank using bottleneck software 1.2.02 (Piry et al., 1999).The allele frequency distribution was established to see absence or presence of bottleneck effect. For qualitative graphical representation of allele frequency distribution another test, mode shift graph (Luikart et al., 1998) was used to identify potential bottlenecks. With the use of Mode shift graph allele frequency classes of ten microsatellite markers was obtained. L-shaped distribution curve of the allele frequency graph indicates no recent bottleneck effect and a shift from L-shaped distribution is suggestive of a recent genetic bottleneck.
 

Chicken represents a valuable genetic resource and diversification of the poultry production is one of the viable options to enhance the food production (Kaushik et al., 2023). Poonchi chicken is an indigenous breed reared mainly in remote areas of Rajouri and Poonch, Jammu and Kashmir (UT). Severe bottlenecks and population decline often lead to the reduction of most important factor i.e. genetic variation in a population (Mundy et al., 1997; Groombridge et al., 2000). Documentation have been done by researchers to acknowledge the importance of variation for survival (McAlpine 1993; Keller et al., 1994), the clutch size (McAlpine 1993) and growth rate (Alvarez et al., 1989). In contrast to it some researchers observed no or little effect of these parameters. (Ardern and Lambert 1997). However, like many other indigenous breeds, Poonchi chicken is facing threats from the widespread use of commercial breeds. Since very less information regarding this indigenous population is available, it is very important to characterize, promote and conserve this breed. Therefore, the present study was carried out for bottleneck analysis of Poonchi chicken using microsatellite markers.
       
Bottleneck occurs when populations experience severe temporary reduction in size (Barani et al., 2024). Bottleneck hypothesis was investigated using bottleneck software 1.2.02. The result for Bottleneck analysis under three microsatellite evolution models are presented in Table 2. Result showed that the number of alleles in the present study ranged from 6 to 9 for recommended microsatellite markers under study. It can be seen that the expected heterozygosity ranged between 0.73 (LEI0094) to 0.859 (MCW0111). Three tests IAM, TPM and SMM were performed to evaluate the Poonchi chicken for mutation drift equilibrium. All three models in Sign test showed expected numbers of loci with heterozygosity excess in Poonchi chicken as 5.97 (P<0.05), 5.93 (P<0.05), 5.90 (P<0.05) respectively. Whereas for Wilcoxon rank test the IAM, TPM and SMM revealed significant values of (P<0.05) deviation indicating that all the loci deviate from mutation-drift equilibrium (Table.3). Result show that the observed heterozygosity excess (He) is more than expected heterozygosity excess (Hee). The higher values of He then Hee indicate the presence of recent bottleneck effect in the Poonchi chicken. Probable reasons for genetic bottleneck in Poonchi chicken population are natural calamities/flood (that occurs during the 2014 causing huge loss in genetic resources and population size drastically reduced which leads to loss of genetic diversity), inbreeding and small population size (Poonchi chicken is reared in small flocks and there is no systematic breeding knowledge among farmers, inbreeding occurs in the population causing reduction of genetic diversity over time and increasing the likelihood of genetic bottlenecks) and geographical relocation (Poonchi bird is unique in nature and after independence some part of natural habitat goes to Pakistan, that left with a small group of individuals which subsequently establishes a new population; hence the genetic variation is limited to Poonchi chicken leading to a genetic bottleneck). Similar results were observed by De la Para et al., (2024) in light Brahma breed.
 

 

       
Bottleneck effect in Poonchi chicken can be improved or removed by introduction of genetically diverse Poonchi chicken population by the farmers or exchange of breeding birds among themselves. Farmers should be trained for scientific breeding management among Poonchi chicken to control inbreeding.
 
Mode-shift test
 
Mode shift test was used as another method of qualitative test for allele frequency. Luikart et al., (1998) proposed mode shift test for qualitative graphical representation of allele frequency distribution. The graph obtained depicted a slight shift from normal L-shaped curve indicating recent bottleneck effect in Poonchi chicken. (Fig 2). A normal L shaped distribution of the allele frequency graph is an indication of no recent bottleneck. When there is some shift or change in L-shaped distribution of the studied populations it indicates recent genetic bottleneck. The result suggested that the Poonchi chicken population had undergone an effective recent genetic bottleneck (last 15-20 generations). The main reason for genetic bottleneck is indiscriminate and unplanned crossbreeding which has led to the loss of effective alleles due to small size population, inbreeding or mating which occur among relatives and consequent to it causes genetic drift. Similar to these results were reported by De la Para et al., 2024 in various Chilean Chicken breeds namely Kollonka, Ketro, Trintre, Cogotepelado, Light Brahma and Barred Plymouth Rock. Comparative to this a normal L-shaped curve was observed by De la Para et al., (2024) in Kollonca de aretes breed, in Punjab Brown chicken  by Vij et al., (2006), in Ankleshwar poultry population of India by Pandey et al., (2005), in Kaunayen chicken of Manipur by Sharma et al., (2017) indicating that these breeds have not experienced any recent genetic bottleneck.
 

The presence of genetic bottleneck calls for an urgent need of implementing an effective well planned breeding policy to preserve and promote this native breed which is well suited for local conditions of Jammu and Kashmir. Genetic heterozygosity and inbreeding are indeed inversely proportional, meaning that as inbreeding increases within a population, genetic heterozygosity decreases. Although, the overall heterozygosity deficiency in the population is negative but the value is very close to zero which indicates loss of substantial heterozygosity in the population. This work is the first study done on Poonchi chicken using specific microsatellite markers indicating genetic bottleneck effect.

There is no conflict of interest for authors to declare.