The family Accipitridae (such as hawks and eagles) represents a large radiation of predatory bird with an almost global distribution (Raposo do Amaral et al. 2006). The common buzzard (Buteo buteo) is one of the most numerous and widespread raptor species, especially in Eurasia (Anonymous, 2017). Among them, Buteo buteo from Iran, is one of the most broadly distributed birds throughout of the north and northwest Iran, but has been experiencing a significant decline in population size during the past century (Mansoori, 2013). Furthermore, it is very popular as captive birds in Arabian countries around the Persian Gulf, which makes it a suitable object for illegal hunting. Molecular genetic investigations of wide-ranging species with several recognized subspecies provide an opportunity to understand the ecological, behavioral and evolutionary patterns responsible for differentiation and evaluate the support for sub-specific status (Hull et al. 2008). The conservation genetic is an interface science with the aim of using molecular genetic techniques to determine and maintain biodiversity (Diz and Presa, 2009). Microsatellite analysis makes it possible to determine the genotypes of individuals by examining the variation of the number of repetitions of sequences that are several nucleotides long (Mahmut et al. 2001). The Buteo buteo from Iran is under conservation but there are no evidence of microsatellite markers used to evaluate population genetic parameters in this species. Using microsatellites to determine the genetic diversity of Buteo buteo from Iran as one of the important species of bio-cycle of wilderness, will provide important information that can be applied in conservation genetic strategies. In order to investigate genetic diversity of this population, 10 microsatellite markers were amplified and the population genetic parameters were estimated.
MATERIALS AND METHODS
Characteristics and habitats of Buteo buteo from Iran
Common buzzard from Iran (genus Buteo, species Buteo buteo) has a variety of coat color (light brown to dark brown) with an average height of 50 cm (Figure 1).
Figure 1 Common Buzzard (Buteo buteo)
It is a medium body size rapt or similar to eagle, which is localized in south areas of Caspian Sea and also distributed in North-East of Iran. The nests are usually found on the shore rocks, trees and scaly lands (Mansoori, 2013). The International Union for Conservation of Nature (IUCN) red list has been mentioned this bird as least concern but nowadays, illegal hunting and disturbance of the natural habitats are issues about this species.
Sampling and DNA extraction
Blood samples were collected from 50 Buteo buteobirdkept in the rehabilitation center of Pardisan Park, Tehran, managed by the Department of Environment of Iran. DNA was extracted from samples using Bioneer Extraction Mini Kit.
In the present study, 10 sets of primers were used to evaluate microsatellite markers Bbu10, Bbu14, Bbu17, Bbu22, Bbu30, Bbu33, Bbu34, Bbu49, Bbu51 and Bbu59 (Johanson et al. 2005). The accession number, length, sequence of primers, annealing temperature and number of plexes from the microsatellite markers are displayed in Table 1. The PCR reactions were conducted in 25 µL (DNA 50 ng/µL) using AmpliqonTaq Polymerase 2X master mix Red. The PCR program was 95 ˚C for 15 minutes, followed by 35 cycles of 95 ˚C for 30 seconds, annealing temperature for 40 seconds and 30 seconds of 72 ˚C, and one cycle of 72 ˚C for 5 minutes. Genotyping was done on an automatic capillary sequence (genetic analyzer ABI, 3130) and analyzed with the Gene Mapper v3.2 software. All individuals were separately screened at least twice and if there was problem with the data, the process was done again. Only genotypes with unambiguous results were included in the final data set.
The dataset was checked with Micro-checker 2.23. (Van Oosterhount et al. 2004). Estimating observed (Ho) and expected (He) heterozygosities, potential deviations from Hardy-Weinberg equilibrium (HWE) and AMOVA based on conventional F-statistics (Fst) were calculated with gene ALE × 6.2 software (Peakall and Smouse, 2012). The number of alleles (Na) and effective number of alleles (Ne) were estimated by gene ALE × 6.2 software (Peakall and Smouse, 2012).
RESULTS AND DISCUSSION
The allele length, Na, Ne, Ho, He and Fst estimated for the microsatellite markers of Buteo buteo from Iran are displayed in Table 2.
The total data set of 50 successfully genotyped individuals of Buteo buteo from Iran yielded an average Na of 11.8 and average Ne of 8.7. The Bbu30 locus had the highest Na (18) and Bbu59 had the lowest Na (6); Johanson et al. (2005) reported 17 and 5 alleles for the same loci, respectively on 90 individuals of the same species from Germany. The frequency of the alleles per locus are summarized in Table 3 and nearly all alleles have frequency lower than 0.95, demonstrating widespread genetic polymorphism in these loci. The Fst is shown in Table 2; the average Fst was 0.21 ranging from 0.104 in Bbu22 to 0.313 in Bbu17.
Genetic diversity in a population could be estimated by Ho and He.
Table 1 Locus name, accession number, length, primers, annealing temperature and plex number of Buteo buteo microsatellite markers (Johanson et al. 2005)
Table 2 Amplified length, Na, Ne, Ho, He and Fst estimated for the microsatellite markers of Buteo buteo from Iran
Na: number of alleles; Ne: effective number of alleles; Ho: observed heterozygosity and He: expected heterozygosity.
The average Ho and He in Buteo buteo from Iran were 0.691 and 0.754, respectively. The highest Ho was 0.860 and the lowest was 0.514 in Bbu30 and Bbu33 locus, respectively. In addition, Bbu30 and Bbu22 had the highest and the lowest He, 0.830 and 0.638, respectively. Johanson et al. (2005) reported Ho ranged from 0.133 to 0.99 in a population of Buteo buteo. None of the loci showed significant deviation from Hardy-Weinberg equilibrium. Genetic diversity is a base material for the evolution of a population (Norris et al. 1999), so maintenance of the genetic diversity plays the main role in most conservation programs. Microsatellite markers are genetic markers that are broadly used in conservation management strategies (Liu et al. 2009). These markers are particularly valuable due to their co-dominance nature and they are therefore more useful than genetic mutations (Liu and Cordes, 2004). In a population, allele enrichment is more valuable than heterozigosity (Diz and Presa, 2009), and microsatellite markers could determine the allele number and frequency in a population. In the present study, all 10 primer sets were amplified successfully and nearly all of the loci had multiple alleles. Locus number Bbu30 had 18 alleles and Bbu22 had 5 alleles; the most and the least numbers of alleles respectively. Johanson et al. (2005) evaluated 60 microsatellite markers in 90 samples of Buteo buteo from different parts of Eastern West-Phalia and not all of the primers had reliable results, but most of the loci were genetically polymorphic. Twenty six novel microsatellite markers were characterized by Hull et al. (2007) in Swainson's hawks. The number of alleles per locus ranged from six to 49 with an average of 18.9. Honnen et al. (2010) evaluated nuclear microsatellite markers in white-tailed sea eagle from central Europe.
Table 3 Allele frequency per locus of microsatellite markers from Buteo buteo from Iran
***: no allele.
The results showed that the population could be subdivided into two main genetic clusters. The total data set yielded 65 different alleles with an average number of 9.3 per locus. The amount of heterozygosity was estimated in the two forms of observed and expected heterozigosity (Ho and He, respectively). Nine loci out of the 10 microsatellite markers had Ho higher than 0.5 with an overall Ho of 0.69. The overall He was 0.75. Honnen et al. (2010) reported 0.59 and 0.70 for the overall Ho and He, respectively. The amount indicated that Buteo buteo population of Iran, have high variability. The He reported for Buteo swainsensis ranged from 0.44 to 0.96 while the Ho ranged from 0.40 to 0.95 (Hull et al. 2008). In the present study the Ho ranged from 0.51 to 0.86 and He ranged from 0.63 to 0.83. The overall Fst estimated 0.21, ranged from 0.104 (Bbu22) to 0.290 (Bbu49), the Fst showed that there were genetic differences between individuals from Buteo buteo from Iran. Hull et al. (2008b) evaluated 21 microsatellite markers from different populations of Buteo lineatus and reported that Fst was 0.18, which means the population had genetic differences. In another study, 19 microsatellite loci were analyzed to determine genetic diversity and structure of Buteo swainson and Fst estimate suggested limited differentiation among Swainson's Hawks with isolation by distance (Hull et al. 2008c). Otherwise 17 loci revealed obvious differences between subpopulations of Buteo jamaicensis, which would be the consequence of geographic distances (Hull et al. 2008d). Buteo galapagoensis is one the most inbred hawk in the world living in a small isolated island. The analysis of microsatellite showed low Fst, which indicated a high resemblance between individuals of the studied populations (Bollmer et al. 2006).
The results of the present study showed that polymorphic microsatellite markers are able to evaluate genetic diversity in Buteo buteo from Iran. They had high genetic polymorphism with heterozygosity, besides overall and individual Fst indicated differentiation of markers in the population. The microsatellite markers had not been suffered from natural selection or small population size, and all loci were in Hardy Weinberg Equilibrium. The common buzzard population of the rehabilitation center of Pardisan Park included individuals from different parts of the North and North-East Iran, which is likely the main reason for the high genetic diversity between individuals in the population. Because of the genetic structure and diversity of this population, it seems that there is good opportunity to establish a base population, by recording and labeling birds to provide information to determine conservation strategies. The data obtained from the present study could be merged with data from other populations of Common buzzard from Iran, also by morphological and other genetic markers such as mitochondria genome, it could make a useful dataset for conservation genetic management.
This work was supported by Islamic Azad University, Karaj Branch under grant number 40629. The authors would like to thank for the laboratory support of this university, too. Also they thank the management of the rehabilitation center of Pardisan Park, Iran for the samples they have provided.