MICROSATELLITE DNA POLYMORPHISM IN SELECTIVELY CONTROLLED APIS MELLIFERA CARNICA AND APIS MELLIFERA CAUCASICA POPULATIONS FROM POLAND

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1 MICROSATELLITE DNA POLYMORPHISM IN SELECTIVELY CONTROLLED APIS MELLIFERA CARNICA AND APIS MELLIFERA CAUCASICA POPULATIONS FROM POLAND Stanimila R. Nikolova 1, Malgorzata Bienkowska 2, Dariusz Gerula 2 and Evgeniya N. Ivanova 1 * 1 Section of Genetics, Department of Developmental Biology, Faculty of Biology, University of Plovdiv Paisii Hilendarski, Plovdiv, Bulgaria 2 Research Institute of Horticulture, Apiculture Division, Pulawy, Poland *Corresponding author: e.ivanova@gmail.com Short title: polymorphism in honeybee populations from Poland Received November 2, 2014, Revised January 20, 2015; Accepted January 21, 2015 Abstract: Genetic polymorphism in selectively controlled honeybee populations of A. m. carnica and A. m. caucasica in Poland, was characterized by microsatellite DNA analysis. All honeybee samples were analyzed for nine microsatellite loci: Ac011; A024; A043; A088; Ap226; Ap238; Ap243; Ap249 and Ap256, which were found to be polymorphic in both populations. The mean number of alleles per locus was for A. m. carnica and for A. m. caucasica. Average observed and expected heterozygosity values were calculated as and in A. m. carnica and as and in A. m. caucasica, respectively. For the nine microsatellite loci, a total of 76 alleles were found in both populations. Thirtyfive private alleles were observed in A. m. carnica and 20 in A. m. caucasica. Information about allele frequencies, F ST values and genotypic differentiation is given. Nei s genetic distance between studied populations of A. m. carnica and A. m. caucasica was calculated as Key words: Honey bee; Apis mellifera; microsatellite DNA; genetic polymorphism; Poland INTRODUCTION European honeybee populations show considerable differences in morphological, behavioral and biological characters across their natural range in the world. Many of these geographically and biologically distinct populations have been recognized as subspecies by Ruttner (1992) that have been clustered into four main lineages: C (Carnica group); M (North and Western European group); A (African group); and the O group (Oriental group). Generally, about 27 subspecies and numerous ecotypes of Apis mellifera have been described (Ruttner, 1988; Sheppard et al., 1997; Sheppard and Meixner, 2003; Meixner et al., 2010; De La Rua et al., 2009; Meixner et al., 2013). A. m. carnica and A. m. caucasica subspecies that are currently the most widely bred in Poland belong to the C and O lineage groups. Initially these two subspecies were studied mainly by classical morphometry (Gromisz, 1978; 1981), then by analyzing the venation of forewings (Tofilski, 2004; 2008; Gerula et al., 2009) and by alloenzyme analysis (Ivanova et al., 2011; 2012). There is no study about their genetic polymorphism based on microsatellite DNA analysis.

2 In this study, polymorphism in honeybee populations of A. m. carnica and A. m. caucasica selectively controlled in Poland was studied using 9 microsatellite loci. The objective of the study was to investigate and characterize genetic variability among both subspecies and to provide information about allele frequencies, number of alleles per locus, levels of polymorphism, observed and expected heterozygosity and genetic distance. MATERIALS AND METHODS Biological material Honeybee workers from A. m. carnica and A. m. caucasica colonies in the Apiculture Division of Research Institute of Horticulture in Pulawy, Poland, with instrumentally inseminated queens, were used for this study. Five colonies per population and 6 to 10 individuals per colony were tested. Collected worker bees were transported to the laboratory alive, kept at C and then moved to tubes with absolute alcohol until use for DNA extraction. DNA extraction and PCR protocol and microsatellite loci DNA extraction, PCR protocol and microsatellite DNA analysis were done as described by Nikolova (2011). All honeybee samples were analyzed for nine microsatellite loci: Ac011; A024; A043; A088; Ap226; Ap238; Ap243; Ap249 and Ap256 (Table 1). Statistical analyses Population genetic statistics were computed using GENEPOP package software version 1.2 (Raymond and Rousset, 1995). F ST values were calculated according to Weir and Cockerham (1984). The exact test for Hardy-Weinberg equilibrium and genotypic differentiation were performed using GENEPOP. Unbiased estimates and standard deviations of heterozygosity were calculated according to Nei (1987). RESULTS All nine microsatellite loci studied were polymorphic in both of the populations. The number of alleles observed and expected heterozygosity per locus for both populations are presented in Table 2. The mean number of alleles per locus was for A. m. carnica and for A. m. caucasica. The average observed heterozygosity values were calculated as in A. m. carnica and in A. m. caucasica. Additionally, the average expected heterozygosities for the studied populations were calculated as in A. m. carnica and in A. m. caucasica. Information about allele frequencies and private alleles for the populations is presented in Table 3. Thirty-five private alleles were observed in A. m. carnica and 20 in A. m. caucasica. There were no private alleles in the Ap249 locus for A. m. caucasica. The highest number of private alleles (6) was observed in A. m. carnica for A024 and A088 loci. A test for the Hardy-Weinberg equilibrium was performed for both populations at nine microsatellite loci. We detected significant deviation (P<0.001) from Hardy-Weinberg equilibrium at 16 out of 76 population combinations of loci. Almost all deviations were in favor of homozygosites, except at Ap226 in A. m. carnica and Ac011 in A. m. caucasica. The heterozygosity level within a subpopulation (F IS ), the heterozygosity level in total populations (F IT ) and the degree of genetic differentiation of subpopulations (F ST ) are presented in Table 4. All 9 loci illustrated an excess of heterozygosity in both populations. The mean F IT amounted to (from to 0.561). Mean F IS within populations (P<0.001) was (from to ). The fixation coefficients of subpopulations for the loci studied within the total populations, measured as an F ST value, varied from (Ap226) to (Ap243), with a mean of (Table 4). The mean gene flow (Nm) value was calculated as It was

3 calculated as for Ap243 and as for Ap226. Nei s genetic distance between the studied populations of A. m. carnica and A. m. caucasica was calculated as DISCUSSION Gene heterozygosity is a suitable parameter for investigating genetic variation. Ott (2001) gave a definition that a polymorphic locus must have a heterozygosity of at least In this aspect, all 9 microsatellite loci studied in our investigation had high polymorphism with a mean expected heterozygosity of for A. m. carnica and for A. m. caucasica, showing a high degree of genetic diversity and relative high selection potential. Microsatellite studies on honeybee populations have been generally carried out for European and African honeybee subspecies (Frank et al., 1998, 2001), whereas recent studies have been published for island populations and Mediterranean honeybee populations (Dall Olio et al., 2007; Franck et al., 2001; Bodur et al., 2007). According to these studies, the expected heterozygosity levels were highest among African honeybee populations between 0.76 and 0.90 (Franck et al., 2001) and lowest among western Mediterranean honeybees between 0.26 and 0.68 (Garnery et al., 1998; Franck et al., 2001). Additionally, the gene diversity for North Mediterranean honeybees varied from and (Dall Olio et al., 2007). Lebanon honeybees including Middle Eastern honeybee populations were studied using microsatellite loci and the gene diversity for these populations was estimated to be 0.65 (Franck et al., 2000). Bodur et al. (2007) found gene diversity values between 0.54 and 0.68, which was very similar to Middle Eastern populations. In our study, locus-based gene diversity values ranged between (Ap243) for A. m. caucasica and (A088) for A. m. carnica. Ap243 revealed the smaller value of for gene diversity in A. m. caucasica (O lineage) and higher value of in A. m. carnica (C lineage). At the same locus, Solignac et al. (2003) estimated a higher gene diversity for M lineage (0.56) and lower gene diversity for C lineage (0.07). We calculated the gene diversity for the A024 locus in A. m. carnica and A. m. caucasica as and 0.755, respectively. Solignac et al. (2003) estimated gene diversities of 0.29 and 0.61 for M and C lineages, respectively. The gene diversity for Ap256 was detected as and for A. m. carnica and A. m. caucasica bees, respectively, in our study. For the same locus, the gene diversity value was estimated at 0.61 for M lineage, 0.79 for C and 0.75 for A lineages (Solignac et al., 2003). The gene diversity estimations for the Ac011 locus were and in our study and reported as 0.80 for M lineage, 0.43 for C and 0.80 for A lineages by Solignac et al. (2003) and as by Chaline et al. (2002) in UK honeybee populations. Additionally, F ST values have been determined for lineages and different populations by many studies (Frank et al., 2000; 2001; Garnery et al., 1998; Dall Olio et al., 2007; Bodur et al. 2007). According to Hartl and Clark (2007), F ST levels between 0 and 0.05 indicate slight genetic differentiation; levels between 0.05 and 0.15 indicate moderate genetic differentiation; levels between 0.15 and 0.25 indicate high genetic differentiation and levels larger than 0.25 designate highly significant genetic differentiation. According to this information, the F ST values for most of the loci in our study ( ) demonstrated slight and moderate levels of heterozygosity, except for the Ap243 (0.247) locus where high genetic differentiation in both populations was established. Franck et al. (2000) revealed that the lineage pairwise F ST values for A and M lineages were smaller than 0.1, whereas C lineage pairwise values were higher than 0.1 levels. Furthermore, Franck et al. (2001) illustrated that pairwise F ST values for A lineage were between

4 0.01 and 0.12, for M lineage smaller than 0.1 and for C lineage between 0.17 and According to Garnery et al. (1998), among western European populations (Portugal, France, Spain, Sweden and Belgium), pairwise F ST was calculated between and (Garnery et al., 1998). Dall Olio et al. (2007) estimated the pairwise F ST value as and for A. m. ligustica clustered in C lineage and as and for the mellifera group. Bodur et al. (2007) estimated pairwise F ST values between 0.0 and for Turkish honeybee populations using nine different microsatellite loci. The gene flow (Nm) values provide information about the genetic divergence or genetic similarity of subpopulations due to gene flow. If the Nm value is smaller than 2, there is still considerable genetic differentiation among subpopulations. In our study, Nm was greater than 2 for most of the loci studied. This indicated small genetic differentiations among the populations. In this study, private alleles for both populations are described. As their frequencies were more than 5%, all 35 private alleles observed in A. m. carnica and 20 private alleles observed in A. m. caucasica, together with the most frequent alleles in the populations (Table 3), could be successfully used as suitable genetic markers. The results of this research provide new information concerning the genetic variability in A. m. carnica and A. m. caucasica selectively reared in Poland. Comparative data about genetic polymorphism in carnica and caucasica honeybees from Poland based on microsatellite DNA analysis are reported and discussed here for the first time. The results could be useful for selection and conservation purposes. Authors contributions: Stanimila R. Nikolova performed the microsatellite DNA work, Malgorzata Bienkowska contributed in organization of breeding work on honey bees of A. m. carnica and A. m. caucasica from Poland and done instrumental insemination, Dariusz Gerula contributed in the implementation of breeding programs, sampled the bees and contributed in the morphometrical analysis, Evgeniya N. Ivanova organized the experiment, worked on microsatellite analysis and prepared the article. Conflict of interest disclosure: The authors declare no conflict of interest. REFERENCES Bodur, C., Kence, M. and Kence, A. (2007). Genetic structure of honeybee, Apis mellifera L. (Hymenoptera:Apidae) populations of Turkey inferred from microsatellite analysis. J. Apic. Res. 46 (1), Chaline, N., Ratnieks, F. and Burke T. (2002). Anarchy in the UK: Detailed analysis of worker reproduction in a naturally-occurring British anarchistic honeybee, Apis mellifera, colony using microsatellite markers. Mol. Ecol. 11, Dall Olio, R., Marino, A., Lodesani, M., Moritz, RF. (2007). Genetic characterization of Italian honey bees, Apis mellifera ligustica, based on microsatellite DNA polymorphisms. Apidologie 38 (2): De La Rua, P., Jaffé, R., Dall Olıo, R., Muňoz, J. and Serrano J. (2009). Biodiversity, conservation and current threats to European honeybees. Apidologie (special issue) 40, Franck, P., Garnery, L., Loıseau, A., Oldroyd, B.P., Hepburn, H.R. and Solıgnac M. (2001). Genetic diversity of the honeybee in Africa: microsatellite and mitochondrial data. Heredity 86, Franck, P., Garnery, L., Solıgnac, M. and Cornuet, J. M. (1998). The origin of west European subspecies of honey bees (Apis mellifera): new insights from microsatellite and mitochondrial data. Evolution 52, Franck, P., Garnery, L., Solıgnac, M. and Cornuet J. M. (2000). Molecular confirmation of a fourth lineage in honeybees from the Near East. Apidologie 31, Garnery, L., Franck, P., Baudry, E., Vautrın, D., Cornuet, J.M. and Solıgnac M. (1998). Genetic diversity of the west European honey bee (Apis mellifera mellifera and A. m. iberica). I. Mitochondrial DNA. Genetics Selection Evolution 30, Gerula, D., Tofilski, A., Wegrzynowicz, P. and Skowronek, W. (2009). Computer-assisted discrimination of honeybee subspecies used for breeding in Poland. J. Apic. Sci. 53(2),

5 Gromisz, M. (1981). Morphological evaluation of colony population in breeding apiary. Pszczelnicze Zeszyty Naukowe 25: (In Polish with English summary) Gromisz, M. (1978). Morphological features of Caucasian honeybees imported to Poland in the years of Pszczelnicze Zeszyty Naukowe 22, (In Polish with English summary) Hartl, D. and Clark, A. (2007). Principles of population genetics. In: Inbreeding, population, subdivision and migration. (Eds. D. Hartl and A. Clark), Sinauer Associates Inc. Publishers, Sunderland, Massachussetts. Ivanova, E., Bouga, M., Staykova, T., Mladenovic, M., Rasic, S., Charistos, L., Hatjina, F. and Petrov, P. (2012). The genetic variability of honey bees from the Southern Balkan Peninsula, based on alloenzymic data. J. Apic. Res. 51 (4), Ivanova, E.N., Bienkowska, M. and Petrov, P. (2011). Allozyme polymorphism and phylogenetic relationships in Apis mellifera subspecies selectively reared in Poland and Bulgaria. Folia Biol-Krakow 59 (3-4), Meixner, M. D., Pinto, M. A., Bouga, M., Kryger, P., Ivanova, E. and Fuchs, S. (2013). Standard methods for characterising subspecies and ecotypes of Apis mellifera. J. Apic. Res. 52 (4)1-28. Meixner, M.D., Costa, C., Kryger, P., Hatjina, F., Bouga, M., Ivanova, E.N. and Büchler, R. (2010). Conserving diversity and vitality for honey bee breeding. J. Apic. Res. 49 (1), Nei, M. (1987). Molecular Evolutionary Genetics. In: DNA polymorphism within and between populations. (Eds. M. Nei), Columbia University Press, New York. Nikolova, S. (2011). Genetic variability of local Bulgarian honey bees Apis mellifera macedonica (rodopica) based on microsatellite DNA analysis. J. Apic. Sci. 55 (2), Ott, J. (2001). Analysis of Human Genetic Linkage (revised edition). In: Genetic loci and genetic polymorphism. (Eds. J. Ott), Johns Hopkins University Press, Baltimore, Maryland. Raymond, M. and Rousset, F. (1995). GENEPOP (Version-1.2) A population genetics software for exact tests and ecumenicism. J. Hered. 86 (3), Ruttner, F. (1988). Biogeography and Taxonomy of Honeybees. Springer-Verlag, 284 pp. Berlin Heidelberg, Germany. Ruttner, F. (1992). Naturgeschichte der Honigbienen. Ehrenwirth, 357 pp. Munich, Germany. Sheppard, W.S. and Meixner, M.D. (2003). Apis mellifera pomonella, a new honey bee subspecies from Central Asia. Apidologie 34, Sheppard, W.S., Arias, M.C., Meixner, M. and Grech, A. (1997). Apis mellifera ruttneri, a new honey bee subspecies from Malta. Apidologie 28, Solignac, M., Vautrin, D., Loiseau, A., Mougel, F., Baudry, E., Estoup, A., Garnery, L., Haberl, M. and Cornuet, J.M. (2003). Five hundred and fifty microsatellite markers for the study of the honeybee (Apis mellifera L.) genome. Mol. Ecol. Notes 3 (2), Tofilski, A. (2004). DrawWing, a program for numeral description of insect wings. J. Insect Sci. 4, Tofilski, A. (2008). Using geometric morphometrics and standard morphometry to discriminate three honeybee subspecies. Apidologie 39, Weir, B. S. and Cockerham, C. C. (1984). Estimating F-statistics for the analysis of population structure. Evolution 38,

6 Table 1. Characteristics of microsatellite loci used for STR analyses of A. m. carnica and A. m. caucasica populations. Name of the locus Am, unified nomenclature for A. mellifera microsatellite markers Am001 to Am552 Accession no. in EBI Size of the sequenced allele (bp) Motifs, repeats between primers Annealing temperature ( o C) Ac AJ (CT) A AJ (CT) A AJ (CT) A AJ (CT) 10 (GGA) Ap AJ (CT) Ap AJ (AT) 6 (GT) 3 (AT) 7 (GA) Ap AJ (TCC) Ap AJ (GA) 6 (GA) Ap AJ (GA) 12 AT(GA) MgCl 2 (mm)

7 Table 2. Number of alleles (Na), heterozygosity observed (Ho) and expected (He) per locus for A. m. carnica and A. m. caucasica populations. Locus Population Ac 011 A024 A043 A088 Ap226 Ap238 Ap243 Ap249 Ap256 A.m. carnica Na Ho He A.m. caucasica Na Ho He

8 Table 3. Allele frequencies at the microsatellite loci studied. Private allele frequencies are marked with (*). Locus Allele/n A.m.carnica A.m.caucasica Ac * * * * * * A * * * * * * * * A * * A * * * * * * * * * Ap * * * * * * Ap * * *

9 * * Ap * * * * * * Ap * * * * Ap * * * * * * * * *

10 Table 4. The heterozygosity level of individual loci within subpopulation (F IS ), in total populations (F IT ), the degree of genetic differentiation of subpopulations (F ST ) and gene flow value (Nm). Locus F IS F IT F ST Nm Ac A A A Ap Ap Ap Ap Ap Mean