Introduction The honey bee Apis mellifera L. has been genetically subdivided in 26 subspecies in long time evolution (Ilyasov, Poskryakov 2006). This level of genetic divergence does not provide isolation between subspecies. Hybridisation of honey bee subspecies occurred throughout Europe with transportation between countries of different subspecies of honey bees due to spreading of hybrid origin population. Successful breeding of honey bees is possible using clean-breeding lines. Beekeeping may be successful only when one breed of honey bees is kept. Dark European bees Apis mellifera mellifera L. are preferred for beekeeping in North Eurasia countries. This honey bee subspecies is belonging to the evolutionary branch M which adapted to live in a continental climate with long cold winter (Ilyasov et al. 2007). Isolated populations of A.m.mellifera remain in small islets in Russia, Switzerland, Denmark, Sweden, Norway, France and Spain (Nikonorov et al. 1998, Jensen et al. 2005). Only genetic control of introduced honey bees in this areas can to provide safety its gene-pool (Nikolenko, Poskryakov 2002). The most popular genetic markers for monitoring the gene pool of honey bees are the gene ND2 and intergenic locus COI-COII of mitochondrial DNA, and many microsatellite loci and single nucleotide polymorphism loci (SNP) of nuclear DNA (Garnery et al. 1992; Nikolenko, Poskryakov 2002; Whitfield et al. 2006; Whitfield et al. 2006; Pinto et al. 2014). Our research based on investigation the nucleotide polymorphism of gene vitellogenin (Vg) of honey bees. The vitellogenin is 180 kDa phospholipoglycoprotein and a major precursor of egg yolk (Chen et al. 1997; Sappington, Raikhel 1998; Tufail, Takeda 1998). Honey bees have only one copy of gene Vg whereas other insects have more (Kent et al. 2011). Nucleotide sequences of six from the seven exons (from 2 to 7) were deposited in GenBank (http://www.ncbi.nlm.nih.gov
) (Fig. 1). Fig. 1. Localisation of the gene vitellogenin of the honey bee on the fourth chromosome and its exon-intron structure.\ We sequenced six exons of the gene vitellogenin of honey bees of A.m.mellifera of evolutionary branch M from twelve isolates from Ural region of Russia. Comparative analysis of sequences of Vg of northern bees from Ural (evolutionary branches M) with southern bees from Caucasus (evolutionary branches C) allowed us to discover 26 single nucleotide differences between them. Comparative analysis of sequences of full length DNA of Vg of honey bees of three evolutionary branches A, M and C allowed clustering into three major population groups of A. mellifera (Whitfield et al. 2006) from Africa (the branch A), from Mediterranean (the branch C) and from Western and Northern Europe (the branch M). Applying single nucleotide differences in exons of gene Vg can to help to monitor and solve the problem of intraspecies hybridisation of honey bees. Materials and methods We collected the worker bees of A.m.mellifera from twelve isolates in Ural region of Russia from Bashkortostan Republic (Kagarmanovo, Kaga, Sermenevo, Galiakberovo, Yaumbaevo, Irgizly, Kustarevka, Sabanchi, Uyadybash) and from Permskii krai (Nytva, Porshakova 1, Porshakova 2) (Fig. 2). Fig. 2. Geographic distribution isolates of honey bees in Ural region in Russia. DNA was extracted from thoracic tissue using “DNA extran 2” kit (Syntol) (http://www.syntol.ru
). PCR was conducted in 15 μL reactions containing 1 U Taq DNA polymerase (Syntol) 2-4 mM of MgSO4, 200 μM of each dNTP, 0.5 μM of each primer (Kent et al. 2011) and 20-100 ng of DNA (Tab. 1). PCR amplification for the exones 2-7 of the gene Vg was performed as follows: 30 cycles at 1 min at 94 °C, 1 min at 55 °C and 2 min at 72 °C. PCR purification and Sanger sequencing on an automatic sequencer using both forward and reverse primers carried out by Syntol. All polymorphic sites were manually checked for errors. All sequences were aligned using MEGA 4.1 using the published Apis mellifera genome as a reference (LG4, NC_007073.3, AADG06005159.1) (http://hymenopteragenome.org/beebase
). The single nucleotide polymorphisms (SNPs) analysed herein were discovered from the alignments as allelic differences between A. mellifera subspecies. Table 1. Primers for PCR of vitellogenin exons of honey bee Apis mellifera (Kent et al. 2011) Vg exon 2 F 5'-tcttgttcgttccaggttcc-3' R 5'-gacagtttcagccgacttcc-3' Vg exon3 F 5'-cctttcgatccattccttga-3' R 5'-gtcaaaacggattggtgctt-3' Vg exon 4 F 5'-tcgaaggggaagaatttcaa-3' R 5'-acgagcaattcctcaacacc-3' Vg exon 5 F 5'-gtcggacaatttcacgtcct-3' R 5'-gttcgagcatcgacacttca-3' Vg exon 6 F 5'-agagccagggatacgtcaaa-3' R 5'-gagtcatctcgaggctcacc-3' Vg exon 7 F 5'-ttctggctgaggtcaggatt-3' R 5'-aatttcgaccacgactcgac-3' Results Nucleotide sequences of the six exons (from 2 to 7) of the gene Vg of honey bees from the Ural region have been deposited in the GenBank: second exon (KJ572309 - KJ572320), third exon (KJ645883 - KJ645894), fourth exon (KJ572297 - KJ572308), fifth exon (KJ572285 - KJ572296), sixth exon (KJ532136 - KJ532147), seventh exon (KJ532124 - KJ532135). All analysed honey bees were homozygous for the gene Vg. Comparative analysis of sequences of Vg of honey bees from Ural with reference sequence from GenBank was allowed us to found 18 transitions (90%) and 2 transversions (10%). In the second exon of Vg we are found 2 transitions, in the third exon - 3 transitions, in the fourth exon - 2 transitions, in the fifth exon - 4 transitions and 2 transversions, in the sixth exon - 1 transition and in the seventh exon - 6 transitions. All transversions of gene Vg were led to amino acid substitution from Ley to Arg and Ile to Ser. One of the 18 transitions was led to amino acid substitution from Ala to Thr. The deletion of 9 nucleotide long was found in honey bee from isolate Uyadybash. Same deletion was occurred in bees from GenBank JN557265, JN557266, JN557273, JN557274, JN557275, JN557276 (Tab. 2). Table 2. Sites of nucleotide substitution of the gene Vg of honey bees from Ural region relative to the reference sequence from GenBank Gene Vg Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Polymorphic sites Samples 526 574 1373 1418 1793 2443 2458 3978 4528 4533 4554 4555 4800 4812 5229 5608 5677 5680 5692 5878 5935 Reference sequence Vg GenBank A A T A T T T C C G A T G A G T T C C T T RB, Beloretskii, Kagarmanovo G G C A T С С C C G A T A A G T T T T T T RB, Beloretskii, Kaga G G C A T T T C C G A T G A A*** - C T T T C RB, Beloretskii, Sermenevo G G C A T С T C C G A T G G A*** C C T T T C RB, Burzyanskii, Galiakberovo G G C G T T T T A* G A T A A A*** C C T T C T RB, Burzyanskii, Yaumbaevo G G C A C С С C A* C** G C G A A*** C C T T C T RB, Burzyanskii, Irgizly G G C A T T T C C G A T A A G C C T T T C RB, Tatyshlinskii, Kustarevka A A C A T T T C C G A T G A A*** C C T T C T RB, Tatyshlinskii, Sabanchi G G C A T T T C C G A T G G A*** C T C C T C RB, Tatyshlinskii, Uyadybash G G C A T T T C C G A T A G A*** C C T T T C PK,Krasnovisherskii, Porshakova 1 A G C A T T T C C G A T G A A*** C C T T T T PK,Krasnovisherskii, Nytva G G C A T T T C A* G A T G A A*** C C T T C T PK,Krasnovisherskii, Porshakova 2 G G C A T С С C A* C** G C G A A*** C C T T C T RB - Bashkortostan republic; PK - Permskii krai. * - substitution of Ley to Ile; ** - substitution of Arg to Ser; *** - substitution of Ala to Thr. Symbol «-» no data gaps. Table 3 The single nucleotide differences in the nucleotide sequence of the gene Vg of honey bees between subspecies of evolutionary branches M and C Gene Vg Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Position Branch 964 997 1039 1415 1460 1901 1970 1976 2788 2887 2888 2920 2938 3981 4242 4288 4316 4500 4508 4509 5114 5210 5225 5306 5321 5328 М T C C T C C G C T A A C T T T C A G G G C C C G T A С C T T C T T A T C T C T C C A T G A A A T T T A A G Fig. 3. Dendrogram of genetic relationship of honey bees of tree evolution branches based on comparative analysis of sequences of gene Vg. Discussion Comparative analysis of sequences of Vg of honey bees belonging to two evolutionary branches M and C found 26 single nucleotide differences (SNP) between them (Tab. 3). These 26 SNPs can be used to recognize A.m.mellifera from other honey bee subspecies of the evolutionary branch C. In the second exon of Vg there were 3 SNPs, in the third exon - 5 SNPs, in the fourth exon - 5 SNPs, in the fifth exon - 7 SNPs, in the sixth exon - 6 SNPs, in the seventh exon - none were found. Comparative analysis of sequences from full length DNA of Vg of honey bees in evolutionary branches A, M and C had allowed us to construct the dendrogram. On the dendrogram honey bee isolates were differentiated into three major population groups of A. mellifera (Whitfield et al. 2006) from Africa (the branch A), from Mediterranean (the branch C) and from Western and Northern Europe (the branch M). The honey bee samples from Ural, Spain and Poland were clustered together and had belonged to the evolutionary branch M. The honey bee samples from Egypt, Germany, Croatia and Slovenia were clustered together under evolutionary branch C. Bees from South Africa were clustered separately from all groups under evolutionary branch A (Fig. 3). Only two samples of the honey bees from South Africa were grouped with honey bees of the evolutionary branch C that can be explained by hybrid origin of these samples. Comparative analysis of the sequence of gene Vg of honey bees from the Ural has allowed us to cluster different honey bee subspecies into three groups similar to the earlier works (Ruttner 1988; Whitfield et al. 2006). This has also allowed us to find 26 SNP which can be used to differentiate A.m.mellifera and to help solve the problem of the intraspecific hybridization of the honey bees in the Russia and North European countries.