GENETICS CHARACTERS INTRAPEDIGRYS TYPES OF CARPATHIAN BREED BEES
Introduction. Today we observed of tendency to reduction of honey bee population in the world what according to honey gathering. For example, in Ukraine, according to statistical data for 7 inhabitants of Poltava region accounts for 1 bee familie, compared with that of 300 years ago, it reached within 3 bee families per citizen. Science and practice open many secrets according biology of bees, allowing bee efficient manage vital functions for humans. But despite the opening of beekeeping is the only farm animal that over 100 years has not been the intervention of human hands to create a new breeds of honeybees. Genetic intensify the search in the field of beekeeping conditions in Ukraine needs to initiate breeding program as planned waste zoning and aspects of reproduction of bees.
The aime of research: determining the characteristics of four intrapedigrees types in the Carpathian bee breed using the methods of population and molecular genetics.
Materials and Methods: Sampling was performed from top five lines: Sinevir, Rakhiv, Vuchkivskyy and Hoverla bee colonies were taken 10 bee worker. For molecular genetic analysis used 20 insects of each Carpathian bees breed type with observance of the principle of representativeness. DNA extraction performed from homogenate tissues using the standard commercial kit «DNA Sorb B», «Amplisense», this some modifications  during sample preparation. Reaction mix purification from bees wax leading this octane. The structure of the primers used for genotyping of bees and their code designations are: OPA-1(3’- CAG GCC CTT C -5’); OPA-4 (3’- AAT CGG GCT G -5’); B15 (3’- GGA GGG TGT T -5’); S1 (3’-AGC AGC AGC AGC AGC AGC C-5’). The program amplification of RAPD - primers: 1 cycle: 940 - 3min .; 2-35 cycle: 940 - 1 min., 360 - 30s., 720 - 1hv.36 cycle (final elongation): 720 - 10 min. The program amplification with primers S1: 1 cycle of 94 ° C - 4min 2 - 31 cycle: 57 ° C - 2 minutes; 72 ° C - 4min; 94 ° C - 1 minute, 32 cycle: 57 ° C - 3 minutes; 72 ° C - 7 minutes. Electrophoretic separation of amplified sections performed in 2% agarose gel in Tris borate buffer conditions. Size of amplification products control was carried out using molecular weight marker 1 kb - Ledder plus ( «Fermentas», Vilnius, Latvia). Processing of the profiles was performed in a standard computer program GELSTAT . Genetic distances were calculated in terms of genetic similarity indices obtained GELSTAT program as follows: Dxy = - lnI
Building a kladohramm performed according to the values of genetic distances TREE program and MEGA 4 [5; 6]. Statistical analysis of amplicon frequencies, heterozygosity, linage similarity, etc., performed by Fisher's algorithm .
Results and discussion. Molecular genetic studies on four primers made it possible to analyze 95 DNA fragments of different lengths, matching the same number of anonymous genetic loci of the genome of bees. Apply primer in RAPD - 15 made it possible to identify 18 amplification products in a range of molecular sizes from 410 to 1000 b. p. It noted that the DNA fragment size 410 b.p. 100% met all the members of Carpathian bees breed and described one genetic monomorfic locus. DNA size band 445 b. p. elektrophoregramme was found in only 20% of bees Vuchkivskiy type in the absence of individuals in other populations. Statistical comparisons (Fisher's criterion) frequency distribution of DNA fragments obtained with primer in -15 revealed a significant number of types of identification markers intrapedigrees of Carpathian bees. The largest number of DNA - fragments set for Vuchkivsksy type whose size is reached within the following limits: 1000, 630, 580 and 485b.p. DNA fragment of 1000 b.p. general was absent in bees Rakhiv type and Synevir, and its frequency in the population of Representatives Hoverla was 0.600 (p <0.001). As individuals, the members of such Synevir, observed no amplicon size 630 b. p. A fragment of a molecular weight of 710 b. p. observed with a frequency of 0.600 to 0.400 bees and type Rahiv, Goverla and 0,100 individuals in such Vuchkivskyy (p <0,05; p <0,01), respectively. Bees type Rahiv, Goverla and can be identified among other types of Carpathian breed presence significantly higher frequency amplification product whose size is 655, 515 b. p., 830 b. p, 530 b. p., respectively. Statistical analysis of the frequency distribution of the products of amplification bees four types derived from molecular genetic analysis of four primers in PCR was performed to identify the most characteristic identification of DNA fragments bees each of type . Based on these characteristics were constructed genetic formula intrapedigrys types of Carpathian breed. According genetic formulas the highest number of specific DNA - fragments characterized by bees for types of Sinevir and Vuchkivskyy, that the overwhelming number of such markers has been found Sinevir system ISSR-S1 (four amplicons) and for the type of system was Vuchkivskyy informative method of RAPD B-15 primer (six DNA fragments). Type Rahiv different from the others by the presence of six DNA markers, such as bees of Hoverla characterized only four specific genetic loci.
For the main parameters of population parameters highest level of genetic diversity characterized types Goverla and Rahiv because in terms of total heterozygosity significantly different from the type of bees from Sinevir and Vuchkivskyy values of 0.362 and 0.354, respectively (p <0,01; p <0,001). Moreover, the type of bees Hoverla observed the largest number of polymorphic loci - 54.9%, with a minimum of meaning in a population of individuals Sinevir type, and the lowest value of intrapedigrys similarity (number of DNA fragments in the same study group) were observed for a sample of bees type Rakhiv (0.665, p <0.001).
Determining the genetic distances between breeds in genealogical structure can be used as a methodical approach predicting the effectiveness of a combination of lines and types for heterosis effect on purebred descendants basis.The maximum value of genetic distance algorithm M. Ney was established between the types Synevir and Rakhiv (0.435), slightly less than this value typical of the combination VUChK - Synevir (0.426) and VUChK - Rakhiv (0.423). The smallest genetic distance calculated between individuals and types Hoverla ‒ Vuchkivskyy, which indicates their high genetic relationship and the undesirability of crossing the representatives of these types together. Application of unweighted pair-group clustering based on the calculated distances made it possible to analyze the nature of the genetic relationships between intrapedigrys types of Carpathian breed in graphic terms.
According dendrogram representatives Synevir types and Rahiv presented by individual branches, indicating their genetic identity. The Goverla and Vuchkivskyy type of bees and united in a common underklaster, due not only to the minimum calculated measure of genetic distance between data types, but confirmed the historical part of the establishment pedigree group Hoverla, based line mares are exactly the type Vuchkivskyy and lost insect genealogical group of Kolochavskiy type.
Conclusion Determining genetic specificity linage types of Carpathian breed bees allowed to obtain the following results: 1) chosen for the study of molecular genetic markers is sufficiently informative for determining the unique, specific features of each breed group and the identification of any sample Carpathian bees with the opportunity to consider linage certain type; 2) received genetic formula of Carpathian bees linage types are proof of the impact of breeding activities and can form the basis the protection of intellectual property of their authors; 3) used molecular genetic markers may serve as a forecasting tool optimal compatibility of linage types to obtain heterosis effect in their offspring.
The prospect of the research of this area is to select as a methodological tool for measuring genetic polymorphism Carpathian breed more accurate, reproducible and standardized markers, locus-specific micro satellite analysis, STR, research of structural genes single nucleotide polymorphism, SNP analysis, accumulation and formation the databases to assess the state of development, management and preservation unique of Carpathian bees gene pool.
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