RT-PCR Analysis of Caucasian and Mugla Honey Bees by SNP Markers of Chalkbrood Disease

Öz

Two different honey bee subspecies’ genotypes obtained from Ordu, Apiculture Research Institute were analyzed by SNP markers using Real-Time PCR-HRM. Genomic DNA samples analysed with 10 SNP primers those were used for identification of chalkbrood disease resistance genes and two SNP primers those were obtained from honey bee genom sequencing. Result of SNP analyses, four primers (AMB-00858574, AMB-01151447, AMB00631190, AMB-00686140) amplified in Caucasian honey bee and six (AMB-00858574, AMB-00612262, AMB-01151447, AMB-00631190, AMB-00674355, AMB-00686140) primers amplified in Mugla honey bee. Four amplicons are similar for this subspecies. Result of electrophoresis analyses, five primers (AMB00858574, AMB-00612262, AMB-01151447, AMB-00631190, AMB-00686140) form a band in Caucasian honey bee, seven amplicons (AMB-00858574, AMB00612262, AMB-01151447, AMB-00631190, AMB-00902548, AMB-00674355, AMB-00686140) form a band in Mugla honey bee and four amplicons (AMB00858574, AMB-01151447, AMB-00631190, AMB-00686140) similar for this subspecies. As a result of this study, in Caucasian and Muğla honeybee for identification of disease resistance and evaluability as a identification key for this subspecies was emerged the capacity of association of single nucleotide polymorphisms to resistance to chalkbrood in two important honeybee genotypes in country of Apis mellifera using RT-PCR for HRM analysis.

Anahtar Kelimeler

• Honey Bee
• SNP
• Mugla bee
• Caucasian bee
• Real-Time PCR
• HRM
• chalkbrood disease

Kafkas ve Muğla Bal Arılarının Tebeşir Hastalığının SNP Belirteçleri ile RT-PCR Analizi

Öz

Ordu, Arıcılık Araştırma Enstitüsü'nden sağlanan iki farklı bal arısı alt türüne ait genotipler Real-Time PCR-HRM kullanılarak SNP markörleri ile analiz edilmiştir. Kireç hastalığı direnç genlerinin tanımlanmasında kullanılan 10 SNP primeri ve bal arısı genom dizilemesinden elde edilen iki SNP primeri ile analiz edilen genomik DNA örneklerinde; Kafkas bal arısında amplifiye edilmiş dört primer (AMB-00858574, AMB-01151447, AMB 00631190, AMB-00686140) ve altı (AMB-00858574, AMB-00612262, AMB-01151447, AMB-00631190, AMB-00674355, AMB-00686140) Muğla bal arısında amplifiye edilmiştir. Bu alt tür için dört amplikon benzer olup elektroforez analizleri sonucunda, Kafkas bal arısında beş primer (AMB 00858574, AMB-00612262, AMB-01151447, AMB-00631190, AMB-00686140), yedi amplikon (AMB-00858574, AMB 00612262, AMB-01151447, AMB -00631190, AMB-00902548, AMB-00674355, AMB-00686140) Muğla bal arısında dört amplikon (AMB 00858574, AMB-01151447, AMB-00631190, AMB-00686140) bir bant oluşturmuştur. Bu çalışma sonucunda, Kafkas ve Muğla bal arısı genotiplerinde hastalık direncinin belirlenmesi ve bu alt tür için bir tanımlama anahtarı olarak değerlendirilebileceği, HRM analizi için RT-PCR kullanarak Apis mellifera'nın önemli iki bal arısı genotipinde tek nükleotid polimorfizmlerinin kireç direnci ile ilişkilendirme kapasitesi ortaya çıkarılmıştır.

Anahtar Kelimeler

• Bal arısı
• Mugla arısı
• Kafkas arısı
• SNP
• RT-PCR
• HRM
• kireç hastalığı

Kaynakça

1. Aronstein, K., Colby, D., & Holloway., B. (2015).Validation of genetic markers associated with chalkbrood resistance. Trends Entomology, 11, 47-53.
2. Chapman, N. C., Harpur, B. A., Lim, J., Rinderer, T. E., Allsopp, M. H., Zayed, A., & Oldroyd, B. P. (2015). A SNP test to identify Africanized honeybees via proportion of african ancestry. Molecular Ecology Resources, 15, 1346-1355. https://doi.org/10.1111/1755-0998.12411.
3. Chavez-galarza, J., Henriques, D., Johnstone, J. S., Azevedo, J. C., Patton, J. C., Muñoz, I., De la rúa, P., & Pinto, M. A. (2013). Signatures of selection in the Iberian honey bee (Apis mellifera iberiensis) revealed by a genome scan analysis of single nucleotide polymorphisms (SNPs). Molecular Ecology, 22, 5890-5907. https://doi.org/10.1111/mec.12537.
4. Donaldson, M. E., Christina M. Davy, Craig K. R. Willis, Scott McBurney, Allysia Park, Christopher J. Kyle. (2017) Profling the immunome of little brown myotis provides a yardstick for measuring the genetic response to white-nose syndrome. Evol. Appl. 10, 1076–1090.
5. Elbers, J. P., Brown, M. B. & Taylor, S. S. (2018) Identifying genome-wide immune gene variation underlying infectious disease in wildlife populations: A next generation sequencing approach in the gopher tortoise. BMC Genom. 19, 64.
6. Gerdts, J. R., Roberts, J. M. K., Simone-Finstrom, M., Ogbourne, S. M, & Tucci, J. (2021). Genetic variation of Ascosphaera apis and colony attributes do not explain chalkbrood disease outbreaks in Australian honey bees. Journal of Invertebra Pathology. 180, 107540. https://doi.org/10.1016/j.jip.2021.107540.
7. Gupta, P., Conrad, T., Spötter, A., Reinsch, N., & Bienefeld, K. (2012). Simulating a base population in the honey bee for molecular genetic studies. Genetics Selection Evolution, 44(14). https://doi.org/10.1186/1297-9686-44-14 Han, F. (2012). Genome wide analysis of genetic variation in honeybee, Apis mellifera [Degree project]. Biology Education Centre and Department of Medical Bio chemistry and Microbiology Uppsala University, Uppsala.
8. Han, F., Wallberg, A., & Webster, M. T. (2012). From where did the Western honeybee (Apis mellifera) originate?. Ecology and Evolution, 2(8), 1949-1957. https://doi.org/10.1002%2Fece3.312.

9. Henriques, D., Jara , L., Chávez-Galarza, J., Rufino Amaro, J., De la Rúa, P., & Pinto, M. A. (2013). Introgression levels of the Italian and carniolan honey bee subspecies into the black honey bee: a comparison between microsatellite and single nucleotide polymorphism (SNP) markers. Instituto Politécnico de Bragança Biblioteka Digital.
10. Henriques D, Lopes AR, Chejanovsky N, Dalmon A, Higes M, Jabal-Uriel C, Le Conte Y, Reyes-Carreño M, Soroker V, Martín-Hernández R, Pinto MA. (2021) A SNP assay for assessing diversity in immune genes in the honey bee (Apis mellifera L.). Sci Rep. 28;11(1):15317. doi: 10.1038/s41598-021-94833-x. PMID: 34321557; PMCID: PMC8319136.
11. Holloway, B., Tarver, M. R., & Rinderer, T. (2013). Fine mapping identifies significantly associating markers for resistance to the honey bee brood fungal disease, Chalkbrood. Journal of Apicultural Research 52(3), 134-140. https://doi.org/10.3896/IBRA.1.52.3.04.
12. Holloway, B., Sylvester, H. A., Bourgeois, L., & Rinderer, T. (2015). Association of single nucleotide polymorphisms to resistance to chalkbrood in Apis mellifera. Journal of Apicultural Research, 51(2), 154-163. https://doi.org/10.3896/IBRA.1.51.2.02.
13. Holloway, Beth, Sylvester, H, Bourgeois, L., Rinderer, T. (2012). Association of single nucleotide polymorphisms to resistance to chalkbrood in Apis mellifera. Journal of Apicultural Research, 51, 154-163. https://doi.org/10.3896/IBRA.1.51.2.02.
14. Iglesias, M. S., & Grzelczak, M. (2020). Using gold nanoparticles to detect single-nucleotide polymorphisms: toward liquid biopsy. Beilstein Journal of Nanotechnology. 11, 263-284. https://doi.org/10.3762%2Fbjnano.11.20 Johnson, L. S. (2011). Apis mellifera (Honey Bee) a teacher’s companion [MEd Prodject]. Antioch University New England, Antioch.
15. Komar, A. A. (2009). Single Nucleotide Polymorphisms; Methods in Molecular Biology; Humana Press: New York City, NY, U.S.A. https://doi.org/10.1007/978-1-60327-411-1.
16. Kongchum, P., Palti, Y., Hallerman, E. M., Hulata, G. & David, L. (2010) SNP discovery and development of genetic markers for mapping innate immune response genes in common carp (Cyprinus carpio). Fish Shellfsh Immunol. 29, 356–361.
17. Kosch, T.A., C. N. S. Silva, L. A. Brannelly, A. A. Roberts, Q. Lau, G. Marantelli, L. Berger, L. F. Skerratt. (2019) Genetic potential for disease resistance in critically endangered amphibians decimated by chytridiomycosis. Anim. Conserv. 22, 238–250.
18. Kwak, M. M., Velterop, O., & Boerigter, E. J. M. (1996). Insect diversity and the pollination of rare plant species. In: A. Matheson, S. L. Buchmann, C. O’Toole, P. Westrich, & I. H. Williams (eds), The conservation of bees. Academic Press.
19. Liu, Yuanzhen, Yan, L. Li, Zhiguo, Huang, Wei-Fone, Liu, X, Su, Songkun. (2016). Larva-mediated chalkbrood resistance-associated single nucleotide polymorphism markers in the honey bee Apis mellifera. Insect molecular biology. 25. 10.1111/imb.12216.
20. Pinto, M. A., Henriques, D., Chávez-Galarza, J., Kryger, P., Garnery, L., van der Zee, R., Dahlee, B., Soland-Reckeweg, G., de la Rúa, P., Dall' Olio, R., Carreckj, N. L., & Johnston, J. S. (2014). Genetic integrity of the dark european honeybee (Apis mellifera mellifera) from protected populations: a genomewide assessment using SNPsand mtDNA sequence data, Journal of Apicultural Research, 53(2), 269-27. https://doi.org/10.3896/IBRA.1.53.2.08.
21. Rehner, Steve & Evans, Jay. (2009). Microsatellite loci for the fungus Ascosphaera apis: Cause of honey bee chalkbrood disease. Molecular Ecology Resources. 9. 10.1111/j.1755-0998.2008.02455.x.
22. Schork, N. J., Fallin, D., & Lanchbury., S. (2000). Single nucleotide polymorphisms and the future of genetic epidemiology, Clinical Genetics, 58, 250-264. https://doi.org/10.1034/j.1399-0004.2000.580402.x
23. Seçgin, Z. (2015). Domates genotiplerinde farklı besi ortamlarında kallus gelişimi ve genetik farklılığın RAPD markörleri ile belirlenmesi, [Yüksek Lisans Tezi], Ondokuz Mayıs Üniversitesi, Fen Bilimleri Enstitüsü, Samsun. https://tez.yok.gov.tr/UlusalTezMerkezi/.
24. Shi, Y. Y., Sun, L. X., Huang, Z. Y., Wu, X. B., Zhu, Y. Q., Zheng, H. J., & Zeng, Z. J. (2013). A SNP based high-density linkage map of Apis cerana reveals a high recombination rate similar to Apis mellifera. PLoS ONE, 8(10), e76459. https://doi.org/10.1371/journal.pone.0076459.
25. Tunca, I. R. (2009). Determination and comparison of genetic variation in honeybee (Apis mellifera l.) populations of turkey by random amplified polymorphic dna and microsatellite analyses, [Doktora Tezi], Orta Doğu Teknik Üniversitesi, Fen Bilimleri Enstitüsü, ANKARA. https://tez.yok.gov.tr/UlusalTezMerkezi/.
26. Wallberg, A., Wellhagen, G., Dahle, B., Kawata, M., Haddad, N., Simões, Z. L. P., Allsopp, M. H., Kandemir, I., De la Rúa, P., Pirk, C. W., & Webster, T. W. (2014). A worldwide survey of genome sequence variation provides insight into the evolutionary history of the honeybee Apis mellifera. Nature Genetics, 46, 1081-1088. https://doi.org/10.138/ng.3077.