Determining the genetic diversity of silkworm lines in Turkey

Studies involving molecular genetic characterization of silkworms are quite limited. Therefore, with this study, pure and hybrid silkworm lines raised in Turkey was characterized for the first time by microsatellite DNA markers. Samples were collected from 7 different silkworm lines, of which the pure lines were obtained from the Provincial Directorate of Agriculture and Forestry in Bursa and the hybrid ones from Silkworm Cocoon Association of Turkey (Kozabirlik). DNA samples were extracted from silkworm eggs. Genotyping was done by combining a total of 16 microsatellite markers in a single multiplex. Two hundred and eighty-one alleles were observed for 16 microsatellite loci. The highest allele number was observed for the T01CTA07R locus (30), while the highest effective allele number (17.61) and the highest polymorphic information content (0.94) were observed for the FL0612 locus. The expected heterozygosity values (He) were in the range of 0.72 (K02) to 0.95 (FL0612). The highest and lowest observed heterozygosity values (Ho) ranged between 0.52 and 1.00. When all lines were considered a single population, only three of the sixteen loci were in the HWE. As a result of this study, successful genotyping was performed with 16 microsatellite loci combined in a single multiplex using 3 different fluorescent dyes. The high polymorphism values obtained for the loci used showed that these loci can be used effectively in the genetic identification of silkworm breeds or lines. This study is expected to fill in the gaps in the related literature as well as to contribute to the conservation of silkworm local genetic resources.

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1. Duran K, Özdemir D, Namlıgöz SE. The enzymatic degumming of silk fibers. Tekstil ve Konfeksiyon 2007; 17 (3): 182-186 (article in Turkish with an English abstract).
2. Lie Y, Song W, Shi S, Liu Y, Pan M et al. Mitochondrial genome nucleotide substitution pattern between domesticated Silkmoth, Bombyx Mori, and its Wild Ancestors, Chinese Bombyx Mandarina and Japanese Bombyx Mandarina. Genetics and Molecular Biology Brazil 2010; 33 (1): 186-189.
3. Özgür, M. Türkiye’de ipekböcekçiliği. Ankara Üniversitesi Dil ve Tarih Coğrafya Fakültesi, Coğrafya Araştırmaları Dergisi 1996; 12: 95-106 (in Turkish).
4. Başkaya, Z. Development and distribution terms the place, distribution, problems and solutions of Bilecik district in silkworm breeding in Turkey. Doğu Coğrafya Dergisi 2013; 18 (30): 257-286 (article in Turkish with an English abstract).
5. Odabaş E, Günbey ÖB, Zengin Y, Sarıkaya AH. Journey of Silkworm in The World and Anatolia. Journal of Animal Science and Products (JASP) 2020; 3 (1): 75-84 (article in Turkish with an English abstract).
6. Yıldırım, MA. Sericulture education in the Ottoman Empire: The opening of Harır Dârüttalım And Dârülharırs. International Periodical For The Languages, Literature and History of Turkish or Turkic Volume 8/5 Spring 2013; p. 577- 594. (article in Turkish with an English abstract)
7. Tsenov P, Vasileva J, Arkova-Pantaleeva D. International testing of different silkworm hybrids in Bulgaria, II. technological characteristics. Journal of Animal Science 2008; 45 (1): 80-83.
8. Taşlıgil, N. From past to date sericulture in Bursa. Marmara Coğrafya Dergisi 1996; 706285: p. 237-246. (article in Turkish with an English abstract)
9. Top, T.B. Türkiye ipekböcekçiliğinde Kozabirliğin rolü. Tarımsal Ekonomi ve Politika Geliştirme Enstitüsü, Tepge Bakış Temmuz 2011 / ISSN: 1303-8346 / Nüsha: 13 (in Turkish).
10. Tautz D, Renz M. Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Research 1984; 12: 4127-4139. doi: 10.1093/nar/12.10.4127
11. Li YC, Korol AB, Fahima T, Beiles A, Nevo E. Microsatellites: genomic distribution, putative functions and mutational mechanisms: Molecular Ecology 2002; 11 (12): 2453-65. doi: 10.1046/j.1365-294x.2002.01643.x
12. Meglécz E, Anderson SJ, Bourguet D, Butcher R, Caldas A et al. Microsatellite flanking region similarities among different loci within insect species. Insect Molecular Biology 2007; 16(2): 175-185. doi: 10.1111/j.1365-2583.2006.00713.x
13. Weber JL, May PE. Abundant class of human DNA polymorphisms genomics. American Journal of Human Genetics 1989; 44 (3): 388-396.
14. Li M, Shen L, Xu A, Miao X, Hou C et al. Genetic diversity among Silkworm (Bombyx mori L., Lep., Bombycidae) germplasms revealed by microsatellites. Canadian Science Publishing Genome 2005; 48 (5): 802-810. doi: 10.1139/g05- 053
15. Prasad MD, Muthulakshmi M, Madhu M, Archak S, Mita K et al. Survey and analysis of microsatellites in the silkworm, Bombyx mori: frequency, distribution, mutations, marker potential and their conservation in heterologous species. Genome 2005; 169 (1): 197-214. doi: 10.1534/genetics.104.031005
16. Vijayan K, Nair CV, Urs SR. Assessment of Genetic diversity in the tropical mulberry silkworm (Bombyx mori L.) with mtDNA-SSCP and SSR markers. Emirates Journal of Food and Agriculture. 2010; 22 (1): 71-83. doi:10.9755/ejfa.v22i2.4895
17. Reddy KD, Abraham EG, Nagaraju J. Microsatellites in the silkworm, Bombyx Mori: abundance, polymorphism, and strain characterization. Genome 1999; 42 (6): 1057-1065. doi:10.1139/g99-027
18. Itoh M, Takeda S, Yamamoto H, Izumi S, Tomino S et al. Cloning and sequence analysis of membrane-bound alkaline phosphatase cDNA of the silkworm, Bombyx Mori. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 1991; 1129 (1): 135-138. doi:10.1016/0167-4781(91)90229-f
19. Fujiwara H, Ishikawa H. Structure of the Bombyx Mori rDNA: initiation site for its transcription. Nucleic Acids Research 1987; 15 (3): 1245-1258. doi: 10.1093/nar/15.3.1245
20. Cheng-Xiang H, Mu-Wang L, Yue-Hua Z, He-Ying Q, PingJiang S et al. Analysis of SSR fingerprints in introduced silkworm germplasm resources. Agricultural Sciences in China 2007; 6 (5): 620-627. doi:10.1016/S1671-2927(07)60091-1
21. Michaille JJ, Mathavan S, Gaillard J, Garel A. The complete sequence of mag, a new retrotransposon in Bombyx Mori. Nucleic Acids Research 1990; 18 (3): 674. doi: 10.1093/ nar/18.3.674
22. Zhang L, Huang YP, Miao XX, Qian M, Lu C. Microsatellite markers application on domesticated silkworm and wild silkworm. Insect Science 2005; 12 (6): 413-419. doi:10.1111/ j.1744-7917.2005.00052.x
23. Ohta T, Kobayashi M, Hirose S. Cloning of a cDNA for DNA Supercoiling factor reveals a distinctive Ca(2+) - binding protein. Journal of Biological Chemistry 1995; 30; 270 (26): 15571-15575. doi: 10.1074/jbc.270.26.15571
24. Weir BS, Cockerham CC. Estimating F-statistics for the analysis of population structure. Evolution 1984; 38 (6): 1358- 1370.
25. Wright S. Evolution in Mendelian populations. Genetics 1931; 16 (2): 97-159.
26. Peakall R, Smouse PE. GENALEX 6: genetic analysis in excel. Population Genetic Software for Teaching and Research, Molecular Ecology Notes. 2006; 6 (1): 288-295. doi:10.1111/ j.1471-8286.2005.01155.x
27. Peakall R, Smouse PE. GenAlEx 6.5: genetic analysis in excel. Population Genetic Software for Teaching and Research – an Update, Oxford Journals, Bioinformatics (Oxford, England) 2012; 28 (19): 2537-2539. doi: 10.1093/bioinformatics/bts460
28. Yeh FC, Yang RC, Boyle TBJ, Ye ZH, Mao JX. POPGENE: the user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Center Edmonton, AB, Canada, University of Alberta 1997; 8 (10): 295-301.
29. Kalinowski ST, Taper ML, Marshall TC. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology 2007; 16 (5): 1099-1106. doi: 10.1111/j.1365-294X.2007.03089
30. Nei M, Tajima F, Tateno Y. Accuracy of estimated phylogenetic trees from molecular data. Journal of Molecular Evolution 1983; 19: 153-170. doi: 10.1007/BF02300753
31. Goudet J. FSTAT (Version 2.9.3.). A program to estimate and test gene diversities and fixation indices. Lausanne, Switzerland: University of Lausanne. 2001.
32. Excoffier L, Lischer HEL. Arlequin Suite Ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Molecular Ecology Resources 2010; 10: 564-567. doi: 10.1111/j.1755-0998.2010.02847.x
33. Belkhir K, Borsa P, Goudet J, Chikhi L, Bonhomme F. GENETIX: logiciel sous WindowsTM pour la genetique des populations. Laboratoire Genome et Populations, CNRS UPR 9060, Universite Montpellier II, France 2001 (in French).
34. Falush D, Stephens M, Pritchard JK. Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 2003; 164 (4): 1567- 1587. doi: 10.1093/genetics/164.4.1567
35. Falush D, Stephens M, Pritchard JK. Inference of population structure using multilocus genotype data: dominant markers and null alleles. Molecular Ecology Notes 2007; 7: 574-578. doi: 10.1111/j.1471-8286.2007.01758.x
36. Hubisz MJ, Falush D, Stephens M, Pritchard JK. Inferring weak population structure with the assistance of sample group information. Molecular Ecology Resources 2009; 9 (5): 1322- 1332. doi: 10.1111/j.1755-0998.2009.02591.x
37. Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics 2000; 155 (2): 945-959. doi: 10.1093/genetics/155.2.945
38. Kopelman NM, Mayzel J, Jakobsson M, Rosenberg NA, Mayrose I. CLUMPAK: a program for identifying clustering modes and packaging population structure inferences across K. Molecular Ecology Resources 2015; 15 (5): 1179-1191. doi: 10.1111/1755-0998.12387
39. Evanno G, Regnaut S, Goudet J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology 2005; 14 (8): 2611-2620. doi: 10.1111/j.1365-294X.2005.02553.x
40. Earl DA, Von Holdt BM. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation Genetics Resources 2012; 4 (2): 359-361.
41. Buhroo ZI, Ganai NA, Malik MA, Kamili AS. DNA polymorphism and genetic variation among bivoltine silkworm (Bombyx mori L.) genotypes revealed by RAPD markers. Biotechnology Journal International 2017; 20 (4): 1-12. doi:10.9734/BJI/2017/35046
42. Hou CX, Li MW, Zhang YH, Qian HY, Sun PJ et al. Analysis of SSR fingerprints in introduced silkworm germplasm resources. Agricultural Sciences in China 2007; 6 (5): 620-627.
43. Kim YK, Kang PD, Kim MJ, Ryu KS, Park JS, Kim I. Microsatellite analysis of silkworm strains (Bombyx mori) of Japan origin preserved in Korea. International Journal of Industrial Entomology 2014; 28 (2): 39-50.
44. Kim KY, Kang PD, Lee KG, Oh HK, Kim MJ et al. Microsatellite analysis of the silkworm strains (Bombyx mori): high variability and potential markers for strain identification. Genes & Genomics 2010; 32: 532-543.
45. Kim KY, Kang PD, Ryu KS, Kim KH, Sung GB et al. Microsatellite analysis of the silkworm strains (Bombyx mori) originated from China. Korean Society of Sericultural Science 2012; 25 (1): 81-92.
46. Liu YQ, Qin L, Li YP, Wang H, Xia RX et al. Comparative genetic diversity and genetic structure of three Chinese Silkworm species Bombyx mori L. (Lepidoptera: Bombycidae), Antheraea pernyi Guérin-Meneville and Samia cynthia ricini Donovan (Lepidoptera: Saturniidae). Systematics, Morphology and Physiology, Neotropical Entomology 2010; 39 (6): 967- 976. doi:10.1590/S1519-566X2010000600019
47. Chakraborty S, Muthulakshmi M, Vardhini D, Jayaprakash P, Nagaraju J et al. Genetic analysis of Indian Tasar silkmoth (Antheraea mylitta) populations. Scientific Reports 2015; 5 (1): 1-14. doi:10.1038/srep15728