Determination of DNA Cytosine Methylation Polymorphism Using RAPD Markers in Some Nigella sativa L. Genotypes

Öz

Epigenetics is defined as heritable changes in gene expression and function without DNA base changes. Methylation in DNA and chemical modification in histone proteins are the two most studied epigenetic mechanisms in plants. There exist low-throughput and high-throughput DNA methylation detection techniques in epigenetic research. One of the low-throughput techniques is an enzyme-based approach based on the presence or absence of DNA cytosine methylation. Using this approach, DNA cytosine methylation in random genes was investigated for Nigella sativa L. varieties with biological activities and therapeutic potential. This study, touch-down polymerase chain reactions methylation sensitiverandom amplified polymorphic DNA (TD-MS RAPD) technique was used to investigate cytosine methylation differences among genotypes of five different Nigella sativa L. species; Çameli, Eskişehir, Konya, Suriye and Şanlıurfa. Genomic DNA samples extracted from the seedlings of these genotypes were treated with MspI, a relative methylation-insensitive restriction enzyme and HpaII, a methylation-sensitive restriction enzyme before touch-down polymerase chain reactions. Among 8 oligonucleotide primers used, one primer (OPB-12) resulted in methylation polymorphisms among the five genotypes. The TD-MS-RAPD-PCR method is a simple and cost-effective method that requires basic equipment. This method can be easily implemented using a normal DNA thermal cycler and DNA gel electrophoresis system. However, the level of methylation polymorphisms detected with this method is very low in black cumin. It was concluded that there is a low level of polymorphism among five different black cumin genomes, which is thought to be due to the low 5’-CCGG-3’ contents of the black cumin genome.

Anahtar Kelimeler

• Black cumin
• Epigenetic
• MspI-HpaII

Bazı Nigella sativa L. Genotiplerinde RAPD Markırları Kullanılarak DNA Sitozin Metilasyon Polimorfizminin Belirlenmesi

Öz

Epigenetik, DNA baz değişimi olmaksızın gen ifadesi ve fonksiyonundaki kalıtsal değişimler olarak tanımlanmaktadır. DNA'da metilasyon ve histon proteinlerindeki kimyasal modifikasyonlar bitkilerde en çok çalışılan iki epigenetik mekanizmadır. Epigenetik araştırmalarda düşük işlem hacimli ve yüksek işlem hacimli DNA metilasyon saptama teknikleri vardır. Düşük işlem hacimli tekniklerden bir tanesi de enzim tabanlı DNA sitozin metilasyonunun varlığına ya da yokluğuna dayanan bir yaklaşımdır. Bu yaklaşım kullanılarak biyolojik aktiviteleri ve terapötik potansiyeli olan Nigella sativa L. genotipleri için rastgele genlerde DNA sitozin metilasyonu araştırılmıştır. Bu çalışmada, beş farklı Nigella sativa L. türüne ait genotipler (Çameli, Eskişehir, Konya, Suriye, Şanlıurfa) arasındaki sitozin metilasyon farklılıklarını araştırmak için touch-down polimeraz zincir reaksiyonları metilasyon duyarlı-rastgele arttırılmış polimorfik DNA (TD-MS RAPD) tekniği kullanılmıştır. Bu genotipler fidelerinden izole edilen genomik DNA örnekleri touch-down polimeraz zincir reaksiyonlarından önce metilasyona duyarsız olan MspI restriksiyon enzimi ve metilasyona duyarlı HpaII restriksiyon enzimi ile muamele edilmiştir. Kullanılan 8 oligonükleotid primerinden bir primer (OPB-12), beş genotip arasında metilasyon polimorfizmleri ile sonuçlanmıştır. TD-MS-RAPD-PZR yöntemi basit ve temel cihazları gerektiren uygun maliyetli yöntemdir. Bu yöntem normal bir DNA termal döngü cihazı ve DNA jel elektroforez sistemi kullanılarak kolayca uygulanabilmektedir. Ancak bu yöntemle saptanan metilasyon polimorfizmlerinin düzeyi çörek otunda çok düşüktür. Beş farklı çörek otu genomu arasında düşük düzeyde polimorfizm olduğu sonucuna varılmış olup bu durum çörek otu genomunun 5’-CCGG-3’ içeriklerinin düşük olmasından kaynaklandığı düşünülmektedir.

Anahtar Kelimeler

• Çörek otu
• Epigenetik
• MspI-HpaII

Kaynakça

1. Abel-Salam BK, 2012. Immunomodulatory Effects of Black Seeds and Garlic on Alloxan-İnduced Diabetes in Albino Rat. Allergol Immunopathol (Madr), 40 (6): 336-340.
2. Ahmad A, Husain A, Mujeeb M, Khan SA, Najmi A, Siddique NA, Damanhouri ZA, Anwar F, 2013. A Review on Therapeutic Potential of Nigella Sativa: A Miracle Herb. Asian Pacific Journal of Tropical Biomedicine, 5 (3): 337-352.
3. Aydin A, Ince AG, Uygur Gocer E, Karaca M, 2018. Single Cotton Seed DNA Extraction without the Use of Enzymes and Liquid Nitrogen. Fresenius Environmental Bulletin and Advances in Food Sciences, 27: 6722-6726.
4. Bestor TH, 2000. Chromatin Challenges During DNA Replication and Repair. Human Molecular. Genetics, 9: 2395-2402.
5. Bird A, 2002. DNA Methylation Patterns and Epigenetic Memory. Genes and Development, 16: 6-21.
6. Cokus SJ, Feng S, Zhang X, Chen,Z, Merriman B, Haudenschild CD, Pradhan S, Nelson SF, Pellegrini M, Jakopsen SE, 2008. Shotgun Bisulphite Sequencing of the Arabidopsis Genome Reveals DNA Methylation Patterning. Nature, 452, 215-219.
7. Ince AG, Karaca M, 2011a. Early Determination of Sex in Jojoba Plant by CAPS Assay. Journal of Agricultural Science and Technology, 149: 327-336.
8. Ince AG, Karaca M, 2011b. Genetic Variation in Common Bean Landraces Efficiently Revealed By Td-DAMD-PCR Markers. Plant Omics, 4: 220-227.

9. Ince AG, Karaca M, Aydin A, Elmasulu SY, Turgut K, 2015.Microsatellites for Genetic and Taxonomic Research on Thyme (Thymus L.). Turkish Journal of Biology, 39: 147-159.
10. Ince AG, Karaca M, Onus AN, 2010a. Genetic Relationships within and Between Capsicum Species. Biochemical Genetics, 48: 83-95.
11. Ince AG, Karaca M, Onus AN, 2010b. Differential Expression Patterns of Genes Containing Microsatellites in Capsicum Annuum L. Molecular Breeding, 25: 645-658.
12. Karaca M, Ince AG, 2008. Minisatellites as DNA markers to classify bermudagrasses (Cynodon spp.): Confirmation of minisatellite in amplified products. Journal of Genetics 87: 84-86.
13. Karaca M, Ince AG, 2018. Primer Pairs for Rice (Oryza Sativa L.) Bisulfite Sequencing Studies. Journal Plant Science Phytopathol, 2: 091-098.
14. Karaca M, Ince AG, Aydin A, Ay ST, 2013. Cross-Genera Transferable E-Microsatellite Markers For 12 Genera of the Lamiaceae Family. Journal of the Science of Food and Agriculture, 93: 1869-1879.
15. Karaca M, Ince AG, Elmasulu SY, Onus AN, Turgut K, 2005. Coisolation of Genomic and Organelle DNAs from 15 Genera and 31 Species of Plants. Analytical Biochemistry, 34: 353-355.
16. Karaca M, Ince AG, Gocer EU, Aydin A, 2016. Exonic and İntronic DNA Methylation Differences in A Fiber Specific Gene of Pima Cotton (Gossypium Barbadense L.). Journal Science Engineering Research, 3: 478-486.
17. Osabe K, Clement JD, Bedon F, Pettolino FA, Ziolkowski L, Llewellyn D, Jean Finnegan EJ, Wilson LW, 2014. Genetic and DNA Methylation Changes in Cotton (Gossypium) Genotypes And Tissues. PLoS ONE 9: e86049.
18. Salmon A, Clotault J, Jenczewski E, Chable V, Manzanares-Dauleux, MJ, 2008.Brassica oleracea Displays a High Level of DNA Methylation Polymorphism. Plant Science, 174, 61-70.
19. Tchurikov NA, 2005. Molecular Mechanisms of Epigenetics. Biochemistry, 70: 406-423.
20. Tiwari JK, Saurabh S, Chandel P, Singh BP, Bhardwaj V, 2015.Assessment of Genetic And Epigenetic Variations in Potato Somatic Hybrids By Methylation-Sensitive ISSR and RAPD Markers. Bangladesh Journa Botany, 44: 45-50.
21. Wang B, Zhang M, Fu R, Qian X, Rong P, Zhang Y, Jiang P, Wang J, Lu X, Wang D, Ye W, Zhu X, 2016. Epigenetic Mechanisms of Salt Tolerance and Heterosis in Upland Cotton (Gossypium Hirsutuml.) Revealed by Methylation-Sensitive Amplified Polymorphism Analysis. Euphytica, 208: 477-491.
22. Welsh J, McCelland M, 1990. Fingerprinting Genomes Using PCR with Arbitrary Primers. Nucleic Acids Research, 18: 7213-7218.
23. Williams JGK, Kubelik ARK, Livak J, Rafalski JA, Tingey SV, 1990. DNA Polymorphism Amplified by Arbitrary Primers is Useful as Genetic Markers, Nucleic Acid Res.earch, 18: 6531-6535.
24. Wu CT, Morris JR, 2001. Genes, Genetics and Epigenetics: A Correspondence. Science, 293: 1103-1105.