ORIGINAL ARTICLE
Optimization of DNA extraction methods for genomic analysis of rice root-knot nematode (Meloidogyne graminicola) using PCR (polymerase chain reaction) and sanger sequencing
| 1 | Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, Indonesia |
A - Research concept and design; B - Collection and/or assembly of data; C - Data analysis and interpretation; D - Writing the article; E - Critical revision of the article; F - Final approval of article
CORRESPONDING AUTHOR
Siwi Indarti
Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, Indonesia
Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, Indonesia
Submission date: 2022-08-26
Acceptance date: 2022-10-17
Online publication date: 2023-01-09
Journal of Plant Protection Research 2023;63(1):50–58
HIGHLIGHTS
- One nematode can be used for DNA extraction by freezing treatment before extraction.
- The commercial kit method is recommended for quick results of nematode DNA extraction.
- CTAB and SDS methods are recommended if the time is available and costs are limited.
KEYWORDS
TOPICS
ABSTRACT
The root-knot nematode Meloidogyne graminicola is an economically important pest in rice
production. The identification of a nematode species is an important basis in nematode
management to reduce yield losses by extracting nematode DNA as an early step in molecular
identification. This study aimed to investigate the optimal extraction method and
number of M. graminicola for nematode genomic analysis based on PCR (polymerase chain
reaction) and Sanger sequencing. The DNA extraction methods used in this study were the
CTAB, SDS, and commercial kit (GeneAidTM Tissue/Blood DNA Mini Kit). The results
revealed that the three DNA extraction methods could be used to analyze the nematode genomics
based on PCR and Sanger sequencing using one nematode, both in a second-stage
juvenile and a female, equipped with the process of nematode destruction by freezing. This
finding was shown by the amplification of all DNA templates with Mg-F3 and Mg-R2 primers
through PCR with a size of 370 bp, while Sanger sequencing obtained 372 bp.
ACKNOWLEDGEMENTS
The authors would like to thank Gadjah Mada University
for research funded by RTA (Rekognisi Tugas
Akhir-Student Final Project Recognition) with Contract
No. 3550/UN1.P.III/Dit-Lit/PT.01.05/2022.
RESPONSIBLE EDITOR
Renata Dobosz
CONFLICT OF INTEREST
The authors have declared that no conflict of interests exist.
REFERENCES (35)
1.
Aboul-Ftooh N., Maaty A., Oraby H.A.S. 2019. Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bulletin of the National Research Centre 43: 25. DOI: https://doi.org/10.1186/s42269....
2.
Alaey M., Naderi R., Verzaei A., Khalighi A., Salami A. 2005. Comparing study betweenfour different methods of genomic DNA extraction from Cyclamen persicum Mill. International Journal of Agriculture and Biology 7 (6): 882–884.
3.
Amani J., Kazemi R., Abbasi A.R., Salmanian A.H. 2011. A simple and rapid leaf genomic DNA extraction method for polymerase chain reaction analysis. Iranian Journal of Biotechnology 9 (1): 69–71.
4.
Braun-Kiewnick A., Kiewnick S. 2018. Real-time PCR: A great tool for fast identification, sensitive detection and quantification of important plant-parasitic nematodes. European Journal of Plant Pathology 152: 271–283. DOI: https://doi.org/10.1007/s10658....
5.
Chen H., Rangasamy M., Tan S.Y., Wang H., Siegfried B.D. 2010. Evaluation of five methods for total DNA extraction from Western corn rootworm beetles. PLoS ONE, 5: e11963. DOI: https://doi.org/10.1371/journa....
6.
Enroth C.H., Fehler A.O., Poulsen L.D., Vinther J. 2019. Excess primer degradation by Exo I improves the preparation of 3′ cDNA ligation-based sequencing libraries. BioTechniques 67: 110–116. DOI: https://doi.org/10.2144/btn-20....
7.
Eurofins Genomic. 2019. DNA sequencing results guide. Eurofins Genomics. Anzinger Str. 7a I 85560 Ebersberg I Germany.
8.
Farrell R.E. 2010. RNA Methodologies: A Laboratory Guide for Isolation and Characterization. 4th edition. Academic Press is an imprint of Elsevier, 191 pp. DOI: https://doi.org/10.1016/C2009-....
9.
Farmawati D.A., Wirajana I.N., Yowani S.C. 2015. Perbandingan kualitas dna dengan menggunakan metode boom original dan boom modifikasi pada isolat Mycobacterium tuberculosis. Jurnal Kimia [Journal of Chemistry] 9 (1): 41–46. DOI: https://doi.org/10.24843/JCHEM....
10.
Htay C., Peng H., Huang W., Kong L., He W., Holgado R., Peng D. 2016. The development and molecular characterization of a rapid detection method for rice root-knot nematode (Meloidogyne graminicola). European Journal of Plant Patholology 146: 281–291. DOI: https://doi.org/10.1007/s10658....
11.
Indarti S., Soffan A., Andrasmara M.M.F. 2020. Short communication: first record of Hirschmanniella mucronata (Nematoda: Pratylenchidae) in Yogyakarta, Indonesia. Biodiversitas 21: 2068–2073. DOI: https://doi.org/10.13057/biodi....
12.
Jue E., Witters D., Ismagilov R.F. 2020. Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits. Scientific Reports 10: 1940. DOI: https://doi.org/10.1038/s41598....
13.
Khan M.R., Ahamad F. 2020. Incidence of root knot nematode Meloidogyne graminicola and resulting crop losses in paddy rice in Northern India. Plant Disease 104: 186–193. DOI: https://doi.org/10.1094/PDIS-1....
14.
Min J.H., Woo M.K., Yoon H.Y., Jang J.W., Wu J.H., Lim C.S., Kim Y.K. 2014. Isolation of DNA using magnetic nanoparticles coated with dimercaptosuccinic acid. Analytical Biochemistry 447: 114–118. DOI: https://doi.org/10.1016/j.ab.2....
15.
Mirsam H., Kurniawati F. 2018. Laporan pertama di Sulawesi Selatan: karakter morfologi dan molekuler nematoda puru akar yang berasosiasi dengan akar padi di Kabupaten Wajo, Sulawesi Selatan. Jurnal Perlindungan Tanaman Indonesia 22: 58–65. DOI: https://doi.org/10.22146/jpti.....
16.
Mondino E.A., Covacevich F., Studdert G.A., Pimentel J.P., Berbara R.L.L. 2015. Extracting DNA of nematodes communities from Argentine Pampas agricultural soils. Anais da Academia Brasileira de Ciências 87: 691–697. DOI: https://doi.org/10.1590/0001-3....
17.
Montgomery D.C. 2011. Design and Analysis of Experimen. 7th edition. Jhon Wiley & Sons, New York, USA.
18.
Natarajan V.P., Zhang X., Morono Y., Inagaki F., Wang F. 2016. A modified SDS-based DNA extraction method for high quality environmental DNA from seafloor environments. Frontiers in Microbiology 7: 986. DOI: https://doi.org/10.3389/fmicb.....
19.
Netscher C., Erlan A. 1993. Root-knot nematode, Meloidogyne graminicola, parasitic on rice in Indonesia. Afro-Asian Journal of Nematology 3: 90–95.
20.
Nisa R.U., Tantray A.Y., Shah A.A. 2022. Shift from morphological to recent advanced molecular approaches for the identification of nematodes. Genomics 114: 110295. DOI: https://doi.org/10.1016/j.ygen....
21.
Nurjayadi M.Y., Munif A., Suastika G. 2015. Identifikasi nematoda puru akar, Meloidogyne graminicola, pada tanaman padi di Jawa Barat. Jurnal Fitopatologi 11: 113–120. DOI: https://doi.org/10.14692/jfi.1....
22.
Oliveira C.M., Souza Junior I.T., Almeida N.O., Feritas M.A., Rocha M.R., Petrofeza S. 2020. Development of quantitative detection method for Meloidogyne incognita by qPCR. Bioscience Journal 36: 76–86. DOI: https://doi.org/10.14393/BJ-v3....
23.
Rahman R.M., Munif A., Kurniawati F. 2018. Deteksi dan identifikasi nematoda Aphelenchoides besseyi dari benih padi. Jurnal Fitopatologi Indonesia 14: 39. DOI: https://doi.org/10.14692/jfi.1....
24.
Reddy P.R., Raju N. 2012. Gel Electrophoresis and Its Applications, Gel Electrophoresis – Principles and Basics. Dr. Sameh Magdeldin (ed.). ISBN: 978-953-51-0458-2. InTech [Available on: http://www.intechopen.com/book...].
25.
Seesao Y., Audebert C., Verrez-Bagnis V., Merlin S., Jerome M., Viscogliosi E., Dei-Cas E., Aliouat-Denis C.M., Gay M. 2014. Monitoring of four DNA extraction methods upstream of high-throughput sequencing of Anisakidae nematodes. Journal of Microbiological Methods 102: 69–72. DOI: https://doi.org/10.1016/j.mime....
26.
Sengupta D.J., Cooksoon B.T. 2010. A general approach for improving cycle-sequencing that facilitates a robust one-step combined amplification and sequencing method. Journal of Molecular Diagnostics 12 (3): 272–277. DOI: https://doi.org/10.2353/jmoldx....
27.
Sloan S., Jenvey C.J., Piedrafita D., Preston S., Stear M.J. 2021. Comparative evaluation of different molecular methods for DNA extraction from individual Teladorsagia circumcincta nematodes. BMC Biotechnology 21: 35. DOI: https://doi.org/10.1186/s12896....
28.
Smaha D., Mokrini F., Imren M., Mokabli A., Dababat A.A. 2019. Morphological and molecular identification of cyst nematode species (Heterodera spp.) in Algerian cereal fields. Journal of Plant Protection Research 59 (3): 400–411. DOI: https://doi.org/10.24425/jppr.....
29.
Southey J.F. 1986. Laboratory Methods for Work with Plant and Soil Nematodes. Her Majesty’s Stationary Office, London, UK.
30.
Suparningtyas J.F., Pramudyawardhani O.D., Purwoko D., Tajuddin T. 2018. Analisis filogenetik beberapa klon karet dengan marka AFLP (Amplified Fragment Length Polymorphism). Jurnal Bioteknologi & Biosains Indonesia 5 (1): 8–19. DOI: https://doi.org/10.29122/jbbi.....
31.
Tasma I.M. 2015. Pemanfaatan teknologi sekuensing genom untuk mempercepat program pemuliaan tanaman. Jurnal Penelitian dan Pengembangan Pertanian 34 (4): 159–168. DOI: http://dx.doi.org/10.21082/jp3....
32.
Tesarova B., Zouhar M., Prysanek P. 2003. Development of PCR for specific determination of root knot nematode (Meloidogyne incognita). Plant Protection Science 39: 23–28. DOI: https://doi.org/10.17221/3823-....
33.
Travers K.J., Chen-Shan Chin, Rank D.R., Eid J.S., Turner S.W. 2010. A flexible and efficient template format for circular consensus sequencing and SNP detection. Nucleic Acids Research 38 (15): 1–8. DOI: https://doi.org/10.1093/nar/gk....
34.
Trzewik A., Nowak K.J., Orlikowska T. 2016. A simple method for extracting dna from rhododendron plants infected with phytophthora spp. for use in pcr. Journal of Plant Protection Research 56 (1): 104–109. DOI: https://doi.org/10.1515/jppr-2....
35.
Xin Y., Xie J., Nan B., Tang C., Xiao Y., Wu Q., Lin Y., Zhang X., Shen H. 2021. Freeze-Thaw pretreatment can improve efficiency of bacterial DNA extraction from meconium. Frontiers in Microbiology 12: 75368. DOI: https://doi.org/10.3389/fmicb.....
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