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ISSN : 1225-8504(Print)
ISSN : 2287-8165(Online)
Journal of the Korean Society of International Agriculture Vol.35 No.4 pp.271-277
DOI : https://doi.org/10.12719/KSIA.2023.35.4.271

An Improved Method for Cultivar Identification in Radish (Raphanus sativus L.) Using M13-Tailed Primers

Hyewon Yu*, Yunjeong Jeong*, Junho Lee**, Wonbyoung Chae*†
*Department of Environmental Horticulture, Dankook University, Cheonan 31116, Republic of Korea
**Vegetable Division, National Institute of Horticultural & Herbal Science, Rural Development Administration, Wanju 55365, Republic of Korea
† Corresponding author (Phone) +82-41-550-3642 (E-mail) wbchae75@dankook.ac.kr
October 11, 2023 October 23, 2023 October 23, 2023

Abstract


Radish is an important root vegetable in the world, and many cultivars have been developed with various molecular marker systems to identify these cultivars. Recently developed markers for radish cultivar identification require only 11 primer pairs, but they still use conventional PCR with different annealing temperatures and time-consuming gel electrophoresis. To improve the genotyping method, we applied touchdown PCR with 11 primers with M13 tails among 105 radish cultivars. Touchdown PCR successfully generated amplicons in all 11 M13-tailed primers with a condition of annealing temperature starting from 55℃, decreased by 1°C and 33 cycles at 53°C. The 11 M13-tailed primers followed by fragment analysis produced 71 amplicons, which produced more amplicons than gel electrophoresis that produced 23 amplicons. Especially, simple sequence repeats produced more amplicons, 12 on average, than the other marker types. The present study requires less effort and provides more accurate results compared to genotyping using gel electrophoresis. Besides, a database can be established using digitized genotyping results among radish cultivars.


Simple sequence repeats , M13-tailed primers , Fragment analysis , Touchdown PCR , Genotyping

M13-tailed primer를 이용한 무 품종 구분 분자표지 개선법

유혜원*, 정윤정*, 이준호**, 채원병*†
*단국대학교 환경원예조경학과 환경원예학전공
**국립원예특작과학원 채소과

초록

    INTRODUCTION

    Radish (Raphanus sativus L.) belongs to the Brassicaceae family, which is cultivated throughout the world as an important root vegetable crop, particularly in East Asia (Wang and He, 2005). Believed to be originated in the Mediterranean coast and West Asia, radish was introduced into Korea prior to the Unified Silla Kingdom (Jung et al., 2014). Radish is rich in various nutrients such as proteins, lipids, fiber, vitamins, iron, magnesium, and calcium. Radish is known for their high yield per cultivated area and extensively used as a key ingredient for kimchi. Many cultivars have been developed in radish by not only multinational companies but small companies or independent breeders.

    Many molecular markers have been established for cultivar identification in radish (Choe et al., 2002;Choi et al., 2008;Liu et al., 2008;Bae et al., 2015;Lee and Park, 2017). However, these markers required expensive equipment and a large number of primer pairs, which need significant time and cost. Recently, the marker system for identifying radish cultivars was developed, which required only 11 primer pairs and, simple PCR and gel electrophoresis (Hong et al., 2023). However, the process of confirming PCR products through gel electrophoresis faces challenges where gels may distort, making identification of amplicons difficult. Moreover, the differences in 1 - 3 base pairs of amplicons are difficult to detect visually, leading to decreased reliability.

    M13-tailed primers along with universal primers labeled with fluorescent tags offer a cost-effective and reliable genotyping method as compared to individually labeling each primer with a fluorescent tag (Schuelke, 2000) and can improve the readability and usability of markers including simple sequence repeats (SSRs) (Lorenz et al., 2001). This approach also allows for efficient and accurate genotyping when compared to conventional gel electrophoresis. Additionally, the digitized data obtained from fragment analysis can be stored, eliminating the need for repetitive PCR comparisons with new samples.

    This study was conducted to enhance the efficiency and reliability of existing genotyping methods for radish cultivar identification (Kang et al, 2016;Hong et al, 2023) using the M13-tailed primer-based genotyping approach.

    MATERIALS AND METHODS

    1. Plant material and DNA extraction

    One hundred five radish cultivars from 15 companies (Table 1) were used for genotyping with M13-tailed primers. Radish cultivars were grown from seeds in a greenhouse for 2-3 weeks, and leaf samples of approximately 3 - 4 cm in length were collected for DNA extraction. The DNA was extracted using the CTAB extraction method (Doyle and Doyle, 1990), and the quantity and quality of the extracted DNA were confirmed using a spectrophotometer (DS-11, DeNOVIX, Wilmington, USA).

    2. The design of M13-tailed primers and touchdown PCR

    The 11 primer pairs (Table 2) were subjected to M13- tailed primers by adding the same sequence (TGT AAA ACG ACG GCC AGT) to the 5' end of forward primers. The PCR reaction was carried out in total volume of 20μl containing 10ng/μl of genomic DNA, 0.5μl of Accu- Power® Taq PCR premix (K–2037, Bioneer, Daejeon, Korea), 0.1μM of M13-tailed primer (or 0.5μM for conventional PCR), and 0.5μM of the reverse primer and the universal primer tagged with FAM at the 5’ end. Touchdown PCR was used to amplify the primers with an initial denaturation step at 95? for 5 minutes, followed by denaturation at 94? for 30 seconds, annealing temperature started at 55 °C for 30 seconds and extension at 72? for 30 seconds. The annealing temperature was decreased every cycle by 1°C until 53°C. A total of 35 cycles were performed and a final extension at 72? for 5 minutes.

    3. Fragment analysis and gel electrophoresis

    Fragment analysis of amplicons was performed in NICEM at Seoul National University, on the ABI 3730xl Genetic Analyzer (Applied Biosystems, Waltham, Massachusetts, USA) with the GeneScan 500 ROX size standard. Size separation of the amplicons was carried out using Open-Source Independent Review and Interpretation System (OSIRIS version 2.16, National Library of Medicine, MD, USA). Peaks with relative fluorescence units (RFU) lower than 5000 did not counted. In addition, the PCR products were separated on both a 1.5% agarose gel at 225V for 90 minutes for visual detection of the amplicons.

    RESULTS

    1. M13-tailed primers with the touchdown PCR

    The melting temperatures of 11 primers were different, ranging from 53 to 55 °C (Table 2). In order to test whether these primers can be amplified in the same PCR condition, touchdown PCR was applied with the annealing temperatures ranging from 55°C to 53°C. All 11 M13-tailed primers were successfully produced amplicons using the touchdown PCR among 105 cultivars (Table 3). The amplicon sizes in Table 3 were presented except for 18bp of M13-tailed sequences.

    2. Fragment analysis compared to conventional gel electrophoresis

    The amplicon sizes lower than 100 bp and RFU 5000 were did not counted as the number of alleles (NA) (Figure 1). A total of 71 amplicons were produced from the 11 M13-tailed primers (Table 3). On average, 6.45 amplicons per primer were produced by fragment analysis and the maximum and minimum number of amplicons were 14 (RsSSR 108) and two (RsInD22), respectively (Table 3).

    Amplicons from M13-tailed primers showed differences of approximately 18 base pairs (bp) compared to conventional PCR (Figure 1), which were attributed to the addition of the 18 bp of M13 tail sequences. For examples, amplicons from RsInD79 (figure 1A) and RsInD25 primers (Figure 1B) were located in gel electrophoresis slightly lower than those from their M13-tailed primers, which were 161 or 181 bp and 207 or 220 bp, respectively, by fragment analysis.

    The SSRs produced more amplicons than other markers. Three SSRs produced the highest number of amplicons, 12 on average, followed by IBP and InDel markers with 5.3 and 3.8 amplicons, respectively (Table 3). Besides, fragment analysis showed better the separation of amplicons than gel electrophoresis (Table 2 & 3). For examples, three amplicons were observed in gel electrophoresis but there were actually six picks in fragment analysis using the primer RsSSR100 (Figure 2A). Similarly, two amplicons in both ‘Saepureunbom’ and ‘Matkkal’ cultivars were observed using the primer RsSSR108 in gel electrophoresis but six and five peaks, respectively, were observed in fragment analysis (Figure 2B).

    DISCUSSION

    To date, many molecular marker systems have been developed and applied to identify radish cultivars and to assess their genetic diversity using amplified fragment length polymorphism (Choe et al., 2002), random amplified polymorphic DNA (RAPD) (Choi et al., 2008), RAPD, ISSR and sequence-related amplified polymorphism (Liu et al., 2008), and SSR (Bae et al., 2015;Lee and Park, 2017). However, these systems required many primers for genotyping. Recently developed markers required only 11 primer pairs for cultivar identification but still suffer from time-consuming gel electrophoresis (Hong et al., 2023). Besides, melting temperatures (Tm) of the primers were different, which made difficult for multiplex PCR. Therefore, we improved the genotyping method by adopting touchdown PCR and adding M13-tails on the 11 primers.

    Touchdown PCR successfully generated amplicons in all 11 primers (Table 3) with various Tm (Table 2) in a condition of annealing temperature starting from 55 to 53°C, decreased by 1°C, and followed by 33 cycles in 53°C. The same PCR condition can make possible to adopt multiplex PCR for these primers. Besides, M13- tailed primers followed by fragment analysis showed better separation compared to gel electrophoresis in the previous study (Hong et al., 2023). This is especially true for SSRs having short, tandemly repeated from di- to pentanucleotide motifs, and difficult to detect polymorphism visually by gel electrophoresis. For example, only two amplicons could be observed using the primer, RsSSR108, in the previous study (Hong et al., 2023) but 14 amplicons be observed in the present study (Table 3) using same 105 cultivars.

    The present study using the 11 M13-tailed primers and touchdown PCR can improve existing radish cultivar identification methods since it requires less time and provide more accurate results compared to traditional PCR and gel electrophoresis method (Hong et al., 2023). In addition, the genotyping results of individual radish cultivar can be digitized and stored as database. The genotyping cost can be further reduced by analyzing PCR products by four universal primers labeled with different fluorescent dyes such as HEX (green), NED (yellow) and ROX (red) in addition to FAM (blue) in a reaction of fragment analysis.

    적 요

    1. 무는 중요한 뿌리채소 작물로 품종 판별을 위해 최근 11 개로 구성된 분자표지가 개발되었으나 PCR시 annealing temperature가 각기 다르고 agarose gel 전기영동법을 이용하는 등 불편한 점이 있어 개선이 필요함

    2. 11개의 forward primer에 M13-tail 서열을 부착하여 reverse primer 및 universal primer와 함께 touchdown PCR 을 수행한 후 fragment analysis를 진행하였음

    3. Touchdown PCR을 통해 하나의 PCR 조건에서 모든 11 개의 primer로부터 amplicon이 성공적으로 생성되었음

    4. 기존 gel 전기영동은 23개의 amplicon을 생성한 반면 fragment analysis를 통해 71개의 amplicon이 확인되었음

    5. 본 연구를 통해 개발된 M13-tailed primer는 기존 primer에 비해 전기영동에 드는 시간이 줄어 좀 더 간편하고 fragment analysis를 통해 더 정확한 결과를 얻을 수 있으며, amplicon의 크기에 대한 수치화가 가능한 장점이 있음

    ACKNOWLEDGMENTS

    This study was funded by a grant, Project No: PJ01504301 (RS-2020-RD009140) “Development of customized breeding lines through the establishment of radish and kimchi cabbage germplasm evaluation system and core collections” from National Institute of Horticultural and Herbal Science, Rural Development Administration.

    Figure

    KSIA-35-4-271_F1.gif

    The comparison of gel electrophoresis between primers with and without M13 tails, and peaks from fragment analysis. Primers RsInD79 (A) and RsInD25 (B) were applied to five radish cultivars ‘Daedeulbo’ (1), ‘Baekja’ (2), ‘Sinbong’ (3), ‘Manchudeapung’ (4), and ‘Supertogwang’ (5). N indicates the negative control.

    KSIA-35-4-271_F2.gif

    The comparison of amplicons from gel electrophoresis (left)and peaks from fragment analysis (right) using the same primers with M13 tails. Primers RsSSR100 (A) and RsSSR108 (B) were applied to radish cultivars, ‘Sunbaekok’ (1) or ‘Shinnamgangaltari’ (2) and ‘Saepureunbom’ (3) or and ‘Matkkal’ (4), respectively. N indicates the negative control.

    Table

    The list of 105 radish cultivars used for genotyping with M13-tailed primers.

    Information on 11 primer pairs applied to 105 radish cultivars (Hong et al., 2023).

    The number of alleles (NA) and amplicon sizes produced from 11 M13-tailed primer pairs.

    Reference

    1. Bae, K. , Sim, S. , Hong, J. , Choi, K. , Kim, D. , Kwon, Y. 2015. Development of genomic SSR markers and genetic diversity analysis in cultivated radish (Raphanus sativus L.). Hortic. Environ. Biotechnol. 56:216–224.
    2. Choe, Y. , Park, D. , Shin, H. , Kwon, Y. , Yoon, W. , Moon, J. 2002. Studies for similarity evaluation of radish and Chinese cabbage cultivars. Hortic. Sci. Technol. 20:160–167. (In Korean)
    3. Choi, W. , Lee, S. , Yoo, S. , Lee, S. , Kang, S. , Ko, H. 2008. Genetic relationship by RAPD analysis of Korean wild radish and local cultivars in radish. Hortic. Sci. Technol. 26:427–431. (In Korean)
    4. Doyle, J.J. , Doyle, J.L. 1990. Isolation of plant DNA from fresh tissue. Focus. 12:13–15.
    5. Hong, H. , Lee, J. , Chae, W. 2023. An economic method to identify cultivars and elite lines in radish (Raphanus sativus L.) for small seed companies and independent breeders. Horticulturae. 9:140.
    6. Jung, U. , Oh, J. , Kim, Y. , Ahn, C. , Lee, K. , Choi, S. , Lim, Y. , Park, S. , Choi, K. et al.2014. Development of highly uniform variety for processing using SSR markers in radish (Raphanus sativus L). J Plant Biotechnol. 41:56–63.
    7. Kang, E. , Ha, S. , Ko, H. , Yu, H. , Chae, W. 2016. Reproductive traits and molecular evidence related to the global distribution of cultivated radish (Raphanus sativus L.). Plant Syst. Evol. 302:1367–1380.
    8. Kim, H. , Hong, C. , Im, S. , Choi, S. , Lim, Y. 2014. Development of molecular markers and application for breeding in Chinese cabbage. Hortic. Sci. Technol. 32:745–752.
    9. Lee, O. , Park, H. 2017. Assessment of genetic diversity in cultivated radishes (Raphanus sativus) by agronomic traits and SSR markers. Sci. Hortic. 223:19–30.
    10. Lorenz, E. , Frees, K.L. , Schwartz, D.A. 2001. M13-tailed primers improve the readability and usability of microsatellite analyses performed with two different allele sizing methods. Bio Techniques. 31:24-26,28.
    11. Liu, L. , Zhao, L. , Gong, Y. , Wang, M. , Chen, L. , Yang, J. , Wang, Y. , Yu, F. , Wang, L. 2008. DNA fingerprinting and genetic diversity analysis of late–bolting radish cultivars with RAPD, ISSR and SRAP markers. Sci. Hortic. 116:240–247.
    12. Mun, J. , Chung, H. , Chung, W. , Oh, M. , Jeong, Y. , Kim, N. , Ahn, B. , Park, B. , Park, S. et al.2014. Construction of a reference genetic map of Raphanus sativus based on genotyping by whole?genome resequencing. Theor Appl Genet. 128:259– 272.
    13. Rohlf, F.J. 2000. NTSYS–pc: Numerical Taxonomy and Multivariate Analysis System Version 2.1. Applied Biostatistics Inc. New York, NY, USA. p. 43.
    14. Schuelke, M. 2000. An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology. 18:233– 234.
    15. Wang, J.L. , Liu, L. , Zhu, X. , Zhia, L. , Gong, Y. 2012. Development and characterization of cDNA library based novel ESTSSR marker in radish (Raphanus sativus L.). Scientia Horticulturae. 140:164–172.
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