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Application of RAPD and SI-typing techniques to confirm genetic integrity of radish (Raphanus sativus L.) varieties
Scientia Horticulturae 119 (2009) 352–356
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Application of RAPD and SI-typing techniques to confirm genetic integrity of radish (Raphanus sativus L.) varieties Jung-Ho Kwak *, Suhyoung Park, Moo Kyoung Yoon Vegetable Research Division, National Horticultural Research Institute, 475 I-mok Dong, Jang-an Gu, Suwon 440-706, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 January 2007 Received in revised form 25 August 2008 Accepted 27 August 2008
The random amplified polymorphic DNA (RAPD) and self-incompatibility (SI) typing techniques were applied to settle a lawsuit between a Seed Company and a farmer concerning seed impurity in radish (Raphanus sativus L.). After sowing ‘‘Halra Woldong’’ (HW) a winter radish variety, two morphologically different radish plants grew. Genomic DNA from each of the two types were prepared and analyzed using ninety-six RAPD markers and four variety-specific markers were found. The polymorphic RAPD band patterns were compared with those of the twenty Korean radish varieties. The genetic integrity of the two varieties in question was identified and further confirmed by SI-typing of S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes. ß 2008 Elsevier B.V. All rights reserved.
Keywords: RAPD markers Self-incompatibility typing Investigative application Korean radish variety Lawsuit
1. Introduction Commercial seed companies are the most common source of vegetable seeds for Korean farmers. However, as more commercial seeds become available, seed purity or seed genetic integrity becomes an issue. Seed impurity or contamination can cause severe economic damage to the farmers and may lead to lawsuit requiring expert’s investigation in differentiating among the plants or varieties. Traditionally, evaluation was based on the crop’s morphological characters, which are somehow not accurate, and most often could not provide clear identification of the variety. Since the most of horticultural phenotypes were easily influenced by environments of cultivation, and often inherited quantitatively, which makes cultivar identification often complicates among similar cultivars (Liu et al., 2008). With the advances in DNA technology, application of molecular techniques like random amplified polymorphic DNA (RAPD) marker coupled with selfincompatibility typing (SI-typing) allow precise, objective and rapid cultivar identification without any effect of the environment. The RAPD technique, originally developed to distinguish genetic variations of bacteria, plants or even human beings was used in this study. It is done by establishing genomic DNA polymorphism using arbitrary primers (Welsh and McClelland, 1990; Williams et al., 1990). In identifying cultivars, RAPD procedure does not usually require too much genetic or
* Corresponding author. Tel.: +82 31 240 3579; fax: +82 31 240 3594. E-mail address: [email protected] (J.-H. Kwak). 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.08.029
physiological information of the plants. Instead, the plant’s genomic DNA serves as material for analysis and the final results is obtained relatively fast and economical. Self-incompatibility (SI) prevents self-fertilization and promotes out-crossing in hermaphrodite plants (de Nettancourt, 1997). In Brassica crops for instance, SI is sporophytically controlled by a single, highly polymorphic S-locus consisting of a series of multiple alleles namely: the S-locus glycoprotein (SLG), the S-locus receptor kinase (SRK), and the S-locus protein 11 (SP11)/S-locus cys-rich protein (SCR) genes (Nasrallah and Nasrallah, 1993; Takayama and Isogai, 2003). SI technique has been used to produce uniform F1 hybrid seeds while preventing self-pollination between parental inbred lines in a restricted seed production field. Therefore, the genetic information of the resulting F1 seedlings must contain SI-related genetic traits from their parents. Since most Korean seed companies used the SI technique in producing commercial F1 seeds of Brassica crops, application of this concept makes it possible to trace the parents of F1 or identify among varieties. This study used RAPD markers, and SI-typing techniques to identify and confirm the genetic integrity of radish variety; ‘‘Halra Woldong’’ (HW). 2. Materials and methods 2.1. Plant material A mega farm complex located in Wando, Jeollanam-do, S. Korea has been doing a massive planting of winter radish using only one radish variety HW from 2005 to 2006. During this period, it was
J.-H. Kwak et al. / Scientia Horticulturae 119 (2009) 352–356
353
Table 1 List of the 20 Korean radish varieties and their identifications Numbers
Name
Company
Seasonal type
SI type
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Dae Keun Baek Yang Sam Yang Young San Myong San Baek Kyung Pal Gwang Tae Baek Daehyoung Chuseok Baek Ja Gil Cho Seong Gong Young Dong Ha Chu Hannong Yeorum Cheong Gwang Daeburyong Yeorum Hanoldaehyungbom Biandaehyungbom Suji
Nongwoobio Nongwoobio Nongwoobio Dongbu Hannong Syngenta Korea Syngenta Korea Syngenta Korea Heungnong Seminis Korea Seminis Korea Nongwoobio Nongwoobio Nongwoobio Dongbu Hannong Dongbu Hannong Syngenta Korea Seminis Korea Dongbu Hannong Kyungshin Nongwoobio
Autumn radish Autumn radish Autumn radish Autumn radish Autumn radish Autumn radish Autumn radish Autumn radish Autumn radish Autumn radish Spring/Summer radish Summer radish Summer radish Summer radish Summer radish Summer radish Summer radish Spring radish Spring radish Summer radish
S8S17 S26S? S21S30 S4S17 n.d.a n.d.a S8S26 S4S21 n.d.a S8S26 S5S17 S8S8 S1S17 S8S21 n.d.a n.d.a n.d.a n.d.a n.d.a n.d.a
This table is modified from Lim et al. (2006). a n.d. means no data available.
noted that some of the radishes in the field produced stalks/ flowers. Knowing such abnormal behavior of the plants to be seed contamination and/or impurity, the farmer filed a lawsuit against the seed company who supplied the radish seeds. Being the expert team assigned by the Kwangju district court for the case #2006KAKI 54, the team visited the field and performed morphological evaluation of the plantings. Radish cultivars were categorized into two distinct groups based on their morphological characteristics such as: (a) the putative autumn radish variety, which produces stalks/flowers in winter season; (b) the winter radish variety, which does not produce stalks/ flowers even in winter season. The plants, which produced stalks/ flowers, were tagged as the ‘S’ group and those that did not produce stalk/flowers as the ‘N’ group. After proper recording of the observation, fifty radish plants from each group were randomly selected and used as source of leaf samples to be used for molecular evaluation. Leaf samples were collected and brought immediately to the laboratory. During this transportation, they are kept under 4 8C to prevent any DNA degradation. Genomic DNAs of 20 Korean radish varieties were included in the analysis to serve as reference materials (Table 1), and they were kind gifts from Dr. Cho, Kang-Hee at Horticultural Biotechnology Division of National Horticultural Research Institute (Suwon, S. Korea). 2.2. Genomic DNA extraction Genomic DNA of each plant samples was extracted from young radish leaves using DNeasy Plant Kit (Qiagen, Germany) following the manufacturer’s instructions. The genomic DNA from each group was pooled to construct a stalking/flowering (S) and a nonstalking/flowering (N) bulks. These two bulk-samples were used for bulked segregant analysis (BSA) (Michelmore et al., 1991) coupled with the RAPD marker technique. 2.3. Random amplified polymorphic DNA (RAPD) procedure RAPD amplification was performed using 96 random primers obtained from Operon (36 decamers, Operon Technologies, U.S.A.), Wako (36 dodecamers, Wako, Japan), and UBC (24 decamers, University of British Columbia, Canada). The reaction mixture
(25 mL) contained 0.2 mL of Top-Taq DNA polymerase (5 units mL1, CoreBioSystem, Korea), 2.5 mL of Top-Taq PCR buffer (10), 2.0 mL of dNTP mixture (2.5 mM), 1.0 mL of RAPD primer (5 pmol mL1), 0.5 mL of template DNA (50 ng mL1), and 18.8 mL of deionized water. Amplification was carried out in a DNA thermal cycler (PTC200, MJ research. U.S.A.) programmed for an initial denaturation cycle (95 8C for 3 min) followed by 40 cycles of 40 s denaturation at 94 8C, 1 min annealing at 40 8C, and 2 min elongation at 72 8C with a final extension at 72 8C for 5 min. RAPD products were observed on a 1.5% agarose gel with ethidium bromide staining and ultraviolet light irradiation. For the reproducibility check, RAPD profiles were confirmed by three independent assays using three independent DNA extractions. 2.4. Procedure for the SI-typing of radish variety SI-typing of radish varieties was done through the PCR technique using a set of primer pairs for each of the genes. To classify the type of SRK gene, srk3 (50 -GCTTTCATATTACCGGGCATCGATGA-30 ) and srk5 (50 -TGATGAGTTTATGAATGAGGTGA-30 ) primer set was used (Delorme et al., 1995). The SLG gene was classified using a new primer set (50 -ATGAAAGGGGTACAGAACAT30 and 50 -ACGGACCGCTCCTTTGYATTTCAWMACKT-30 ) designed by Dr. Kim, Sunggil at Dongbu Hannong Chemicals. The PCR reaction mixture (25 mL) contained 0.3 mL of Excel-Taq DNA polymerase (5 units mL1, CoreBioSystem, Korea), 2.5 mL of Excel-Taq PCR buffer (10), 2.0 mL of dNTP mixture (2.5 mM), 0.3 mL of each sense/antisense primer (5 pmol mL1), 0.5 mL of template DNA (50 ng mL1), and 19.1 mL of deionized water. Amplification was carried out in a DNA thermal cycler (PTC200, MJ research. U.S.A.) programmed for an initial denaturation cycle (94 8C for 5 min) followed by 35 cycles of 30 s denaturation at 94 8C, 30 s annealing at 55 8C, and 1 min elongation at 72 8C with a final extension at 72 8C for 5 min. The PCR products were observed on a 1.5% agarose gel with ethidium bromide staining and ultraviolet light irradiation. The amplified bands were recovered from the gel using QIAquick Gel Extraction Kit (Qiagen, Germany) following the manufacturer’s instructions. The purified PCR product was sent for DNA sequencing (Genotech, S. Korea). The final sequencing results were analyzed using NCBI BLAST service
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J.-H. Kwak et al. / Scientia Horticulturae 119 (2009) 352–356 Table 2 DNA sequences of the RAPD primers used
Fig. 1. RAPD band patterns amplified with seven arbitrary primers using ‘S’ and ‘N’ bulks as template DNA. DNA size marker was used in lane 1 and white arrows indicate variety-specific RAPD bands.
(National Center for Biotechnology Information, http://www.ncbi. nlm.nih.gov/). 3. Results and discussion 3.1. Field evaluation of radish plantings From the morphological evaluation of the plants, it was observed that about 9.1% of the radish plantings produced stalks/flowers. According to the farmer, two morphologically different radish plants emerged during the mid-development stage after planting the HW radish variety. Some plants produced stalks/ flowers while the other plants did not produce stalks/flowers. An interesting observation was noted from the ‘S’-group in that all the flowers that were produced are white, while the ‘N’-group was left in the field, not harvested and then eventually produced all purple flowers. 3.2. RAPD analyses of S/N bulks and 20 Korean radish varieties The RAPD analyses of ‘S’ and ‘N’ groups against the 20 Korean radish varieties were conducted using the 96 commercially available RAPD primers that were arbitrary decamers or dodecamers. After RAPD amplifications, each RAPD band pattern was
Name
Sequence
Company
C01 E14 A11 703
TTCGAGCCAG TGCGGCTGAG GATGGATTTGGG CCAACCACCC
Operon, U.S.A. Operon, U.S.A. Wako, Japan UBC, Canada
analyzed on a 1.5% agarose gel (data not shown). Majority of the RAPD primers showed similar RAPD band patterns, but seven primers (C01, C03, C05, E14, A07, A11, and 703) gave distinctive band patterns as shown in Fig. 1. These primers were then used for further RAPD reproducibility test. Concentration of template DNA was the most critical factor to obtain reproducible RAPD bands as well as standardization of each sample concentration. By using 6 template DNA concentration (5, 10, 15, 20, 25, and 50 ng) in preliminary experiments, it was found that the 25 ng condition produced optimum number of reproducible bands. It is reported that 5 ng of template DNA resulted to the maximum number of reproducible bands (Sarkhosh et al., 2006), but in this study, RAPD band did not obtained in some samples by using the 5 ng condition. Although the 25 ng of template DNA did not show the maximum number of reproducible bands, the total number of RAPD bands produced was at optimum level with the 25 ng condition. Finally, four primers were confirmed as true polymorphic RAPD markers by three independent assays using three independent DNA extractions (Table 2). To identify ‘S’ group variety, RAPD band patterns were compared and analyzed between the 20 Korean radish varieties and ‘S’/‘N’ bulks using the four identified primers (C01, E14, A11, and 703). Since the seed company of HW withdrew all their HW seeds from Korean market and did not cooperate for our investigation, the most typical 20 Korean radish varieties were selected as reference varieties without HW variety. Several radish varieties showed similar RAPD band patterns with that of ‘S’ bulk (Fig. 2). Because the Korean radish varieties #2 and #10 showed the highest band pattern similarity with S bulk, the two varieties were further analyzed with 72 RAPD primers, respectively. The summary of the resulting band patterns (Fig. 3) clearly indicate that the RAPD band pattern of ‘B’ bulk (variety #10; Baek Ja radish)
Fig. 2. RAPD band patterns amplified with four arbitrary primers using 20 Korean radish varieties and ‘S’/‘N’ bulks as template DNA (A: primer C01, B: primer E14, C: primer A11, and D: primer 703). DNA size marker was used in each lane 1 and white stars indicate similar RAPD band patterns with that of ‘S’ bulks.
J.-H. Kwak et al. / Scientia Horticulturae 119 (2009) 352–356
355
Fig. 3. RAPD band patterns amplified with arbitrary primers (E1 to 608) using ‘S’ and ‘B’ bulks as template DNA. DNA size marker was used in side lanes. ‘S’: S bulk and ‘B’: Korean variety #10 (Baek Ja variety).
Fig. 4. RAPD band patterns amplified with 4 arbitrary primers using S, N, and B bulks as template DNA. DNA size marker was used in lane 1 and white stars indicate variety-specific RAPD bands.
is exactly the same with that of the ‘S’ bulk. RAPD band patterns were compared among ‘S’, ‘N’, and ‘B’ bulks by the amplifications through the identified four RAPD primers (Fig. 4). All the band patterns of ‘S’ and ‘B’ bulk groups were the same, but not with those of the ‘N’ bulk group as expected. Since the reference genomic DNA of HW radish was not obtainable, it is unable to match the RAPD band patterns of ‘N’ bulk group against those of ‘HW’ bulk. It was only assumed that ‘N’ bulk is a pure HW variety through its morphological features. Based on the results, the expert team made a preliminary conclusion that the variety of ‘S’ bulk could be the Korean variety #10 (Baek Ja radish). The seeds of this Baek Ja variety may have been mixed into the pure HW seed lot. However, it could not be confirmed whether the seed company or the farmer is responsible for the seed mixture. 3.3. Self-incompatibility (SI) type analysis of Korean variety #10, ‘S’, and ‘N’ bulks To confirm the preliminary conclusion, SI-typing technique was employed. In Brassica crops, SI expression is controlled by SLG and SRK genes’ activities, and pollen SI is determined by the expression of SCR/SP11 gene (Delorme et al., 1995; Nasrallah and Nasrallah, 1993; Sakamoto et al., 1998; Shiba et al., 2001; Suzuki et al., 1999;
Fig. 5. SI-typing of ‘S’, ‘N’, and ‘B’ with SRK and SLG gene specific primers. 1 kb ladder was used in side lanes. White arrows indicate successfully amplified PCR products.
Takasaki et al., 2000). To date, more than 100 types of SI related genes were reported and registered at the NCBI database for Brassica oleracea, B. campestris, and Raphanus sativus L. (Lim et al., 2002; Nou et al., 1993; Sakamoto et al., 1998; Suzuki et al., 1999). Once the SI related gene is amplified by PCR using appropriate primers, the PCR product’s sequence could be determined, and then the amplified SI gene’s type could be identified with the NCBI database through their DNA sequences. The SRK and SLG gene-specific primer sets were used to determine SI types of ‘S’, ‘N’, and ‘B’ bulks. The SRK genes were easily amplified by PCR, but not the SLG genes. Hence, after the first PCR amplification of the SLG gene, the product was purified, and then used as the template DNA for the second PCR as a nested PCR. The band patterns of each SI types were confirmed for the two genes (Fig. 5). These PCR products were extracted and purified from the agarose gels, and then sent for sequencing (Genotech, S. Korea). The final sequence results of the amplified SRK and SLG genes were compared with the NCBI database (balstn service with default parameters). There is a great similarity between ‘S’ and ‘B’ bulks but no similarity between ‘N’ and ‘B’ or ‘S’ and ‘N’ bulks (Table 3). The data confirmed the preliminary conclusion that the
Table 3 DNA sequence comparison of ‘S’, ‘N’ and ‘B’ bulks (NCBI Blast service) Samplea
blastn result
S-srk N-srk B-srk S-slg N-slg B-slg
Raphanus Raphanus Raphanus Raphanus Raphanus Raphanus
a
sativus sativus sativus sativus sativus sativus
haplotype haplotype haplotype haplotype haplotype haplotype
S8 srk S1 srk S8 srk S26 slg S4 slg S26 slg
Score (bits)
Identities
Size (bp) compared
1265 1372 1493 1185 1011 1154
673/678 749/760 768/773 627/633 566/585 620/634
700 800 800 651 600 645
S: stalking/flowering plant type; N: non-stalking/flowering plant type; B: Baek Ja radish variety; srk: S-locus receptor kinase; slg: S-locus glycoprotein.
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‘N’ bulk is entirely a different radish variety to the ‘S’ bulk, and that the ‘S’ bulk is a very similar variety to the ‘B’ bulk, which is the Baek Ja radish. From the results of this study, RAPD and SI-typing techniques proved to be useful molecular approaches for investigative applications. Considering that these techniques are very simple and convenient; they are reliable tools that could be used in providing critical court evidence to resolve lawsuits between farmers and seed companies. References de Nettancourt, D., 1997. Incompatibility in angiosperms. Sex. Plant Reprod. 10 (4), 185–199. Delorme, V., Giranton, J.L., Hatzfeld, Y., Friry, A., Heizmann, P., Ariza, M.J., Dumas, C., Gaude, T., Cock, J.M., 1995. Characterization of the S locus genes, SLG and SRK, of the Brassica S3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene. Plant J. 7 (3), 429–440. Lim, S.-H., Cho, J., Lee, J., Cho, Y.-H., Kim, B.-D., 2002. Identification and classification of S haplotypes in Raphanus sativus by PCR-RFLP of the S locus glycoprotein (SLG) gene and the S locus receptor kinase (SRK) gene. Theor. Appl. Genet. 104 (8), 1253–1262. Lim, S.-H., Kim, K.-T., Park, S., Cho, H.-J., Park, H.-Y., Hwang, S.-Y., Yoon, M.-K., Mok, I.-G., Woo, J.-G., Oh, D.-G., 2006. Identification of S haplotypes in commercial F1 hybrid cultivars of radish by PCR-RFLP analysis. Korean J. Breed. Sci. 38 (3), 167– 172. Liu, L.W., Zhao, L.P., Gong, Y.Q., Wang, M.X., Chen, L.M., Yang, J.L., Wang, Y., Yu, F.M., Wang, L.Z., 2008. DNA fingerprinting and genetic diversity analysis of latebolting radish cultivars with RAPD ISSR and SRAP markers. Sci. Hortic. 116 (3), 240–247.
Michelmore, R.W., Paran, I., Kesseli, R.V., 1991. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. U.S.A. 88 (21), 9828–9832. Nasrallah, J.B., Nasrallah, M.E., 1993. Pollen[mdash]stigma signaling in the sporophytic self-incompatibility response. Plant Cell 5 (10), 1325–1335. Nou, S.I., Watanabe, M., Isogai, A., Hinata, K., 1993. Comparison of S-alleles and Sglycoproteins between two wild populations of Brassica campestris in Turkey and Japan. Sex. Plant Reprod. 6 (2), 79–86. Sakamoto, K., Kusaba, M., Nishio, T., 1998. Polymorphism of the S-locus glycoprotein gene (SLG) and the S-locus related gene (SLR1) in Raphanus sativus L. and self-incompatible ornamental plants in the Brassicaceae. Mol. Gen. Genet. 258 (4), 397–403. Sarkhosh, A., Zamani, Z., Fatahi, R., Ebadi, A., 2006. RAPD markers reveal polymorphism among some Iranian pomegranate (Punica granatum L.) genotypes. Sci. Hortic. 111 (1), 24–29. Shiba, H., Takayama, S., Iwano, M., Shimosato, H., Funato, M., Nakagawa, T., Che, F.S., Suzuki, G., Watanabe, M., Hinata, K., Isogai, A., 2001. A pollen coat protein, SP11/ SCR, determines the pollen S-specificity in the self-incompatibility of Brassica species. Plant Physiol. 125 (4), 2095–2103. Suzuki, G., Kai, N., Hirose, T., Fukui, K., Nishio, T., Takayama, S., Isogai, A., Watanabe, M., Hinata, K., 1999. Genomic organization of the S locus: Identification and characterization of genes in SLG/SRK region of S(9) haplotype of Brassica campestris (syn. rapa). Genetics 153 (1), 391–400. Takasaki, T., Hatakeyama, K., Suzuki, G., Watanabe, M., Isogai, A., Hinata, K., 2000. The S receptor kinase determines self-incompatibility in Brassica stigma. Nature 403 (6772), 913–916. Takayama, S., Isogai, A., 2003. Molecular mechanism of self-recognition in Brassica self-incompatibility. J. Exp. Bot. 54 (380), 149–156. Welsh, J., McClelland, M., 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18 (24), 7213–7218. Williams, J.G., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey, S.V., 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18 (22), 6531–6535.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Application of RAPD and SI-typing techniques to confirm genetic integrity of radish (Raphanus sativus L.) varieties Jung-Ho Kwak *, Suhyoung Park, Moo Kyoung Yoon Vegetable Research Division, National Horticultural Research Institute, 475 I-mok Dong, Jang-an Gu, Suwon 440-706, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 January 2007 Received in revised form 25 August 2008 Accepted 27 August 2008
The random amplified polymorphic DNA (RAPD) and self-incompatibility (SI) typing techniques were applied to settle a lawsuit between a Seed Company and a farmer concerning seed impurity in radish (Raphanus sativus L.). After sowing ‘‘Halra Woldong’’ (HW) a winter radish variety, two morphologically different radish plants grew. Genomic DNA from each of the two types were prepared and analyzed using ninety-six RAPD markers and four variety-specific markers were found. The polymorphic RAPD band patterns were compared with those of the twenty Korean radish varieties. The genetic integrity of the two varieties in question was identified and further confirmed by SI-typing of S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes. ß 2008 Elsevier B.V. All rights reserved.
Keywords: RAPD markers Self-incompatibility typing Investigative application Korean radish variety Lawsuit
1. Introduction Commercial seed companies are the most common source of vegetable seeds for Korean farmers. However, as more commercial seeds become available, seed purity or seed genetic integrity becomes an issue. Seed impurity or contamination can cause severe economic damage to the farmers and may lead to lawsuit requiring expert’s investigation in differentiating among the plants or varieties. Traditionally, evaluation was based on the crop’s morphological characters, which are somehow not accurate, and most often could not provide clear identification of the variety. Since the most of horticultural phenotypes were easily influenced by environments of cultivation, and often inherited quantitatively, which makes cultivar identification often complicates among similar cultivars (Liu et al., 2008). With the advances in DNA technology, application of molecular techniques like random amplified polymorphic DNA (RAPD) marker coupled with selfincompatibility typing (SI-typing) allow precise, objective and rapid cultivar identification without any effect of the environment. The RAPD technique, originally developed to distinguish genetic variations of bacteria, plants or even human beings was used in this study. It is done by establishing genomic DNA polymorphism using arbitrary primers (Welsh and McClelland, 1990; Williams et al., 1990). In identifying cultivars, RAPD procedure does not usually require too much genetic or
* Corresponding author. Tel.: +82 31 240 3579; fax: +82 31 240 3594. E-mail address: [email protected] (J.-H. Kwak). 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.08.029
physiological information of the plants. Instead, the plant’s genomic DNA serves as material for analysis and the final results is obtained relatively fast and economical. Self-incompatibility (SI) prevents self-fertilization and promotes out-crossing in hermaphrodite plants (de Nettancourt, 1997). In Brassica crops for instance, SI is sporophytically controlled by a single, highly polymorphic S-locus consisting of a series of multiple alleles namely: the S-locus glycoprotein (SLG), the S-locus receptor kinase (SRK), and the S-locus protein 11 (SP11)/S-locus cys-rich protein (SCR) genes (Nasrallah and Nasrallah, 1993; Takayama and Isogai, 2003). SI technique has been used to produce uniform F1 hybrid seeds while preventing self-pollination between parental inbred lines in a restricted seed production field. Therefore, the genetic information of the resulting F1 seedlings must contain SI-related genetic traits from their parents. Since most Korean seed companies used the SI technique in producing commercial F1 seeds of Brassica crops, application of this concept makes it possible to trace the parents of F1 or identify among varieties. This study used RAPD markers, and SI-typing techniques to identify and confirm the genetic integrity of radish variety; ‘‘Halra Woldong’’ (HW). 2. Materials and methods 2.1. Plant material A mega farm complex located in Wando, Jeollanam-do, S. Korea has been doing a massive planting of winter radish using only one radish variety HW from 2005 to 2006. During this period, it was
J.-H. Kwak et al. / Scientia Horticulturae 119 (2009) 352–356
353
Table 1 List of the 20 Korean radish varieties and their identifications Numbers
Name
Company
Seasonal type
SI type
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Dae Keun Baek Yang Sam Yang Young San Myong San Baek Kyung Pal Gwang Tae Baek Daehyoung Chuseok Baek Ja Gil Cho Seong Gong Young Dong Ha Chu Hannong Yeorum Cheong Gwang Daeburyong Yeorum Hanoldaehyungbom Biandaehyungbom Suji
Nongwoobio Nongwoobio Nongwoobio Dongbu Hannong Syngenta Korea Syngenta Korea Syngenta Korea Heungnong Seminis Korea Seminis Korea Nongwoobio Nongwoobio Nongwoobio Dongbu Hannong Dongbu Hannong Syngenta Korea Seminis Korea Dongbu Hannong Kyungshin Nongwoobio
Autumn radish Autumn radish Autumn radish Autumn radish Autumn radish Autumn radish Autumn radish Autumn radish Autumn radish Autumn radish Spring/Summer radish Summer radish Summer radish Summer radish Summer radish Summer radish Summer radish Spring radish Spring radish Summer radish
S8S17 S26S? S21S30 S4S17 n.d.a n.d.a S8S26 S4S21 n.d.a S8S26 S5S17 S8S8 S1S17 S8S21 n.d.a n.d.a n.d.a n.d.a n.d.a n.d.a
This table is modified from Lim et al. (2006). a n.d. means no data available.
noted that some of the radishes in the field produced stalks/ flowers. Knowing such abnormal behavior of the plants to be seed contamination and/or impurity, the farmer filed a lawsuit against the seed company who supplied the radish seeds. Being the expert team assigned by the Kwangju district court for the case #2006KAKI 54, the team visited the field and performed morphological evaluation of the plantings. Radish cultivars were categorized into two distinct groups based on their morphological characteristics such as: (a) the putative autumn radish variety, which produces stalks/flowers in winter season; (b) the winter radish variety, which does not produce stalks/ flowers even in winter season. The plants, which produced stalks/ flowers, were tagged as the ‘S’ group and those that did not produce stalk/flowers as the ‘N’ group. After proper recording of the observation, fifty radish plants from each group were randomly selected and used as source of leaf samples to be used for molecular evaluation. Leaf samples were collected and brought immediately to the laboratory. During this transportation, they are kept under 4 8C to prevent any DNA degradation. Genomic DNAs of 20 Korean radish varieties were included in the analysis to serve as reference materials (Table 1), and they were kind gifts from Dr. Cho, Kang-Hee at Horticultural Biotechnology Division of National Horticultural Research Institute (Suwon, S. Korea). 2.2. Genomic DNA extraction Genomic DNA of each plant samples was extracted from young radish leaves using DNeasy Plant Kit (Qiagen, Germany) following the manufacturer’s instructions. The genomic DNA from each group was pooled to construct a stalking/flowering (S) and a nonstalking/flowering (N) bulks. These two bulk-samples were used for bulked segregant analysis (BSA) (Michelmore et al., 1991) coupled with the RAPD marker technique. 2.3. Random amplified polymorphic DNA (RAPD) procedure RAPD amplification was performed using 96 random primers obtained from Operon (36 decamers, Operon Technologies, U.S.A.), Wako (36 dodecamers, Wako, Japan), and UBC (24 decamers, University of British Columbia, Canada). The reaction mixture
(25 mL) contained 0.2 mL of Top-Taq DNA polymerase (5 units mL1, CoreBioSystem, Korea), 2.5 mL of Top-Taq PCR buffer (10), 2.0 mL of dNTP mixture (2.5 mM), 1.0 mL of RAPD primer (5 pmol mL1), 0.5 mL of template DNA (50 ng mL1), and 18.8 mL of deionized water. Amplification was carried out in a DNA thermal cycler (PTC200, MJ research. U.S.A.) programmed for an initial denaturation cycle (95 8C for 3 min) followed by 40 cycles of 40 s denaturation at 94 8C, 1 min annealing at 40 8C, and 2 min elongation at 72 8C with a final extension at 72 8C for 5 min. RAPD products were observed on a 1.5% agarose gel with ethidium bromide staining and ultraviolet light irradiation. For the reproducibility check, RAPD profiles were confirmed by three independent assays using three independent DNA extractions. 2.4. Procedure for the SI-typing of radish variety SI-typing of radish varieties was done through the PCR technique using a set of primer pairs for each of the genes. To classify the type of SRK gene, srk3 (50 -GCTTTCATATTACCGGGCATCGATGA-30 ) and srk5 (50 -TGATGAGTTTATGAATGAGGTGA-30 ) primer set was used (Delorme et al., 1995). The SLG gene was classified using a new primer set (50 -ATGAAAGGGGTACAGAACAT30 and 50 -ACGGACCGCTCCTTTGYATTTCAWMACKT-30 ) designed by Dr. Kim, Sunggil at Dongbu Hannong Chemicals. The PCR reaction mixture (25 mL) contained 0.3 mL of Excel-Taq DNA polymerase (5 units mL1, CoreBioSystem, Korea), 2.5 mL of Excel-Taq PCR buffer (10), 2.0 mL of dNTP mixture (2.5 mM), 0.3 mL of each sense/antisense primer (5 pmol mL1), 0.5 mL of template DNA (50 ng mL1), and 19.1 mL of deionized water. Amplification was carried out in a DNA thermal cycler (PTC200, MJ research. U.S.A.) programmed for an initial denaturation cycle (94 8C for 5 min) followed by 35 cycles of 30 s denaturation at 94 8C, 30 s annealing at 55 8C, and 1 min elongation at 72 8C with a final extension at 72 8C for 5 min. The PCR products were observed on a 1.5% agarose gel with ethidium bromide staining and ultraviolet light irradiation. The amplified bands were recovered from the gel using QIAquick Gel Extraction Kit (Qiagen, Germany) following the manufacturer’s instructions. The purified PCR product was sent for DNA sequencing (Genotech, S. Korea). The final sequencing results were analyzed using NCBI BLAST service
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Fig. 1. RAPD band patterns amplified with seven arbitrary primers using ‘S’ and ‘N’ bulks as template DNA. DNA size marker was used in lane 1 and white arrows indicate variety-specific RAPD bands.
(National Center for Biotechnology Information, http://www.ncbi. nlm.nih.gov/). 3. Results and discussion 3.1. Field evaluation of radish plantings From the morphological evaluation of the plants, it was observed that about 9.1% of the radish plantings produced stalks/flowers. According to the farmer, two morphologically different radish plants emerged during the mid-development stage after planting the HW radish variety. Some plants produced stalks/ flowers while the other plants did not produce stalks/flowers. An interesting observation was noted from the ‘S’-group in that all the flowers that were produced are white, while the ‘N’-group was left in the field, not harvested and then eventually produced all purple flowers. 3.2. RAPD analyses of S/N bulks and 20 Korean radish varieties The RAPD analyses of ‘S’ and ‘N’ groups against the 20 Korean radish varieties were conducted using the 96 commercially available RAPD primers that were arbitrary decamers or dodecamers. After RAPD amplifications, each RAPD band pattern was
Name
Sequence
Company
C01 E14 A11 703
TTCGAGCCAG TGCGGCTGAG GATGGATTTGGG CCAACCACCC
Operon, U.S.A. Operon, U.S.A. Wako, Japan UBC, Canada
analyzed on a 1.5% agarose gel (data not shown). Majority of the RAPD primers showed similar RAPD band patterns, but seven primers (C01, C03, C05, E14, A07, A11, and 703) gave distinctive band patterns as shown in Fig. 1. These primers were then used for further RAPD reproducibility test. Concentration of template DNA was the most critical factor to obtain reproducible RAPD bands as well as standardization of each sample concentration. By using 6 template DNA concentration (5, 10, 15, 20, 25, and 50 ng) in preliminary experiments, it was found that the 25 ng condition produced optimum number of reproducible bands. It is reported that 5 ng of template DNA resulted to the maximum number of reproducible bands (Sarkhosh et al., 2006), but in this study, RAPD band did not obtained in some samples by using the 5 ng condition. Although the 25 ng of template DNA did not show the maximum number of reproducible bands, the total number of RAPD bands produced was at optimum level with the 25 ng condition. Finally, four primers were confirmed as true polymorphic RAPD markers by three independent assays using three independent DNA extractions (Table 2). To identify ‘S’ group variety, RAPD band patterns were compared and analyzed between the 20 Korean radish varieties and ‘S’/‘N’ bulks using the four identified primers (C01, E14, A11, and 703). Since the seed company of HW withdrew all their HW seeds from Korean market and did not cooperate for our investigation, the most typical 20 Korean radish varieties were selected as reference varieties without HW variety. Several radish varieties showed similar RAPD band patterns with that of ‘S’ bulk (Fig. 2). Because the Korean radish varieties #2 and #10 showed the highest band pattern similarity with S bulk, the two varieties were further analyzed with 72 RAPD primers, respectively. The summary of the resulting band patterns (Fig. 3) clearly indicate that the RAPD band pattern of ‘B’ bulk (variety #10; Baek Ja radish)
Fig. 2. RAPD band patterns amplified with four arbitrary primers using 20 Korean radish varieties and ‘S’/‘N’ bulks as template DNA (A: primer C01, B: primer E14, C: primer A11, and D: primer 703). DNA size marker was used in each lane 1 and white stars indicate similar RAPD band patterns with that of ‘S’ bulks.
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Fig. 3. RAPD band patterns amplified with arbitrary primers (E1 to 608) using ‘S’ and ‘B’ bulks as template DNA. DNA size marker was used in side lanes. ‘S’: S bulk and ‘B’: Korean variety #10 (Baek Ja variety).
Fig. 4. RAPD band patterns amplified with 4 arbitrary primers using S, N, and B bulks as template DNA. DNA size marker was used in lane 1 and white stars indicate variety-specific RAPD bands.
is exactly the same with that of the ‘S’ bulk. RAPD band patterns were compared among ‘S’, ‘N’, and ‘B’ bulks by the amplifications through the identified four RAPD primers (Fig. 4). All the band patterns of ‘S’ and ‘B’ bulk groups were the same, but not with those of the ‘N’ bulk group as expected. Since the reference genomic DNA of HW radish was not obtainable, it is unable to match the RAPD band patterns of ‘N’ bulk group against those of ‘HW’ bulk. It was only assumed that ‘N’ bulk is a pure HW variety through its morphological features. Based on the results, the expert team made a preliminary conclusion that the variety of ‘S’ bulk could be the Korean variety #10 (Baek Ja radish). The seeds of this Baek Ja variety may have been mixed into the pure HW seed lot. However, it could not be confirmed whether the seed company or the farmer is responsible for the seed mixture. 3.3. Self-incompatibility (SI) type analysis of Korean variety #10, ‘S’, and ‘N’ bulks To confirm the preliminary conclusion, SI-typing technique was employed. In Brassica crops, SI expression is controlled by SLG and SRK genes’ activities, and pollen SI is determined by the expression of SCR/SP11 gene (Delorme et al., 1995; Nasrallah and Nasrallah, 1993; Sakamoto et al., 1998; Shiba et al., 2001; Suzuki et al., 1999;
Fig. 5. SI-typing of ‘S’, ‘N’, and ‘B’ with SRK and SLG gene specific primers. 1 kb ladder was used in side lanes. White arrows indicate successfully amplified PCR products.
Takasaki et al., 2000). To date, more than 100 types of SI related genes were reported and registered at the NCBI database for Brassica oleracea, B. campestris, and Raphanus sativus L. (Lim et al., 2002; Nou et al., 1993; Sakamoto et al., 1998; Suzuki et al., 1999). Once the SI related gene is amplified by PCR using appropriate primers, the PCR product’s sequence could be determined, and then the amplified SI gene’s type could be identified with the NCBI database through their DNA sequences. The SRK and SLG gene-specific primer sets were used to determine SI types of ‘S’, ‘N’, and ‘B’ bulks. The SRK genes were easily amplified by PCR, but not the SLG genes. Hence, after the first PCR amplification of the SLG gene, the product was purified, and then used as the template DNA for the second PCR as a nested PCR. The band patterns of each SI types were confirmed for the two genes (Fig. 5). These PCR products were extracted and purified from the agarose gels, and then sent for sequencing (Genotech, S. Korea). The final sequence results of the amplified SRK and SLG genes were compared with the NCBI database (balstn service with default parameters). There is a great similarity between ‘S’ and ‘B’ bulks but no similarity between ‘N’ and ‘B’ or ‘S’ and ‘N’ bulks (Table 3). The data confirmed the preliminary conclusion that the
Table 3 DNA sequence comparison of ‘S’, ‘N’ and ‘B’ bulks (NCBI Blast service) Samplea
blastn result
S-srk N-srk B-srk S-slg N-slg B-slg
Raphanus Raphanus Raphanus Raphanus Raphanus Raphanus
a
sativus sativus sativus sativus sativus sativus
haplotype haplotype haplotype haplotype haplotype haplotype
S8 srk S1 srk S8 srk S26 slg S4 slg S26 slg
Score (bits)
Identities
Size (bp) compared
1265 1372 1493 1185 1011 1154
673/678 749/760 768/773 627/633 566/585 620/634
700 800 800 651 600 645
S: stalking/flowering plant type; N: non-stalking/flowering plant type; B: Baek Ja radish variety; srk: S-locus receptor kinase; slg: S-locus glycoprotein.
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‘N’ bulk is entirely a different radish variety to the ‘S’ bulk, and that the ‘S’ bulk is a very similar variety to the ‘B’ bulk, which is the Baek Ja radish. From the results of this study, RAPD and SI-typing techniques proved to be useful molecular approaches for investigative applications. Considering that these techniques are very simple and convenient; they are reliable tools that could be used in providing critical court evidence to resolve lawsuits between farmers and seed companies. References de Nettancourt, D., 1997. Incompatibility in angiosperms. Sex. Plant Reprod. 10 (4), 185–199. Delorme, V., Giranton, J.L., Hatzfeld, Y., Friry, A., Heizmann, P., Ariza, M.J., Dumas, C., Gaude, T., Cock, J.M., 1995. Characterization of the S locus genes, SLG and SRK, of the Brassica S3 haplotype: identification of a membrane-localized protein encoded by the S locus receptor kinase gene. Plant J. 7 (3), 429–440. Lim, S.-H., Cho, J., Lee, J., Cho, Y.-H., Kim, B.-D., 2002. Identification and classification of S haplotypes in Raphanus sativus by PCR-RFLP of the S locus glycoprotein (SLG) gene and the S locus receptor kinase (SRK) gene. Theor. Appl. Genet. 104 (8), 1253–1262. Lim, S.-H., Kim, K.-T., Park, S., Cho, H.-J., Park, H.-Y., Hwang, S.-Y., Yoon, M.-K., Mok, I.-G., Woo, J.-G., Oh, D.-G., 2006. Identification of S haplotypes in commercial F1 hybrid cultivars of radish by PCR-RFLP analysis. Korean J. Breed. Sci. 38 (3), 167– 172. Liu, L.W., Zhao, L.P., Gong, Y.Q., Wang, M.X., Chen, L.M., Yang, J.L., Wang, Y., Yu, F.M., Wang, L.Z., 2008. DNA fingerprinting and genetic diversity analysis of latebolting radish cultivars with RAPD ISSR and SRAP markers. Sci. Hortic. 116 (3), 240–247.
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