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Identification of 205 Current Rice Cultivars in Japan by Dot-Blot-SNP Analysis

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Identification of 205 Current Rice Cultivars in Japan by Dot-Blot-SNP Analysis Breeding Science 60: 447–453 (2010) doi:10.1270/jsbbs.60.447 Note Identification of 205 current rice cultivars in Japan by dot-blot-SNP analysis Hideki Sato1), Takashi Endo1,3), Sachiko Shiokai2), Takeshi Nishio2) and Masayuki Yamaguchi*1,4) 1) National Agricultural Research Center for Tohoku Region, Daisen Research Station, 3 Shimofurumichi, Yotsuya, Daisen, Akita 014- 0102, Japan 2) Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya-machi, Tsutsumidori, Aoba, Sendai, Miyagi 981-8555, Japan 3) Present address: Miyagi Prefectural Furukawa Agricultural Experiment Station, 88 Fukoku Furukawa, Ousaki, Miyagi 989-6227, Japan 4) Present address: National Agricultural Research Center for Tohoku Region, 4 Akahira, Shimo-Kuriyagawa, Morioka, Iwate 020-0198, Japan Using 77 single-nucleotide polymorphic (SNP) markers and dot-blot analysis, we examined 218 rice cultivars, respectively occupying 99% and 92% of the planted areas of non-glutinous and glutinous rice for three con- secutive years from 2003 to 2005 in Japan. Among them, 205 cultivars were identified at one time by the genotypes of 18 markers, but 13 cultivars belonged to six groups in which cultivars were indistinguishable from each other. The 205 cultivars were individually distinguished from the others using combinations of up to six markers. This result was considered to be useful for the identification of Japanese commercial rice cul- tivars, monitoring the contamination of rice with other cultivars, and rice breeding using these cultivars. Key Words: rice, identification of rice cultivar, SNP, dot-blot. Introduction powerful tools for genetic analysis, since they are distributed over the rice genome at a very high frequency and the poly- As rice cultivars are discriminated by commercial brands, morphism is preserved firmly in alternate generations. Various and the demand for cultivar identification has increased in methods have been used to detect SNPs: DNA sequencing order to prevent rice seed contamination, reports on the iden- (Kwok et al. 1994), MALDI-TOF mass spectrometry (Haff tification of rice cultivars using DNA markers have become and Smirnov 1997), denaturing capillary electrophoresis more common (Akagi 2000, Ogasawara and Takahashi (Wenz et al. 1998), cleaved amplified polymorphic sequence 2000, Shinmura et al. 2005). Although genetic polymor- (CAPS) analysis using mismatched polymerase chain reac- phism between japonica and indica rice cultivars is easily tion (PCR) primers (Michaels and Amasino 1998), single- detected at a high frequency by DNA markers, the frequency strand conformation polymorphism (SSCP) analysis (Gonen among japonica cultivars is very low (Kono et al. 2000). Ac- et al. 1999, Kozlowski and Krzyzosiak 2001, Tahira et al. cording to the genealogy of current rice cultivars, many were 2002), allele-specific PCR by 3′ locked nucleic acid primer derived from crossing with cultivar ‘Koshihikari’, which is (Latorra et al. 2003), mismatch cleavage analysis using var- the most popular and the most-grown cultivar in Japan. ious nucleases (Till et al. 2004), and microarray technology ‘Hitomebore’, ‘Akitakomachi’ and ‘Hinohikari’ which are (Wang et al. 2005). Among them, dot-blot analysis can de- derived from crossing with ‘Koshihikari’, and are the second, tect SNPs at relatively low initial investment. After PCR, the third and fourth most-grown in Japan, are reported to exhibit process requires an incubator, which is standard equipment 80.8%, 80.0% and 61.3% of the ‘Koshihikari’ genome, re- in most laboratories. The dot-blot method is more effective spectively (Yamamoto et al. 2010). Therefore, it was as- when treating a large number of samples. Furthermore, since sumed that many of the cultivars were closely related to this method is conducted via specific PCR and hybridiza- each other, and thus difficult to be distinguished. Single- tion, it possesses higher reliability. nucleotide polymorphism (SNP) markers are considered Polymorphisms used to be difficult to detect with dot-blot analysis using SNP markers (hereafter referred to as “dot- Communicated by M. Yano blot-SNP analysis”) because of mismatch hybridization of Received August 10, 2010. Accepted October 14, 2010. the oligonucleotides used as probes. However, this problem *Corresponding author (e-mail: [email protected]) was effectively mitigated by the addition of competitive 448 Sato, Endo, Shiokai, Nishio and Yamaguchi oligonucleotides in the hybridization mixture (Shirasawa et tinous): ‘Koshihikari’-‘Yumegokochi’-‘Saga1’, ‘Hinohikari’- al. 2006). In this way, the identification of a single nucle- ‘Morinokumasan’, ‘Hitomebore’-‘Satonouta’, ‘Dewasansan’- otide substitution, insertion or deletion has become possible, ‘Ginginga’, ‘Koganemochi’-‘Miyakoganemochi’ and and this method is considered to be useful for genetic analy- ‘Shigahabutae-mochi’-‘Shin-habutaemochi’. Although sis. In this study, we used dot-blot-SNP analysis to identify ‘Satonouta’ was distinguished from ‘Hitomebore’ by head- the non-glutinous and glutinous rice cultivars that are grown ing date and ‘Saga1’ had significantly shorter culm length almost all rice fields in Japan. than ‘Koshihikari’-‘Yumegokochi’, we were unable to dis- tinguish cultivars in the other four groups by observation in Rice cultivars the field (data not shown). This study used 172 non-glutinous and 46 glutinous rice cultivars, planted on over 50 ha in Japan. These cultivars Specification of one rice cultivar from others were chosen from the list of rice cultivars compiled in 2003 We also determined the smallest number of SNP markers by the General Food Policy Bureau of the Ministry of Agri- that are needed to distinguish a certain rice cultivar from culture, Forestry and Fisheries. The rice cultivars used in others. Using five markers, all of the cultivars were distin- this study respectively occupied 99% and 92% of the planted guished from the others used in this study, except for the cul- areas of non-glutinous and glutinous rice over three consec- tivar ‘Hitomebore’, which required six markers. utive years from 2003 to 2005 in Japan. Table 2 shows an example of the identification of the cul- tivar ‘Akitakomachi’. Only ‘Akitakomachi’ represents a PCR and dot-blot analyses using SNP markers variant type in the five SNP markers, R2382, S3010, Genomic DNA of the 218 cultivars was extracted from C30024, S13781 and NK10. Only one SNP marker was fresh rice leaves according to Thomson and Henry (1995) needed to identify each of the cultivars ‘Milky queen’, and used for PCR as described below. DNA fragments in- ‘Shintaishomochi’ and ‘Takasagomochi’: namely, Wx-mq, cluding SNPs were amplified by PCR. The PCR primers and S10045 and Ehd1, respectively. Thus, the results of dot-blot probes are listed in Supplemental Table 1. Most of the dot- analysis can confirm whether a picked cultivar is truly the blot analysis procedures were conducted according to specified one; we were able to distinguish one cultivar from Shirasawa et al. (2006). Ten microliters of the PCR reaction the others using a small number of SNP markers. mixture contained 2 × Green GoTaq® DNA polymerase Yamamoto et al. (2010) applied large amounts of SNPs (Promega KK, USA), 0.2 μM of primer and 10–100 ng of found between ‘Koshihikari’ and ‘Nipponbare’ to 151 repre- DNA. PCR was conducted as follows: 1-min pre-denaturation sentative Japanese cultivars grown during the past 150 years at 94°C; 40 cycles of 30-sec denaturation at 94°C, 30-sec and revealed the dynamics of the genome composition. In annealing at 58°C, 30-sec extension at 72°C, and 1-min final the present study, we used 218 current cultivars that were extension at 72°C. The PCR products were alkali-denatured grown in almost all rice fields in Japan in 2003–5, and could with 8.4 μl of 25 mM EDTA and 1.6 μl of 5 N NaOH, and identify 205 cultivars by 77 SNP markers. This result will be dot-blotted on a nylon membrane using a Multi-pin Blotter useful as a dataset for the discrimination of Japanese com- (ATTO Corporation, Japan). The hybridization temperature, mercial rice cultivars, monitoring the contamination of rice washing temperature, and concentration of the washing with other cultivars in Japan, and rice breeding involving buffer are shown in Supplemental Table 1. DIG- or biotin- crosses of the rice cultivars examined in this study. labeled probes were detected by a chemiluminescent reac- tion following the protocol provided by Roche Diagnostics Acknowledgments K. K. (Switzerland). We are grateful to the National Institute of Agrobiological Rice cultivar identification by dot-blot-SNP analysis Sciences Genebank, National and Prefectural Agricultural Using 77 previously developed SNP markers (Shiokai et Research Centers and Agricultural Experiment Stations, al. 2010, Shirasawa et al. 2006), we examined 218 rice cul- Japan Tobacco Inc., Nakajima Yoshio Shoten Co., Ltd. and tivars (172 non-glutinous and 46 glutinous rice cultivars) by Mr. Jiro Ozeki for providing seeds of the rice cultivars. This dot-blot analysis (Supplemental Table 2). Out of the 218 work was supported in part by the Research Project for Uti- total cultivars, 205 were distinguishable using the 77 SNP lizing Advanced Technologies in Agriculture, Forestry, and markers. The 18 markers shown in Table 1 were the lowest Fisheries. number necessary to distinguish all 205 cultivars from each other at one time. Among non-glutinous and glutinous culti- vars, the smallest numbers of markers needed for the identi- fication at one time were 18 and 8,
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