Identification and Purity Test of Super Hybrid Rice with SSR Molecular Markers

Transcription

1 Rice Science, 2005, 12(1): Identification and Purity Test of Super Hybrid Rice with SSR Molecular Markers XIN Ye-yun 1,2, ZHANG Zhan 2, XIONG Yi-ping 2, YUAN Long-ping 1,2 ( 1 School of Life Science and Technology, Central South University, Changsha , China; 2 China National Hybrid Rice Research and Developmental Center, Changsha , China) Abstract: Five super hybrid rice combinations, i.e. HYS-1/R105, Pei ai 64S/E32, Liangyoupeijiu (Pei ai 64S/9311), 88S/0293, and J23A/Q611, and their parental lines were tested by means of SSR analysis. A total of 144 SSR primer pairs distributed on 12 rice chromosomes were used, out of which 47 detected polymorphism among the tested rice lines. Among all these primers, RM337 and RM154 produced polymorphic patterns in four or more of the tested experimental materials respectively, and they could distinguish among most rice genotypes tested. Twenty-four primer pairs, two on each rice chromosome, were selected to make a reference SSR marker-based fingerprinting for the rice lines. For most of the primer pairs, F 1 hybrids mainly showed complementary pattern of both parents, which could be very useful to distinguish the F 1 from its parental lines. In addition, 5 primer pairs were selected as special primer pairs for five hybrid rice combinations respectively. By combining the rapid, simple method on DNA extraction, it is suggested that SSR technique has wide prospective in variety authentication and purity identification. Key words: SSR; molecular marker; super hybrid rice; identification; purity DNA marker is a new approach based on DNA polymorphism among tested genotypes, and thus applicable to biological research. It offers many advantages over other categories of markers such as morphological, cytological or biochemical markers. For example, DNA marker can cover the whole genome and, therefore, is much larger in quantity. There is more polymorphism in DNA markers, which are able to reveal the variation and allelism. Many DNA markers are co-dominant and can differentiate between the homozygous and heterozygous genotypes. Furthermore, DNA markers are neutral, and they have no effect on phenotype, no epistatic effect, and are not influenced by environmental conditions and developmental stages. Therefore, DNA marker is simple, quick, less environmentally conditioned, and experimentally reproducible well. It has been applied widely in the identification, registration of plant variety, and in monitoring of the seed purity and the authenticity with high accuracy, high reliability and low cost [1-3]. At present, the main DNA markers are RFLP, RAPD, AFLP and SSR [4-6]. SSR has much more polymorphism than most of other DNA markers, and is co-dominant and large in quantity. Therefore, SSR has become an ideal molecular marker in identification of plant variety [7-12]. Yu et al [13] established the DNA fingerprint of Ning 2A and Ning Received: 1 January2005; Accepted: 9 March B with SSR markers, and differentiated the two parental lines from other rice varieties by using two SSRs. Zhan et al [14] tested six hybrid combinations and their parents by using 178 SSRs, of which 52 showed stable polymorphic patterns in one or more hybrid combinations, and two SSRs could be used to detect the purity of V46 and Jinyou 207 F 1 seeds. Peng et al [15] selected 26 SSR markers on 12 rice chromosomes to detect nine major hybrid rice combinations and their parents. They found that all of the restorer lines and most of the sterile lines could be distinguished as well as the purity of each single seed of Shanyou 63 and Liangyoupeijiu could be monitored effectively. The result of seed purity from SSR marker analysis was very close to that from field purity test. This study was initiated to identify the pioneer super hybrid rice combinations, together with their parental lines developed most recently, using SSR markers, to provide the DNA fingerprint for these rice hybrids and their parental lines, and to establish the basis for identification and monitoring of seed purity for these hybrid rice combinations. MATERIALS AND METHODS Plant materials Five super hybrid rice combinations were selected for this study: HYS-1/R105, Pei ai 64S/E32,

2 8 Rice Science, Vol. 12, No. 1, 2005 Liangyoupeijiu (Pei ai 64S/9311), 88S/0293, and J23A/Q611. In addition, the nine parental lines for the above five combinations were also included, i.e. HYS-1, R105, Pei ai 64S, E32, 9311, 88S, 0293, J23A and Q611. Among the super hybrid rice combinations tested, two pioneer super hybrids, Liangyoupeijiu and Pei ai 64S/E32, reached the yield goal (10.5 t/ha) of the first phase super rice program set by Ministry of Agriculture of China in Especially, Pei ai 64S/E32 created a record yield of 17.1 t/ha in Yongsheng County, Yunnan Province in 1999, and this combination has been planted recently in China over 1.3 million ha each year. A new combination 88S/0293 yielded 12 t/ha on eight locations, with 6.7 ha for each, during and, therefore, become a promising combination for the second phase of the development of super hybrid rice. The yields of another two hybrids HYS-1/R105 and J23A/Q611 were t/ha, and these two hybrids were considered to have great yield potential as first and second rice crop, respectively. DNA extraction Three methods were used to extract total genomic DNA, i.e. FastDNA Kit, CTAB, and Acid & Alkali DNA preparation. The FastDNA Kit from BIO 101, Inc. was used following the manufacturer s instructions. The CTAB protocol was practiced as follows: about 0.1 g of young leaf tissue for each sample was homogenized in liquid nitrogen, and incubated at 60 for min with 500 µl of CTAB buffer (100 mmol/l ph 8.0 Tris-HCL,1.4 mol/l NaCl, 20 mmol/l EDTA, 1% PVP-360). Then 500μL 24:1 of chloroform:isoamyl alcohol mixture was added and blended thoroughly for 5 min. After centrifugation (5 min, r/min at 4 ), aqueous layer was pipetted into a new eppendorf tube and an approximately equal volume of cold isopropanol was added. After storage at -20 for min, precipitated DNA was centrifuged followed by washing with 70% ethanol, vacuum drying, and final storage in double distilled water (ddh 2 O). Acid & Alkali method [14] was also used for DNA extraction with modifications. 40 µl of ddh 2 O was added to 1.5 ml Eppendorf tube containing one shoot grown at 37 and cut into pieces. Then the sample was incubated at 100 for 5 min. After 40 µl of NaOH (0.25 mol/l) was added and heated at 100 for exactly 30 s, 80 µl of HCl (0.5 mol/l) and 40 µl of Tris buffer (0.5 mol/l, ph 7) was added for neutralization. The mixture was placed at 100 for 2 min. After centrifugation, 3 5 µl of aqueous DNA sample was ready for one PCR reaction. PCR amplification One hundred and forty-four SSR primer pairs (Proligo LLC.) were selected in this study. The volume of the reaction mixture was 25 µl which consisted of 1 µl (20 ng) DNA sample, 0.25 µl (40 µmol/l) primers, 2.5 µl 10 PCR buffer, 0.5 µl dntps (10 mmol/l), 2 µl MgCl 2 (20 mmol/l), 0.1 µl Taq (5 U/ µl) and µl ddh 2 O. The PTC-200 Peltier Thermal Cycler was used and programmed for 35 cycles of 94 (1 min), 55 (1 min), 72 (2 min), then followed by post-extension at 72 for 10 min. PCR products ( µl) were used for electrophoresis on 2% agarose gels stained with ethidium bromide at V/cm for min, and photographed under UV light using Gel Doc 2000 system (BIO RAD, Gel Doc 1000). RESULTS Different methods for DNA extraction For three methods of total DNA extraction in this study, FastDNA Kit consumed comparatively less time (only 3 4 hours) and produced high quality DNA. Especially with the yellow seedling grown in dark at 37 for 4 to 6 days, better results of PCR amplifications could be obtained with this method. Clear bands could be observed using CTAB method even though it involves more steps. The simplest method, acid & alkali DNA preparation, also could produce clear bands if DNA was extracted within only 30 min and PCR was performed in a timely manner. SSR analysis This study selected 144 SSR primer pairs on 12 rice chromosomes to genotype experimental materials.

3 XIN Ye-yun, et al. Identification of Super Hybrid Rice with SSR Molecular Marker 9 The result showed that 109 primer pairs (75.7%) had DNA amplifications, in which 47 (32.6%) primers showed stable polymorphism. The rest of the primers failed to amplify DNA in one PCR condition, however, could still amplified DNA bands among the materials tested if the PCR conditions were modified. Of all primers pairs used in this study, RM337 and RM154 produced highly polymorphic patterns in four or more of the tested experimental materials. It demonstrated that RM337 amplified five bands with size of 170, 190, 250, 440 and 530 bp among different hybrids and their parents. Banding patterns for RM154 displayed high polymorphism, with four indicative bands of 160, 180, 190 and 200 bp in addition to the non-specific band. As a result, five hybrid rice combinations and their parental lines could be distinguished easily by using only these two primer pairs (RM 337 and RM154). It was found that the most of SSRs showed polymorphisms in two or more experimental materials. In theory, the more polymorphic primers, the more accurate in plant identification. Therefore, 24 primer pairs (two on each of the 12 rice chromosomes) created 78 bands of different sizes (3.25 bands per marker) and, thus were selected as the critical primers for fingerprinting these hybrid combinations and their parental lines. This fingerprinting study could be used in variety authentication, registration, and plant variety protection through pedigree analysis and linkage analysis for various traits such as yield. Application of SSR markers in monitoring of seed purity In order to monitor the seed purity of the two-line hybrids, the following two factors have to be considered: sterile line selfing affected by low temperature, and cross-pollination or mechanical mixture. For most SSRs F 1 hybrids showed complementary banding pattern of both parents. It was valuable to distinguish the F 1 from their male and female parents. As shown in Table 1, this study screened SSRs for individual hybrid combination and their parental lines. RM337 RM bp 440 bp 250 bp 190 bp 170 bp 200 bp 190 bp 180 bp 160 bp M M Fig. 1. Amplification polymorphism of primers RM337 and RM154. Lane 1, HYS-1; Lane 2, R105; Lane 3, HYS-1/R105; Lane 4, Pei ai 64S; Lane 5, E32; Lane 6, Pei ai 64S/E32; Lane 7, 9311; Lane 8, Liangyoupeijiu (Pei ai 64S/9311); Lane 9, 88S; Lane 10, 0293; Lane 11, 88S/0293; Lane 12, J23A; Lane 13, Q611; Lane 14, J23A/Q611. Table 1. The polymorphic primers for the hybrids and their parents. Hybrids Polymorphic primers HYS-1/R105 RM545, RM251, RM519, RM218, RM250, RM565, RM551, RM206, RM337, RM219, RM202, RM21, RM286, RM587, RM505 Pei ai 64S/E32 RM34, RM164, RM585, RM251, RM429, RM519, RM154, RM520, RM565, RM234, RM337, RM152, RM264, RM286, RM21, RM304, RM587, RM219 Liangyoupeijiu RM34, RM251, RM585, RM429, RM258, RM519, RM154, RM472, RM520, RM565, RM234, RM337, RM152, RM219, RM206 88S/0293 RM154, RM251, RM34, RM520, RM505, RM234, RM337, RM519, RM250 J23A/Q611 RM154, RM587, RM585, RM295, RM337, RM202, RM481, RM106

4 10 Rice Science, Vol. 12, No. 1, 2005 RM246 RM bp RM154 RM250 RM251 RM565 RM551 RM252 RM164 RM26 RM587 RM585 RM234 RM429 RM337 RM152 RM219 RM205 RM258 RM228 RM286 RM202 RM19 RM519 M M Fig. 2. Amplication results of 24 primer pairs (two on each of the 12 rice chromosomes). Lane 1, HYS-1; Lane 2, R105; Lane 3, HYS-1/R105; Lane 4, Pei ai 64S; Lane 5, E32; Lane 6, Pei ai 64S/E32; Lane 7, 9311; Lane 8, Liangyoupeijiu (Pei ai 64S/9311); Lane 9, 88S; Lane 10, 0293; Lane 11, 88S/0293; Lane 12, J23A; Lane 13, Q611; Lane 14, J23A/Q611.

5 XIN Ye-yun, et al. Identification of Super Hybrid Rice with SSR Molecular Marker 11 Primer pair of RM250 was used for identification of the purity with single seed of HYS-1/R105 (Fig. 3). In addition, four other SSRs were selected for a specific hybrid rice combination, i.e. RM 250 for HYS-1/R105, RM 337 for Pei ai 64S/E32 and J23A/Q611, RM 429 for Liangyoupeijiu, RM228 for 88S/0293. DISCUSSION This study revealed that RM337 and RM154 among 144 SSRs showed high polymorphism and allowed us to create the fingerprints of five super hybrid rice combinations. Twenty-four SSRs were selected as an effective set of markers for this fingerprinting job. Based on the complementary banding patterns between the hybrids and their parents, feasible methods were developed to monitor the seed purity of different hybrid rice combinations. Together with the quick and simple method for DNA extraction, SSR analysis was proved to be a promising approach in variety authentication and purity monitoring. This marker technology will provide corporate managers and breeders with legal evidence of commercial hybrid seeds in the seed market and help to protect plant proprietary rights of the new super hybrid rice combinations. In this study, the following results were also observed: 1) An extra band was often observed in addition to the DNA bands from the two parental lines. 2) In our study it was difficult to find a SSR marker to differentiate between 9311 and These two M Fig. 3. Primer pair of RM250 was used for identification of the purity with single seed of HYS-1/R105 (DNA extracted by a simple, rapid method with Acid & Alkali [14] ). parental lines might be close in their pedigrees. 3) Promising results were often achieved such as clear bands, under the electrophoretic conditions: V/cm and 2.5 % EB staining. ACKNOWLEDGEMENTS The author would like to thank the help from Dr. Li Jiming for manuscript modification. The whole experimental work was supported by AoE Project, The Chinese University of Hong Kong. REFERENCES 1 Zhao Z Q, Zheng H L, Zhang C G. Development of molecular markers and their application in botany research. Fujian Sci&Technol Trop Crops, 2000, 25(4): (in Chinese with English 2 Fang X J, Liu S H, Jiang S Y. Progress on identification of seed purity and authenticity using DNA molecular markers. J Agric Biotech, 2000, (2): (in Chinese with English 3 Liang M S, Zeng Y, Zhou X, Hou L J, Li X. Genetic markers and their applications in identifying crop cultivars. Chinese Bull Bot, 2001, 18(3): (in Chinese with English 4 Williams J G, Kubelik A R, Livak K J, Rafalski J A, Tingey S V. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl Acids Res, 1990, 18(22): Chen H, Chen M L, Zhu L H, Qian Q, Min S K. Purity identification of hybrid rice Shanyou 63 by RAPD method. Chinese Sci Bull, 1996, 41(9): (in Chinese) 6 Chen Y H, Jia J H, Li C Y, Jin D M, Wang B. Rice seed ientification by computerized AFLP-DNA fingerpringting analysis. In: Prospects on Rice Genetics and Breeding for the 21 st Century International Rice Genetics and Breeding Symposium. China Agricultural Science and Technology Press, (in Chinese with English 7 Panaud O, Chen X, McCouch S R. Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.). Mol Gen Genet, 1996, 252(5): McCouch S R, Chen X, Panaud O, Temnykh S, Xu Y, Cho Y G, Huang N, Ishii T, Blair M. Microsatellite marker development, mapping and applications in rice

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