ASSESSMENT OF PURITY OF RICE crop. It is estimated that for every 1% impurity in the hybrid seed, the yield reduction is 100 kg per hectare HYBRIDS USING MICROSATELLITE (Mao et al., 1996). Purity of hybrid seed lots is assayed AND STS MARKERS conventionally by a GOT on a representative sample of the seed that is to be marketed. The GOT involves J. Yashitola, T. Thirumurugan, R. M. Sundaram, growing plants to maturity and assessing several mor- M. K. Naseerullah, M. S. Ramesha, N. P. Sarma, phological and floral characteristics that distinguish and Ramesh V. Sonti* the hybrid. In China, hybrid rice seeds are generally harvested Abstract in late August to early September and a GOT is con- The estimation of hybrid rice seed purity is done conventionally ducted on a representative sample from each seed lotby the grow out test (GOT), which is based on the assessment of in Hai-nan island, which is a natural winter nurserymorphological and floral characteristics in plants grown to maturity. (Yan, 2000). The seeds are contracted for distributionFor seed companies, large amounts of capital are locked up in the by the end of the year, at which time the seed qualityform of hybrid seed stock while awaiting the results of the GOT. data are not available. Legal disputes can arise if it turnsWith the objective of replacing the GOT with DNA based assays, out that the contracted seed do not have the requiredcytoplasmic male sterile (CMS), restorer, and hybrid lines have been level of purity (Yan, 2000).screened by means of microsatellite and sequence tagged site (STS)polymorphisms. A simple procedure for detecting heterozygosity and In India, hybrid seed production is generally takenpurity has been standardized and uses 6-d-old rice (Oryza sativa L.) up in the dry (rabi) season (January–April) because theseedlings, which could be used for detection of off-types in hybrid weather conditions (low humidity, lack of rainfall at theseed lots. The extent of heterozygosity within parental lines of rice time of flowering, etc.) are congenial for production ofhybrids was assessed and the results suggest that a single, appropriately good quality seeds (Vijaykumar, 1996). Hybrid rice ischosen microsatellite marker should be sufficient for assessing hybrid generally cultivated in the wet (kharif) season (June–seed purity. October), which coincides with the South-West mon- soon that brings rainfall to large parts of the country. The hybrid seed produced is not used immediately forR ice is the staple food for a large segment of the Asian population. It has been estimated that riceproduction in India as well as several other Asian coun- raising the crop because it is necessary to check for the purity by GOT in the succeeding season. This entails a lot of cost in terms of locked-up capital and attendanttries must double by the year 2025 to meet the require- problems of storage. Moreover, GOT can be subjective;ments of the increasing population (Hossain, 1996; Par- several aspects of plant phenotype (morphology, yield,oda, 1998). As successfully demonstrated in the People’s etc.) can be affected by environmental conditions. Fur-Republic of China, hybrid rice technology appears to ther, there is also the possibility that adverse climaticbe a feasible and readily available option for raising the conditions (like heavy rain or wind) can damage oryield potential. In China, the area planted to hybrid rice destroy the crop and make it difficult to collect data.is around 15 million hectares, which constitutes about Thus, there is a need for an assay to assess genetic purity50% of the total rice area (Jirong, 2000). Besides China, of hybrid seeds that is both accurate and faster, so seedrice hybrids are being grown successfully in India, Viet- produced in the dry season can be released for commer-nam, and the Philippines. Many other rice growing coun- cial cultivation in the ensuing wet season. DNA-basedtries in Asia and Latin America are also expected to markers can be applied for this purpose because theyadopt hybrid rice technology soon. In 1997, about 3000 can be used for assessing precisely the genotype of aMg of hybrid seed was produced in India and the area plant.planted to hybrid rice was around 120 000 hectares In rice, microsatellites are abundant and well distrib-(Krishnaiah et al., 1998). It is anticipated that the area uted throughout the genome (Akagi et al., 1996; Mc-under hybrids in India will increase substantially and Couch et al., 1996, 1997; Wu and Tanksley, 1993). Theycontribute towards increasing rice production. are valuable as genetic markers because they are codom- In a self-pollinated crop like rice, one of the chal- inant, detect high levels of allelic diversity, and are as-lenges is the production and supply of adequate quanti- sayed efficiently by the polymerase chain reactionties of pure hybrid seed to the farmers. Maintenance of (PCR) (McCouch et al., 1997). The current level ofhigh level of genetic purity of hybrid is essential to average genome-wide coverage provided by microsatel-exploit the moderate level of heterosis observed in this lites in rice, one marker every 6 centimorgans (Temnykh et al., 2000), is sufficient to be useful for assessment ofJ. Yashitola and Ramesh V. Sonti, Centre for Cellular and Molecular hybrid seed purity and for genotype identification. TheBiology, Uppal Road, Hyderabad-500 007, India; T. Thirumurugan,R.M. Sundaram, M.S. Ramesha, and N.P. Sarma, Directorate of Rice use of microsatellite markers for assessing seed purityResearch, Rajendranagar, Hyderabad-500030, India; M.K. Naseerul- is already reported to be routine among tomato seedlah, E.I.D. Parry (India) Limited, Devanahalli Road, Off Old Madras producers (Smith and Register, 1998).Road, Bangalore-560 049, India. Received 10 Apr. 2001. *Correspond-ing author (firstname.lastname@example.org). Abbreviations: GOT, grow out test;CMS, cytoplasmic male sterile;Published in Crop Sci. 42:1369–1373 (2002). PCR, polymerase chain reaction; STS, sequence tagged site. 1369
1370 CROP SCIENCE, VOL. 42, JULY–AUGUST 2002 Similar to microsatellites, an STS is a short stretch of Table 1. Rice lines analyzed in this study.genomic sequence that can be detected by PCR and is Parental and hybrid lines Developed atmapped to a specified site as a landmark in the genome. CMS linesIn this paper, we have screened several microsatellite IR58025A International Rice Researchand STS markers to identify polymorphisms that distin- Institute (IRRI), Manila,guish certain CMS, restorer, and hybrid lines of rice. the Philippines IR62829A IRRI, Manila, the PhilippinesThe usefulness of these polymorphisms for determining Restorer lineshybrid seed purity is reported. MTU9992 Agricultural Research Station (ARS), Maruteru, India IR40750 IRRI, Manila, the Philippines Materials and Methods C2OR Tamil Nadu Agricultural University (TNAU),Rice Lines Coimbatore, India KMR3 University of Agricultural Two CMS lines, IR58025A and IR62829A, and six restorer Sciences (USA), V. C.lines, MTU9992, IR40750, C20R, KMR3, Ajaya, and BR827- Farm, Mandya, India Ajaya Directorate of Rice Research35, of rice and their hybrids in a set of six combinations (Table (DRR), Hyderabad, India1) were analyzed in this work. BR827-35 IRRI, Manila, The Philippines Hybrids†DNA Isolation APRH2 (IR62829A ϫ MTU9992) ARS, Maruteru, India DRRH1 (IR58025A ϫ IR40750) DRR, Hyderabad, India Genomic DNA was isolated, as per the protocol of Kochert CORH2 (IR58025A ϫ C2OR) TNAU, Coimbatore, Indiaet al. (1989), from leaves of 18- to 20-d-old rice plants grown KRH2 (IR58025A ϫ KMR3) UAS, V. C. Farm, Mandya, Indiain the greenhouse and used in a microsatellite and STS poly- CNRH3 (IR6289A ϫ Ajaya) Rice Research Station,morphism survey of parental and hybrid lines. For single seed- Chinsurah, India.ling assays to estimate hybrid seed purity, seeds were germi- Sahyadri (IR58025A ϫ BR827-35) Regional Agricultural Research Station, Karjat,nated at 32ЊC in the dark on moistened filter paper in Petri Indiadishes. Six-day-old seedlings were separated from the endo- Otherssperm with a forceps and homogenized with a pestle in a 1.5- T(N)1 Introduced from Taiwan andmL tube containing 200 L of extraction buffer made up of maintained at DRR, Hyderabad, India5% (w/v) Chelex-100 (Bio-Rad Laboratories, USA) in steriledistilled water (Chunwongse et al., 1993). The homogenate † The CMS and restorer lines from which the hybrids were obtained arewas incubated at 95ЊC for 10 min and pelleted in a microcentri- indicated in parentheses.fuge. The supernatant (ෂ50 L) contained enough DNA (datanot shown) for at least 10 PCRs performed as described below. 1 min at 55ЊC, 2 min at 72ЊC, and 5 min at 72ЊC for final extension (RM markers); 35 cycles of 30 s at 94ЊC, 30 s at 55ЊC, and 1 min at 72ЊC (OSR markers). For STS markers,PCR Amplification and Detection of Polymorphisms the PCR profile was 35 cycles of 1 min at 94ЊC, 1 min at 55ЊC, A total of 13 oligonucleotide primer pairs flanking microsa- and 2 min at 72ЊC (pTA248); 35 cycles of 1 min at 93ЊC, 1 mintellite repeat sequences were used in the present study. Six at 57ЊC, and 2 min at 72ЊC (RG235 and RG365); 30 cycles ofprimer pairs (RM1, RM8, RM9, RM10, RM19, and RM21) 1 min at 94ЊC, 1 min at 58ЊC, and 4 min at 72ЊC (F8); 30 cycleswere chosen from the published sequences of Panaud et al. of 1 min at 94ЊC, 1 min at 52ЊC, and 2 min at 72ЊC (F43). For(1996); three primer pairs (RM122, RM164, and RM203) were assessing genetic purity of seedlings, PCRs were carried outselected from the sequences of Wu and Tanksley (1993); one as above with the exception that 0.6 U of Taq Polymeraseprimer pair (RM206) was chosen from Chen et al. (1997) and and 5 L of single seedling extracted DNA samples werethree primer pairs (OSR2, OSR17, and OSR28) were from used in 15-L reaction volumes. PCR amplified products wereAkagi et al. (1996). All the sequences targeted by the 13 resolved on 2 or 3% (for RM loci), 3% (for OSR loci), orprimers, except OSR17, have been mapped previously (Akagi 1.2% (for STS markers) agarose gels, stained with ethidiumet al., 1996; Chen et al., 1997; Panaud et al., 1996; Wu and bromide and visualized under UV.Tanksley, 1993). RM1, RM9, and OSR2 on chromosome 1,RM8 on chromosome 2, RM203 on chromosome 3, RM122and RM164 on chromosome 5, RM10 on chromosome 7, Results and DiscussionOSR28 on chromosome 9, RM21 and RM206 on chromosome Microsatellite and STS Polymorphisms in Parental11, and RM19 on chromosome 12. For STS markers, five and Hybrid Lines of Riceoligonucleotide primer pairs namely pTA248 (chromosome11; Ronald et al., 1992), F8 (chromosome 2; Nair et al., 1995), Genomic DNA was isolated from two CMS lines, sixF43 (chromosome 8; Nair et al., 1996), RG235 and RG365 restorer lines and six hybrids (Table 1). All six hybrids(chromosomes 12 and 2 respectively; Ghareyazie et al., 1995) are in commercial cultivation at various locations inwere used. India. Thirteen microsatellite and five STS markers In polymorphism surveys, DNA samples (50 ng) were am- were used in the analysis of these lines. Summarizedplified in 25-L reaction volumes containing 1ϫ PCR buffer results of these studies are given in Table 2. As expected,[10 mM Tris.HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01%( v/v) gelatin] (Perkin-Elmer, USA), 0.2 mM of each dNTPs only one allele was detected in a hybrid when the parents(Amersham Pharmacia Biotech, Sweden), 10 pmol of each were monomorphic for a particular microsatellite–STSprimer and 1U of Taq polymerase. Samples were overlaid locus and two alleles (one allele per parent) were pres-with mineral oil and PCR was carried out in a Thermal cycler ent in a hybrid when polymorphism was detected be-(Perkin-Elmer-480, USA). For microsatellite markers, the ba- tween the CMS and restorer lines (see Fig. 1 for a repre-sic PCR profile was 5 min at 94ЊC, 35 cycles of 1 min at 94ЊC, sentative example of one microsatellite and one STS
NOTES 1371Table 2. Frequency of heterozygosity at microsatellite and STS of Rice Research (DRR) and another from a seed-lot loci in rice hybrids.† of the National Seeds Corporation (NSC), which is in- Frequency of heterozygosity volved in the multiplication and marketing of the Rice varieties Microsatellite STS DRRH1 hybrid. Genomic DNA was isolated from 50P1 P2 H markers markers seedlings of the DRRH1 hybrid from each of the aboveIR62829A MTU9992 APRH2 2/13 0/5 two separate sources and PCR analysis was performedIR58025A IR40750 DRRH1 3/13 1/5 by means of the RM164 microsatellite marker. FigureIR58025A C2OR CORH2 3/13 0/5IR58025A KMR3 KRH2 2/13 2/5 2 indicates that some of the seeds obtained from theIR62829A Ajaya CNRH3 5/13 1/5 DRR farm are off types. Three out of 50 seeds analyzedIR58025A BR827-35 Sahyadri 1/13 3/5 from this source were off types, while no off types were† A total of 13 microsatellite loci and five STS loci were analyzed as observed amongst the other set of 50 seeds from the described in Materials and Methods; P1 ϭ CMS line; P2 ϭ Restorer NSC. In PCR analysis with the pTA248 marker, the line; H ϭ Hybrid. three seeds identified as off types by means of RM164polymorphism). Of the 13 microsatellite loci analyzed, were confirmed to be off types and all other seeds werefive (RM1, RM19, RM21, RM164, and RM206) were confirmed to be hybrids (data not shown). The confir-polymorphic and eight (RM8, RM9, RM10, RM122, RM mation of genotypes predicted by one marker with anal-203, OSR2, OSR17, and OSR28) were monomorphic ysis by an unlinked marker (RM164 and pTA248 mapfor the lines that were screened. Of the five STS loci to chromosomes 5 and 11, respectively; Wu and Tank-analyzed, three (pTA248, F8, and F43) were polymor- sley 1993 and Ronald et al 1992) indicates that this is aphic and two (RG235 and RG365) were monomorphic very reliable method for assessing hybrid seed purity.for the lines that were screened. The frequency of heter-ozygosity for a hybrid ranged from 7.7% (1/13) to a Estimation of the Frequency of Heterozygositymaximum of 38.5% (5/13) with respect to microsatellite at Microsatellite Loci in Rice Varietiesloci and 0% (0/5) to 60% (3/5) for STS loci. At least Genomic DNAs were isolated from rice varietiesone polymorphism was detected with this set of markers T(N)1, Ajaya (Restorer), IR58025A, and IR62829Afor each of the parental combinations being used in (CMS lines) to estimate the frequency of heterozygosityhybrid rice production. There are currently at least 351 at two microsatellite loci, RM1 and RM164. PCR analy-well distributed and mapped microsatellite markers in sis was performed individually on genomic DNA iso-rice (Cho et al., 2000; Temnykh et al., 2000), constituting lated from fifty seedlings of each of these four lines.a large source of markers for detecting polymorphisms Heterozygosity was assessed by agarose gel electropho-between parental lines of hybrids. resis and ethidium bromide staining. For T(N)1 and Ajaya, all of the seedlings used were homozygous forDetection of Off Types among Hybrids in Single these two loci indicating that the percent heterozygosity,Seedling Assays as analyzed by this method, is less than 0.02 at these Seeds of the DRRH1 hybrid were obtained from two loci. For the open pollinated IR62829A line, no hetero-separate sources. One of these sources is the Directorate zygosity was detected at the RM1 locus among the 50 seedlings that were screened. However, two out of 50 seedlings were found heterozygous with respect to RM164 marker suggesting that a certain amount of polli- nation has occurred with donors other than the Main- tainer line. The pollen parent of the off-type plantsFig. 1. Microsatellite and STS marker polymorphism between paren- Fig. 2. Single seedling assay for detecting hybrid seed purity. Polymor- tal lines and a rice hybrid. Polymorphism between CMS (IR58- phism between CMS (IR58025A), hybrid (DRRH1) and restorer 025A), hybrid (DRRH1) and restorer (IR40750) lines of rice at (IR40750) lines of rice at RM164 microsatellite locus (Lanes 2–4). RM164 microsatellite locus (Lanes 2–4) and for pTA248 STS locus DNA was isolated from single seedlings of the DRRH1 hybrid, (Lanes 5–7). Molecular weight marker (Lane 1) is a 1-kilobase PCR analysis was performed and genotype assessed (Lanes 5–13) DNA ladder. Electrophoresis on agarose gels and detection is as as described in Materials and Methods. Off types are in Lanes 7 and described in Materials and Methods. 12. Molecular weight marker (Lane 1) is a 1-kilobase DNA ladder.
1372 CROP SCIENCE, VOL. 42, JULY–AUGUST 2002appears to have polymorphism at the RM164 locus but so that the seeds can be marketed for commercial culti-not at RM1 locus in respect of IR62829A. With respect vation in the immediate season. This will result in con-to the other CMS line IR58025A, no heterozygosity was siderable savings for the seed industry, especially inobserved at both RM 1 and RM 164 loci. India, where large amounts of capital are locked up in A minimum isolation distance of 300 m is prescribed the form of stored seed. Estimates for the current seasonfor multiplication of CMS lines (Virmani, 1993). At this indicate as much as 3200 Mg of hybrid seed being mar-stage, we are not aware if this was strictly followed keted of which, at least 80% contribution is from privateduring multiplication of the IR62829A seeds (they were seed industry. This amounts to approximately US$7.2obtained from a field station) or whether there was an million of locked up capital for almost a year. The costsinbuilt heterozygosity in the original seed stock. We have of storage for a whole season and cost of acquiring landfurther examined an additional 50 seeds of IR62829A, and growing the crop for the GOT can also be avoided.obtained from a different source and found no heterozy- Besides the above, the assays described here would begosity among these seeds at the RM164 locus (data not much more accurate for determining hybrid seed purityshown). This result is consistent with the possibility that than morphological characteristics as they would be di-the growing conditions being employed for rice CMS rectly assessing the genotype.line multiplication are affecting the frequency of pollina-tion from sources other than the maintainer line. Acknowledgments These results indicate that the markers used for as-sessing hybrid seed purity should be selected carefully This work was supported, in part, by a grant to R.V.S. from E.I.D. Parry Limited, Chennai, India.after taking into consideration the varieties grown inadjacent fields that can serve as potential pollen donorseither during CMS line multiplication or hybrid seed Referencesproduction. The marker(s) selected for assessing hybrid Akagi, H., Y. Yokozeki, A, Inagaki, and T. Fujimura. 1996. Microsatel-seed purity should be monomorphic between the CMS lite DNA markers for rice chromosomes. Theor. Appl. Genet. 93:line and potential rogue donors but polymorphic be- 1071–1077. Chen, X., S. Temnykh, Y. Xu, Y.G. Cho and S.R. McCouch. 1997.tween CMS and restorer lines. Detection of the expected Development of a microsatellite framework map providing genomeheterozygosity will then be an indicator of hybrid seed wide coverage in rice (Oryza sativa L.). Theor. Appl. Genet. 95:production. These specific markers can be identified in 553–567.polymorphism surveys conducted either by means of Cho, Y.G., T. Ishii, S. Temnykh, X. Chen, L. Lipovich, S.R. McCouch,microsatellite or STS markers on CMS, restorer, and W.D. Park, N. Ayres, and S. Cartinhour. 2000. Diversity of microsa- tellites derived from genomic libraries and GenBank sequences inpotential rogue donor lines. rice (Oryza sativa L.) Theor. Appl. Genet. 100:713–722. By using the criteria described above, we suggest that Chunwongse, J., G.B. Martin, and S.D. Tanksley. 1993. Pre-germina-a single polymorphic marker should suffice to ascertain tion genotypic screening using PCR amplification of half-seeds.hybrid seed purity in rice. Additional markers can be Theor. Appl. Genet. 86:694–698. Ghareyazie, B., N. Huang, G. Second, J. Bennett, and G.S. Khush.used to ascertain purity, depending on consideration of 1995. Classification of rice germplasm. I. Analysis using ALP andthe cost for conducting the assay. Multiplex PCR with PCR-based RFLP. Theor. Appl. Genet. 91:218–227.two different markers would substantially reduce the Hossain, M. 1996. Economic prosperity in Asia: Implications for ricecosts of using two markers. For example, we have mul- research. p. 3–16. In G.S. Khush (ed.) Rice Genetics III, Proc.tiplexed the RM206 microsatellite marker and the Third Intl. Rice Genet. Symp., Los Banos, Manila, the Philippines. ˜ 16–20 Oct. 1995. International Rice Rearch Institute, Manila, thepTA248 STS marker for assessing purity of the DRRH1 Philippines.hybrid in single seedling assays (data not shown). Under Jirong, T. 2000. Talents in China. Hunan Agric. Sci. Technol. Newsl.our conditions, we estimate a cost of approximately US$ 1:2.0.75/marker/seedling; this includes the costs of reagents Kochert, G., S.D. Tanksley, and J.P. Price. 1989. RFLP training coursefor DNA isolation, PCR, and agarose gel electrophore- laboratory manual. p. 5–6. Rockefeller Program on Rice Biotech- nology, Cornell Univ., Ithaca, NY.sis. This would increase to about US$ 0.85, if two mark- Krishnaiah, K., B.C. Viraktamath, and M.I. Ahmed. 1998. Hybrid riceers are multiplexed. These estimates do not include costs research network - A key for success story in India. Hybrid Riceof labor and overheads, as these are likely to be more Newsl. 1:6–7.variable depending on the location and organization. Mao, C.X., S.S. Virmani, and I. Kumar. 1996. Technological innova- In India, hybrid seed production is contracted to farm- tions to lower the costs of hybrid rice seed production. p. 111–128. In S.S. Virmani et al. (ed.) Advances in hybrid rice technology. Proc.ers by seed companies and the produce from a single Third Intl. Symp. on Hybrid Rice, Directorate of Rice Research,farmer (2–10 Mg at an average of 2 Mg haϪ1 ) is being Hyderabad, India.considered as one seed lot for purity purposes. A sample McCouch, S.R., X. Chen, O. Panaud, S. Temnykh, Y. Xu, Y.G. Cho,of 400 seeds is collected randomly from each seed lot N. Huang, T. Ishii, and M. Blair. 1997. Microsatellite marker devel- opment, mapping and application in rice genetics and breeding.for conducting the GOT (Verma, 1996). A similar sam- Plant Mol. Biol. 35:89–99.ple size can be used for estimating seed purity by the McCouch, S.R., O. Panaud, X. Chen, Y. Xu. 1996. Development ofDNA assay. We estimate that three technicians working microsatellite markers and characterization of simple sequencetogether in a modestly equipped laboratory can com- length polymorphims in rice (Oryza sativa L.). p. 549–559. In G.S.plete the assaying of 400 seedlings in approximately 30 Khush (ed) Rice genetics III, Proc. Third Intl. Rice Genet. Symp., Los Banos Manila, the Philippines. 16–20 Oct. 1995. International ˜work-hours. By deploying a suitable number of person- Rice Research Institute, Manila, the Philippines.nel and equipment, it should be possible to complete Nair, S., J.S. Bentur, U. Prasad Rao, and M. Mohan. 1995. DNAthe DNA test within 15 to 20 d from the time of harvest, markers tightly linked to gall midge resistance gene (Gm 2 ) are
NOTES 1373 potentially useful for marker-aided selection in rice breeding. Smith, J.S.C., and J.C. Register III. 1998. Genetic purity and testing Theor. Appl. Genet. 91:68–73. technologies for seed quality: a company perspective. Seed Sci.Nair, S., A. Kumar, M.N. Srivastava, and M. Mohan. 1996. PCR based Res. 8:285–293. DNA markers linked to a gall midge resistance gene, Gm 4t has Temnykh, S., W.D. Park, N. Ayres, S. Cartinhour, N. Hauck, L. Lipo- potential for marker-aided selection in rice. Theor. Appl. Genet. vich, Y.G. Cho, T. Ishii, and S.R. McCouch. 2000. Mapping and 92:660–665. genome organization of microsatellite sequences in rice (OryzaPanaud, O., X. Chen, and S.R. McCouch. 1996. Development of micro- sativa L.). Theor. Appl. Genet. 100:697–712. satellite markers and characterization of simple sequence length Verma, M.M. 1996. Procedures for Grow-Out Test (GOT). Seed Tech. polymorphism (SSR) in rice (Oryza sativa L.). Mol. Gen. Genet. New. 26:1–4. 252:597–607. Vijaykumar, R. 1996. Hybrid Rice Seed Production-Preliminary con-Paroda, R.S. 1998. Priorities and opportunities of rice production siderations. p. 73–77. In M.I. Ahmed et al. (ed.) Hybrid rice technol- and consumption in India for self-sufficiency. p. 357–390. In N.G. ogy. Directorate of Rice Research, Hyderabad, India. Dowling et al. (ed.) Sustainability of rice in the global food system. Virmani, S.S. 1993. Hybrid rice. Adv. Agron. 57:377–462. International Rice Research Institute, Manila, the Philippines. Wu, K.S., and S.D. Tanksley. 1993. Abundance, polymorphism andRonald, P.C., B. Albano, R. Tabien, L. Albenes, K.S. Wu, S.R. genetic mapping of microsatellites in rice. Mol. Gen. Genet. 241: McCouch, and S.D. Tanksley. 1992. Genetic and Physical analysis 225–235. of the rice bacterial blight disease resistance locus, Xa 21. Mol. Yan, W. 2000. Crop heterosis and herbicide. United States patent Gen. Genet. 236:113–120. number 6,066,779.