1. 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 lot
by the grow out test (GOT), which is based on the assessment of in Hai-nan island, which is a natural winter nursery
morphological and floral characteristics in plants grown to maturity. (Yan, 2000). The seeds are contracted for distribution
For seed companies, large amounts of capital are locked up in the by the end of the year, at which time the seed quality
form of hybrid seed stock while awaiting the results of the GOT. data are not available. Legal disputes can arise if it turns
With the objective of replacing the GOT with DNA based assays, out that the contracted seed do not have the required
cytoplasmic 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 taken
purity has been standardized and uses 6-d-old rice (Oryza sativa L.)
up in the dry (rabi) season (January–April) because the
seedlings, which could be used for detection of off-types in hybrid weather conditions (low humidity, lack of rainfall at the
seed lots. The extent of heterozygosity within parental lines of rice time of flowering, etc.) are congenial for production of
hybrids was assessed and the results suggest that a single, appropriately good quality seeds (Vijaykumar, 1996). Hybrid rice is
chosen 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 for
R ice is the staple food for a large segment of the
Asian population. It has been estimated that rice
production 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 attendant
tries 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 climatic
be a feasible and readily available option for raising the conditions (like heavy rain or wind) can damage or
yield 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 purity
50% of the total rice area (Jirong, 2000). Besides China, of hybrid seeds that is both accurate and faster, so seed
rice 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-based
tries in Asia and Latin America are also expected to markers can be applied for this purpose because they
adopt hybrid rice technology soon. In 1997, about 3000 can be used for assessing precisely the genotype of a
Mg 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). They
contribute 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 reaction
ties of pure hybrid seed to the farmers. Maintenance of (PCR) (McCouch et al., 1997). The current level of
high 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 of
J. Yashitola and Ramesh V. Sonti, Centre for Cellular and Molecular hybrid seed purity and for genotype identification. The
Biology, 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 purity
Research, Rajendranagar, Hyderabad-500030, India; M.K. Naseerul- is already reported to be routine among tomato seed
lah, 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 (sonti@ccmb.ap.nic.in).
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

2. 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 at
mapped to a specified site as a landmark in the genome. CMS lines
In this paper, we have screened several microsatellite IR58025A International Rice Research
and 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 Philippines
The usefulness of these polymorphisms for determining Restorer lines
hybrid 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 Research
35, of rice and their hybrids in a set of six combinations (Table (DRR), Hyderabad, India
1) 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, India
et al. (1989), from leaves of 18- to 20-d-old rice plants grown KRH2 (IR58025A ϫ KMR3) UAS, V. C. Farm, Mandya,
India
in 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 India
dishes. Six-day-old seedlings were separated from the endo- Others
sperm with a forceps and homogenized with a pestle in a 1.5- T(N)1 Introduced from Taiwan and
mL tube containing 200 ␮L of extraction buffer made up of maintained at DRR,
Hyderabad, India
5% (w/v) Chelex-100 (Bio-Rad Laboratories, USA) in sterile
distilled water (Chunwongse et al., 1993). The homogenate † The CMS and restorer lines from which the hybrids were obtained are
was incubated at 95ЊC for 10 min and pelleted in a microcentri- indicated in parentheses.
fuge. The supernatant (ෂ50 ␮L) contained enough DNA (data
not 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 min
tellite repeat sequences were used in the present study. Six at 57ЊC, and 2 min at 72ЊC (RG235 and RG365); 30 cycles of
primer pairs (RM1, RM8, RM9, RM10, RM19, and RM21) 1 min at 94ЊC, 1 min at 58ЊC, and 4 min at 72ЊC (F8); 30 cycles
were 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 out
selected from the sequences of Wu and Tanksley (1993); one as above with the exception that 0.6 U of Taq Polymerase
primer pair (RM206) was chosen from Chen et al. (1997) and and 5 ␮L of single seedling extracted DNA samples were
three primer pairs (OSR2, OSR17, and OSR28) were from used in 15-␮L reaction volumes. PCR amplified products were
Akagi et al. (1996). All the sequences targeted by the 13 resolved on 2 or 3% (for RM loci), 3% (for OSR loci), or
primers, except OSR17, have been mapped previously (Akagi 1.2% (for STS markers) agarose gels, stained with ethidium
et 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, RM122
and RM164 on chromosome 5, RM10 on chromosome 7, Results and Discussion
OSR28 on chromosome 9, RM21 and RM206 on chromosome Microsatellite and STS Polymorphisms in Parental
11, and RM19 on chromosome 12. For STS markers, five and Hybrid Lines of Rice
oligonucleotide primer pairs namely pTA248 (chromosome
11; Ronald et al., 1992), F8 (chromosome 2; Nair et al., 1995), Genomic DNA was isolated from two CMS lines, six
F43 (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 in
were 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. Summarized
plified 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–STS
primer 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

3. NOTES 1371
Table 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 50
P1 P2 H markers markers seedlings of the DRRH1 hybrid from each of the above
IR62829A MTU9992 APRH2 2/13 0/5 two separate sources and PCR analysis was performed
IR58025A IR40750 DRRH1 3/13 1/5 by means of the RM164 microsatellite marker. Figure
IR58025A C2OR CORH2 3/13 0/5
IR58025A KMR3 KRH2 2/13 2/5
2 indicates that some of the seeds obtained from the
IR62829A Ajaya CNRH3 5/13 1/5 DRR farm are off types. Three out of 50 seeds analyzed
IR58025A 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 RM164
polymorphism). Of the 13 microsatellite loci analyzed, were confirmed to be off types and all other seeds were
five (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 map
for 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 a
phic 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 Heterozygosity
maximum of 38.5% (5/13) with respect to microsatellite at Microsatellite Loci in Rice Varieties
loci and 0% (0/5) to 60% (3/5) for STS loci. At least Genomic DNAs were isolated from rice varieties
one polymorphism was detected with this set of markers T(N)1, Ajaya (Restorer), IR58025A, and IR62829A
for each of the parental combinations being used in (CMS lines) to estimate the frequency of heterozygosity
hybrid 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 for
Detection 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 plants
Fig. 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.

4. 1372 CROP SCIENCE, VOL. 42, JULY–AUGUST 2002
appears 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 in
observed 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 season
for 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 private
during multiplication of the IR62829A seeds (they were seed industry. This amounts to approximately US$7.2
obtained from a field station) or whether there was an million of locked up capital for almost a year. The costs
inbuilt heterozygosity in the original seed stock. We have of storage for a whole season and cost of acquiring land
further 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 be
gosity among these seeds at the RM164 locus (data not much more accurate for determining hybrid seed purity
shown). 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 in
adjacent fields that can serve as potential pollen donors
either during CMS line multiplication or hybrid seed References
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5. NOTES 1373
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