Published online 22 January 2007
Published in Crop Sci 47:207-211 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY-NOTES
A Mitochondrial Repeat Specific Marker for Distinguishing Wild Abortive Type Cytoplasmic Male Sterile Rice Lines from their Cognate Isogenic Maintainer Lines
P. Rajendrakumar,
A. K. Biswal,
S. M. Balachandran,
M. S. Ramesha,
B. C. Viraktamath and
R. M. Sundaram*
Biotechnology Lab., Crop Improvement Section, Directorate of Rice Research, Rajendranagar, Hyderabad–500 030, Andhra Pradesh, India
* Corresponding author (rms_28{at}rediffmail.com)
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ABSTRACT
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The realization of yield potential of rice (Oryza sativa L.) hybrids developed through the "three-line system" largely depends on genetic purity of parental lines. Cytoplasmic male sterile (CMS) lines often get contaminated with cognate isonuclear maintainer lines during multiplication. We report identification of a rice mitochondrial DNA sequence, possessing a repeat motif that is polymorphic between the wild abortive (WA)-CMS lines and their cognate isonuclear maintainer lines. We have designed a primer pair flanking the unique repeat and two fragments of different size were amplified in all WA-CMS and their maintainer lines through PCR. The PCR assay was able to precisely detect the impurities in a commercial seed lot of a popular CMS line, IR58025A. The possibility of utilizing this marker as a replacement for the morphology-based (grow-out test) GOT in accurate detection of contaminants in CMS seed stocks is discussed.
Abbreviations: CMS, cytoplasmic male sterile • GOT, grow-out test • RAPD, random amplified polymorphic DNA • RFLP, restriction fragment length polymorphism • SNPs, single nucleotide polymorphisms • SSRs, simple sequence repeats • WA, wild abortive
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INTRODUCTION
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ONE OF THE READILY AVAILABLE and economically viable technological options for meeting the projected global demand of rice is exploitation of heterosis through large-scale cultivation of rice hybrids. Currently, two different technologies are adopted for development of rice hybrids. One is the "three-line system" involving a CMS line, maintainer line, and restorer line. The other system is the "two-line system" wherein only two lines are involved in hybrid seed production. The three-line system is the most stable and popular system deployed for developing hybrids in many crops including rice. Three-line rice hybrids are cultivated in many countries including China, Vietnam, India, and Bangladesh. Among the CMS lines used for development of three-line rice hybrids, lines based on WA cytoplasm originally derived from a wild rice are the most popular in all countries where hybrid rice is cultivated on a commercial scale (Yuan, 1995).
Success of hybrid rice technology depends on efficient seed production technology to ensure timely supply of genetically pure seeds. It has been estimated that every 1% mixture in hybrid seed will result in yield reduction of 100 kg ha–1 (Mao et al., 1996). The required levels of hybrid seed purity can be ensured by using pure parental lines in hybrid seed production. The most commonly observed contamination in hybrid seed production is that of maintainer line getting admixed with CMS line (Yashitola et al., 2004). To exploit the moderate level of heterosis as reported in a self-pollinated crop like rice, utmost genetic purity (99.9%) of the CMS line should be ensured. Presence of maintainer line plants in foundation seed stock may result in appearance of completely sterile plants in the next generation leading to drastic reduction in yield. Since they are isonuclear, it is not possible to distinguish CMS and maintainer lines before flowering. Removal of impurities requires more labor for rouging (removal of off-type plants based on visual inspection), which adds to the cost of seed production in addition to a significant reduction in the quality of hybrid seed. The traditional method of the morphology-based GOT usually followed to assess genetic purity has many limitations (Yashitola et al., 2002). Assays involving PCR-based DNA markers have been suggested for CMS purity assessments (Yashitola et al., 2004) as a replacement for GOT. Restriction fragment length polymorphism (RFLPs) that distinguish WA-CMS lines from their maintainers have been reported (Narayanan et al., 1996; Sane et al., 1997), but these are not ideally suited for rapid and large-scale screening since the process is laborious, time consuming, expensive, and hazardous. Random amplified polymorphic DNA (RAPD) markers that distinguish WA-CMS and maintainer lines of rice have been described (Sane et al., 1997; Jena and Pandey, 1999; Ichii et al., 2003), but their utilization in routine screening is not feasible due to their low reproducibility. A PCR marker based on a mitochondrial DNA sequence of rice capable of distinguishing WA-CMS lines from their maintainer lines was recently reported by Yashitola et al. (2004). However, this marker amplifies a 386-bp band in the CMS line and no band in the maintainer line, which necessitates a multiplex PCR assay with another nuclear genome specific marker to serve as a positive control.
Availability of complete sequence of rice mitochondrial genome in public domain (http://www.ncbi.nlm.nih.gov/GenBank/index.html) has opened up new avenues for marker development. In a preliminary study, we designed 35 oligonucleotide primer pairs that flank microsatellite repeats present within or upstream (
500 bp) of mitochondrial genes responsible for cellular respiration and used them for screening a set of rice lines which included land races, cultivated varieties, and wild relatives of rice for polymorphism. Twelve out of the 35 primer pairs tested were polymorphic (unpublished data, 2006). One of the polymorphic markers (RMT6) was found to amplify a (AT)6 repeat present upstream of the gene coding for the nad5 subunit. Since earlier studies had indicated a role for mitochondrial genes and their upstream sequences in CMS (Dai et al., 1978; Kubo and Kadowaki, 1997; Yashitola et al., 2004) we examined the amplification of RMT6 marker among different types of CMS lines of rice and their maintainers. The marker was observed to be polymorphic between all WA-CMS lines and their maintainers amplifying two fragments in CMS line and one fragment in maintainer line. The amplicons were sequenced and primers were redesigned to obtain a clear amplification of two distinct fragments in CMS and maintainer lines. We report the utility of this PCR-based marker in distinguishing WA-CMS and maintainer lines in detection of contaminants through single seed–seedling based assay as a replacement for GOT.
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MATERIALS AND METHODS
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Extraction of DNA from Rice Genotypes
Total rice genomic DNA was isolated from the rice lines under study (Table 1) from leaves of 18- to 20-d-old greenhouse grown rice plants by following the protocol of Kochert et al. (1989). For single plant genotyping, DNA was isolated from 2-cm-long leaf pieces obtained from 20-d-old seedlings as per the protocol of Zheng et al. (1995). Mitochondrial DNA was isolated from 18- to 20-d-old etiolated seedlings as per the procedure of Mulligan et al. (1988).
PCR Amplification
PCR was performed with RMT6 mitochondrial microsatellite primer pair (RMT6F 5'-GATGGTTTGGAAGGCTG-3'; RMT6R 5'-GGGTTTAGAGTCGCCAC-3') on rice genomic DNA and mitochondrial DNA template under conditions described by Panaud et al. (1996) with slight modifications. PCR was performed in 10-µL reactions containing 1x PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, 0.01% [v/v] gelatin), 25 to 50 ng of template DNA, 2 pmol of each primer, 125 µM (each) deoxyribonucleotides, and 1 unit of Taq polymerase. The thermal profile followed was 94°C for 5 min (initial denaturation), followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and a final extension of 5 min at 72°C. PCR was performed with the reaction composition described above, with rice genomic DNA and mitochondrial DNA as template. PCR was also performed using drrcms primer pair (drrcmsF 5'-ACCTTTGGGCGATGGTT-3' and drrcmsR 5'-GGGTTTAGAGTCGCCAC-3') with the reaction conditions described above. All PCR products were separated on 3% (w/v) agarose gels (agarose, Biogene, Kimbolton, UK), stained with ethidium bromide and visualized under UV.
DNA Sequencing and Analysis
The 197 and 211-bp fragments amplified using the primer pair RMT6F and RMT6R in CMS line and a 209-bp fragment amplified in maintainer line were gel eluted and purified with Qiaquick Gel Extraction Kit (Qiagen, Hilden, Germany), cloned in TOPO-TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced using an ABI Prism 3700 automated DNA sequencer (PerkinElmer, Wellesley, MA). Homology searches were performed by BLASTn algorithm (Altschul et al., 1997) through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast). DNA sequences were aligned using the software ClustalW (Higgins et al., 1994). Based on sequence information, primer pairs were designed using Genetool Lite software (Bio Tools Inc., AB).
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RESULTS AND DISCUSSION
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Identification of a Mitochondrial DNA Sequence Possessing a Repeat Motif Specific to CMS Lines
PCR was performed with RMT6 primer pair on total genomic DNA isolated from the rice lines IR58025A (CMS), IR58025B (maintainer), and DRRH1 hybrid (derived from a cross between IR58025A and the restorer line IR40750R). Two amplicons (197 and 211 bp) were obtained when template DNA was used from either CMS line or hybrid, while maintainer line yielded a 209-bp product (Fig. 1
). Identical results were obtained when mitochondrial DNA was used as template, which confirmed that the amplified fragments were indeed from mitochondria. To develop a marker that can clearly amplify two distinct fragments in CMS and maintainer lines, we sequenced and analyzed all three fragments. A BLAST search of the sequences indicated homology (99% nucleotide sequence identity) to a region of rice mitochondrial DNA (DDBJ accession no. DQ167807) that is 5' to the nad5 subunit (Tian et al., 2006). The three fragments were polymorphic with respect to an AT repeat motif. The 197-bp fragment possessed the repeat motif (AT)6 while 209- and 211-bp fragments possessed (AT)11 and (AT)14, respectively. Apart from differential number of AT repeats, these fragments also had few single nucleotide polymorphisms (SNPs) (Fig. 2
). The 197-, 209-, and 211-bp sequences have been deposited in GenBank with accession numbers DQ529240, DQ529241, and DQ641914, respectively.
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Fig. 1. PCR amplification of a DNA sequence specific to cytoplasmic male sterile (CMS) lines of rice. PCR was performed with RMT 6 mitochondrial microsatellite marker. Lane 1, 100-bp ladder; Lane 2, PCR-amplified product of a CMS line (IR58025A); Lane 3, the hybrid (DRR H1); and Lane 4, maintainer line (IR58025B). An extra DNA band, which is present only in the hybrid and CMS line but absent in the maintainer line, is indicated by the arrow.
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Fig. 2. Alignment of nucleotide sequences of DNA fragments amplified from wild abortive cytoplasmic male sterile (WA-CMS) lines and their cognate isonuclear maintainer lines of rice using RMT 6 mitochondrial microsatellite marker. CMS-specific DNA fragments (211 and 197 bp) were amplified in CMS lines while the maintainer line had a 209-bp fragment. The repeat motifs are indicated in bold while the SNPs are underlined. Arrows indicate the binding sites of the drrcms marker.
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A Mitochondrial Repeat Specific PCR Marker Capable of Distinguishing WA-CMS and Maintainer Lines
PCR amplification using RMT6 primer pair was found to be highly sensitive to reaction conditions. The 197-bp fragment in CMS line showed strong amplification, while the 211-bp fragment was found to be faint and difficult to distinguish from the 209-bp fragment amplified in maintainer line. To avoid amplification of 211-bp fragment in CMS lines and to develop a reliable PCR assay for distinguishing CMS and maintainer lines, a new primer pair (drrcmsF and drrcmsR) was designed by targeting an SNP (Fig. 2) between the 197- and 211-bp fragments such that a 130-bp fragment is amplified in the CMS line and a 142-bp band in maintainer lines. PCR was performed with drrcms marker using the template DNA from a CMS line (IR58025A), its cognate isonuclear maintainer line (IR58025B), and the hybrid DRRH1. This marker could unambiguously distinguish CMS (or the hybrid) from maintainer line (Fig. 3
). Since the hybrid also derived its cytoplasm from the CMS line, it also exhibited an amplification pattern similar to the CMS line. PCR was also performed with template DNA from 15 other cognate pairs of WA-CMS and maintainer lines and similar results were obtained with the marker showing clear polymorphism between CMS and maintainer lines (Fig. 4
).
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Fig. 3. PCR amplification of a DNA sequence specific to cytoplasmic male sterile (CMS) lines of rice. PCR was performed with drrcms marker. Lane 1, 100-bp ladder; Lane 2, PCR-amplified product of a CMS line (IR58025A), Lane 3, the hybrid (DRR H1); and Lane 4, maintainer line (IR58025B).
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Fig. 4. PCR assay for distinguishing cytoplasmic male sterile (CMS) and maintainer lines of rice. PCR was performed with drrcms marker. M, 100-bp DNA ladder. Lanes 1 and 11, IR58025A and B; Lanes 2 and 12, IR69628A and B; Lanes 3 and 13, IR68888A and B; Lanes 4 and 14, IR68897A and B; Lanes 5 and 15, CRMS31A and B; Lanes 6 and 16, DRR2A and B; Lanes 7 and 17, Pusa5A and B; Lanes 8 and 18, PMS10A and B; Lanes 9 and 19, DMS3A and B; Lanes 10 and 20, DMS4A and B.
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This marker could also distinguish two other diversified CMS lines, DMS3A (derived from O. nivara S.D. Sharma and Shastry cytoplasm) and DMS4A (derived from O. rufipogon Griff. cytoplasm), from their corresponding maintainer lines. Interestingly, these lines also showed amplification pattern similar to WA-CMS and maintainer lines (Fig. 4).
Utility of drrcms Marker in Purity Assessment of Seeds of IR58025A
A predetermined mixture of 400 seeds of CMS line IR58025A was made with its maintainer line IR58025B and seeds were planted individually in an experimental farm at the Directorate of Rice Research, Hyderabad. Each plant was given a code number and leaf material was collected from 20-d-old seedlings for DNA extraction. The DNA from individual seedlings was then used for PCR amplification using the drrcms marker. After resolving the amplified fragments in agarose gels, impurities among 400 coded lines were identified based on amplicon sizes. The genotypes of some of the plants analyzed are shown in Fig. 5
. All 400 plants were grown to maturity and data related to pollen and spikelet fertility were recorded. Out of 400 plants analyzed, 12 plants were identified as impure (i.e., IR58025B) on the basis of DNA marker-based genotyping. At maturity, these plants were found to set seed (i.e., pollen fertile), indicating a perfect correlation between results based on marker genotype and phenotype. Similar results were also observed in independent coded tests conducted in the wet season 2005 and the impurities among WA-CMS plants could be accurately detected at seed/seedling stage itself based on marker analysis.
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Fig. 5. Purity analysis of seed stock of popular CMS line IR58025A. Genomic DNA was isolated individually from 400 plants and PCR was performed using drrcms marker. Data from a representative set of 20 plants are shown. M, 100-bp DNA ladder; Lane A, CMS line; Lane B, maintainer line; Lanes 1–20, test samples. The CMS lines show a 130-bp fragment and maintainer line shows a 142-bp fragment. Asterisk indicates plants determined to be maintainer lines.
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The marker (drrcms) developed in the present study has a clear advantage over the marker (cms) developed by Yashitola et al. (2004) as it can distinguish WA-CMS lines from their cognate maintainers based on differential fragment sizes compared to the cms marker, which requires multiplexing with another nuclear genome specific marker. Further, unlike the nuclear simple sequence repeats (SSRs) which sometimes show heterozygous amplification, the mitochondrial SSR marker (drrcms) shows only a single band and is more easier to analyze in seed lots for detection of contaminants. Another notable feature of drrcms marker is its ability to distinguish CMS lines derived from O. nivara and O. rufipogon cytoplasmic background from their maintainers.
In conclusion, the drrcms marker described here can be used in a PCR assay to reliably detect contamination of maintainer and other male fertile lines in seed lots of WA-CMS lines. This is the first report of a PCR-based marker that amplifies distinct fragments in CMS and their cognate isonuclear maintainer lines. Application of this marker in purity testing of commercial CMS seed stocks can be of immense help for hybrid seed industry in ensuring timely supply of pure CMS lines for production of pure hybrid seeds.
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ACKNOWLEDGMENTS
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The authors thankfully acknowledge the support and help received from Dr. S.V. Subbaiah, Project Director, Directorate of Rice Research for conducting this research. We thank Dr. V. Dinesh Kumar, Senior Scientist (Biotechnology) and Ms. K.N. Yamini, Senior Research Fellow, Directorate of Oilseeds Research for their help and assistance in cloning.
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NOTES
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Drs. Rajendrakumar and Biswal contributed to this work equally.
Received for publication June 7, 2006.
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ABSTRACT
INTRODUCTION
