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A sequence specific PCR marker for distinguishing rice lines on the basis of Wild Abortive cytoplasm from their cognate maintainer lines.

RICE IS A major food and carbohydrate source for more than one third of the world's population. More than 90% of the world's rice is produced and consumed in Asia. As demographic estimates forecast doubling of population of rice eaters in these countries by 2025, the demand for rice is expected to outstrip production. A readily adoptable technological option for raising the genetic yield ceiling is the exploitation of the phenomenon of heterosis or hybrid vigor through large-scale cultivation of [F.sub.1] hybrids. The discovery of a male-sterility inducing cytoplasm designated 'Wild Abortive' (WA) cytoplasm from a wild rice in China has been exploited through a three-line hybrid breeding system involving cytoplasmic male sterile (CMS/A) lines and their cognate isonuclear maintainer (B) and restorer (R) lines. The majority of the rice hybrids that are currently under commercial cultivation in the world derive their cytoplasm from the WA source (Yuan, 1995).

Any impurities in the hybrids would reduce the expected yield. It has been estimated that every 1% mixture of female line seed in the hybrid seed results in yield reduction of 100 kg per hectare (Mao et al., 1996). The Indian seed act prescribes that, for hybrid rice, the purity should be 98% (Verma, 1996), while in the People's Republic of China it is mandated that the purity of hybrid rice should be at least 96% (Yan, 2000). To ensure the required levels of purity in hybrid seed, the parental lines that are utilized in hybrid seed production should have a very high (about 99%) level of purity.

One of the common admixtures that is observed during hybrid seed production is that of maintainer lines with those of the CMS lines. Because these are isonuclear, it is not possible to distinguish between them until they flower. The availability of appropriate DNA markers would greatly facilitate the screening of seed lots for contamination of the CMS line with male fertile lines. RFLPs that distinguish WA-CMS lines from their maintainers have been identified (Narayanan et al., 1996; Sane et al., 1997). However, RFLP markers are not ideally suited for routine large-scale screening. Random amplified polymorphic DNA (RAPD) markers that distinguish CMS (WA cytoplasm) and maintainer lines of rice have been described (Sane et al., 1997; Jena and Pandey, 1999). However, low reproducibility has made it practically impossible to apply them in a routine manner for distinguishing CMS and maintainer lines.

In a previous study (Yashitola et al., 2002), we screened 13 oligonucleotide primer pairs that flank microsatellite repeat sequences and identified several markers which are polymorphic between certain CMS and restorer lines being utilized in hybrid rice production in India. Here we report that one of these 13 oligonucleotide primer pairs, the RM9 marker, also detects a polymorphism between the isonuclear CMS and maintainer lines. We have sequenced the polymorphic fragment and identified a region of mitochondrial DNA that is specific to CMS lines of rice containing the WA cytoplasm. On the basis of this sequence, specific oligonucleotide primers were developed that can be used in a PCR assay to distinguish a number of CMS lines derived from WA cytoplasm from the cognate maintainer and other male fertile lines.


Isolation of DNA from Rice Lines

The rice lines used in this study are listed in Table 1. Total rice genomic DNA was isolated by the protocol of Kochert et al. (1989) from leaves of 18- to 20-d-old greenhouse grown rice plants. 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) except that phenol:chloroform:isoamylalcohol (25:24:1) was used for DNA purification instead of chloroform only.

PCR Amplification

PCR was performed with RM9 microsatellite primers (Panaud et al., 1996; Forward: 5'-GGTGCCATTGTCGTC CTC-3'; Reverse: 5'-ACGGCCCI'CATCACCTTC-3') on rice genomic DNA template under conditions described by Panaud et al. (1996) with slight modifications. PCR was performed in 25-[micro]L reaction volumes containing 1 x PCR buffer [10 mM Tris. HCl (pH 8.3), 50 mM KCl, 1.5 mM Mg[Cl.sub.2], 0.01% (v/v) gelatin], 50 to 100 ng of template DNA, 5 pmol of each primer, 200 [micro]M (each) deoxyribonucleotides, and 1 unit of Taq polymerase. PCR conditions were 95[degrees]C for 7 min (initial denaturation), followed by 35 cycles of denaturation at 94[degrees]C for 1 min, annealing at 55[degrees]C for 1 min, extension at 72[degrees]C for 2 min, and a final extension of 5 min at 72[degrees]C.

PCR using cms primers (cmsF 5'-ACTTTTTGTTTTTGT GTAGG-3' and cmsR 5'-TGCCATATGTCGCTTAGACT TTAC-3') either alone or multiplexed with other primers (RG136F, 5'-TCCCAGAAAGCTACTACAGC-3'; RG136R, 5' GCAGACTCCAGqWGACTTC-3'; Huang et al., 1997) were performed with the reaction composition described above, with rice genomic DNA as template. The PCR conditions were an initial denaturation at 95[degrees]C for 7 min followed by 35 cycles of 94[degrees]C for 30 s, 44[degrees]C for 1 min, and 72[degrees]C for 2 min. A final extension was given at 72[degrees]C for 7 min. All the PCR products were separated on 1% (w/v) agarose gels, stained with ethidium bromide and visualized under UV.

DNA Sequencing and Analysis

A 323-bp PCR product that was amplified by means of RM9 microsatellite primers on template DNA only from CMS line or hybrid but not from maintainer line was gel eluted and purified with a Qiaquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to the Manufacturer's instructions. The sequence of this fragment was obtained with RM9 primers using an ABI Prism 3700 automated DNA sequencer (Perkin Elmer, Foster City, CA). The 323-bp sequence has been deposited in GenBank with accession #AY295770. Homology searches were performed by the BLAST algorithm (Altschul et al., 1997) through the National Center for Biotechnology Information (; verified 27 January 2004).


Identification of a Nucleotide Sequence That Is Specific to CMS Lines of Rice

PCR was performed with RM9 primers on genomic DNA isolated from the rice lines IR58025A (CMS), 58025B (maintainer), and DRRH1 hybrid (derived from a cross between IR58025A and restorer line 40750R). Multiple amplification products were observed when the PCR products were separated on an agarose gel (Fig. 1). One particular DNA fragment of about 330 bp was obtained as a PCR product only when template DNA was from either the CMS line or the hybrid. PCR was also performed with template DNA from four other cognate pairs of CMS and maintainer lines (IR62829A and IR62829B, PMS8A and PMS8B, PMS10A and PMS10B, IR68897A and IR68897B). In each case, the about 330-bp band was amplified when template DNA was from the CMS line and was not observed when template DNA was from the maintainer line. This DNA fragment was eluted from the gel, purified and the resulting 323-nucleotide DNA sequence determined (Fig. 2). A BLAST search of the sequence indicated homology (97% nucleotide sequence identity) to a region of the rice mitochondrial DNA (DDBJ accession #D21251) that is 5' to the rps3-rpl16-nad3-rps12 gene cluster (Nakazono et al., 1995). This homology extends from nucleotides 37-319 of the CMS-specific DNA and corresponds to 1014-1294 nucleotide region of the 7500-bp sequence in accession D21251. Interestingly, nucleotides 1 to 36 and 320 to 323 of the 323-bp sequence (indicated in bold letters in Fig. 2) do not exhibit significant similarity to the sequence of rice mitochondrial DNA in accession D21251.


A Sequence Specific PCR Marker for Distinguishing CMS and Maintainer Lines of Rice

PCR amplification of the 323-bp fragment using RM9 primers was found to be highly sensitive to reaction conditions and sometimes was not repeatable. To develop a reliable PCR assay for distinguishing CMS and maintainer lines, one primer (cmsF; 5'-ACTTTTTGTT TTTGTGTAGG-3') was designed such that it binds to the 36 bp unique sequence described above. The second primer (cmsR; 5'-TGCCATATGTCGCTTAGACTT TAC-3') was the complement of nucleotide sequence 1384 1361 of sequence accession #D21251. PCR was performed with the cms primers on template DNA from a CMS line (IR58025A) and it's cognate maintainer line (IR58025B). A 386-bp fragment was amplified from the CMS line and the derived hybrid (DRRH1) but not from the maintainer line (Fig. 3).


Thc cms primers were multiplexed, in a single PCR assay with a second set of primers (RG136F and R), which amplify a monomorphic fragment from both CMS and maintainer lines. The RG136 primer sequences are linked to the xa13 disease resistance gene on chromosome 8 (Huang et al., 1997). The about 1.1-kb product amplified with RG136 primers was obtained from 13 different CMS lines derived from WA cytoplasm as well as their cognate maintainer lines (Table 1), whereas the 386-bp product amplified by cms primers was obtained from all thirteen CMS lines but not from any of the cognate maintainer lines (Fig. 4). The presence of a monomorphic fragment in CMS and maintainer lines served as a positive control for successful PCR. In addition to the maintainer lines, this 386-bp fragment was also not amplified from the five male fertile restorer lines (Table 1) that were analyzed in this study.


In addition to testing different CMS lines of WA cytoplasm background, other diversified CMS lines derived from O. nivara (DMS3A), O. rufipogon (DMS4A), O. perennis (IR66707A) were also tested with the cms and RG136 primers. It was observed that, although all of the CMS lines of WA cytoplasm showed the 386-bp amplicon, no other CMS line amplified the specific marker.

Coded Test of cms Primers to Distinguish CMS and Maintainer Lines of Rice

A designed mixture of seeds of CMS line IR58025A was made with those of its maintainer line IR58025B and 108 seeds of this mixture were planted individually in an experimental farm at the Directorate of Rice Research, Hyderabad. Each of the plants was given a code number and leaf material was collected at 20-d-old stage from these seedlings for DNA extraction. The DNA from individual seedlings was then used for PCR amplification using the cms primers and the RG136 primers in a multiplex PCR. After agarose gel electrophoresis and ethidium bromide staining, the fertile plants among the 108 coded lines were identified on the basis of the banding pattern and recorded. The genotypes of some of the plants analyzed are shown in Fig. 5. All 108 plants were grown to maturity and bagged during flowering (to avoid cross pollination) and pollen fertility was recorded. Out of the 108 plants studied, the 18 that were detected as impure (i.e., IR58025B) on the basis of DNA marker data set seed at maturity (i.e., pollen fertile). Similar results were obtained in independent coded tests conducted in two different growing seasons wherein it was possible to distinguish accurately WA-CMS plants from cognate maintainer lines using this PCR assay. These results show that the cms primers can be reliably used to detect seed mixtures among WA-CMS lines.


In summary, an admixture of maintainer line in seed stock of CMS line is a serious problem in production of pure CMS lines. The maintainer lines are self-fertile and can perpetuate through selfed seed, leading to contamination of the hybrid seeds. This type of contamination is frequently observed during hybrid rice seed production and leads to a reduction in the expected yield and poor performance of the hybrid in the field. The majority of the CMS lines that are employed in commercial production of hybrid rice are developed through nuclear diversification of WA cytoplasmic background. The oligonucleotide primers described in this manuscript can be used in a PCR assay by hybrid rice breeders or seed companies to reliably detect contamination of the maintainer and other male fertile lines within seed stocks of the WA-CMS line. By ensuring purity of the CMS lines, a major source of contamination of the hybrid seeds can be avoided to obviously benefit the seed industry and farmers.

Abbreviations: CMS, cytoplasmic male sterile; WA, wild abortive.
Table 1. Rice lines analyzed in this study.

               Cytoplasmic male sterile lines

Line                                        Source

  IR58025A (wild abortive
    cytoplasm; WA)          International Rice Research
                              Institute (IRRI), Philippines
  IR62829A (WA)                              IRRI
  IR68886A (WA)                              IRRI
  IR68888A (WA)                              IRRI
  IR68897A (WA)                              IRRI
  PMS8A (WA)                Punjab Agricultural University
                              (PAU), Ludhiana, Punjab, India
  PMS10A (WA)                                 PAU
  PMS11A (WA)                                 PAU
  PMS12A (WA)                                 PAU
  Pusa5A (WA)               Indian Agricultural Research Institute
                              (IARI), Pusa, New Delhi, India
  DRR2A (WA)                Directorate of Rice Research
                              (DRR), Hyderabad, India
  DRR3A (WA)                                  DRR
  CRMS31A (WA)              Central Rice Research Institute
                              (CRRI), Cuttack, Orissa, India
  DMS3A (O. nivara
    cytoplasm)                                DRR
  DMS4A (O. rufipogon
    cytoplasm)                                DRR
  IR66707A (O. perennis
    cytoplasm)                               IRRI

                      Maintainer lines

Line                                        Source

  IR58025H                                   IRRI
  IR62829B                                   IRRI
  IR68886B                                   IRRI
  IR68888B                                   IRRI
  IR68897B                                   IRRI
  PMS8B                                       PAU
  PMS10B                                      PAU
  PMS11B                                      PAU
  PMS12B                                      PAU
  Pusa5B                                     IARI
  DRR2B                                       DRR
  DRR3B                                       DRR
  CRMS31B                                    CRRI
  DMS3B                                       DRR
  DMS4B                                       DRR
  IR66707B                                   IRRI

                         Restorer lines

Line                                        Source

  IR40750R                                   IRRI
  BR827-35                                   IRRI
  Ajaya                                       DRR
  C20R                      Tamilnadu Agricultural University
                              (TNAU), Coimbatore, Tamil
                              Nadu, India
  KMR3R                     University of Agricultural Sciences
                              (UAS), Mandya, Karnataka, India


Hybrid                                      Source

  DRRHI (IR58025A x
    IR40750R)                                 DRR
  KRH2 (IR58025A x
    KMR3R)                                    UAS
  Sahyadri (IR58025A x
    BR827-35)               Regional Agricultural Research Station,
                              Karjat Maharashtra, India


We thank Mehar Sultana for oligonucleotide synthesis and N. Nagesh for sequencing.


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J. Yashitola, R. M. Sundaram, S. K. Biradar, T. Thirumurugan, M. R. Vishnupriya, R. Rajeshwari, B. C. Viraktamath, N. P. Sarma, and Ramesh V. Sonti *

J. Yashitola, M.R. Vishnupriya, R. Rajeshwari and Ramesh V. Sonti, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500007, India; R.M. Sundaram, S.K. Biradar, T. Thirumurugan, B.C. Viraktamath and N.P. Sarma, Directorate of Rice Research, Rajendranagar, Hyderabad-500030, India. Received 20 May 2003. * Corresponding author (;
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Title Annotation:Genomics, Molecular Genetics & Biotechnology
Author:Yashitola, J.; Sundaram, R.M.; Biradar, S.K.; Thirumurugan, T.; Vishnupriya, M.R.; Rajeshwari, R. Vi
Publication:Crop Science
Date:May 1, 2004
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