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Development of a DNA marker for distinguishing CMS lines from fertile lines in rice (Oryza sativa L.)

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Abstract

The main objective of the present study was to identify mitochondrial DNA based marker, which can distinguish male sterile and fertile counterparts of the cytoplasmic male sterile (CMS) lines used in production of rice hybrids. Amplified fragment length polymorphism (AFLP) analysis in CMS lines: IR58025A & IR62829A and their respective maintainers: IR58025B & IR62829B identified a polymorphic DNA fragment of about 510 bp size that was present in both CMS (A) and absent in their maintainer (B) lines. Sequencing followed by database analysis of the polymorphic fragment indicated about 97% similarity with mitochondrial NADH gene subunits of rice, maize and wheat. Based on the variable sequence regions, a site specific primer pair (BF-STS-401) was designed. PCR analysis showed that BF-STS-401 could amplify a strong band of 464 bp size in CMS and a faint band of the same size in maintainer line. To act as a positive control and avoid possible errors in PCR, BF-STS-401 was multiplexed with a new primer pair (BF-STS-402), derived from mitochondrial atp9 subunit of rice, producing monomorphic amplification indiscriminately in both CMS and maintainer lines. Both the primer pairs in combination clearly differentiated CMS lines from their corresponding maintainer lines. This primer combination was validated in a set of diverse genotypes consisting of different sources of CMS lines, restorer lines, hybrids, varieties and mixed samples from private seed companies. Our results suggested that the multiplex primer pairs developed in this study can be effectively utilized to assess the genetic purity in commercial seed lots of CMS lines and hybrids of rice.

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Abbreviations

AFLP:

Amplified fragment length polymorphism

CMS:

Cytoplasmic male sterility

NADH:

Nicotinamide adenine dehydrogenase

PCR:

Polymerase chain reaction

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Acknowledgements

This work was supported by the Barwale Foundation, Hyderabad, India. We thank Mr. Dinesh C. Joshi, Executive Director for his support and encouragement; Dr. Bharat R. Char and Dr. M. Venugopal, Maharashtra Hybrid Seed Company Ltd., Jalna, Maharashtra, India for DNA sequencing and marker validation; Dr. M.G. Gangashetti, Pioneer Overseas Corporation, Hyderabad for providing leaf samples for validation; Mr. Pawan Khera, Mr. K.R. Kamalnath Reddy and Ms. Anupama Yadav for their technical support. The experiments carried out complied with the regulations prescribed by the Government of India.

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  1. Research and Training Centre, Barwale Foundation, No. 8-2-703, A.G. Heights, Road No. 12, Banjara Hills, Hyderabad, Andhra Pradesh, 500034, India

    Nirmala Rajendran, Ramkumar Gandhimani, Sukhpal Singh & Kadirvel Palchamy

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  1. Nirmala Rajendran
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  2. Ramkumar Gandhimani
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  3. Sukhpal Singh
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  4. Kadirvel Palchamy
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Correspondence to Kadirvel Palchamy.

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Rajendran, N., Gandhimani, R., Singh, S. et al. Development of a DNA marker for distinguishing CMS lines from fertile lines in rice (Oryza sativa L.). Euphytica 156, 129–139 (2007). https://doi.org/10.1007/s10681-007-9360-3

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  • DOI: https://doi.org/10.1007/s10681-007-9360-3

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