Development and Application of SCAR Markers for Discriminating Cytoplasmic Male Sterile Lines from Their Cognate Maintainer Lines in Indica Rice

LU Chao1, LIU Jian1, JIANG Jian-hua1, Caleb Manamik BRERIA1, TAN He-lin1, Masahiko ICHII2, HONG De-lin1

doi:10.1016/S1672-6308(13)60122-5

2013 , Vol. 20 >Issue 3: 191 - 199

DOI: https://doi.org/10.1016/S1672-6308(13)60122-5

Research Paper

Development and Application of SCAR Markers for Discriminating Cytoplasmic Male Sterile Lines from Their Cognate Maintainer Lines in Indica Rice

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1State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China; 2Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan

Revised date: 2012-12-31

Online published: 2013-03-27

Supported by

This research was financially supported by the National High-Tech Research and Development Program of China (Grant No. 2010AA101301), the Program of Introducing Talents of Discipline to Universities (Grant No. B08025), the ‘948’ Program of Ministry of Agriculture, China (Grant No. 2006-G8[4]-31 -1), and the Key Project of Scientific Base Qualification Platform of Ministry of Education, China (Grant No. 505005).

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Abstract

The DNA fragments about 1 600 bp were amplified using random amplified polymorphism DNA (RAPD) primer OPA12 with the templates of mitochondrial DNA of Zhenshan 97A and Zhenshan 97B, and were sequenced. The nucleotide sequences and lengths of the fragments from Zhenshan 97A and Zhenshan 97B showed no difference. The precise length of the fragment was 1 588 bp. Sequence characterized amplification region (SCAR) primers were then developed to discriminate the cytoplasmic male sterile (CMS) lines and their maintainer lines. A specific 1 588 bp fragment could be amplified with SCAR primers, CHI19F2/CHI19R2 and CHI20F3/CHI23R3, in the mitochondrial DNA of Zhenshan 97A, but not Zhenshan 97B. Furthermore, the specific fragment could be also amplified from the total DNA from green leaf tissues of Zhenshan 97A with SCAR primers, but not Zhenshan 97B. With the corresponding primers, the specific fragment could also be amplified from the total DNA of green leaves of other two CMS lines with wild abortive type cytoplasm (CMS-WA), namely Zhenpin A and Tianfeng A, but not in their maintainer lines. Moreover, using total DNA as template, each of the four pairs of SCAR primers could also be used to amplify the 1 588 bp fragment in CMS-ID (Indonesia paddy type) line Ⅱ-32A, but not in II-32B, and the specific fragment was amplified from the DNA of both F1 and F2 seedlings of Shanyou 63. The results of detecting the genetic purity of a man-made mixture of the seeds of Zhenshan 97A using CHI20F3/CHI23R3 were completely consistent with the phenotypes. Taken together, these results indicated that the specific 1 588 bp-fragment amplified by CHI20F3/CHI23R3 was the unique amplification products of CMS mitochondrial DNA, and could be used to distinguish CMS-WA and CMS-ID lines from their corresponding maintainer lines at the seedling stage.

Key words： Oryza sativa; cytoplasmic male sterile line; maintainer line; genetic purity; sequence characterized amplification region marker

Cite this article

LU Chao1, LIU Jian1, JIANG Jian-hua1, Caleb Manamik BRERIA1, TAN He-lin1, Masahiko ICHII2, HONG De-lin1 . Development and Application of SCAR Markers for Discriminating Cytoplasmic Male Sterile Lines from Their Cognate Maintainer Lines in Indica Rice[J]. Rice Science, 2013 , 20(3) : 191 -199 . DOI: 10.1016/S1672-6308(13)60122-5

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