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Development of molecular markers specific to petaloid-type cytoplasmic male sterility in tuber mustard (Brassica juncea var. tumida Tsen et Lee)

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Abstract

To establish a simple and rapid method for isolating mitochondrial DNA (mtDNA) from Brassica vegetables, the effects of different factors on mtDNA extraction were investigated firstly. A new protocol includes five steps: organelle isolation, deoxyribonuclease treatment, lysis, RNase treatment, and deproteinization. Results indicate that a 15 min-lysis time can achieve higher mtDNA yields from etiolated seedlings. Moreover, it is found that the inflorescence of the cytoplasmic male sterile (CMS) line is unfit for the isolation of mtDNA. The mtDNA isolated using this method is intact and pure, and can be used for further molecular analysis. Subsequently, the genomic and transcriptional differences of atps and coxs genes on the mitochondria between the petaloid-type CMS line and its maintainer line have been identified. RFLP analysis revealed that out of the five atps and three coxs genes, except of atp4 and cox3, the others mtDNA protein coding genes exhibited polymorphisms, respectively. This results suggest that atps and coxs genes are located in a long mtDNA fragment, and the mtDNA evolves rapidly in structure between the CMS line and its maintainer line in tuber muster. Northern blot analysis showed that the expression level of these genes in flower bud is higher than that of leaf and flower, and that, alternative splicing have been found among the atp6, atp8 and cox3 genes, respectively. Our results modified a efficient protocol for isolating the mtDNA, and provided some novel molecular markers indicating the CMS trait in tuber mustard. The comparative analysis presented in this study allows a more comprehensive understanding of the molecular mechanism on CMS in Brassica crops.

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Abbreviations

ATP:

Adenosine triphosphate

CMS:

Cytoplasmic male sterility

COX:

Cytochrome oxidase

GMS:

Genic male sterile

mtDNA:

Mitochondrial DNA

ORF:

Open reading frame

PCR:

Polymerase chain reaction

RFLP:

Restriction fragment length polymorphism

RT-PCR:

Reverse transcription polymerase chain reaction

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Acknowledgments

This research was partially supported by the Breeding Project of Sci-Tech Foundation of Zhejiang Province (2012C12903-6-1), and the Nature Science Foundation of Zhejiang Province (Y3090294). The authors gratefully acknowledge Dr. Gergely Gulyas and Dr. Gen Hattori for their helpful advice, and we particularly thank Dr. Johnson Cameron for the stimulating discussions and for critically reading the manuscript.

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Authors and Affiliations

  1. Laboratory of Cell & Molecular Biology, Institute of Vegetable Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, People’s Republic of China

    Xiaolin Yu, Yapei Liu, Yanxia Lv, Zhenning Liu, Zhujun Chen, Gang Lu & Jiashu Cao

  2. Laboratory of Horticultural Plant Growth & Quality Regulation, Ministry of Agriculture, Hangzhou, 310058, People’s Republic of China

    Xiaolin Yu, Yapei Liu, Yanxia Lv, Zhenning Liu, Zhujun Chen, Gang Lu & Jiashu Cao

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  1. Xiaolin Yu
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  2. Yapei Liu
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Correspondence to Xiaolin Yu.

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Yu, X., Liu, Y., Lv, Y. et al. Development of molecular markers specific to petaloid-type cytoplasmic male sterility in tuber mustard (Brassica juncea var. tumida Tsen et Lee). Mol Biol Rep 41, 769–778 (2014). https://doi.org/10.1007/s11033-013-2916-5

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  • DOI: https://doi.org/10.1007/s11033-013-2916-5

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