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Identification of mitochondrial DNA sequence variation and development of single nucleotide polymorphic markers for CMS-D8 in cotton

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Abstract

Cytoplasmic male sterility (CMS), which is a maternally inherited trait and controlled by novel chimeric genes in the mitochondrial genome, plays a pivotal role in the production of hybrid seed. In cotton, no PCR-based marker has been developed to discriminate CMS-D8 (from Gossypium trilobum) from its normal Upland cotton (AD1, Gossypium hirsutum) cytoplasm. The objective of the current study was to develop PCR-based single nucleotide polymorphic (SNP) markers from mitochondrial genes for the CMS-D8 cytoplasm. DNA sequence variation in mitochondrial genes involved in the oxidative phosphorylation chain including ATP synthase subunit 1, 4, 6, 8 and 9, and cytochrome c oxidase 1, 2 and 3 subunits were identified by comparing CMS-D8, its isogenic maintainer and restorer lines on the same nuclear genetic background. An allelic specific PCR (AS-PCR) was utilized for SNP typing by incorporating artificial mismatched nucleotides into the third or fourth base from the 3′ terminus in both the specific and nonspecific primers. The result indicated that the method modifying allele-specific primers was successful in obtaining eight SNP markers out of eight SNPs using eight primer pairs to discriminate two alleles between AD1 and CMS-D8 cytoplasms. Two of the SNPs for atp1 and cox1 could also be used in combination to discriminate between CMS-D8 and CMS-D2 cytoplasms. Additionally, a PCR-based marker from a nine nucleotide insertion–deletion (InDel) sequence (AATTGTTTT) at the 59–67 bp positions from the start codon of atp6, which is present in the CMS and restorer lines with the D8 cytoplasm but absent in the maintainer line with the AD1 cytoplasm, was also developed. A SNP marker for two nucleotide substitutions (AA in AD1 cytoplasm to CT in CMS-D8 cytoplasm) in the intron (1,506 bp) of cox2 gene was also developed. These PCR-based SNP markers should be useful in discriminating CMS-D8 and AD1 cytoplasms, or those with CMS-D2 cytoplasm as a rapid, simple, inexpensive, and reliable genotyping tool to assist hybrid cotton breeding.

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Authors and Affiliations

  1. Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces, NM, 88003, USA

    Hideaki Suzuki, Jiwen Yu, Fei Wang & Jinfa Zhang

  2. Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, 87131, USA

    Hideaki Suzuki

  3. State Key Laboratory in Cotton Biology, Cotton Research Institute, Chinese Academy of Agricultural Science, 455000, Anyang, Henan, China

    Jiwen Yu

  4. Department of Soil and Crop Sciences, Texas A&M University, College Station, TX, 77843, USA

    Fei Wang

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  1. Hideaki Suzuki
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  2. Jiwen Yu
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  3. Fei Wang
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  4. Jinfa Zhang
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Corresponding author

Correspondence to Jinfa Zhang.

Additional information

Communicated by A. Schulman.

This work is dedicated to the memory of Dr. James McD. Stewart (Department of Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, AR, USA) as the developer of the CMS-D8 system in cotton and for his numerous contributions to genetic and genomic studies of the system.

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Suzuki, H., Yu, J., Wang, F. et al. Identification of mitochondrial DNA sequence variation and development of single nucleotide polymorphic markers for CMS-D8 in cotton. Theor Appl Genet 126, 1521–1529 (2013). https://doi.org/10.1007/s00122-013-2070-4

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  • DOI: https://doi.org/10.1007/s00122-013-2070-4

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