Development of SCAR Markers for Early Identification of Cytoplasmic Male Sterility Genotype in Chili Pepper (Capsicum annuum L.)

  • Kim, Dong Hwan (Department of Plant Science, College of Agriculture and Life Sciences, and Center for Plant Molecular Genetics and Breeding Research, Seoul National University) ;
  • Kim, Byung-Dong (Department of Plant Science, College of Agriculture and Life Sciences, and Center for Plant Molecular Genetics and Breeding Research, Seoul National University)
  • Received : 2005.08.05
  • Accepted : 2005.09.07
  • Published : 2005.12.31

Abstract

We previously used Southern blot analysis to detect restriction-length polymorphisms between male fertile and cytoplasmic male sterile (CMS) cytoplasms at the coxII and atp6 loci of the mtDNA of Capsicum annuum L. Two copies of atp6 were found in each male fertile and CMS pepper lines. Interestingly, one of the copies of atp6 in CMS pepper was a 3'-truncated pseudogene. The open reading frame of the coxII gene was the same in the fertile (N-) and CMS (S-) lines. However, the nucleotide sequence in the S-cytoplasm diverged from that in the N-cytoplasm 41 bp downstream of the stop codon. To develop CMS-specific sequence-characterized amplified region (SCAR) markers, inverse PCR was performed to characterize the nucleotide sequences of the 5' and 3' flanking regions of mitochondrial atp6 and coxII from the cytoplasms of male fertile (N-) and CMS (S-) pepper plants. Based on these data, two CMS-specific SCAR markers, 607 and 708 bp long, were developed to distinguish N-cytoplasm from S-cytoplasm by PCR. The CMS-specific PCR bands were verified for 20 cultivars containing either N- or S-cytoplasm. PCR amplification of CMS-specific mitochondrial nucleotide sequences will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of $F_1$ seed lots. The strategy used in this report for identifying CMS-specific markers could be adopted for many other crops where CMS is used for F1 seed production.

Keywords

atp6;coxII;Cytoplasmic Male Sterility(CMS);N (S)-Cytoplasm;SCAR Marker

Acknowledgement

Supported by : Korea Science and Engineering Foundation (KOSEF)

References

  1. Hanson, M. and Bentolila, S. (2004) Interactions of mitochondrial and nuclear genes that affect male gametophyte development. Plant Cell 16, S154-S169 https://doi.org/10.1105/tpc.015966
  2. Kim, D. H. and Kim, B. D. (2005) The organization of mitochondrial apt6 gene region in male fertile and CMS lines of pepper (Capsicum annuum L.). Curr. Genet. (in press)
  3. Kim, D. H., Kang, J. G., Kim, S., and Kim, B. D. (2001) Identification of coxII and atp6 regions as associated to CMS in Capsicum annuum L. by using RFLP and long and accurate PCR. J. Kor. Soc. Hort. Sci. 42, 121-127
  4. Lee, S. J., Jeung, J. U., Cho, S. K., Um, B. Y., Chung, W. I., et. al. (2002) Diversity and varietal classification of Hibiscus syriacus L. with the heterogeneity within retrotransposonlike elements. Mol. Cells 13, 362-368
  5. Makaroff, C. A. and Palmer, J. D. (1988) Mitochondrial DNA rearrangements and transcriptional alterations in the malesterile cytoplasm of Ogura radish. Mol. Cell. Biol. 8, 1474-1480
  6. Makaroff, C. A., Apel, I. J., and Palmer, J. D. (1989) The atp6 coding region has been disrupted and a novel reading frame generated in the mitochondrial genome of cytoplasmic malesterile radish. J. Biol. Chem. 264, 11706-11713
  7. Schnable, P. S. and Wise, R. P. (1998) The molecular basis of cytoplasmic male sterility and fertility restoration. Trends Plant Sci. 3, 175-180 https://doi.org/10.1016/S1360-1385(98)01235-7
  8. Senda, M., Mikami, T., and Kinoshita, T. (1993) The sugar beet mitochondrial gene for the ATPase alpha-subunit: sequence, transcription and rearrangements in cytoplasmic male-sterile plants. Curr. Genet. 24, 164-?170 https://doi.org/10.1007/BF00324681
  9. Satoh, M., Kubo, T., Nishizawa, S., Estiati, A., Itchoda, N., et al. (2004) The cytoplasmic male-sterile type and normal type mitochondrial genomes of sugar beet share the same complement of genes of known function but differ in the content of expressed ORFs. Mol. Gen. Genomics 272, 247-256 https://doi.org/10.1007/s00438-004-1058-9
  10. Peterson, P. A. (1958) Cytoplasmically inherited male sterility in Capsicum. Am. Nat. 92, 111-119 https://doi.org/10.1086/282017
  11. Dieterich, J. H., Braun, H. P., and Schmitz, U. K. (2003) Alloplasmic male sterility in Brassica napus (CMS 'Tournefortii- Stiewe') is associated with a special gene arrangement around a novel atp9 gene. Theor. Appl. Genet. 269, 723-731
  12. Engelke, T. and Tatlioglu, T. (2004) The fertility restorer genes X and T alter the transcripts of a novel mitochondrial gene implicated in $CMS_1$ in chives (Allium schoenoprasum L.). Mol. Gen. Genomics 271, 150-160 https://doi.org/10.1007/s00438-003-0966-4
  13. Kadowaki, K., Suzuki, T., and Kazama, S. (1990) A chimeric gene containing the 5′ portion of atp6 is associated with cytoplasmic male-sterility of rice. Mol. Gen. Genet. 224, 10-16
  14. Siculella, L. and Palmer, J. D. (1988) Physical and gene organization of mitochondrial DNA in fertile and male-sterile sunflower. CMS-associated alterations in structure and transcription of the atpA gene. Nucleic Acids Res. 16, 3787-3799 https://doi.org/10.1093/nar/16.9.3787
  15. Sparks, R. B. and Dale, R. M. K. (1980) Characterization of $^3H$- Hlabelled supercoiled mitochondrial DNA from tobacco suspension culture cells. Mol. Gen. Genet. 180, 351-355 https://doi.org/10.1007/BF00425847
  16. Schuster, W. and Brennicke, A. (1987) Nucleotide sequence of the Oenothera ATPase subunit 6 gene. Nucleic Acids Res. 15, 9092 https://doi.org/10.1093/nar/15.21.9092
  17. Young, E. G. and Hanson, M. R. (1987) A fused mitochondrial gene associated with cytoplasmic male sterility is developmentally regulated. Cell 50, 41-49 https://doi.org/10.1016/0092-8674(87)90660-X
  18. Jang, I., Moon, J. H., Yoon, J. B., Yoo, J. H., Yang, T. J., et al. (2004) Application of RAPD and SCAR markers for purity testing of F1 hybrid seed in chili pepper (Capsicum annuum). Mol. Cells 18, 295-299
  19. Yoo, I. W. (1990) The inheritance of male sterility and its utilization for breeding in pepper (Capsicum spp.). Kyung Hee University, South Korea, Ph. D. Dissertation
  20. Fang, G., Hammar, S., and Grumet, R. (1992) A quick and inexpensive method for removing polysaccharides from plant genomic DNA. BioTechniques 13, 52-54
  21. Hanson, M. R. (1991) Plant mitochondrial mutations and male sterility. Annu. Rev. Genet. 25, 461-486 https://doi.org/10.1146/annurev.ge.25.120191.002333
  22. Sato, Y. (1998) PCR amplification of CMS-specific mitochondrial nucleotide sequences to identify cytoplasmic genotypes of onion (Allium cepa L.). Theor. Appl. Genet. 96, 367-370 https://doi.org/10.1007/s001220050750
  23. Spassova, M., Moneger, F., Leaver, C. J., Petrov, P., Antanassov, A., et al. (1994) Characterization and expression of the mitochondrial genome of a new type of cytoplasmic male-sterile sunflower. Plant Mol. Biol. 26, 1819-1831 https://doi.org/10.1007/BF00019495
  24. Bland, M. M., Levings, C. S. III, and Matzinger, D. F. (1987) The ATPase subunit 6 gene of tobacco mitochondria contains an unusual sequence. Curr. Genet. 12, 475-481 https://doi.org/10.1007/BF00419555
  25. Newton, K. (1988) Plant mitochondrial genomes: organization, expression and variation. Ann. Rev. Plant Physiol. Plant Mol. Biol. 39, 503-532 https://doi.org/10.1146/annurev.pp.39.060188.002443