Research Article

Analysis of mitochondrial DNA using amplified fragment length polymorphism markers of isonuclear allocytoplasmic male sterile wheat accessions and their maintainer lines

Published: October 30, 2013
Genet. Mol. Res. 12 (4) : 5207-5214 DOI: 10.4238/2013.October.30.5

Abstract

To produce a good F1 hybrid variety wheat crop, it is necessary to explore novel cytoplasmic male sterility (CMS) lines and their maintainer line. This study aimed to identify cytoplasmic variation in three isonuclear-alloplasmic male sterile lines Aegilops kotschyi (Ae.kots) -90-110, Aegilops ventricosa (Ae.ven) -90-110, and Triticum spelta (T.spelta) -90-110 and their maintainer line, A-90-110, at the molecular level. Mitochondrial DNA (mtDNA) was isolated using a combination of centrifugation and density gradient ultracentrifugation, sucrose sedimentation, lysis with sodium dodecyl sulfate (SDS), potassium proteinase, and phenol/chloroform extraction methods. To detect mtDNA purity, specific primers were designed for nuclear (β-actin) and mitochondrial (COXIII) genes. Results indicated that the mtDNA was pure, and therefore suitable for polymerase chain reaction (PCR) and genetic analysis. Comparative analysis of mtDNA was conducted using amplified fragment length polymorphism (AFLP) markers. Reproducible polymorphisms were detected between the Aegilops and Triticum species and the male sterile lines. Four specific primers were screened from 64 AFLP marker primers, which provided the molecular basis for further studies investigating specific cytoplasmic male sterility characteristics.

To produce a good F1 hybrid variety wheat crop, it is necessary to explore novel cytoplasmic male sterility (CMS) lines and their maintainer line. This study aimed to identify cytoplasmic variation in three isonuclear-alloplasmic male sterile lines Aegilops kotschyi (Ae.kots) -90-110, Aegilops ventricosa (Ae.ven) -90-110, and Triticum spelta (T.spelta) -90-110 and their maintainer line, A-90-110, at the molecular level. Mitochondrial DNA (mtDNA) was isolated using a combination of centrifugation and density gradient ultracentrifugation, sucrose sedimentation, lysis with sodium dodecyl sulfate (SDS), potassium proteinase, and phenol/chloroform extraction methods. To detect mtDNA purity, specific primers were designed for nuclear (β-actin) and mitochondrial (COXIII) genes. Results indicated that the mtDNA was pure, and therefore suitable for polymerase chain reaction (PCR) and genetic analysis. Comparative analysis of mtDNA was conducted using amplified fragment length polymorphism (AFLP) markers. Reproducible polymorphisms were detected between the Aegilops and Triticum species and the male sterile lines. Four specific primers were screened from 64 AFLP marker primers, which provided the molecular basis for further studies investigating specific cytoplasmic male sterility characteristics.