Abstract：The purpose of this study is to identify cytoplasmic type of isonucleur allocytoplasm cytoplasmic male sterile lines with Aegilops kotschyi, Ae. Ventricosa cytoplasm, Triticum spelta cytoplasm and their maintainer lines (90-110 and 8222) and to improve its directional genetic improvement in the research and application of hybrid wheat(Triticum aestivum L.). Sequence characterized amplified region(SCAR) molecular marker technique and amplified fragment length polymorphism(AFLP) marker molecular technique were used to analyze the wheat mitochondrial DNA. Three isonucleur allocytoplasm cytoplasmic male sterile lines with Aegilops kotschyi, Ae. Ventricosa cytoplasm, Triticum spelta cytoplasm and their maintainer lines were selectively amplified using 64 primer combinations EcoRⅠ-NNN/MseⅠ-NNN, and the results showed that 113 out of 682 bands were polymorphic. Primer E-AGG/M-CTA was found to amplify a special 300 bp fragment in the cytoplasmic male sterile lines with Aegilops Kotschyi cytoplasm. Specific fragment was collected and sequenced, SCAR molecular markers were redesigned with Primer Premier 5.0, and SCAR marker(YW1) amplified a fragment in 3 isonuclear alloplasmic cytoplasmic male sterile lines and their maintainer lines. SCAR marker(YW2) was successfully designed and validated, and it did not amplify fragment in isonuclear alloplasmic cytoplasmic male sterile lines with Ae. Ventricosa cytoplasm, Triticum spelta cytoplasm and their maintainer lines (90-110 and 8222), only one specific 198 bp fragment was amplified with YW2 in ms(Kots)-90-110 and ms(Kots)-8222 which showed sequence similarity was as high as 99%, and it was sequence of nicotinamide adenine dinucleotide(NADH) dehydrogenase gene (GenBank: EU534409.1). Oxidoreductase (complexⅠ) is the entry enzyme of mitochondrial oxidative phosphorylation, it was closely associated with the cytoplasmic male sterility in wheat. This SCAR marker can be used in molecular marker-assisted breeding for identifying wheat male sterile lines and provide theoretical and technical support for identifying cytoplasmic types of wheat.
