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Transcript profile and post-transcriptional processing of the mitochondrial orf156 were compared among the nucleus-cytoplasm (NC) hybrids of tetraploid and hexaploid wheat and their parental lines. These NC hybrids, in which the plasmon from the maternal Aegilops squarrosa parent is combined with the nuclear genomes of the paternal parents, and their parental lines were previously shown to have heteroplasmic copies of the polycistronic transcriptional unit nad3-orf156 in different sub-stoichiometries. Despite the heteroplasmic conditions, only single transcript types were detected among reverse transcription-PCR products of the orf156 mRNA in the respective lines. In the NC hybrids only a major maternal copy was transcribed and edited, likewise in the parental lines only single major copies were transcribed and edited. RNA gel blot analysis using the orf156 probe showed that the NC hybrids had the transcripts similar to the parental lines but unlike the maternal line. Primer extension analysis further revealed the orf156 transcripts with different 5' termini, which most probably resulted from the alternative post-transcriptional cleavages of the precursor mRNA. These cleaved transcripts were polymorphic not only among the lines but also among four different tissues. The cleavages were highly selective and the paternal-type cleavages predominated in the NC hybrids, irrespective of the tissues studied. This seemingly neutral polymorphisms in wheat could make this system convenient for further analysis of mitochondrial gene expression in plants.

Structural heterogeneity depicted as heteroplasmy of the mitochondrial (mt) transcriptional unit of nad3-orf156 (atp8) was studied in a nucleus-cytoplasm (NC) hybrid of Triticum timopheevi with the D plasmon from the maternal Aegilops squarrosa and compared with that of the parental lines. The tetraploid NC hybrid and the parental lines both showed varying degrees of heteroplasmy in this mtDNA region. The G plasmon of the paternal T. timopheevi possessed five sequence types, while two sequence types were detected in the D plasmon of Ae. squarrosa. The NC hybrid possessed all the five sequence types identical to those of the paternal parent in a 30% relative stochiometry. The remaining 70% comprised only one of the two maternal sequence types, suggestive of strong and selective NC interaction. No novel sequence types were detected and the relative stoichiometries of the paternal sequence types were conserved in the NC hybrid. No paternal-identical or -related sequences were detected in the maternal D plasmon. These results provide evidence of the paternal transmission of the mtDNA and possibly account for the origin of the observed mtDNA heteroplasmy in the NC hybrid.

A mitochondrial (mt) transcriptional unit, nad3-orf156, was studied in the nucleus-cytoplasm hybrids of wheat with D/D2 plasmons from Aegilops species and their parental lines. A comparative RFLP analysis and sequencing of the random PCR clones revealed the presence of seven sequence types and their polymorphic sites were mapped. All the hybrids possessed the paternal copies besides the maternal copies. More paternal copies were present in the D2 plasmon hybrids, whereas more maternal copies were present in the D plasmon hybrids. Two major copies were present with different stoichiometries in the maternal Aegilops parents. However, only a major D plasmon copy was detected in the hybrids, irrespective of their plasmon types. The hexaploid wheat parent (AABBDD genome) possessed the major D plasmon copy in ~5% stoichiometry, while no D plasmon-homologous copies were detected in the tetraploid wheat parent (AABB genome). The results suggest that the observed mtDNA heteroplasmy is due to paternal contribution of mtDNA. The different copy stoichiometry suggests differential amplification of the heteroplasmic copies among the hybrids and the parental lines. All editing sites and their editing frequencies were conserved among the lines, and only the maternal pattern of editing occurred in the hybrids.