Short communicationAn apple atp9 pseudogene is maintained at high copy number in ‘Golden Delicious’-type mitochondria but is present substoichiometrically in ‘Delicious’-type mitochondria
Highlights
► An apple cultivar ‘Golden Delicious’ mitochondria contains one intact atp9 gene and two truncated copies (termed φatp9-1 and φatp9-2). ► The φatp9-1 sequence is maintained at high copy number in the six ‘Golden Delicious’-cytotype cultivars. ► In the eight ‘Delicious’-cytotype cultivars, however, φatp9-1 is present substoichiometrically. ► A homologous recombination was a possible cause to generate φatp9-1 using the short repeat in a common ancestral mitochondrial genome of ‘Golden Delicious’ and ‘Delicious’. ► The φatp9-2 was revealed to be present in high abundance in all 14 cultivars.
Introduction
A fundamental characteristic of higher plant mitochondrial DNA (mtDNA) is its propensity to recombine across dispersed repetitive sequences (Knoop, 2004, Mackenzie, 2007). Extensive recombination frequently generates considerable variation in genomic organization. Such recombination events are also likely to be involved in the creation of mitochondrial chimeric genes and pseudogenes (Conklin and Hanson, 1994, Fauron et al., 2004). In apples (Malus × domestica Borkh.), mtDNA has been used to characterize the cytoplasmic diversity of a wide range of cultivars and landraces (Ishikawa et al., 1992, Kato et al., 1993). The use of mitochondrial cox1 (cytochrome c oxidase subunit 1) and atp9 (ATP synthase F1 subunit 9) gene probes detected restriction fragment length polymorphisms (RFLPs), which enabled classification of a large number of apple genotypes into four cytoplasmic groups: ‘Golden Delicious’ type, ‘Delicious’ type, ‘McIntosh’ type, and ‘Dolgo Crab’ type.
In order to understand the molecular basis of the changes in the mitochondrial genome leading to these diverse cytoplasmic types, it is necessary to analyze the genomic regions in each of the four mtDNA types that can be used to distinguish apple germplasm. Wakatsuki et al. (2011) have recently shown that in two apple cultivars, ‘Golden Delicious’ and ‘Delicious,’ the cox1 reading frame exists as one full-length version (intact copy) and one truncated version (pseudocopy), and that the intact cox1 and pseudocopy have an 1115 bp segment in common. They also suggested that recombination events may have occurred within the 1115 bp repeats to create the two distinct mitochondrial genome organizations characteristic of the ‘Golden Delicious’ and ‘Delicious’ cytotypes. In this paper, we present an analysis of the rearrangements involving the atp9 loci of the ‘Golden Delicious’ and ‘Delicious’ cytotype cultivars and rootstocks.
Section snippets
Plant material and nucleic acid preparation
Leaf samples of apple cultivars and rootstocks (Table 1) were obtained from the collections at the National Institute of Fruit Tree Science, National Agriculture and Food Research Organization, Japan and the Field Center for Northern Biosphere, Hokkaido University, Japan. The preparation of total genomic DNA and total RNA from green leaves has been described previously (Wakatsuki et al., 2011).
Hybridization and sequence analysis
Restriction enzyme digestion, agarose gel electrophoresis, Southern and Northern blot analysis, DNA
‘Golden Delicious’ atp9 locus
A previous study showed that hybridization of HindIII-digested apple DNA with the pea atp9 probe produced a 4.8 kb fragment in gels of the ‘Golden Delicious’ and ‘Delicious’ cytotype cultivars, and an additional 9.2 kb fragment in ‘Golden Delicious’ cytotype cultivars (Kato et al., 1993). Kato et al. (1995) subsequently showed that the 4.8 kb fragment from ‘Delicious’ contained an intact atp9 gene and the first and second exons of the nad5 gene (nad5 ex1 and nad5 ex2).
Here, we isolated the 4.8 kb
Acknowledgements
Leaf samples were kindly supplied by National Institute of Fruit Tree Science, Tsukuba, Japan and Field Science Center for Northern Biosphere, Experiment Farm, Hokkaido University, Sapporo, Japan. This work was supported in part by Grants-in-Aids for Scientific Researches from Ministry of Education, Science, and Culture, Japan.
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Cited by (2)
Cytoplasmic diversity and possible maternal lineages in apples as revealed by analysis of the mitochondrial cox1 and atp9 loci
2013, Scientia HorticulturaeCitation Excerpt :Kato et al. (1993) defined four apple cytoplasmic types within the cultivar and landrace accessions, based on RFLP studies using mitochondrial cox1 and atp9 gene probes: the cytotypes have been described as ‘Golden Delicious’ type, ‘Delicious’ type, ‘McIntosh’ type, and ‘Dolgo Crab’ type. We have recently characterized mitochondrial genome rearrangements implicated in the origin of the cytoplasmic types (Wakatsuki et al., 2011; Kato et al., 2012). The rearrangements were suggested to be the consequence of homologous recombination events involving the cox1 and atp9 loci.
Cytoplasmic genome diversity in the cultivated apple - Short communication
2015, Horticultural Science
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These authors contributed equally to this work.