Redox control of gene expression is suggested as the common feature of those chloroplast and mitochondrial proteins that are encoded in situ. Recent evidence is consistent with this hypothesis, and its underlying assumptions and predictions are described.
Abstract Chloroplasts and mitochondria originated as bacterial symbionts. The mitochondrial plastid DNAs and their plastid origins are linked with gray line. The positions of six InDel markers M1 to M6 are labeled with red triangles. The same color indicates the same genotype for polymorphic sites among parental lines and F 1 hybrids.
A total of polymorphic sites, including SNPs and 46 InDels, were identified between the mitochondrial sequences of the two parental inbred lines Fig. The NGS mitochondrial reads mapped onto the polymorphic sites had an average coverage of Among the polymorphic sites, , 46, and six were identified in intergenic regions, introns, and exons, respectively. All six exonic SNPs were non-synonymous substitutions that resulted in amino acid changes. Molecular markers were developed based on the polymorphic sites identified in the chloroplast and mitochondrial sequences.
For the chloroplast sequence, a pair of dCAPS markers were designed based on the single SNP identified between the chloroplast genomes of the two parental inbred lines Fig. For the mitochondrial sequence, six targets with more than a bp InDel between the two parental lines were selected among the polymorphic sites Fig. Overall, seven polymorphic sites, one from the chloroplast genome and six from the mitochondrial genome, were used to design molecular markers for the investigation of the inheritance patterns of the chloroplasts and mitochondria Table 3.
The molecular markers were used for genotyping of the two parental inbred lines and their two reciprocal F 1 hybrids. The chloroplast-derived markers revealed that the F 1 hybrids shared an identical genotype with their maternal parent in each cross Fig.
By contrast, all six of the mitochondria-derived markers were used to reveal identical genotypes between the F 1 hybrids and their paternal parent Fig. Validation of molecular markers to confirm inheritance pattern of organelles in cucumber.
The maternal inheritance of chloroplast genome and paternal inheritance of mitochondrial genome were confirmed from all six reciprocal crosses of the four parental lines. Summarized inheritance patterns of chloroplast and mitochondria in cucumber.
Organelle inheritance from parental inbred lines to their two reciprocal F 1 progeny was elucidated through whole-organelle genome- and polymorphic marker-based genotyping, confirming that cucumber chloroplasts were inherited maternally while mitochondria were inherited paternally. Colors indicate genotypes.
Here, we generated approximately 1 Gb of NGS data, which is about three-fold the haploid genome coverage of Cucumis sativus These data were used to assess the genetic diversity and inheritance of the organellar genomes.
This quantity of data was sufficient to facilitate the assembly of the chloroplast genomes of the four assessed cucumber lines, which confirmed their maternal inheritance based on the inheritance of one SNP identified between the parental lines. We also identified SNPs and 46 InDels across the mitochondrial genome by mapping NGS reads directly to the reference cucumber mitochondrial genome. Here, we used complete genome sequences to validate the previously reported inheritance patterns of the organellar genomes, which were identified using only a few Restriction Fragment Length Polymorphism RFLP markers 33 , This inheritance pattern was further inspected in the present study using another four cucumber lines and their reciprocal F 1 progenies.
Our method is based on advanced NGS technologies and could be widely used not only for the study of the inheritance patterns of plant organellar genomes, but also for the study of mitochondrial genomes in general, which are usually more difficult to fully assemble than the chloroplast genomes.
In this study, the chloroplast genomes of the four cucumber lines MGL, CFL, their reciprocal F 1 hybrids could be fully assembled; however, the mitochondria genomes could not. This is not only because the chloroplast copy number is generally much higher than that of the mitochondria, but also because the cucumber mitochondrial genome is unusually large and complex The mitochondrial gene sequences are more conserved than the chloroplast genes 40 , 41 ; however, here, we identified 52 polymorphic sites in the mitochondrial gene regions between two cucumber lines but none in the chloroplast gene regions.
The six exonic polymorphic sites in the mitochondrial genome were non-synonymous mutations, which caused amino acid changes in the corresponding protein sequence.
Genes containing the non-synonymous mutations included rps1 , encoding a ribosomal protein; ccmB , encoding an ABC transporter subunit; and nad7 , encoding a NADH dehydrogenase subunit. In Arabidopsis, the chloroplast rps1 gene is involved in heat stress tolerance 42 , and may help to optimize chloroplast integrity under heat stress.
It is therefore possible that the cucumber rps1 gene in the mitochondrial genome might also be involved in the heat stress response. The mitochondrial ccmB is involved in cytochrome c and c1 biogenesis in wheat Nad7 encodes a NADH dehydrogenase, which is involved in the essential respiratory chain. The inhibition of the respiration is a main cause of the production of reactive oxygen species, which can damage cells and tissues The important roles these genes play in plants suggests that future studies should explore how their identified mutations in cucumber affect the phenotypes of these plants.
Many plastid-derived DNA fragments have been identified in plant mitochondrial genomes 45 , As discussed above, the cucumber mitochondrial genes are particularly diverse relative to the diversity observed in other plants 41 , and MTPT is also abundant in the cucumber mitochondrial genome. We can therefore assume that the abundant gene diversity in the cucumber mitochondrial genome is related to its paternal inheritance trait and its high content of MTPT fragments.
Similar cases were reported in the organelle genomes of gymnosperms including the Pinaceae and Taxaceae, which are often paternally inherited The synonymous substitution rates of both chloroplast and mitochondrial genomes were reported to be higher in species displaying paternal inheritance rather than maternal inheritance In animals, the organellar genomes are rarely inherited paternally or biparentally 49 ; however, plants with unique organelle inheritance patterns are not as rare as their animal counterparts.
Most conifers display paternal chloroplast inheritance patterns 50 , 51 , while the chloroplast genomes of alfalfa Medicago sativa and Oenothera spp. Like cucumber, wild bananas Musa acuminata display a maternal chloroplast inheritance and a paternal mitochondrial inheritance Ten markers designed to target polymorphisms in the mitochondrial, chloroplast, and nuclear genomes were successfully applied to validate the genomic inheritance patterns in the F 1 progenies derived from crosses between several cucumber lines.
Plant cytoplasmic organelles are not only involved in photosynthesis and respiration; recent studies have revealed their diverse roles, including in agriculturally important traits such as male sterility 16 , 53 , The cucumber mitochondrial genes may also affect male sterility properties, making the paternal inheritance of the cucumber mitochondrial genome especially important for agriculture.
The independent inheritance of the cucumber chloroplasts and mitochondria must be considered in breeding programs. The genome sequences and markers developed here are therefore expected to be of great value in the cucumber breeding industry. In addition, we expect that our results could be used as a foundation to further the research into plants with unusual organelle inheritance traits, particularly in cucumber itself.
The molecular markers developed in this research could be practically applied to the genotyping of other cucumber inbred lines or samples; for example, combinations of the chloroplast and mitochondrial markers could be used to check the purity of F 1 hybrid cucumber seeds as they are easily detected high-copy targets derived from the maternal and paternal parents, respectively.
All the breeding lines and reciprocal F 1 hybrids were developed in this study. Total genomic DNAs were extracted from fresh leaves using a modified cetyltrimethyl ammonium bromide CTAB method 55 and their quality was examined using agarose electrophoresis and a spectrometer.
The chloroplast genome contigs were extracted, ordered, and merged to generate a single draft sequence, based on the reported reference chloroplast genome sequence of C. The draft chloroplast sequences were manually combined, corrected, and gap-filled using a series of PE read mapping.
The chloroplast genome sequence of each sample was independently assembled. Positions of properly mapped reads were selected using SAMtools ver. Polymorphic sites identified in the chloroplast and mitochondrial sequences were used to design molecular markers for the analysis of the chloroplast and mitochondrial genotypes in other cucumber lines.
For the optimal amplification of each specific PCR product, the cycles and annealing temperatures of the PCR reactions were adjusted for each of the markers. A final concentration of 1. All of the uncropped gel image was included in Supplementary information Supplementary Fig. S4 and S5. In addition, InDel polymorphic sites in the nuclear genome sequences of the two parental lines were identified by mapping the NGS reads to the cucumber reference nuclear genome sequence v2.
These nuclear InDel sites were then used to design markers to validate the heterozygous nuclear genotype of the F 1 hybrids. The four of chloroplast genomes of C.
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All the other authors declare no competing interests. Reprints and Permissions. Kang, BC. Chloroplast and mitochondrial DNA editing in plants. Plants 7, — Download citation. Received : 20 April Accepted : 11 May Published : 01 July Issue Date : July Anyone you share the following link with will be able to read this content:.
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