Abstract
Key message
Almost identical mitochondrial genome sequences of two recently diverged male-fertile normal and male-sterile CMS-T-like cytoplasms were obtained in onions. A chimeric gene, orf725 , was found to be a CMS-inducing gene.
Abstract
In onions (Allium cepa L.), cytoplasmic male-sterility (CMS) has been widely used in hybrid seed production. Two types of CMS (CMS-S and CMS-T) have been reported in onions. A complete mitochondrial genome sequence of the CMS-S cytoplasm has been reported in our previous study. Draft mitochondrial genome sequences of male-fertile normal and CMS-T-like cytoplasms are reported in this study. Raw reads obtained from normal and CMS-T-like cytoplasms were assembled into eight and nine almost identical contigs, respectively. After connection and reorganization of contigs by PCR amplification and genome walking, four scaffold sequences with total length of 339 and 180 bp were produced for the normal cytoplasm. A mitochondrial genome sequence of the CMS-T-like cytoplasm was obtained by mapping trimmed reads of CMS-T onto scaffold sequences of the normal cytoplasm. Compared with the CMS-S mitochondrial genome, the normal mitochondrial genome was highly rearranged with 31 syntenic blocks. A total of 499 single nucleotide polymorphisms (SNPs) or insertions/deletions were identified in these syntenic regions. On the other hand, normal and CMS-T-like mitochondrial genome sequences were almost identical except for orf725, a chimeric gene consisting of cox1 with other sequences. Only three SNPs were identified between normal and CMS-T-like syntenic sequences. These results indicate that orf725 is likely to be the casual gene for CMS induction in onions and that CMS-T-like cytoplasm has recently diverged from the normal cytoplasm by introduction of orf725.
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Acknowledgements
This research was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agriculture, Food and Rural Affairs Research Center Support Program (Vegetable Breeding Research Center), funded by the Ministry of Agriculture, Food and Rural Affairs (710011-03), Golden Seed Project (Center for Horticultural Seed Development, No 213007-05-2-SBB10), and a Grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ013400). The authors thank Ji-wha Hur, Jeong-Ahn Yoo, and Su-jung Kim for their dedicated technical assistance.
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Supplementary Fig. 1.
PCR amplification patterns of four previously developed molecular markers used for identification of cytoplasm types of onions. The 7th nucleotide ‘G’ in the reverse primer of the marker reported by Havey (1995) was changed into ‘A’ to avoid mismatch with complete chloroplast genome sequences of the normal (GenBank accession: KM088013), CMS-S (KM088014), and CMS-T (KM088015) cytoplasms. (TIFF 145 kb)
Supplementary Fig. 2.
Organization of nine contigs assembled from normal and CMS-T-like cytoplasms using trimmed reads produced by next-generation sequencing. Genes transcribed as forward and reverse complements are indicated as boxes on and beneath lines, respectively. Filled and empty boxes indicate exons and introns, respectively. Contig 9 was identified only in the CMS-T-like cytoplasm. (TIFF 110 kb)
Supplementary Fig. 3.
Duplicated sequences in scaffold 1 and scaffold 2. A. Positions of duplicated sequences. Duplicated sequences are shown as hatched boxes. Arrow-shaped boxes indicate 5′-to-3′ direction. Exons and introns are shown as gray and empty boxes, respectively. Horizontal arrows indicate primer-binding sites. B. PCR products of primer pairs designed based on duplicated regions and their flanking sequences shown in Supplementary Fig. 2A. A primer pair of nad3-F1 and rps12 was designed for nad3 and rps12 genes, respectively. (TIFF 250 kb)
Supplementary Fig. 4.
Duplicated sequences in scaffold 1 and scaffold 3. A. Positions of duplicated sequences. Duplicated sequences are shown as hatched boxes. Filled boxes indicate positions of R6 repeats (Supplementary Table 5). Arrow-shaped boxes indicate 5′-to-3′ direction. Horizontal arrows indicate primer-binding sites. B. PCR products amplified using primer pairs designed based on the duplicated regions and their flanking sequences as shown in Supplementary Fig. 3A. A primer pair of nad3-F1 and rps12 was designed based on nad3 and rps12 genes, respectively. (TIFF 186 kb)
Supplementary Fig. 5.
Positions of repetitive sequences larger than 500 base pairs on scaffold sequences. Genes transcribed as forward and reverse complements are indicated as small boxes on and beneath large rectangular boxes, respectively. Exons and introns are shown as dark-gray and empty small boxes, respectively. Repeat sequences are shown as filled boxes in large rectangular boxes. Names of repeats in filled boxes are shown on inverted triangles linked to repeats. Detailed information about repeats is described in Supplementary Table 5. (TIFF 193 kb)
Supplementary Fig. 6.
Distribution of repeat sequences in mitochondrial genome sequences of onions and four other plant species. Dot matrix views of sequence alignments performed by BLAST search (http://blast.ncbi.nlm.nih.gov) against themselves are presented. GenBank accession numbers of complete mitochondrial genome sequences are KU318712 (Allium cepa, CMS-S cytoplasm), JN375330 (Phoenix dactylifera), KX028885 (Cocos nucifera), JQ804980 (Spirodela polyrhiza), KR559021 (Heuchera parviflora), DQ645536 (Zea mays), and BA000029 (Oryza sativa). (TIFF 337 kb)
Supplementary Fig. 7.
Verification of sequence organizations flanking orf725 and cox1 genes using long PCR amplification. A. PCR products of three combinations of primers. Positions of primers are shown in Fig. 2. Sequences of primers are shown in Supplementary Table 1. N, T, S indicate normal, CMS-T-like, and CMS-S mitotypes, respectively. PCR product under the asterisk is a low-copy-number subgenome containing gene organization similar to the corresponding region of CMS-S mitotype. B. Organization of the subgenomic sequence containing orf725 in the CMS-T-like mitotype. Nucleotide sequences of this PCR product were verified by sequencing PCR product which had been further amplified by ten additional cycles. Arrow-shaped boxes indicate 5′-to-3′ direction. Horizontal arrows indicate primer-binding sites. Homologous regions are connected by vertical lines. Filled, hatched, and dotted boxes indicate identical repeat sequences. (TIFF 137 kb)
Supplementary Fig. 8.
Location and probability of transmembrane domains in deduced amino acid sequence of orf725. The output of TMHMM Server (http://www.cbs.dtu.dk/services/TMHMM/) is shown underneath the orf725 diagram. An arrow-shaped box indicates the 5′-to-3′ direction. Gray and black colors indicate cox1-homologous and unknown sequences, respectively. (TIFF 932 kb)
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Kim, B., Yang, TJ. & Kim, S. Identification of a gene responsible for cytoplasmic male-sterility in onions (Allium cepa L.) using comparative analysis of mitochondrial genome sequences of two recently diverged cytoplasms. Theor Appl Genet 132, 313–322 (2019). https://doi.org/10.1007/s00122-018-3218-z
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DOI: https://doi.org/10.1007/s00122-018-3218-z