Abstract
A novel male-sterility trait was identified in a radish (Raphanus sativus L.) population. Although the size of male-sterile anthers was comparable to that of normal flowers, no pollen grain was observed during anther dehiscence. However, dissection of male-sterile anthers revealed an abundance of normal pollen grains. Analysis of segregating populations showed that a single recessive locus, designated RsMs1, conferred male sterility. Based on two radish draft genome sequences, molecular markers were developed to delimit the genomic region harboring the RsMs1. The region was narrowed down to approximately 24 kb after analyzing recombinants selected from 7511 individuals of a segregating population. Sequencing of the delimited region yielded six putative genes including four genes expressed in the floral tissue, and one gene with significant differential expression between male-fertile and male-sterile individuals of a segregating population. This differentially expressed gene was orthologous to the Arabidopsis MYB26 gene, which played a critical role in anther dehiscence. Excluding a synonymous single nucleotide polymorphism in exon3, no polymorphism involving coding and putative promoter regions was detected between alleles. A 955-bp insertion was identified 7.5 kb upstream of the recessive allele. Highly conserved motifs among four Brassicaceae species were identified around this insertion site, suggesting the presence of putative enhancer sequences. A functional marker was developed for genotyping of the RsMs1 based on the 955-bp insertion. A total of 120 PI accessions were analyzed using this marker, and 11 accessions were shown to carry the recessive rsms1 allele.
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Acknowledgements
The authors thank Ji-hwa Heo, Jeong-An Yoo, and Su-jeong Kim for their dedicated technical assistance.
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This study was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) via Golden Seed Project (Center for Horticultural Seed Development, No. 213007–05-5-SBB10) along with a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ013400).
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Seongjun Kim performed experiments and drafted the manuscript. Sunggil Kim organized and coordinated this research project and edited the final manuscript.
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11032_2021_1254_MOESM1_ESM.tif
Supplementary file1 Pedigree of segregating populations used in this study. DCGMS: the original CMS plants identified by Lee et al. (2008). R109: a male-fertile breeding line, R121: a male-fertile breeding containing a dominant Rfd1 allele. MF: male-fertile, MS: male-sterile, Segregating: segregation of male-fertile and male-sterile phenotypes. Heterozygous male-fertile plants in F2 and F3 populations selected by flanking markers were used to produce succeeding segregating populations, respectively. (TIF 89 KB)
11032_2021_1254_MOESM2_ESM.tif
Supplementary file2 Viability tests of pollen grains of the novel male-sterility. A, B. light microphotographs of pollen grains stained with an aniline blue solution (Sweigart 2019). Images were taken at a magnification of 200x. A. Normal male-fertile radish, B. Novel male-sterile radish. Viable and non-viable pollen grains were stained densely and lightly, respectively. (TIF 509 KB)
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Supplementary file3 Collinear genomic regions containing the RsMs1 locus among two radish draft genome sequences and Arabidopsis chromosome 3. Genes showing homology are connected with horizontal lines. (TIF 685 KB)
11032_2021_1254_MOESM4_ESM.tif
Supplementary file4 Phylogenetic relationship of a radish PPR gene (Rs295360) located in the delimited region with other Rf and Rf-like PPRs isolated from other plant species. GenBank accession numbers of Rf proteins are shown in parenthesis. Information about Rf-like PPRs identified in Arabidopsis was obtained from Fujii et al. (2011). (TIF 70 KB)
11032_2021_1254_MOESM5_ESM.tif
Supplementary file5 Gene organization of radish and Arabidopsis in the delimited regions containing the RsMs1 locus. Arrow-shaped boxes indicate genes and 5’-to-3’ orientation. The RsMYB26 and Arabidopsis MYB26 genes are indicated by black boxes. Homologous genes are connected with vertical arrows. (TIF 108 KB)
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Supplementary file6 Transcription of genes associated with anther dehiscence: male-fertile vs. male-sterile bulk RNAs. The RPKM values were obtained from a previous study (Lee et al. 2014). Detailed information about these genes is presented in Supplementary Table 8. (TIF 60 KB)
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Kim, S., Kim, S. An insertion mutation located on putative enhancer regions of the MYB26-like gene induces inhibition of anther dehiscence resulting in novel genic male sterility in radish (Raphanus sativus L.). Mol Breeding 41, 67 (2021). https://doi.org/10.1007/s11032-021-01254-9
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DOI: https://doi.org/10.1007/s11032-021-01254-9