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Cytoplasmic male-sterility (CMS) has been exclusively used to produce F1 hybrid seeds of onion (Allium cepa L.). A single nuclear locus, Ms, is known to restore male-fertility of CMS in onions. Unstable male-sterile onions producing a small amount of pollen grains have been identified in a previous study. When such unstable male-sterile onions were crossed with stable male-sterile onions containing CMS-R cytoplasm, male-fertility was completely restored, although genotypes of the Ms locus were homozygous recessive. Inheritance patterns indicated that male-fertility restoration was controlled by a single locus designated as Ms2. A combined approach of bulked segregant analysis and RNA-seq was used to identify candidate genes for the Ms2 locus. High resolution melting markers were developed based on single nucleotide polymorphisms (SNPs) detected by RNA-Seq. Comparative mapping of the Ms2 locus showed that Ms2 was positioned at the end of chromosome 2 with a distance of approximately 70 cM away from the Ms locus. Although 38 contigs containing reliable SNPs were analyzed using recombinants selected from 1344 individuals, no contig showed perfect linkage to Ms2. Interestingly, transcription levels of orf725, a CMS-associated gene in onions, were significantly reduced in male-fertile individuals of segregating populations. However, no significant change in its transcription level was observed in individuals of a segregating population with male-fertility genotypes determined by the Ms locus, suggesting that male-fertility restoration mechanism of Ms2 might be different from that of the Ms locus.

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.

Key messageA DNA transposon was found in the gene encoding a bHLH transcription factor. Genotypes of the marker tagging this DNA transposon perfectly co-segregated with color phenotypes in large F2:3 populationsA combined approach of bulked segregant analysis and RNA-Seq was used to isolate causal gene for C locus controlling white bulb color in onions (Allium cepa L.). A total of 114 contigs containing homozygous single nucleotide polymorphisms (SNPs) between white and yellow bulked RNAs were identified. Four of them showed high homologies with loci clustered in the middle of chromosome 5. SNPs in 34 contigs were confirmed by sequencing of PCR products. One of these contigs showed perfect linkage to the C locus in F2:3 populations consisting of 2491 individuals. However, genotypes of molecular marker tagging this contig were inconsistent with color phenotypes of diverse breeding lines. A total of 146 contigs showed differential expression between yellow and white bulks. Among them, transcription levels of B2 gene encoding a bHLH transcription factor were significantly reduced in white RNA bulk and F2:3 individuals, although there was no SNP in the coding region. Phylogenetic analysis showed that onion B2 was orthologous to bHLH-coding genes regulating anthocyanin biosynthesis pathway in other plant species. Promoter regions of B2 gene were obtained by genome walking and a 577-bp non-autonomous DNA transposon designated as AcWHITE was found in the white allele. Molecular marker tagging AcWHITE showed perfect linkage with the C locus. Marker genotypes of the white allele were detected in some white accessions. However, none of tested red or yellow onions contained AcWHITE insertion, implying that B2 gene was likely to be a casual gene for the C locus.

A yellow inbred line (SP3B) and a red doubled haploid line (H6) were used to produce a F2 mapping population of onion (Allium cepa L.). Initially, insertion/deletion and cleaved amplified polymorphic sequence markers were developed based on polymorphisms in the reference transcriptome. To improve efficiency of marker development, RNA-seq was carried out to identify single nucleotide polymorphisms (SNPs) between parental lines. High-quality SNPs were selected via customized screening process, and 344 high resolution melting (HRM) markers were developed. In addition, 161 HRM markers were developed based on the SNPs detected using genotyping-by-sequencing. A linkage map consisting of 652 molecular markers distributed in eight linkage groups was constructed. The total length of the linkage map was 749.8 cM, and the average interval between markers was 1.91 cM, the highest resolution among onion linkage maps reported so far. All eight linkage groups were assigned to onion chromosomes by identifying the common homologous loci in other linkage maps. Four major loci (C, I, R, and L) determining onion bulb colors were located on separate chromosomes. Analysis of ‘Santero’, a F1 cultivar resistant to downy mildew, revealed that the length of the chromosome fragment introgressed from Allium roylei, harboring the resistance gene estimated at 27.6 cM at the end of chromosome 3. The high-resolution linkage map constructed in this study and chromosomal locations of major qualitative loci will be used to design an effective selection strategy for onion breeding programs.

Both genomes in chloroplasts and mitochondria of plant cell are usually inherited from maternal parent, with rare exceptions. To characterize the inheritance patterns of the organelle genomes in cucumber ( Cucumis sativus var. sativus ), two inbred lines and their reciprocal F 1 hybrids were analyzed using an next generation whole genome sequencing data. Their complete chloroplast genome sequences were de novo assembled, and a single SNP was identified between the parental lines. Two reciprocal F 1 hybrids have the same chloroplast genomes with their maternal parents. Meanwhile, 292 polymorphic sites were identified between mitochondrial genomes of the two parental lines, which showed the same genotypes with their paternal parents in the two reciprocal F 1 hybrids, without any recombination. The inheritance patterns of the chloroplast and mitochondria genomes were also confirmed in four additional cucumber accessions and their six reciprocal F 1 hybrids using molecular markers derived from the identified polymorphic sites. Taken together, our results indicate that the cucumber chloroplast genome is maternally inherited, as is typically observed in other plant species, whereas the large cucumber mitochondrial genome is paternally inherited. The combination of DNA markers derived from the chloroplast and mitochondrial genomes will provide a convenient system for purity test of F 1 hybrid seeds in cucumber breeding.

Cytoplasmic chloroplast (cp) genomes and nuclear ribosomal DNA (nR) are the primary sequences used to understand plant diversity and evolution. We introduce a high-throughput method to simultaneously obtain complete cp and nR sequences using Illumina platform whole-genome sequence. We applied the method to 30 rice specimens belonging to nine Oryza species. Concurrent phylogenomic analysis using cp and nR of several of specimens of the same Oryza AA genome species provides insight into the evolution and domestication of cultivated rice, clarifying three ambiguous but important issues in the evolution of wild Oryza species. First, cp-based trees clearly classify each lineage but can be biased by inter-subspecies cross-hybridization events during speciation. Second, O. glumaepatula , a South American wild rice, includes two cytoplasm types, one of which is derived from a recent interspecies hybridization with O. longistminata . Third, the Australian O. rufipogan -type rice is a perennial form of O. meridionalis .

Key messageA gene encoding a laccase responsible for chartreuse onion bulb color was identified. Markers tagging this gene showed perfect linkage with bulb colors among diverse germplasm.To identify a casual gene for the G locus determining chartreuse bulb color in onion (Allium cepa L.), bulked segregant RNA-Seq (BSR-Seq) was performed using yellow and chartreuse individuals of a segregating population. Through single nucleotide polymorphism (SNP) and differentially expressed gene (DEG) screening processes, 163 and 143 transcripts were selected, respectively. One transcript encoding a laccase-like protein was commonly identified from SNP and DEG screening. This transcript contained four highly conserved copper-binding domains known to be signature sequences of laccases. This gene was designated AcLAC12 since it showed high homology with Arabidopsis AtLAC12. A 4-bp deletion creating a premature stop codon was identified in exon 5 of the chartreuse allele. Another mutant allele in which an intact LTR-retrotransposon was transposed in exon 5 was identified from other chartreuse breeding lines. Genotypes of molecular markers tagging AcLAC12 were perfectly matched with bulb color phenotypes in segregating populations and diverse breeding lines. All chartreuse breeding lines contained inactive alleles of DFR-A gene determining red bulb color, indicating that chartreuse color appeared when both DFR-A and AcLAC12 genes were inactivated. Linkage maps showed that AcLAC12 was positioned at the end of chromosome 7. Transcription levels of structural genes encoding enzymes in anthocyanin biosynthesis pathway were generally reduced in chartreuse bulk compared with yellow bulk. Concentrations of total quercetins were also reduced in chartreuse onion. However, significant amounts of quercetins were detected in chartreuse onion, implying that AcLAC12 might be involved in modification of quercetin derivatives in onion.

White bulb colors of onion (Allium cepa L.) are determined by the C and I loci which control the so-called recessive and dominant white bulb colors, respectively. To identify the causal gene responsible for the I locus, a combined approach of bulked segregant analysis and RNA-Seq was used in the present study. A total of 68 contigs containing homozygous single nucleotide polymorphisms (SNPs) between red and dominant white bulked RNAs were identified. The position of I locus was found to be located at chromosome 3 by performing comparative analysis of these contigs and using a previously constructed linkage map. After verification of homozygous SNPs by sequencing of PCR products, 12 high resolution melting, one cleaved amplified polymorphic sequence, and one InDel markers were developed. A linkage map flanking the I locus was constructed using these markers. Two tightly linked markers (DW51596 and DW35019) flanking the I locus were identified by analyzing 1457 F4 individuals. A total of 104 and 39 contigs showing more than tenfold increase of expression in red and dominant white bulks, respectively, were identified. Transcriptions of all structural genes encoding enzymes in flavonoid biosynthesis pathway were significantly reduced in the dominant white bulk. Transcription levels of most contigs showing more than tenfold reduced expression in dominant white were also significantly reduced in the recessive white bulbs controlled by the C locus. Genomic DNA sequences of 12 genes encoding transcription factors assumed to regulate flavonoid biosynthesis were analyzed. However, the causal gene for the I locus could not be identified.

Anthocyanins, the pigmented flavonoids responsible for red and blue colors in horticultural products, promote human health by preventing cancers and lowering the risk of cardiovascular disease. Red onions contain several cyanidin- and peonidin-based anthocyanins. In this study, we constructed a single-nucleotide polymorphism (SNP)-based genetic linkage map in an F2 segregating population derived from a cross between the inbred line ‘SP3B’ (yellow bulb) and the doubled haploid line ‘H6′ (red bulb) to identify quantitative trait loci (QTLs) for total anthocyanin content of onion bulbs using a genotyping-by-sequencing (GBS) analysis based on a reference gene set. A total of 101.9 Gbp of raw sequences were generated using an Illumina HiSeq 2500 system and a total of 1625 SNP loci were identified with the criteria of three minimum depths, lower than 30% missing rate, and more than 5% minor allele frequency. As a result, an onion genetic linkage map consisting of 319 GBS-based SNP loci and 34 high-resolution melting (HRM) markers was constructed with eight linkage groups and a total genetic distance of 881.4 cM. In addition, the linkage groups were assigned to corresponding chromosomes by comparison with the reference genetic map OH1×5225 through marker development based on common transcripts. The analysis revealed one major QTL, qAS7.1, for anthocyanin synthesis and two significant QTLs, qAC4.1 and qAC4.2, for anthocyanin content. The QTL qAS7.1, located on chromosome 7 with a phenotypic variation of 87.61%, may be a dihydroflavonol 4-reductase (DFR) gene that determines whether the bulb color is red or yellow. The QTLs qAC4.1 and qAC4.2 are separately positioned on chromosome 4 with R2 values of 19.43% and 26.28%, respectively. This map and QTL information will contribute to marker development and breeding for high anthocyanin content in bulb onion.

A novel cytoplasmic male sterility (CMS) conferred by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its restorer-of-fertility gene (Rfd1) was previously reported in radish (Raphanus sativus L.). Its inheritance of fertility restoration and profiles of mitochondrial DNA (mtDNA)-based molecular markers were reported to be different from those of Ogura CMS, the first reported CMS in radish. The complete mitochondrial genome sequence (239,186 bp; GenBank accession No. KC193578) of DCGMS mitotype is reported in this study. Thirty-four protein-coding genes and three ribosomal RNA genes were identified. Comparative analysis of a mitochondrial genome sequence of DCGMS and previously reported complete sequences of normal and Ogura CMS mitotypes revealed various recombined structures of seventeen syntenic sequence blocks. Short-repeat sequences were identified in almost all junctions between syntenic sequence blocks. Phylogenetic analysis of three radish mitotypes showed that DCGMS was more closely related to the normal mitotype than to the Ogura mitotype. A single 1,551-bp unique region was identified in DCGMS mtDNA sequences and a novel chimeric gene, designated orf463, consisting of 128-bp partial sequences of cox1 gene and 1,261-bp unidentified sequences were found in the unique region. No other genes with a chimeric structure, a major feature of most characterized CMS-associated genes in other plant species, were found in rearranged junctions of syntenic sequence blocks. Like other known CMS-associated mitochondrial genes, the predicted gene product of orf463 contained 12 transmembrane domains. Thus, this gene product might be integrated into the mitochondrial membrane. In total, the results indicate that orf463 is likely to be a casual factor for CMS induction in radish containing the DCGMS cytoplasm.