Cytoplasmic Male Sterility In Onion Research Articles

A Single Nucleotide Polymorphism Unique to the Galanthum-CMS of Onion

Different sources of cytoplasmic male sterility (CMS) are used to produce hybrid onion seed. The most commonly used source of CMS in onion is S cytoplasm (S-CMS), and male fertility is restored by a dominant allele at the nuclear male-fertility restoration locus (Ms). Male-sterile plants possess S cytoplasm and have the homozygous recessive genotype at Ms; seed propagation of male-sterile plants is possible by crossing with a male-fertile maintainer plant or inbred possessing normal (N) male-fertile cytoplasm and the homozygous recessive at the Ms locus (N msms). Some commercially important onion populations possess S-CMS and high frequencies of the dominant Ms allele, eliminating the possibility to develop maintainer lines. An alloplasmic source of CMS (Gal-CMS) was developed by backcrossing the cytoplasm of Allium galanthum into the nuclear background of onion. The advantage of Gal-CMS is that the dominant allele at Ms does not restore male fertility, making this source of CMS useful for the development of male-sterile lines from populations possessing S cytoplasm and dominant allele(s) at Ms. In this research, a single nucleotide polymorphism unique to the cytoplasms of A. galanthum and Gal-CMS was identified, useful to distinguish Gal-CMS from other onion cytoplasms.

Cloning and Expression Analysis of Onion (Allium cepa L.) MADS-Box Genes and Regulation Mechanism of Cytoplasmic Male Sterility.

Flower organ development is one of the most important processes in plant life. However, onion CMS (cytoplasmic male sterility) shows an abnormal development of floral organs. The regulation of MADS-box transcription factors is important for flower development. To further understand the role of MADS-box transcription factors in the regulation of cytoplasmic male sterility onions. We cloned the full-length cDNA of five MADS-box transcription factors from the flowers of onion using RACE (rapid amplification of cDNA ends) technology. We used bioinformatics methods for sequence analysis and phylogenetic analysis. Real-time quantitative PCR was used to detect the expression patterns of these genes in different onion organs. The relative expression levels of five flower development genes were compared in CMS onions and wild onions. The results showed that the full-length cDNA sequences of the cloned MADS-box genes AcFUL, AcDEF, AcPI, AcAG, and AcSEP3 belonged to A, B, C, and E MADS-box genes, respectively. A phylogenetic tree construction analysis was performed on its sequence. Analysis of MADS-box gene expression in wild onion and CMS onion showed that the formation of CMS onion was caused by down-regulation of AcDEF, AcPI, and AcAG gene expression, up-regulation of AcSEP3 gene expression, and no correlation with AcFUL gene expression. This work laid the foundation for further study of the molecular mechanism of onion flower development and the molecular mechanism of CMS onion male sterility.

Identification of Ms2, a novel locus controlling male-fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.), and development of tightly linked molecular markers

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.

Identification of a novel haplotype of the Ms locus controlling restoration of male-fertility and its implication in origination of cytoplasmic male-sterility in onion (Allium cepa L.)

ABSTRACT Seeds of F1 hybrid cultivars of onions have been exclusively produced by utilising cytoplasmic male-sterility (CMS). A single locus, Ms, controls restoration of male-fertility of CMS onions. When a molecular marker tagging the AcPMS1 gene, a candidate gene for the Ms locus, was used to genotype diverse breeding lines, an unusual marker genotype showing an intermediate size of PCR product was identified in one breeding line (ORI854). The male-fertility phenotype of ORI854 was all male-sterile. Full-length genomic DNA sequence (16,464 bp) of this novel AcPMS1 allele from ORI854 was more closely related to a recessive AcPMS1 allele than to a dominant allele. However, there were more than 200 polymorphic sequences between this novel allele and the recessive allele, indicating that this novel AcPMS1 allele might not have recently diverged from the recessive allele. Phylogenetic analysis implied that onion male-sterility might have first appeared in a common ancestor before divergence of this novel allele and recessive allele of AcPMS1. Analysis of full-length cDNA sequences of additional 12 genes showing almost perfect linkage disequilibrium with the Ms locus indicated that no recombination might have happened among 13 genes including AcPMS1 in the novel Ms haplotype after divergence from the recessive Ms haplotype.

Development of a genotyping method for onion (Allium cepa L.) male-fertility based on multiplex PCR

ABSTRACTThe most widely used source of onion cytoplasmic male-sterility (CMS) is controlled by a single nuclear gene and a cytoplasmic factor, and the sterility trait can be recovered by the dominant nuclear gene (Ms). Marker-assisted selection (MAS) has a great potential in the selection of male sterility lines and maintainer lines. In this study, two markers at the Ms loci and one cytoplasm marker were used to a develop multiplexed polymerase chain reaction (PCR) system for MAS of fertility traits in onion. Seven pairs of PCR primers were designed on the basis of the flanking sequences (F1890 and S1887) of the male-fertility restoration (Ms) locus and a mitochondrial sequence (mt472) in onion. One set of compatible primers were selected to develop a multiplex-PCR molecular marker (AcCN), which was established in forty-eight lines. The fertility types of cytoplasm and nuclear were distinguished by one-time PCR detection. The accuracy of the AcCN marker was verified in practical backcross breeding population. The AcCN marker types perfectly matched with their genotypes and phenotypes. It can be concluded that this marker is of great practical significance for MAS in the selection of onion maintainer lines.

Transcriptome de novo assembly and analysis of differentially expressed genes related to cytoplasmic male sterility in onion

Onion (Allium cepa L.) is one of the major vegetables in China and accounts for a large proportion of China's vegetable exports. Onion cytoplasmic male sterility, which is often used in onion breeding, is caused by the interaction between the nuclear genes and the cytoplasm. However, the underlying molecular mechanism of onion cytoplasmic male sterility remains unclear. In this study, we analysed the anther microstructure of the onion cytoplasmic male sterile line SA2 and the onion maintainer line SB2. We found that the pollen abortion in SA2 occurred at the tetrad stage during the microspore development, which was very different from that in SB2. We used the Illumina HiSeq platform to sequence RNA from anthers at the tetrad stage collected from the SA2 and SB2 lines. The RNA sequencing and transcriptome assembly produced 146,413 All-Unigenes. Based on an analysis of the differentially expressed genes, we identified two cytoplasmic control genes, atp9 and cox1, and three nuclear-related genes, SERK1, AG and AMS. These transcriptomic results were also verified by fluorescence quantitative PCR. Our study provides important information about genes related to onion cytoplasmic male sterility, and it will help improve the understanding of the molecular mechanism of onion cytoplasmic male sterility.

Identification of candidate genes associated with fertility restoration of cytoplasmic male-sterility in onion (Allium cepa L.) using a combination of bulked segregant analysis and RNA-seq.

A combination of BSA and RNA-seq was performed to identify candidates for the restorer-of-fertility gene in onion. The AcPMS1 involved in DNA mismatch repair was identified as the best candidate. To identify candidate genes of the restorer-of-fertility gene (Ms) responsible for fertility restoration of onion cytoplasmic male-sterility, a combined approach of bulked segregant analysis and RNA-seq was employed. From 32,674 de novo assembled contigs, 430 perfectly homozygous SNPs between male-fertile (MF) and male-sterile (MS) bulks were identified in 141 contigs. After verifying the homozygosity of the SNPs by PCR amplification and sequencing, the SNPs on 139 of the contigs were genotypes for the two recombinants which contained crossover events between the Ms locus and two tightly linked molecular markers. As a result, 30 contigs showing perfect linkage with the Ms locus in the large-sized segregating population were identified. Among them, 14 showed perfect linkage disequilibrium (LD) with the Ms locus, as determined by genotyping 251 domestic breeding lines. Furthermore, molecular markers tagging the 14 contigs also showed almost perfect LD with each other in 124 exotic accessions introduced from 21 countries, except for one accession which contained a crossover event by which the 14 markers were divided into two groups. After sequencing of the full-length cDNA of the 14 contigs showing perfect LD, the deduced amino acids sequences of the MF and MS alleles were compared. Four genes were shown to harbor putative critical amino acid changes in the known domains. Among them, the gene encoding PMS1, involved in the DNA mismatch repair pathway, was assumed to be the best candidate gene responsible for fertility restoration of male-sterility in onion.

Comparative analysis of the complete chloroplast genome sequences of a normal male-fertile cytoplasm and two different cytoplasms conferring cytoplasmic male sterility in onion (Allium cepaL.)

SummaryThe complete chloroplast genome sequences of a normal male-fertile (N) and two male-sterile (CMS) cytoplasms of onion (Allium cepa L.) were produced by next-generation sequencing (Illumina HiSeq2000). The sizes of the normal (N), CMS-S, and CMS-T chloroplast genomes were 153,529 bp, 153,440 bp, and 153,568 bp, respectively. All chloroplast genome structures consisted of a pair of large inverted repeats (26,450 - 26,468 bp) plus large and small single-copy regions. The content and order of genes in the three chloroplast genomes were identical. Each contained 74 proteincoding, four rRNA, and 30 tRNA genes. Many polymorphisms [352 single nucleotide polymorphisms (SNPs) and 141 insertions or deletions of bases (indels)] were observed between the N and CMS-S cytotypes, but only four SNPs and two indels were found between the N and CMS-T cytotypes, supporting the alloplasmic and autoplasmic origins of CMS-S and CMS-T male-sterility, respectively. A 7.6-fold reduction in mutation rate was observed in the large inverted repeats, while an elevated mutation rate was found in the intergenic region between the ndhF and rpl32 genes. Increased mutation rates were also observed in the ycf1, accD, and ndhF coding regions. A simple PCR marker was developed to distinguish the three cytotypes on the basis of the different lengths of the indels positioned between the petN and psbM genes. A total of 247 domestic breeding lines and 130 exotic accessions of onion, originating from diverse countries, were analysed using this and other previously developed markers to identify variant cytotypes.Two variants of each N and CMS-T cytotype were identified. In addition, two novel cytotypes showing different stoichiometries of the mitochondrial orf725 and cox1 genes were identified in the diverse germplasm.

The Dominant Ms Allele in Onion Shows Reduced Penetrance

The most commonly used source of cytoplasmic male sterility in onion (Allium cepa) is controlled by the interaction of the cytoplasm [male-sterile (S) or normal male-fertile (N)] and one nuclear male-fertility-restoration locus (Ms). Scoring of genotypes at Ms is generally done by testcrossing male-fertile to male-sterile (S msms) plants, followed by scoring of testcross progenies for male-fertility restoration. We identified two N-cytoplasmic families, one that was homozygous dominant and the other segregating at Ms. Plants from each of these two families were individually testcrossed to male-sterile onion. Nuclear restoration of male fertility in testcross progenies was evaluated in the field over 4 years. For male plants homozygous dominant at Ms, we expected testcross families to show 100% male-fertility restoration, but observed mean values between 46% and 100%. For plants segregating at Ms, we again observed lower than expected frequencies of male-fertility restoration. These results demonstrate that the dominant Ms allele shows reduced penetrance, requiring that male-fertility restoration be scored over years to more confidently assign genotypes at Ms.