KR101760932B1 - Molecular Markers related a Restorer-of-Fertility gene and Methods for Selecting of Male-Fertility or Male-Sterility in Onion - Google Patents

Abstract

The present invention relates to a nucleic acid molecule for screening male fertility or male sterility and a male-fertile or male-sterile determination method of onion using the nucleic acid molecule. The present inventors selected five candidate genes for determining the recovery of male sterility in onion. The candidate genes identified by the present inventors can be easily analyzed for genotypes on an agarose gel and can be used universally. Using the discriminant method of the present invention, the efficiency of F ₁ hybrid crossing of the onion can be improved, and in particular, the work which takes more than 4 years in the progeny test can be completed in a single day, It can be improved.

Description

Molecular Markers related a Restorer-of-Fertility gene and Methods for Selecting of Male-Fertility or Male-Sterility in Onion}

The present invention is a molecular marker related to fertility recovery of onions and a method for selecting male-fertility or male-sterility using the same, and more particularly, for onion cytoplasmic male infertility fertility recovery using bulk segregant analysis and RNA sequencing. (fertility restoration of cytoplasmic male-sterility) This relates to the identification of gene-based molecular markers.

Cytoplasmic male-sterility (CMS) is a maternally inherited trait that does not affect female fertility, but affects pollen production. It is still unclear whether CMS has a selective advantage in evolution (Hanson and Bentolila 2004), but CMS has been adopted as a successful reproduction strategy in over 140 species of plants in nature (Laser and Lersten, 1972). Up to now, cases of CMS spontaneously occur due to the generation of non-ideal genes in the mitochondrial genome (Hu et al. 2O14).

CMS is expected to be induced due to the characteristics of several plant mitochondrial genomes (Schnable and Wise L998; Budar et al. 2003; Knoop 2004; Kubo and Newton 2008). Unlike small and stable animal mitochondria, plant mitochondria have large genomes of relatively different sizes, for example Brassica species have genomes of about 200 kb (Palmer and Herbon 1987) and Silene conica have genomes of 11,319 kb. (Sloan et aL. 2012). In addition, plants have very diverse genome composition due to the dynamic rearrangement of genes (Palmer 1988; Park et al. 2013b). Accordingly, the structure of the plant mitochondrial genome has not been clearly established (Backert et at. 1997; Oldenburg and Bendich 2001; Allen et al.20O7).

Frequent repeat sequence-mediated recombination results in dynamic rearrangements (Palmer L988; Small et al. 1989; Albert et al. 1,998; Woloszynska and Trojanowski 2OO9). Genomic rearrangement appears in relatively short repeat sequences (<1 kb), but long repeat sequences affect the formation of various parts by forming various subgenomes due to genome rearrangement. Subgenome copy numbers and stoichiometry also vary, even at the intraspecific level. The specific stoichiometry of subgenomes is generally maintained through generations, but can be changed by a mechanism called substoichiometric shifting (Sakai and Imamura 1993; Bellaoui et al. 1998; Janska et al. 1998; Arrieta-Montiel et al. 200L; Kim et al. 20O7). The increase in the copy number of the low-copy-number subgenome with the CMS-inducing gene, which is indicated by substoichiometric shift, leads to CMS induction within a short period of time (Janska et al. 1998). Tissue culture (Kanazawa et al. 1994) and mutations in certain nuclear genes are known to lead to substoichiometric shifts in plants. Msh1 (Abdelnoor et al. 2006) and RecA (Shedge et al. 2007) genes are involved in DNA repair as well as single-stranded DNA binding protein OSB1 (Zaegel et aL. 2006), and are also involved in the inhibition of mitochondrial genome rearrangement. In many plant species, the male reproduction caused by abnormal mitochondrial genes is mostly restored by the nuclear restorer-of-fertility (Rf) gene. Since the first isolation of the Rf gene from maize (Cui et al. 1.996), the Rf gene has been cloned from several plant species in other plants.

Four Rf genes have been found to encode a pentatricopeptide repeat (PPR) protein (Bentolila et al.2OO2; Brown et aL.2003; Desloire et al.20O3; Koizuka et al.2OO3; Komori et al. 20O4; Klein et al. 2005), aldehyde dehydrogenase (Cui et al. L996), glycine-rich protein (Itabashi et al. 2011) and other unknown proteins (Fujii and Toriyama, 2009). Reported.

In onion, two CMS systems have been reported: CMS-S (Jones and Emsweller 1936) and CMS-T (Berninger 1965). This system has been widely used in the production _{of F 1 hybrid seeds.} Although it is difficult to distinguish the male infertility phenotype of the two systems by visual observation, it has been reported that the genetic mechanisms of fertility recovery are different from each other. One of the Rf genes, the Ms locus, was found to regulate fertility recovery in CMS-S (Jones and Clarke 1943), and three or more independent Rf loci were found to be involved in fertility recovery in CMS-T. (Schweisguth 1973). However, a recent study reported that the male reproduction of CMS-S and CMS-T may be restored by the same Ms locus or other closely related genes (Kim et al., 2014).

Since onion is a biennial crop, and economical F ₁ hybrid seed production is possible only by using CMS, rapid and reliable identification of cytoplasmic type and Rf gene type is fundamental to _{onion F 1 hybrid cultivation.} Accordingly, many molecular markers based on mitochondrial and chloroplast genomic polymorphism have been developed for the identification of cytoplasmic types (Havey 1995; Sato 1998; Engelke et al. 2OO3; Kim et al. 2OO9). In addition, molecular markers associated with the Ms locus have been developed for marker-assisted selection of the Ms genotype (Gokge et al., 2O02; Bang et al., 2013; Park et al., 2O13; Yang et al., 2013; Kim et al., 2014). In the present invention, as a method of combining bulk segregant analysis (BSA, Michelmore et al., 1991) and RNA-Seq, a candidate gene affecting the recovery of male sterility of CMS onions was identified, and the candidate A reliable genetic marker for Ms genotyping was identified based on the polymorphism of the gene.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have made intensive research efforts to identify candidate genes that affect the recovery of male infertility in onions. As a result, the present inventors identified five candidate genes related to the recovery of male infertility in onions using bulk segregant analysis and RNA-Seq. It was confirmed that male-fertile or male-sterile can be discriminated.

Accordingly, an object of the present invention is to provide a nucleic acid molecule for selecting onion male fertility or onion male sterility.

Another object of the present invention is to provide a method for determining male-fertile or male-sterile onions.

Another object of the present invention is to provide a kit for determining male-fertile or male-sterile onions.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention is a nucleic acid molecule for selection of male fertility onions comprising a nucleotide sequence of SEQ ID NO: 1, 3, 5, 7 or 9 of the Sequence Listing Provides.

According to one aspect of the present invention, the present invention is a nucleic acid molecule for selection of male fertility and onion male fertility comprising a nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, or 10 of the Sequence Listing. Provides.

As a result of the present inventors' diligent research efforts to identify candidate genes that affect the recovery of male infertility in onions, five candidate genes related to the recovery of male infertility in onions (Acepa31446) using bulk segregant analysis and RNA-Seq. , Acepa28839, Acepa26780, Acepa27528, Acepa23881) were identified, and when used as a molecular marker for cytoplasmic type analysis, it was confirmed that male-fertility or male-infertility of onions can be discriminated.

In order to identify the fertility recovery gene (Ms ) candidate involved in the recovery of the cytoplasmic male infertility fertility of onions, bulk isolate analysis and RNA-seq were used in combination. Of the 32,674 new combination contigs, 430 perfect homologous SNPs between the male-fertile (MF) and male-sterile (MS) bulks were identified in 141 contigs. As a result of confirming the homozygosity of SNPs by PCR amplification and sequencing, SNPs on 139 contigs were genotypes of two recombinants in which cross-action between the Ms gene and two closely related molecular markers occurred. As a result, 30 contigs were identified that were perfectly related to the Ms gene in a large-scale isolate. Of these, 14 showed complete linkage disequilibrium (LD) with the Ms gene as shown in the genotyping results of 251 domestic cultivars. In addition, the molecular markers tagging 14 contigs were almost completely related to 124 foreign varieties introduced from 21 countries, except for one breed where 14 markers were separated into 2 groups and crossover appeared. Showed an imbalance. After sequencing the full-length cDNA of 14 contig showing complete LD, it was compared with the amino acid sequences of the MF and MS alleles. Four genes (Acepa31446, Acepa28839, Acepa26780, and Acepa27528) had amino acid changes that are expected to be very important in the known domain, and one gene (Acepa23881) had amino acid changes in the conserved region (Figure 5 Reference).

Among these, the PMS1 gene, which is involved in the DNA mismatch repair mechanism, is expected to be the most potent candidate gene related to the recovery of male fertility in onions. Most of the male infertile organic genes present in the mitochondrial genome are chimeric genes, which are caused by a frequently occurring recombinaiton of the mitochondrial genome. Genes present in the nucleus that inhibit the recombination of the mitochondrial genome have been reported, and one of them is the genes involved in DNA mismatch repair, including the PMS1 gene. Therefore, based on this association, AcPMS1 is expected to be the most promising candidate gene among candidate genes.

The present invention provides a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs: 1 to 10 in Sequence Listing.

In the present specification, the term "nucleic acid molecule" has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, are not only natural nucleotides, but also sugar or base sites modified Also includes analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

In some cases, variations in nucleotides do not result in changes in proteins. Such nucleic acids include functionally equivalent codons or codons encoding the same amino acid (e.g., due to the degeneracy of codons, there are six codons for arginine or serine), or codons encoding biologically equivalent amino acids. It includes a nucleic acid molecule.

Considering the above-described mutation having biologically equivalent activity, it is interpreted that the nucleic acid molecule of the present invention also includes a sequence exhibiting substantial identity with the sequence listed in the sequence listing. The actual identity of the above is, for example, at least 80% when the sequence of the present invention and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It means a sequence showing homology of, more preferably 90% homology, and most preferably 98% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv . Appl . Math. 2:482 (1981) Needleman and Wunsch, J. Mol . Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol . Biol . 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc . Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl . BioSci . 8:155-65 (1992) and Pearson et al., Meth . Mol . Biol . 24:307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol . Biol . 215:403-10(1990)) can be accessed from NCBI (National Center for Biological Information), etc. It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. The BLSAT is accessible at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

Nucleic acid molecules comprising the nucleotides of SEQ ID NOs: 1 to 10 _{are markers for segregation of individuals in onion F 1} hybrid breeding programs, that is, male-fertile and male-sterile individuals. It can be used as a useful molecular marker for marker-assisted selection.

According to the present invention, a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NOs: 1 to 10 of the Sequence Listing of the present invention is a CAPS (Cleaved Amplified Polymorphic Sequences) marker or an ILP (intron length polymorphism) marker.

The “CAPS marker” refers to a nucleotide sequence having a genetic difference according to an expression trait that can be used in the CAPS method of degrading DNA using restriction enzymes for genetic differences between individuals and analyzing a result. In the CAPS method, PCR amplification including different restriction enzyme sites between individuals is performed, and the product of the PCR amplification is digested with a restriction enzyme. The decomposed product is subjected to electrophoresis on an agarose or acrylamide gel to analyze the band pattern of the decomposed product.

The “ILP marker” is mainly used as a molecular marker, and the ILP marker is a length polymorphism that appears in introns. It is detected by PCR using a primer designed based on the sequence around the target intron. The PCR amplified product is subjected to electrophoresis on an agarose or acrylamide gel to analyze the band pattern of the PCR product.

In the specification of the present invention, the'molecular marker' for discriminating male-fertile or male-sterile of onion includes the'CAPS marker' and'IPL marker'.

According to an embodiment of the present invention, the molecular marker comprising the nucleotide sequence of the first sequence or the second sequence of the sequence listing is Acepa31446, and the nucleotide sequence of the third sequence or the fourth sequence of the sequence listing The molecular marker is Acepa28839, and the molecular marker including the nucleotide sequence of SEQ ID NO: 5 or 6 is Acepa26780, and the molecule includes the nucleotide sequence of SEQ ID NO: 7 or 8 The marker is Acepa27528, and the molecular marker including the nucleotide sequence of SEQ ID NO: 9 or 10 is Acepa23881. The nucleotide sequence of Sequence Listing 1 to Sequence Listing 10 is a gene showing complete linkage disparity (LD) with the Ms locus (see Table 4).

According to a specific embodiment of the present invention, the nucleotide sequence of the sequence listing first sequence and sequence listing second sequence encodes a DNA mismatch repair protein PMS1, and the sequence listing third sequence and the sequence listing first The nucleotide sequence of SEQ ID NO: 4 encodes mannose-6-phosphate isomerase, and the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 encode protein AAR2 homolog, and the sequence number 7 and sequence The nucleotide sequence of SEQ ID NO: 8 is a 2-hydroxyacyl-CoA lyase protein, and the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 10 encode peroxisome biosynthetic protein 22.

According to another aspect of the present invention, the present invention provides a method for determining male-fertility or male-fertility of onions comprising the following steps:

(a) obtaining gDNA (genomic DNA) of onion; And

(b) analyzing at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 10 of the gDNA.

In the method for determining male-sterile or male-fertility of onion of the present invention, gDNA is obtained from onion, and among the gDNAs, 10 novel molecular markers of the present invention, that is, in the group consisting of SEQ ID NOs: 1 to 10 It is carried out by analyzing the sequence of at least one nucleotide of choice.

According to an embodiment of the present invention, the analysis in step (b) is performed by CAPS (Cleaved Amplified Polymorphic Sequence), ILP (intron length polymorphism), PCR (Polymerase Chain Reaction), RAPD (Randomly Amplified Polymorphic DNA), AFLP (Amplified fragment length polymorphism), Restriction Fragment Lennth Polymorphism (RFLP), DNA Amplification Fingerprinting (DAF), Arbitrarily primed PCR (AP-PCR), Single-strand Conformation Polymorphism (PCR-SSCP), Reverse transcriptase PCR (RT-PCR), ISSR (Inter-simple Sequence Repeat Amplication), Sequence Tagged Site (STS), Expressed sequence tag (EST), Sequence Characterized Amplified Regions (SCAR), DNA sequencing, hybridization by microarray, Northern Blot or This can be done with Southern blot.

According to an embodiment of the present invention, the ILP analysis method comprises: SEQ ID NOs: 11 and 12; Alternatively, it can be carried out using a primer set of SEQ ID NOs: 13 and 14 in the Sequence Listing. The ILP analysis method is a gene analysis method in which a corresponding gene is amplified through a PCR method, and the amplified product is electrophoresed to analyze a band pattern. Onion male-fertile or male-sterile can be determined by observing the size of the band.

When analyzing the first sequence or the second sequence in the sequence listing in step (b) of the male-sterile or male-fertility determination method of the onion of the present invention, the primers used to perform the PCR are sequence listing 11th sequence and the second sequence. It is the 12th sequence. After performing PCR using the primer sequences 11 and 12, it can be determined that the male infertility and male senility according to the difference in the size of the band.

According to an embodiment of the present invention, PCR is performed using a primer set of SEQ ID NO: 11 and SEQ ID NO: 12 on the gDNA obtained from onion, and the Indels size is analyzed to determine the male-sterile or male- Determining fertility: (i) If a product having an Indels size of 34 bp of the PCR product is present, it is judged as male-infertile, and (ii) if the product does not exist, it is judged as male-fertile.

In the case of analyzing the sequence number 3 or 4 in step (b) of the method for determining male-sterile or male-fertility of onion of the present invention, the primers used to perform PCR are SEQ ID NOs: 13 and 14 Sequence.

According to an embodiment of the present invention, PCR was performed using a primer set of SEQ ID NO: 13 and SEQ ID NO: 14 on gDNA obtained from onion, and the Indels size was analyzed to determine the male-sterile or male- Determining fertility: (i) If a product having an Indels size of 13 bp of the PCR product is present, it is judged as male-sterile, and (ii) if the product does not exist, it is judged as male-fertile.

According to another embodiment of the present invention, the CAPS analysis method may be carried out using at least a pair of primer sets selected from the group consisting of SEQ ID NOs: 15 to 20 in Sequence Listing.

The CAPS of the present invention is a gene analysis method for amplifying a gene through a PCR method, decomposing the amplified product by treating a restriction enzyme, and analyzing the band pattern by electrophoresis of the degraded product. Onion male-fertile or male-sterile can be determined by observing the band.

In the case of analyzing the sequence number 5 or 6 in step (b) of the male-sterile or male-fertility determination method of onion of the present invention, the primers used to perform PCR are SEQ ID NO: 15 and sequence number 6 It is sequence 16.

According to another embodiment of the present invention, PCR was performed using a primer set of SEQ ID NO: 15 and SEQ ID NO: 16 on gDNA obtained from onion, and the PCR product was subjected to restriction enzyme Hind III to decompose patterns. Analysis of the onion is male-sterile or male-fertile: (i) If the PCR product is not cut and there is one 808 bp band, it is judged as male-fertile, and (ii) the product is cut and 603 When cut into two bands of bp and 205 bp, it is judged as male-infertile.

When analyzing the 7th or 8th sequence of Sequence Listing in step (b) of the method for determining male-sterile or male-fertility of onion of the present invention, the primers used to perform PCR are SEQ ID NO. It is sequence 18.

According to another embodiment of the present invention, PCR is performed using a primer set of SEQ ID NO: 17 and SEQ ID NO: 18 on the gDNA obtained from onion, and the PCR product is subjected to restriction enzyme Dra I to decompose the pattern. Analysis of the onion to determine the male-fertility or male-fertility: (i) If the PCR product is not cut and there is one 640 bp band, it is judged as male-fertile, and (ii) the product is sheared and 427 If cut into two bands of bp and 207 bp, it is judged as male-infertile.

In the case of analyzing the sequence number 9 or 10 in step (b) of the method for determining male-sterile or male-fertility of onions of the present invention, the primers used to perform PCR are SEQ ID NOs: 19 and 19 It is sequence 20.

According to another embodiment of the present invention, PCR is performed using a primer set of SEQ ID NO: 19 and SEQ ID NO: 20 on gDNA obtained from onion, and the PCR product is subjected to restriction enzyme Pvu II to decompose the pattern. Analysis to determine the male-sterile or male-fertile onion: (i) If the PCR product is not cut and there is one 1,344 bp band, it is judged as male-sterile, and (ii) the product is sheared 936 If cut into two bands of bp and 406 bp, it is judged as male-fertile.

According to another aspect of the present invention, the present invention comprises: (a) a forward primer hybridized to the complementary sequence of SEQ ID NO: 1 or SEQ ID NO: 1; And (b) a kit for male-fertility discrimination of onions comprising a reverse primer hybridized to the sequence listing first sequence or the complementary sequence of the sequence listing sequence 1; or

(a) a forward primer that hybridizes to a sequence complementary to SEQ ID NO: 2 or SEQ ID NO: 2; (b) Provides a kit for male-infertility discrimination of onions comprising a reverse primer hybridized to a sequence listing second sequence or a complementary sequence of the sequence listing sequence 2.

Preferably, the primer is composed of SEQ ID NOs: 11 and 12 in Sequence Listing.

According to another aspect of the present invention, the present invention provides: (a) a forward primer hybridized to the complementary sequence of SEQ ID NO: 3 or 3; And (b) a kit for male-fertility discrimination of onions comprising a reverse primer hybridized to a complementary sequence of SEQ ID NO: 3 or 3; or

(a) a forward primer hybridized to the complementary sequence of SEQ ID NO: 4 or 4; (b) It provides a kit for male-infertility discrimination of onions comprising a reverse primer hybridized to the complementary sequence of SEQ ID NO: 4 or SEQ ID NO: 4.

Preferably, the primer is composed of the 13th and 14th sequences of the Sequence Listing.

According to another aspect of the present invention, the present invention provides: (a) a forward primer hybridized to the complementary sequence of SEQ ID NO: 5 or 5; And (b) a kit for male-fertility determination of onions comprising a reverse primer hybridized to a complementary sequence of SEQ ID NO: 5 or SEQ ID NO: 5; or

(a) a forward primer hybridized to the complementary sequence of SEQ ID NO: 6 or 6; (b) It provides a kit for male-infertility discrimination of onions comprising a reverse primer hybridized to the complementary sequence of SEQ ID NO: 6 or SEQ ID NO: 6.

Preferably, the primer is composed of SEQ ID NOs: 15 and 16 in Sequence Listing.

According to another aspect of the present invention, the present invention provides: (a) a forward primer hybridized to the complementary sequence of SEQ ID NO: 7 or 7; And (b) a kit for male-fertility discrimination of onions comprising a reverse primer hybridized to a complementary sequence of SEQ ID NO: 7 or SEQ ID NO: 7; or

(a) a forward primer hybridized to the complementary sequence of SEQ ID NO: 8 or 8; (b) It provides a kit for male-infertility discrimination of onions comprising a reverse primer hybridized to the complementary sequence of SEQ ID NO: 8 or SEQ ID NO: 8.

Preferably, the primer is composed of SEQ ID NOs: 17 and 18 in Sequence Listing.

According to another aspect of the present invention, the present invention provides: (a) a forward primer hybridized to the complementary sequence of SEQ ID NO: 9 or 9; And (b) a kit for male-fertility discrimination of onions comprising a reverse primer hybridized to a complementary sequence of SEQ ID NO: 9 or 9; or

(a) a forward primer hybridized to the complementary sequence of SEQ ID NO: 10 or 10; (b) It provides a kit for male-infertility discrimination of onions comprising a reverse primer hybridized to the complementary sequence of SEQ ID NO: 10 or SEQ ID NO: 10.

Preferably, the primer is composed of SEQ ID NOs: 19 and 20 in Sequence Listing.

Since the kit of the present invention uses the male-fertile or male-sterile determination method of the onion of the present invention described above, descriptions of the contents in common between the two are omitted in order to avoid excessive complexity of the present specification.

The primer used in the present invention contains a sequence that is substantially complementary to the target nucleic acid sequence. The term “complementary” means that a primer or probe is sufficiently complementary to selectively hybridize to a target nucleic acid sequence under predetermined annealing or hybridization conditions, and is substantially complementary and perfectly complementary. It has the meaning of all inclusive, and preferably means completely complementary. Preferably, the primer is deoxyribonucleotide and is single chain. Primers used in the present invention may include naturally occurring dNMP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides.

As used herein, the term “hybridization” means that two single-stranded nucleic acids form a duplex structure by pairing of complementary nucleotide sequences, in which the complementarity between single-stranded nucleic acid sequences is completely complementary. In the case of a perfect match, or even in the presence of some mismatched bases, the degree of complementarity required for hybridization may vary depending on the conditions of the hybridization reaction, and in particular, may be controlled by temperature.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a nucleic acid molecule for selecting onion male fertility or male sterility, and a method for determining male fertility or male-fertility of onions using the same.

(b) The present inventors selected five genes as candidate genes for determining fertility recovery from male infertility in onions, and in particular, Acepa31446 (AcPMS1) was selected as the best candidate gene.

(c) Candidate genes identified by the present inventors can be easily genotyped on an agarose gel, and can be used universally.

(d) When the method of the present invention is used, _{the efficiency of the F 1} hybrid crossing of onions can be improved, and in particular, when the progeny test, the work that takes more than 4 years can be completed in a single day, so that onion varieties breeding There is an effect that can dramatically improve efficiency.

1A and 1B show the association map of the Ms locus and the marker genotype of the selected recombinant and breeding line. Figure 1a shows the association map of the Ms gene side revealed in the previous study (Park et aL. 2013a). 1B shows the marker genotypes of two recombinants selected from a large-scale isolate and four randomly selected breeding lines. Homogeneous recessive genotypes are indicated by gray boxes. A: homozygous dominant, H: heterozygous, B: homozygous recessive, D: homozygous dominant or heterozygous.
Figure 2 shows the relationship between the 14 molecular markers tagging candidate genes of the Ms locus in foreign varieties. The jnurf13 marker reported in the previous study was also included (Kim 2014). Marker genotypes that do not match the majority of the marker genotypes are indicated by filled boxes. Arrows indicate'PI233186' and contain crossovers dividing the markers into two groups (groups A and B). Accessions number was not separately stated. 1: RF15334, 2: RF23881, 3: RF24123, 4: RF24998, 5: RF25191,6: RF28L84, 7: RF28314, 8: RF31446, 9: RF31869, 10: RF24501, 11: RF26780, 12: RF27463, 13: RF28839, 14: jnurf13, 15: RF27528.
Figure 3 shows the phylogenetic relationship of the protein containing the PPR motif of onion, isolated from other plant species. GenBank accession numbers of Rf genes isolated from other species are indicated in parentheses. The phylogenetic tree was constructed by flanking binding method using the final amino acid sequence. The number displayed on the node represents the probability (%) of bootstrap with 1000 replicates.
4 is a result of comparison of SNP frequencies in four groups associated with the Ms locus or showing complete linkage disparity (LD). A: 9 genes showing complete LD in the breeding line belonging to group A in Fig. 2, B: 5 genes showing complete LD in the breeding line belonging to group B in Fig. 2, C: Ms locus and complete in a large-scale isolated group 15 genes that showed association, and did not show complete LD in the breeding system, and 98 genes that were associated with the D: Ms locus and did not show a complete association with the Ms locus of the isolated group.
Figure 5 shows the MF and MS-specific amino acid change ratios appearing in the top 50 homologous proteins isolated from other plant species. Black and gray bars represent MF and MS-specific amino acid ratios, respectively. Known domains were identified using Pfam search (Finn et al., 2014), and the location was indicated by a horizontal line (horizontal line) in the graph. Changes in amino acids specific to the MS allele and located on the known domain are indicated by an asterisk on the graph.
6A and 6B are rice chromosome maps, showing the location of the Ms locus-associated onion gene and the orthologous gene. The distance unit in the map is kbp (kilobase pair), and was created using MapChart 2.L software (Voorrips 2OO2).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental materials and test methods

Plant material

BSA and RNASeq analysis was performed using _{an F 2:5} isolated group derived between a male-sterile (MS) maternal (506L) and a male-fertile (MF) paternal (H6). . To screen for closely related contigs, in 4,273 isolated plants made in previous studies (Park et al. 2013), the Ms gene and closely related jnurf05 (Park et al., 2013) and OPT (Bang et al., 2O11) Two recombinants (12-510 and 11-248) showing crossovers between markers were selected. The contig was identified where there was a linkage disequilibrium (LD) with the Ms gene using 251 breeding lines owned by six different breeding institutions in Korea (Breaking institutions: Nongwoo Bio, Nonghyup Seedling Center, Onion Nara, National Institute of Horticultural Science, Seeds and People, Onion Research Institute). The male fertility phenotype and cytoplasmic type of these breeding lines were confirmed in previous studies (Kim et al., 2014).

In addition, 124 foreign varieties introduced from 21 countries were analyzed to evaluate the level of linkage disequilibrium (LD) of contig, which represents the complete LD among breeding lines. Of these, 83 varieties were introduced from the Ladle Plant Germplasm System (Agricultural Research Service, Baltimore, MD, USA). The other 41 species are commercially available in various countries.

RNA-Seq analysis and homologous SNP analysis between MF and MS bulks

According to the male fertility phenotype determined in visual examination and the genotype of closely related molecular markers (jnurf05 and OPT) near the Ms locus, 10 individuals were selected from each of the MF and MS isotypes. These two markers were reported in previous studies (Bang et al., 2011; Park et al., 2013). RNA was extracted from pre-flowering flowers of 10 bulk samples of MF and MS plants using an RNA extraction kit (RNeasy Plant Mini Kit, QIAGEN, Valencia, CA, USA). RNA sequencing and SNP analysis were performed in specialized institutions (Phyzen, Seoul, Republic of Korea). That is, a ^{cDNA library was generated from the RNA sample using the Illumina TruSeq ®} RNA sample preparation v2 guide (Illumina, Hayward, CA, USA). The transcriptome was sequenced using HiSeq 2000 (Illumina), and 101 bp paired-end reads were generated. Subsequently, the transcript sequence was stored in the SRA database as “accession numbers SRP056991”. Using Trinity software (Haas et al. 20l3), trimmed raw reads of MF and MS bulk were combined and combined into a new contig. In order to analyze SNPs appearing between bulks using SAMTools software (Li et al., 2OO9), raw reads of MF and MS bulks were mapped to separate contig combinations. Quantification of each contig in MF and MS was performed using RSEM software (Li and Dewey, 2011).

In order to identify flower tissue-specific contigs that show a significant increase in expression in the MF bulk, 30,004 contigs combined in previous leaf tissues were used (Kim et al., 201). As in the present invention, the contig was made using the same parental family (H6). Contig that was not detected in 30,004 contig and expressed otherwise was judged as a flower tissue-specific contig. contigs represent a set of DNA fragments with overlapping sites.

DNA extraction, PCR Increase And PCR Sequencing the output

Total genomic DNA was extracted from leaf or peduncle tissue using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987). In order to identify the homologous SNPs between MF and MS bulks, primer pairs were designed based on exon sequences near 1 or 2 expected introns of 139 screened contigs. Exon-intron presumptive boundaries were determined by comparing them with rice orthologous sequences retrieved from the Rice Genome Annotation Project (Ouyang et al., 2OO7) (data not shown). Information on the primer sequence and some contig is described in Table 1). In the same way, primer sequences were designed based on the exon sequence of 145 contig showing a significant increase in transcription in the MF bulk.

contig contig length SNP number Annotation primer
(5' to 3') RF31446 1,023 One DNA mismatch repair protein PMS1 F: GGCTAAAGGGGTTCGGTTAC
R: AAGAGTGCCTTCATCGGAAA RF28839 1,278 5 Mannose-6-phosphate isomerase F: TTACACAAGATGCACCCAAA
R:GGGGCTCATTAGCATCCAG RF26780 1,227 3 Protein AAR2 homolog F: GGGCCTTACCCTCTAAACCA
R: CGCTCCTTGACAATATGAGGA RF27528 1,704 11 2-hydroxyacyl-CoA lyase F: GCTTGGCCTGCTGTTATGAT
R: GGAGACTCGCCAAAATGAAG RF23881 675 7 Peroxisome biosynthetic protein 22 F: GATCCTCATGGCTTTGGAGA
R: GGGAAGTGAACACGTTTGGT

PCR amplification of contigs was 0.1 μg template, 2.5 μL l0 x PCR buffer, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 0.5 μL dNTPs (10 mM each), and 0.25 μL polymerase mix. (Advantage 2 Polymerase Mix, Clontech, Palo Alto, CA, USA). PCR amplification consists of the following steps: initial denaturation step 95° C., 4 minutes; 10 cycles, 95[deg.] C., 30 seconds, 65[deg.] C. (decrease by 0.8[deg.] C. for each cycle), 30 seconds and 72[deg.] C., 1 minute; 35 cycles, 95°C, 30 seconds, 57°C, 30 seconds, 72°C, 1 minute; And the final 72 ℃, 10 minutes extended reaction. The PCR result was confirmed after staining with ethidium bromide on a 1.5% agarose gel. When a single PCR product was observed, the PCR product was purified using the QlAquick PCR Purification Kit (QIAGEN). The sequencing reaction was performed using Big Dye (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol, and the sequence was obtained using ABI PRISM 3730XL Analyzer (Applied Biosystems).

For PCR amplification of CAPS (cleaved amplified polymorphic sequence) and ILP (intron length polymorphism) markers, 10 μL reaction mixture and 0.25 U Taq polymerase (Prime Tag DNA polymerase; GeNet Bio, Nonsan, Korea) were used for mass sample analysis. . And the same PCR amplification conditions as the above-described PCR conditions were applied. The PCR result of CAPS marker was treated with restriction enzyme at an appropriate temperature for 3 hours. Subsequently, the PCR product or the restriction enzyme treatment product was confirmed on a 1.5% agarose gel. Primer sequences and restriction enzymes are listed in Table 2.

Molecular marker of the present invention

RT-PCR and RACE (rapid amplification of cDNA ends)

In order to obtain contig MF and MS full-length cDNA sequences representing the Ms locus and complete LD, RT-PCR was performed when the full-length cDNA sequence could be obtained from the assembled contig. If only some sequences could be assembled, RACE was performed to obtain the full length cDNA sequence of the MF and MS alleles. According to the RT-PCR method, the same MF and MS bulk RNA used for RNA sequencing analysis and a commercially available cDNA synthesis kit (SuperScriptrM III first-strand synthesis system for RT-PCR, Invitrogen, Carlsbad, CA, USA) was used. To generate cDNA. RT-PCR amplification was performed under the following conditions: initial denaturation step 94° C., 3 minutes; 40 cycles, 94°C, 30 seconds, 65°C, 30 seconds, 72°C, 3 minutes min; And the final extension reaction at 72° C., 10 minutes. RACE was performed using a commercially available kit (SMART RACE cDNA Amplification Kit; Clontech). The PCR product was purified and sequenced by the method described above.

onion PPR Identification of genes, and onions and others From plant species Separate PPR Building a phylogenetic tree of proteins

Through a local BLAST search using BioEdit software, onion contig containing a PPR motif was identified (Hall 1999). Rice Rf1a protein (GenBank accession number ABC4233O) was used as a query. Using BioEdit software (Hall 1999), the amino acid sequence of the onion PPR gene relatively close to the PPR-containing Rf gene was aligned with the PPR-containing Rf gene isolated from corn, rice, radish and petunia. Five Arabidopsis Rf-PPR-like (RFL) genes (Fujii et al. 2011) were also included in the alignment process. The gap that appeared in the alignment was removed by “less stringent selection” using Gblocks software (Castresana 2000). The phylogenetic tree was constructed using MEGA version 4 (Tamura et al. 2007) applying the neighbor-joining method. Node support in the phylogenetic tree was evaluated by 1,000 bootstrap copies.

Experiment result

Selection of contigs closely related to the Ms locus using BSA and RNA-Seq analysis

In order to identify candidate genes related to male reproductive recovery in onions, RNA was extracted from flower tissues of MF and MS bulk samples, and transcriptome sequencing was performed, and as a result, total 4.9 Gb and 5.3 Gb sequences were obtained. After trimming the low quality sequence and pooling the MF and MS sequences, 32,674 contigs were newly assembled. In order to identify SNPs between the bulk of raw reads from MF and MS bulks, they were separated and mapped into assembled contigs. 430 fully homologous SNPs were identified between bulks, and these were distributed in 141 contigs. The SNP was reconfirmed by visual investigation of the aligned read using IGV viewer (Robinson et aL. 2011), and two contigs representing heterozygous SNP were removed (Table 3).

Selection process of contigs showing linkage disequilibrium (LD) with the Ms locus step Screening criteria number of contigs One De novo assembly of contigs from raw reads produced by RNASeq 32,674 2 Contigs containing homozygous SNPs between MF and MS bulks 141 3 Contigs containing reliable SNPs verified by the IGV viewer 139 4 Contigs containing perfectly homozygous SNPs verified by PCR amplification and sequencing of bulked DNAs of MF and MS individuals 128 5 Contigs containing the SNPs showing perfect linkage with the Ms locus in the large-sized segregating population consisting of 4,273 plants 30 6 Contigs containing the SNPs showing perfect LD with the Ms locus in diverse breeding lines 14

In order to identify homozygous SNPs between bulks, a primer pair for the SNP sequence was designed at 139 contigs (primer sequences for some contigs, see Table 1). Putative exon-intron junctions were estimated using rice orthologous genes, and if possible, primers were designed based on exon sequences adjacent to one or two introns. Initially, MF and MS bulk DNA, constituting 10 homologous individuals selected from the isolated group, were used as templates for PCR amplification. If the PCR amplification failed, another primer pair was designed, and if the PCR amplification was successful, the PCR product was immediately sequenced to confirm the homozygous SNP. Except for 11 contigs, it was found that, as expected, fully homogeneous SNPs between bulks appeared in all contigs. In addition, the sequencing results showed that the intron positions in 81.3% of the contigs (61 contigs out of 75 contigs containing at least one intron) matched perfectly with the intron positions in rice orthologs. However, although the positions are not completely random, the chromosomal positions of rice orthologous genes are not correlated with each other on the rice genome (FIGS. 6A and 6B ).

Subsequently, in the screening step, the SNPs on 128 selected contigs were sequenced to genotype the recombinants (12-510) showing the crossover between the Ms locus and the jnurfO5 marker (FIGS. 1A to 1B ). The jnurf05 marker is closely related to the Ms locus as a distance of 0.047 cM (Fig. 1A, Park et al. 20l3). The male fertility phenotype of the '12-510' plant was male fertility; Its Ms genotype was expected to be recessive homozygous. However, in the recombinant, 85 contig of SNP genotypes were heterozygous. This means that 85 genes are associated with the Ms locus at the position in the same direction as the jnurf05 marker (FIG. 1B). Subsequently, another recombinant (11-248) containing a cross between the Ms locus and the OPT marker was analyzed for 43 contig SNPs that showed the recessive homozygous SNP genotype of the 12-510 recombinant. Of these, 30 contigs showed the same genotype as the Ms locus, indicating that it was completely associated with the Ms locus in a large-scale isolate (Table 3).

Next, in four randomly selected breeding lineages (90377, 90143, 90904 and 90027), SNPs of 30 contigs indicating complete association with the Ms locus were genotyped. The Ms and associated marker genotypes of the breeding line are known (Fig. 1B). Sixteen of the 30 contigs had the same genotype as the Ms locus. For large sample analysis, co-dominant CAPS or ILP markers were developed based on SNP and indel polymorphism in 16 contigs. The markers were used to analyze 251 breeding lines in 6 different organs, and the genotypes of all markers except the RF24000 and RF24437 markers matched perfectly with the phenotype of the Ms locus. This indicates that the 14 contigs are almost completely linkage disequilibrium (LD) with the Ms locus.

Additionally, to evaluate the LD levels of these markers in various germplasms, 15 markers were analyzed from 124 foreign cultivars introduced from 21 countries. One cultivar PI233186 was found to have a crossover that divides 15 markers into 2 groups (FIG. 2). In FIG. 2, 9 markers including group A showed complete LDs to each other, but RF27528 (No. 15), one of the five markers of group B, showed several recombinants. The jnurfl3 marker found in previous studies (Kim et al., 2O14) was also included in group B. However, because P1233186 had a normal cytoplasm, the present inventors could not determine the location of the Ms locus.

Analysis of gene expression differences between MF and MS bulk and correlation between Ms locus and onion PPR gene family

If the recessive Ms allele is not transcribed in MS plants, the candidate gene cannot be detected in the SNP assay described above. Therefore, compared to the MS bulk, the contig showing a 10-fold increase in expression in the MF bulk was screened.

Using a step-by-step screening method, the final 145 contigs were selected. Of the 145 contigs, 97 transcripts were judged to be flower tissue-specific because they were not detected in transcripts assembled from leaf tissue. However, although five contigs showed close association with the Ms locus (Acepa02974, Acepa068l0, Acepal3742, Acepa15612 and Acepa27928), after analysis of PCR products amplified from recombinant MF and MS bulk and four breeding lines, SNP analysis There were no contigs showing the Ms locus and full LD as in.

Since most of the Rf genes cloned from other plant species encode the PPR protein, contigs containing the PPR domain were selected from onion transcripts as candidate genes. A local BLAST search was performed using the rice RF1a protein as a query to select 483 contigs having at least one PPR motif. Two contigs (Acepa02436 and Acepa14756) were included in the contig list containing homologous SNPs between MF and MS bulks, but did not show a full association with the Ms gene. In addition, 14 PPR genes that showed at least a 3-fold increase in expression in the MF bulk were analyzed, but none of them showed any association with the Ms locus. 41 PPR genes closely related to Rf and Rf-like PPR genes isolated from other species were analyzed (FIG. 3 ), but these genes did not contain homogeneous SNPs or showed a 2-fold or more increase in expression in MF bulk (data Not stated).

Ms Locus and Indicating LD contigs Full-length cDNA sequence of Anotei Line And Ms At the locus Identification of candidate genes for Korea

Using RT-PCR and RACE, full-length cDNA sequences of 14 contigs were obtained from MF and MS homologous individuals, showing the Ms locus and complete LD in the breeding line. The frequency of SNPs in the 14 gene coding regions was significantly higher than that of other related genes (Fig. 4). In particular, 9 genes belonging to group A in FIG. 2 showed the highest SNP frequency. Because the location of the gene was close to the Ms locus, the frequency of SNPs was higher.

Next, the predicted functions of the 14 genes were analyzed through BLAST-p search using the amino acid sequence (Table 4). The function of the Rf gene, such as the PPR protein, has not been identified. To identify important mutations, the amino acid sequences of 14 genes, MF and MS alleles, were compared. One of the genes, Acepa15334, did not show a polymorphic amino acid sequence between the MF and MS alleles, so it was excluded from subsequent analysis. Of the remaining 13 genes, a conserved region was identified by aligning the amino acid sequences of the top 50 homologous proteins isolated from other plant species with the onion sequence. Amino acid sequence changes that are located in the known domain and specific to the MS allele were searched. Four genes (Acepa31446, Acepa28839, Acepa26780, and Acepa27528) showed significant amino acid changes in known domains (Fig. 5). In the case of the Acepa23881 gene, two amino acid changes were observed in the conserved region even though there was no amino acid change in the known domain (FIG. 5). Based on LD analysis, that is, the annotated function and the change in important amino acids, the gene encoding the DMA mismatch repair protein PMS1 (Acepa31446) was selected as the most potent fertility recovery gene candidate in onions. Hereinafter, the PMS1 gene identified in the present invention is referred to as AcPMS1.

Number of amino acid changes between genes and MF/MS alleles showing complete linkage disequilibrium (LD) with the Ms locus gene Annotation Coding sequence length (bp) Number of amino acid changes Acepa15334 60S ribosomal protein L37a 276 0 Acepa23881 Peroxisome biogenesis protein 22 765 6 Acepa24123 Inactive leucine-rich repeat receptor-like serine/threo nine-protein kinase 2,001 8 Acepa24501 66 kDa stress protein 1,766 4 Acepa24998 Beta-galactosidase 2,487 6 Acepa25191 Uncharacte rized protein 744 11 Acepa26780 Protein AAR2 homolog 1,224 10 Acepa27463 KH domain-containing protein 1,920 6 Acepa27528 2-hydroxyacyl-CoA lyase 1,7O1 5 Acepa28184 Uncharacterized protein 942 10 Acepa28314 Kinesin-13A 2,163 7 Acepa28839 Mannose-6-phosphate isomerase 1,275 13 Acepa31446 DNA mismatch repair protein PMS1 2,640 26 Acepa31869 Serine/threonine-protein kinase EDR1 2,724 17

Argument

Identification of genes representing Ms locus and complete LD using BSA and RNA-Seq

14 genes with Ms locus and complete LD were successfully identified using BSA and RNA-Seq. With the introduction of next-generation sequencing, RNA sequencing has become a powerful tool for large-scale SNP studies of various species (Schneeberger and Weigel 2011; Mutz et al. 2013; Wolf 2013). Due to the lack of reliable data on the genome sequence for this analysis (Edwards et al., 2013), RNA-seq analysis in particular is the study of crop species containing complex or large genomes, especially onions (16,400 Mb/lC). Useful for In cloning causative genes exhibiting important characteristics in corn (Liu et al. 2012) and wheat (Trick et al. 2012), a combination method of BSA and RNA-Seq analysis has been reported to be useful.

In order to identify the SNPs identified in silico and to identify RCR-based markers, primers were designed based on exon sequences close to one or two introns. Since rice is the model most closely related to onions, the exon-intron boundary of each contig was predicted from the information of the rice orthologous gene. About 81% of the onion intron positions were matched with rice orthologous genes. Similarly, the location of 83% of the onion intron was the same as that of the rice intron (Martin et al., 2005), but there was no collinearity between the genomes of onion and rice. The present inventors reconfirmed whether there is no microsynteny between monocotyledonous crops. The position of the rice gene orthologous to the onion gene closely related to the Ms locus is almost random (FIGS. 6A and 6B ). Since the onion genes analyzed in the present invention were all associated with the Ms locus, if the synteny between the rice and onion genomes was preserved, the rice ortholog genes would have formed clusters on the rice genome. Therefore, a separate effort is required in establishing the onion genomic sauce.

Onion CMS Restoration of male and female sexuality Identification of related candidate genes

Candidate genes were screened using two recombinants (12-510 and 11-248) selected from a large population of 4,273 individuals. Using this recombinant, 30 genes located within about 0.55 cM interval between OPT and jnurf05 markers were successfully identified. 16 of the genes were removed through analysis of 231 breeding lines. These breeding lines were obtained from six different organs, but all of these organs are in Korea, and their genetic diversity seems to be very narrow.

Through the analysis of 124 foreign varieties introduced from 21 countries, 14 genes were classified into 2 groups (groups A and B, see FIGS. 2 and 4). In particular, PI233186 contains a crossover between the two groups. The marker genotype of group A was heterozygous, and group B was homozygous recessive. However, it failed to find the male fertility phenotype in these foreign cultivars because the growth conditions did not match. In addition, since P1233186 has a normal cytoplasm, it will take at least 4 years to genotype the Ms locus of P1233186. Although the Ms genotype has not been identified, since RF27528, a marker of group B, contains several recombinants out of 124 accessions, the Ms locus is considered to belong to group A. On the other hand, the markers belonging to group A showed complete LD in all accessions. The SNP frequency appearing in the gene coding sequence of group A was slightly higher than that of group B, and was 3.8 times higher than that of the associated gene (group D, Fig. 4). The high frequency of SNPs in LD blocks containing group A genes is seen as a characteristic of selection over long reproductive periods (Gupta et al., 2005). The SNP frequency gradually decreased with the distance between genes, and the Ms locus became further with the LD decrease (FIG. 4).

Annotated function. Based on the high SNP frequency (1.97 / 100 bp) and significant amino acid changes, AcPMSl is expected to be the most potent candidate gene involved in the recovery of male fertility in onion CMS. This gene encodes the PMS1 protein, which is involved in DNA mismatch repair (MMR). The MMR system is one of the DNA repair pathways and is highly conserved from bacteria to animals/plants. The three homodimeric proteins, MutS, MutL, and MutH, are

It is the composition of the Escherichia coli. MMR system, and MutS recognizes DNA mismatch and recruits MutL. As a result, MutH with endonuclease activity is rerouted in the mismatched portion (Kolodner and Marsischky 1999; Bray and West 2005; Kimura and Sakaguchi 2006). The PMS1 protein is known to form a heterodimer with MLHL and play a role similar to that of MutL in yeast and plants (Bray and West 2005).

In Arabidopsis, two independent PMS1 gene mutants destroyed more than 50% of pollen grains and increased homozygous recombination (Li et al., 2009). A high rate of pollen collapse was also observed in Arabidopsis thaliana bearing the MLH1 gene mutation. The MLH1 gene is a heterodimer-forming partner with PMS1 (Dion et al., 2007). In addition, the Msh1 gene product is homologous to MutS and is known to be involved in the regulation of MMR and plant mitochondrial recombination (Abdelnoor et al., 2003). Msh1 mutations in Arabidopsis lead to extensive mitochondrial genome rearrangements (Arrieta-Montiel et al., 2009). RNAi-induced Msh1 gene inactivation in tobacco and tomatoes leads to genetic cytoplasmic fertility (Sandhu et al., 2007). Despite not supporting the association between the MMR system and CMS clarified and guided, AcPMS1 genes involved if the fertility restoration of CMS onions, AcPMS1 gene is expected to be an excellent tool to study these associations. The AcPMS1 gene is proposed as a strong candidate for the Ms locus in the present invention, and it cannot be ruled out the possibility that the actual causative gene is not found in the screening process of the present invention for identification of isotype SNPs and gene expression differences.

Application of molecular markers indicating Ms locus and complete LD in onion breeding

The shift from open-pollinate varieties to F ₁ hybrids has been a global trend in the vegetable seed market, including onions. Since CMS is the only way to realize _{F 1} hybrid seed production, it has become an essential tool for _{onion F 1 hybrid breeding.} However, the development of various maintainer lines has been limited due to the time-consuming and effort-consuming progeny test. This limitation could be overcome by using reliable molecular markers for genotyping of the Ms locus.

In a previous study, the present inventors reported on a molecular marker (jnurf13) representing the Ms locus and linkage disequilibrium (LD) (Kim et al., 2014), but the marker is based on the indel polymorphism of the intergenic region. It was designed, and the size of indel (12 bp) was very small, so that marker genotyping was possible only on acrylamide gels, and jnurf13 was found to belong to the group B marker in Fig. 2. Therefore, if the AcPMS1 gene is fertility in onions If found to be involved in recovery, a simple PCR marker based on polymorphism on the AcPMS1 gene (RF31446) will be a genotyping marker for the Ms locus, which belongs to group A, and in all tested accessions in 21 countries. In addition, since a relatively large 34 bp indel is used, this marker can be easily genotyped using an agarose gel Therefore, the RF31446 marker of the present invention can be widely used, and the F _{1 of onion} Will greatly increase the efficiency of hybrid breeding. In addition to the experimental application, AcPMS1 And other genes showing complete disparity with the Ms locus will be very useful materials for cloning the Ms locus in the future.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

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<110> Industry Foundation Of Chonnam National University <120> Molecular Markers related a Restorer-of-Fertility gene and Methods for Selecting of Male-Fertility or Male-Sterility in Onion <130> PN150302 <160> 20 <170> KopatentIn 2.0 <210> 1 <211> 14474 <212> DNA <213> Onion AcPMS1 Male-fertile <400> 1 ttggacatac caaaacccta atttcgaaca gtgaacaata tgaatgaaga aattgcctcg 60 tctcctacaa tcaaacccat taacaaatcg gtggtccata gaatctgctc gggtcaagtg 120 attttagatc ttcaatcggc agttaaagag ctgctcgaga atagtttgga tgcaggtgcg 180 acctgtatcg aaatcaattt gaaagagcat ggcgaagaat attttaaggt tgtcgacaat 240 gggtctggta tctctcctga taattttcag gtaatttttg gtgagacttt cggttggttt 300 tctgctgttt ggtttgtgtt cagttattct gttttgatat catatgcatg tatatataaa 360 ttcaaagata catctaatta caagggatgt aaaggtttgt tgaaattttt taggatttca 420 cgcataaacc gcatatttta attttaagaa ctattgaaat tatggattat gaaggaaagt 480 acaactgctt acattgaaaa catgaatgtg gaatgttgct ttgtaacttg gtctgaatgg 540 cagacgccgg tggtgcacat gtagaaatac tggctgaaaa tgataacaaa gtccagaact 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aagtagttaa ttaaaatatt taatagtaaa ataaattata aaatttaatt attagttaat 3180 aaaatagttt aataaaatat ttaagtagtt gaatgaatta taaatagtta aaattataat 3240 tttagttaac aaaatattta aatatttaaa aatagttgaa tgcattataa ttttagttaa 3300 taaaattgtt aaataaaata tttaaatggt tgaatgactt ataaagagtt aaaatttgaa 3360 ttatatttaa taacatattt aaatatttat aaatagttgg atgaattata atttagttaa 3420 taaaatagtt aaataaaata aaatggttat caacactaga tttatcattt ttagtagcaa 3480 ttcatagtga agtttttttt ttacaccaaa aaagctcaga cttgtataag agacgcataa 3540 aaaaggccaa ggtgcaaaaa agccctaggc acaagccttg cgccttaact actttgatgg 3600 agaatacata tgaacattgt gcatgtatgt gattaacatc ttagatagcc aactctgcat 3660 gccgttcaca cacagtatga ctgtgtttgc gttttgttga ttaatagcaa tctctagccg 3720 tggtgcagat tcgtatgcat cagatatgtg ttgtcgttga ttgcatcact caatctggag 3780 aatacatatg aacttcgtgc atgcatgtga tcaacatctt agatagccaa ctcagcatgc 3840 catttagaca tatttggaaa actcaatcat gttttgcatt tatggatttt cacacttgac 3900 catggtttaa agtatcaagg gtgtcacgat aaaaggtgaa ctgttgctta ggaatctcaa 3960 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acacatactt gaactccttg catattggat ggtttcaaaa gtggatgcga gagagtcaga 4860 agacatatgg tcgtgaacta gttgacaata actcgattgt taaggcaaac ccaccaaaat 4920 ttgggatttg gggcccaaaa tggtaagtgc acccattttt atcagatgag tgtatatatt 4980 agacttatta gttgttttca ttattaagaa tagtaaatgt gccttagctg ttagatttgt 5040 tagaacttag aacacaaggc ttgcgtttat gaagccctgg ttcaaaaccg ataatatcaa 5100 ttgcttgttt attatcatta cacacacacg cgcacgaaag cacgcttatc ccttcactca 5160 tagcaattaa tcttggtgca gaaccttgct cgcaaacatc atacttctaa aatagcagat 5220 ttttctgatc tttattcgtt agcgactttt ggatttagag gagaggcatt gagctctctc 5280 tgtgcaattg gagacttgtc tattgaaaca agaaccaaat atgagtctgt tggcacacat 5340 ctgatctacg atcactctgg gtcagtaaaa tctgaaagaa agattgctcg tcaaattggt 5400 accactgtta ctgttgagaa attattctcc accttgccag tacgaagtaa agaattcaac 5460 cgcaacattc gtcgtgaata tggaaagctt atctctttgt tgaatgtaag acatttttcc 5520 ctagaaacct tataatctag ctatgctcta aatgatatga gtgttctttg tgatcaagcc 5580 atcggggttg tctttggatg cttagatgta aaaaaaagtt tgcatttgaa aactaaacaa 5640 gttgttgctt taaatttctt ttattagact atcagtgtat atacctcata gctctatacc 5700 agtaatattt ctttctaatt ctgttagtgg caattaagcc tagggatcta ttccttgttg 5760 gttttatatg aattaaaagt aaaacactaa tcaatttttt gttttgatac ttaatggata 5820 acttgaagaa actaaaagct tttctgtcca gctatacttc atgttgaaat tcacatcaag 5880 cagatatgtt gtcttcaaaa tttgtatgat atttacttta ccgtagtttc atctatctta 5940 tgtgtattga cttgaaaaag taagatgttt agcattgttt attcattttg gtctttcttt 6000 tctccatgtt atttaggcat atgccatcat ggctaaaggg gttcggttac tttgtacaaa 6060 tatttcaggc aaaaacacaa aatcattagt tcttaaaact caaggaagca gctcaattaa 6120 agataatatc atcaccgtat ttggcataaa gacatttaaa tgcctggagc cttttagctt 6180 atgcatatca gacacctgca aagttgaagg ctatctttcg aagcctggca atggctgtgg 6240 tcgtaatttg gtagacagac agtactatta tgttaatgga aggcctgttg atatgcccaa 6300 ggtcagcaaa gttgtgaatg agttatatcg aaattcaaat tccaaacaat atcctattgc 6360 tattataaat tttattgtgc ctaccaaatc atatgatgtt aatgtaacac ctgacaaaag 6420 aaagattttc ttttccgatg aaggcactct tgtgctttca ttaagagaag ctatagaaaa 6480 gatatactct ccaaatcaac gcagttattc tataaatggg gtaaagaaag tcaacgagga 6540 aacatatgag tgtgacatag atgatgccga tgaaaatttg acactaacta gatatgatag 6600 tttatgtgaa gtaaagaagg ttgttaatgg tgatgaaata tcaccatcaa aggatttgtt 6660 tgtcaatgcg cccctggagc agcacggcag tttttctacc tgcagatata aagcttcaac 6720 tagtttttat acaccaaaaa gtattactga tagtaggagc cctattcaat ccctggacat 6780 tttgccaagt aaagatagcc cttcaaactc aaaatttgtg caatcttctc ttacaaagtt 6840 tgtaacgtta aataagagga agcatgaaaa cggctccgat gttttatctg aaatgcctgt 6900 attgagatct gaaacgccat cttgtaaaat tagtaaaact tgtaaagaaa cacatggttt 6960 ggtttcgaga tccatatctt ctgaaatttc aagagattat gtacctaaag ccactgcaga 7020 aagtctagct ggagtatcat gtggacagga aaatgccttt tctgaaggtc accaggtgga 7080 tagagaagat attgataagg tataataatt gtcaagtgca caacactgta cattgttttc 7140 acttctaaaa gaaaccaatc ccgatggtac taaatgtact aaataactct tcttaggttt 7200 agatatttga ttgcttgcta gcatattatg acgccttatt tgctattttt tttttcaaaa 7260 catgaaaatt aaataaaaac cttctccatt ttggatgcag ctcaatgatt gcatgtggaa 7320 ttttttagct aacacatgtg tagtattttc atatttatat ttggtgacca ttacatttag 7380 gtagaactca atccttgtga agtgtctgga taaaaccaat aatttatcaa ccttattacc 7440 ttgatttaat gcatttctag catttcctta gactgataaa tgatcatttt tttaaaaaaa 7500 ttgatgagtt gccagttctg tagacgtcat acttccattt aattattcta ttctggagca 7560 cttcttttca aatatattat tctgaatatt ttatactttt cagggcaatt tggaagtacc 7620 tgaaacttct ttgactgatg atacaaaatt ggtaggtctg tctggagaag ttcatggaac 7680 ttcatccata gaaccaccta gatcatgttc tctaatggaa ccatgtgatg tacttgctct 7740 caaaccttgt tctgctaata aaacatattc tacttatcaa tttaatatcg aagatattag 7800 aaaacggaag aaaatgatct ctagctattt acagttcgat tccacataca atggaacaaa 7860 cattcaaagg tagcttagta gcttaagttg atttttttaa actaatggta gaatgctata 7920 ctgcaaggga cctgctaact tttgagaaac catcctgcat gcatgctctt tggttctact 7980 tttctgatta aacttttgat ggacaatgta gcaagttaga atcatgtata ctgactacag 8040 agcactcaaa agaattttat gaaggataaa tatggaggga aaataatcta cgtgcaaaat 8100 atcatgatct taagtgcagg atggtttcca atgagcaact aaattgatgg ggtttgcctg 8160 caatgtagct aaaatagacc gctctgctat tttatgatct gtcagttttg acatattatt 8220 tcatttcaaa ggcgctataa tgctgcaacc cttgagaatt ctcagccaga aaatgatgaa 8280 ggaaaatcac aagctttagc tgctgctaca agagagctgg agagattttt tagaaaagaa 8340 gattttggaa gaatgcaggt aattatgtat tttttttaaa ttcttgtaac gcaatcgttg 8400 cgatgaatac ccaaaattta ttgtgttgac tgctgagcat atttaacacc catgcacttt 8460 tgaagtattt gttatgcggc atctgcttac gaaaaatagg gaaggtacca aatagctgaa 8520 agttctatta acatacgaac acatacattt accgaacacg gctagtttca aacttcaaat 8580 attcattttt ctctaatttt gattaagcct aatagtttcc acttgcttct ttactaattt 8640 cttttttcat gccaggatgc tttataaaaa atctcaagat gcttattact gttgttgctg 8700 ttcctgctgt tgttgttgat attattgata tgttccactt ttatctgttt attttggtac 8760 aattctcggc ctcaaatgct ttacagcgta cccatcttat cattctcatt tgtaatagag 8820 tggtccctct tatacttgag tgggttatgc cgaaggctag ttttatacca aatatcagca 8880 tcttagcctt ttagtcattt ttggaagctg gttgtcttta gattattctc tgctactgat 8940 agtatgaaag tatgatggaa catgctctct tcctcttttt tgtcgtgcaa cagctgaaac 9000 tgaaatttgt attttttgag aacttaaatt tccttgtact gttttgtcga tataaatgat 9060 ctgcagacca gaaattcatc attttttgta tgagtagttt ggacgttact attatgatct 9120 ttcatctata cacatcacca gtggatatat tattagttta aaactcatca tattttgttt 9180 ggttagaaga tggttcccag atgctgcatc aaaagctttc tgggcaacca tattacttct 9240 cttggtgacc ttttgcattt actggtatca ggttgttggg cagttcaatc ttggttttat 9300 cattggaaaa ctcagtgaag atttatttgt ggtagatcag gttagtgttt atatgttttt 9360 tcacatcagg ctgcgcttac acattgttag taattcattt gttcatctga caatgttaga 9420 tgaaattcta tctcttattg tgcacaccac agttattgaa ggcgcctaca agagcaaatc 9480 caaaagattt tccaaagggt catagactaa aaaagaatca aaatcaacca aagattatcc 9540 ttataaagtt tgttacagtg atatatcaat ttcagttgca attgtttctg tcaaaatttc 9600 atcgattgaa attgttaaat aatagattat ttgttgttta tttctctaag cacatcttct 9660 tgaatattgt aatcaaacga taattgagtc aatcatattc cacacagtcg attcttgtca 9720 atctttaacc aataggcagt aagatagact cttaatcttt gattagatct caactaacca 9780 atttgacgat ctaatgtata aatcctttaa ccaatttcaa ttttaaatgc caagcatttt 9840 tttgtgacga tgatatcaat tatcaacata catgttttga ttttctgaat cagcacttaa 9900 tcatattaca catttaacat acgcaacatt acaaagataa attatatttg tcaatcaatc 9960 atggttcgta gaaaaataga aagcaaataa atattacgtt ttttgacagt ttccccgaat 10020 ttaaaatggt taaggttttg acaattttac atctctttac atctcaactg ttaataaacg 10080 gaattaattt cataacattc aacagcgtct ttctgaaaca ataaaattac cctttaaact 10140 tctattactt caccatattt tgttattgca attttaactt gagtttcaac ttccgataag 10200 ttatttgcac ttcactcatc attatttact tttttaatat gatacgatgt tctatttttg 10260 tttttgtttt ttaaaaaact gtataatgat tatttttttg tattaaaaaa tttatcattt 10320 ttttgataag tcttctgttt gaaataaaaa ttatatttat gaaccaaaat tagttaacta 10380 aaaatatatt agatattata tttttgttag tgtcttgtta acacatatta tgatctaaaa 10440 agttgatttt aattgcatcc agtaagcgat gtaagtttat gcacatcatg tatgtcgatt 10500 tactagtata ttgaagaata gtgacaaata tacctaatgg agaagaaaat aagcgcaaag 10560 ctagcactac aatttcaagg agcaacatag gatgtttcaa aatcaatata tagaaggcga 10620 agtcatagag cagtagcttg ggtgagactg atgaatagaa taaaaaaaga gcgaagggat 10680 ttgtgtatca attcttgaat ttggcaagaa aaattgtggg tttcattttt tttaatgctc 10740 gtcagcgaag aattaggatg agcgaaaaaa gtatactacc atagataccc catacgttga 10800 cttcctaaca ccatttcaaa aaaagaattc caaacatgtt tattttttta agacctagag 10860 gaccaaggtt atgcttgtga actccgagga attatgcagt gtacgcacaa aacatactct 10920 aagtaaatca tatagattaa atgtagatag actgaacctc caaattcgcc aacgaacatc 10980 acatgaacta caaactacac tttccgtgag tagaaaatgt atcatttcaa ccatttatgt 11040 aatattttgt gatacattaa tatttgaaga tttcacaaat ctaataagtt gaatttacgt 11100 ttgactgcat gattaatttc gagtttgtgt atatatgcaa tttgttttta ccaaaacaat 11160 ttcatatata accgttccat ttcttgattt gaataaataa ttggtttttg ttgtggaaaa 11220 caaccaattg gtgtcatacc gaatggtggt gtgaatggta gcttacaccg tggctgtaag 11280 actgagtgaa tatatgacaa aagtaatatt tggtcggtct ttgaaaaaaa aagtgcaagt 11340 ggagggtgga tgcaagagga tttttatttt tcttgtgagt gtagaggtga gcgagtggag 11400 attaaatgtg ggtgtataca attacaccct aaatatatat gatgtgatga gaaatactct 11460 gttgtctttt tcacaattta aaggttgaca ttttataaat tattagtcct agctaattta 11520 gaaattttgt aatttttctc tcgtaatttc aaagggcgac tttttataaa ttatacgtaa 11580 atttgctatt gaatgcaatg catggcacag caatatgaaa aaaaataatt ctgaacccta 11640 aacctgtcgc acataccttt taattgagat ttactataaa aatactgtat gtacatacat 11700 atgataacat ccatcaaaac tacaagagaa ttagttactt catgcaaata aactatatct 11760 ttaaaatatg attatgagag catatgatat gataatctca tttatgtttt taaaatgatg 11820 caaaaccgta taataattag tcaaaaaatg gaaaaggcac accttttact gcagtatgtc 11880 atttttaagc ccaatacggt tcaagttgtg ccttgagtga aaatgacacg tatgccttgc 11940 tgtgccttgc cgtagcctgt tgtttacaac aatggtctac atatcctaga tagtttataa 12000 aaaaaaactg ataaaattca gttctgacac ttctttcatt gtttgttgtt gtgcaatact 12060 agcatgccgc agatgagaaa cacaacttcg aacaactttc agactcaaca gtcctgcatg 12120 tgcagcctct actccagtaa gaatttgact gaatttttca ttactgtaac tgtagttttg 12180 ttaataatct tttatcaagt gcaagacaaa atctggtcat tgaacaattt aaaggaacgt 12240 gtggcatgtg ccaccccagc atggcaacat acgtagttgt accgattatg agttgatttg 12300 aatctcttct ggtctcttat atgtcatttt ttgcttatgg atcgctattt gttcttttat 12360 gaattcttcc aactccaagt tctgttgtat gctagtgatg cataggctat cccttaaaca 12420 tgttcgttga ttgtcatgac tgcttatcaa taagttttag ccttcagttc aatgcaaaaa 12480 ttactggtct tatcttaaat aatatcttat gctgtccttc acgtatgagt ttatactatt 12540 gctttctttc tacagatgtt atatctaccg tttgtacgaa aacatgcagt tttgttggct 12600 atacatgctt atgttacttt tgaattctta ggccagttag attggaatta tcaccagagg 12660 aagaagtaat ggtatctctg catatggaga tcattaggta aggtttattt atatagtgtt 12720 catgcaactc aaatactgaa attatctagt gctagtattt tgtatgttga gtatgcagcc 12780 aagttttgta caagttcaaa ttgaaaagga agcgcgtagt tgtgccaaaa ttgtgattag 12840 aggggaataa tgatataaat taaagagaag cattattaga aggaaaactg taaagtaaac 12900 ggtctaaagg tttgtgaaga tgtgatgaag tataccaatt gtgtggtatg atataagagt 12960 taacaaaggg atcccacttt tgaatattag agggtcaaaa agtttggtgg tccccatcga 13020 aaataaaata aaatttacac tactcacggt gatcccatac cgaaaaacac acctaaattc 13080 ggtgacttcc ctacagaaaa ctttttcata tccaaaagta gagtctcttt aacaatgtcc 13140 tttttttata gatcaacaag tacacatgta aaaaattaat aattcaaata gagtttacaa 13200 attacaattg gaccggttat gattattgta agaaaacctg tttaacaata cactaaaaaa 13260 aactgtatgc atggacacat agatttgggt tacaaaaatt catagattaa agagtgataa 13320 atacccaaga ggaaaatgtt tagtgatcac ttaggagttt gtgtggatgc taaaccccaa 13380 tacaattata taattttgtc agctcacacc actggtaaca gtagagatga attttctctt 13440 tcttgtcaac atgaaaactt gagtactgaa gtatataaag gcataatcat ctcatttact 13500 acgataacaa gttctccatg cacaacctgt cagatcccga aaaatctaaa caatattctt 13560 caattttatc gtatctttga tttaaaaaac atgtttaagc taggaaatta gcacattgtt 13620 tttaggtagc ttgccataga ttttgatgct tcttttttaa ttgctcaact ctggacaatg 13680 acgacgttaa taaagcatat gatctaggta tgtttctgtt atgtcagcac caattttcac 13740 atttataagg ataagataat atggttcacg tctctgtctc cccatactat gatccatatt 13800 ctttcttttt actagacagc aggcgatccc attttgcata gtaaattcat cctcttagtt 13860 tctatgctgt gatctcaatt atacgtactc tctttctgca aggaaaaatg gttttgcttt 13920 aacagaggat atgcttgctc ctcctggtca acgttatttg ctcaaagctg taccattcag 13980 taaaaaaatt acttttgggg ttgaaggtaa actgtatgtt tgaattctgt acctgaacca 14040 ttttccaccc cagaagcttt tatatcatga atcaaagaaa aatcaaatgt atgcatttct 14100 tgcagattta aaggacctta tttcaactct ctctgatagt caaggagagt gttcgattgt 14160 tagcagctac agatctaaca cttgtgattc tctctgccca tcaaaggtcc gtgctatgtt 14220 ggcttctcgt gcatgccaag cttctgttat ggttggtgat gcgctcgcaa aaaatgaaat 14280 gcagaatata ctacgtaact tggcaggtct caagtcccct tggaattgcc ctcatggtag 14340 accaacaatg cgccacctgg ttgatttgag tactcttaaa catcaaagca accttgtaga 14400 atgattcttc aagtttagga cagccactgt cgccaagcaa acttttgcaa attccactgt 14460 tttctgttgt caat 14474 <210> 2 <211> 14900 <212> DNA <213> Onion AcPMS1 Male-sterile <400> 2 ttggacatac caaaacccta atttcgaata gtgaacaata tgaacgaaga aattgcatcg 60 tctcctacaa tcaaacccat taacaaatcg gtggtccata gaatctgctc gggtcaagtg 120 attttagatc ttcaatcggc agttaaagag ctgctcgaga atagtttgga tgcaggtgcg 180 acctgtatcg aaatcaattt gaaagagcat ggcgaagaat attttaaggt tgtcgataat 240 gggtctggta tctctcctga taattttcag gtaatttttg gtgagacttt cggttcattt 300 tctgctgttt ggtttgtgtt cagttattct gctttgatat catatgcatg tatatataaa 360 tacaaagatg catctaatat tacaagggat gtaaagtttt gttgaaactt tttaggattt 420 cacaccgcgg attttaattt taagaactat tgaaatgatg gattatgaag gaaagtacaa 480 ctgcttacat agaagacatg aatgtggaat gttactttgt aacttggtct gaaatggcaa 540 acgccagtgc tgcacatgca gaaatactga ctgaaaatga taataaaatc tagaactgtc 600 tacagcatta tgagatcaca gtaatatcta caatcacaag tacattgtca cataagcaag 660 tgcagatcat taattctagg aacatttgat gtctaaataa ttgaagaaaa catggaatgc 720 attttggttt catgataaaa ctttcctact atttatgact gtagtatagg tgataatgga 780 agagttatta atcgtccctt aaactggaca aatgttacat aatctgtgaa agcagcttgt 840 aacgaacatt gatttcactt ataatgaaat ataaagcaaa gtcgcattaa atttacatta 900 ttgtgaaatg gttcgtacac tcaataacta ttgcagtttt cattcatgcg atataaatgt 960 tttactctgt ttactattgt tttcatataa gaaaaatcgt aattatcagt aaatgaacag 1020 gcatagtagg gatacacttt tgtgttgttt gtcatacatc catcaacata catcgggcct 1080 gagattttaa taaaccatta gatgaatatt ttatgaccgc taatttagta agcacaccct 1140 atcacttaag tgtgagtgaa cgtgtggcat gtatgctgtg tgtgattgag tttgaatatt 1200 aacattcatg taatgcccgc ctcttcacaa ttccattttc ttaaacatac tataattata 1260 atattcttca cattaaaaaa aataatacaa cgatcgctac cagttcttag attttttttt 1320 cttgattgct tgaacaatcg taaatgattg ctgaaaagtc ttgaagtttg caaactaatg 1380 gatggaacat accaagaaaa tgaaatcaca tgataatcgt tgatataaag aatttctaat 1440 atgtatgaaa ttattaaatg aattattcta gagtagtgca atactatttt ggtcttgagc 1500 caacttatga tttaaaacat ttcatcatca ttttagcatg actctcatca tttttctaaa 1560 ttctttgatg agagaacgat caacgtttac taagtactgg gtaaaatttt gaaataggct 1620 aatttttgaa agtaaatgca gatttacgtt gtttttaaaa ctatttgtaa aagcaagttg 1680 gttttttatg agctcatcac taatgtgaat cgcagacata caaattcaaa tataaattta 1740 catagatgca actgcctaat gtgcttatgt aacttttctt cgggagctag atgcttccat 1800 tccattttcc ttatgaaaca accgactgat ttttatcaaa caaccaattt ttgtttttgt 1860 tttttaatca aatcagtcaa acaaacaata aactatccat cgtcaattaa tatcgtgaat 1920 tacaacaaaa aatagtgctt ggtgataacc atgtaaaaag tcgtaatgat aacgtacttt 1980 ataattcaat tgcatttaaa caaccacaca acaatttttt tgaaaagcat ctttgttctc 2040 ttcagatggt tcgacttgag ttattggtag ttgaaaaaga cctgacttcg tgctcttttt 2100 tggatttccg tctttgtcta gtgtttcttt tcacataagt aattattggt cactttggca 2160 gaggatctga tcatgctagt tgatttcgat gaagtaccga atgtaaaata ttttcaaatt 2220 caaacattat ggttgtcttg tgcatttact tgttcatgtc caatatgacc aaattttctt 2280 gaaatcgtct cgatctcatc tactgaaaat gatataattt aatttattag aacggtaaac 2340 acaattgagt ttaaaaaccg aattgaaaga attataatca gaaataaatt tgtacgaatc 2400 aggtctcact caagtgacac gagaagtata tgcacattcc caatacatgc taataaatgg 2460 ttgagtattt gatcatatcg actcttagac tatgatattg gttaccgttg atgtccttca 2520 cactcaatat gaatgtgttt gcatttcgtt gatttatagc aatcttcggt cgtattgcaa 2580 actcgtatgc atcagatatg tgttgttgta gactgcatca ctcgatatgg agaccatagt 2640 tgtcaagctg cgcgcctagg cacctgctgc gtctttcgtg ccgtcgaggt gcctttgcta 2700 ctggaggcgc gcaaaaagtg caataaggtg cgattttttc gagccgccga agcgcaataa 2760 ggcgcgcctc gaagggtcga ctacagtcga cattcaattt tagggttctt cagagttcga 2820 acaaaaaagg actgctgcca tccatcgtcg ccgtcactgc tgccaccact atcgatggtc 2880 tgccgccgcc tagccattgt cgctgccatt gcctcctgtt gctgaacctg atttgacggc 2940 ttgacatgtg atgttttttt acttaaatta atagttatat aaatttgatt aatctagttt 3000 aaaatttaaa tagttatgtt aaatataatt atggctttaa atttaaattt tagttaatta 3060 aaagttaaat tttgaatttt agacttttta gttgatgaat agttaaattt aaattttact 3120 aaataaagta gttaaataaa atatttaata gtaaaataaa ttataaaatt taatttaaaa 3180 tttaatcatt agttaataaa atagtttaat aaaatattta agtagttgaa tgaattataa 3240 atagttaaaa ctataatttt agttaataaa atatttaaaa atagttgaat gcattataat 3300 tttagttaat aaaattgtta aacaaaatat ttaaacgatc aaatggctta taaagagtta 3360 aatttgaatt atatttaata acatatttaa atatttataa atagttggat gaattataat 3420 ttagttaata aaatagttaa ataagatggt tatcaacact agatttatca tttttagtag 3480 taattcatag tgcagttttt ttcccaccaa aaaactcaga cttgtgtaag agatgcataa 3540 aaaaggccaa ggcgcaagcg ttgcgcctta actactttga tggagaatac atatgaacat 3600 tgtgcatgca tgtgatcaac atgttagata gccaactctg catgccgttc acacacaata 3660 tgactgtgtt tgcattttgt tgattaatag aaatctccgg ccgtggtgca gattcatatg 3720 catcagatat gtgttgtcgt tgattgcatc actcaatctg gagaatacat atgaacttcg 3780 tgcatgcatg tgatcaacat cttagatagc caactcagca tgccatttag acatatttgg 3840 agaactcagt catgttttgc atttatggat tttcacactt gaccatggtc taaaagatca 3900 agggtgtcac gataaaaggt gaactgttgc ttaggaatct caatctaacg tttttcgtta 3960 aaccacttga gcatataaat tagatattaa gataatggtt aaattgtttt agataggagt 4020 ttcaaaaaat aatatgtata gacaatggac tatctggcat tgcaagacag tgtcctctca 4080 gtttttgcat ttggagaagt gttcggatga ccaagaggaa gatgtgttca taatcaaagt 4140 tgaaaatgta atcatgaata ttagaacaaa tttattaact catgtgcaaa aagaataata 4200 cacactacac acacatgcac acacacatat atacatacac acatatatac atacacacac 4260 acatacatac acacacacat atacatacac acacatatat acatacacac acacatatat 4320 acatacacac acacatatat acatatatga agcaatatat atgtaaaagc ttgggacaac 4380 cacctatgaa tttagatttc atcagcacaa catccgttaa ttcattatac aagctagtca 4440 aaattgcgca atcccgtaaa taagtagaat atacatataa atgtctaaga atcaataaca 4500 aaaaaaatga ttaagttgtc tctttgagtg gttacgaaaa tggcaagaac atatataatt 4560 aagaatcaca acttgtgctt gatgttcaaa ttctgataca tcaaatgttg gaacattttt 4620 aactttttta ctaagccatg ccaatttaat tattccatct tgtaatttgc ttaaaggtga 4680 tatcacacct aagtattttt cacaatgtcg gtacttgggt gaaacactgt tacaattttt 4740 ctttctttct gatccatgca ttgttaaacg tgtgatcatt agtagatgta tcatagcata 4800 tgctgcctat tagcgtatcc tattttagcc taacaggttc aagtagcaag ttgatgtttt 4860 tttatgcaat tacacccacc ttttgttgga tgagtaatgt atgagcagta tataaaagca 4920 catatacttg aactccttgc atattggatg gtttcaaaag tggatgcgag agagtcagaa 4980 gacatatggt catagactag ttgacaataa cttgattgtt aaggtaaacc catcaaaatt 5040 tgggatttgg ggcccaaaat ggtacgtgca cctatcttta tcagatgagt gtatatatta 5100 gacttattag ttgttttcat tattaagaat agtaaatgtg ccttagctgt tagattcgtt 5160 agaacttaga acacaaggct tgcgtttatg aagccctggt tcaaaaccga taatatcaat 5220 tgcttgttta ttatcattac acacacgtgc acgaaagcac gcttatccct tcactcacag 5280 taattaatct tggtgcagaa ccttgttcgc aaacatcata cttctaaaat agcagatttt 5340 tctgatcttc attcgttagc tacttttgga tttagaggag aggcattgag ctctctctgt 5400 gcaattggag acttgtctat tgaaacaaga accaaatatg agtctgttgg cacacatctg 5460 atctacgatc actctgggtc agtaaaatct gaaaaaaaga ttgctcgtca aattggtacc 5520 actgttactg ttgagaaatt attctccacc ttgccagtac gaagtaaaga attcaaccgc 5580 aacattcgtc gtgaatatgg aaagcttgtc tctttgttga atgtaagaca tatttcccta 5640 gaaaccttat actttagcta tgctctaaat gatacgactg ttctttgtga tcaagccatc 5700 gggttgtctt tggatgctta gatgtaaaaa aagtttgcat ttgaaaacta aacaagtttt 5760 tggtttaaat ttcttttatt agactgtcag tgtatatacc tcatagctct ataccagtaa 5820 tattcctttg taattctgtt agtgtcaatt aagcctaggg atctattcct tgtttgtttt 5880 atatgaatta aaagtaaaac actaatcagt tttttgtttt gatacttaat ggataacttg 5940 aagaaactaa aagcttttct gtccagctat acttcatgtt gaaattcaca ttaagcagat 6000 atatgttgtc ttcaaaattt gtatgatatt tactttacca tagtttcatc tatcttatgt 6060 gtattgactt gaaaaagtaa gatgtttagc actgtttatt cattttggtc tttcttttct 6120 tcatgttctt taggcatatg ccatcatggc taaaggggtt cggttacttt gtacaaatat 6180 ttcaggcaaa aacgcaaaat cattagttct taaaactcaa ggaagcagct caattaaaga 6240 taatatcatc accgtatttg gcataaagac atttaaatgt ctggagcctt ttagcttatg 6300 catatcagac acctgcaaag ttgaaggcta tctttcgaag cctggcaatg gttgtggtcg 6360 taatttggga gacagacagt actattatgt taatggaagg cctgttgata tgcccaaggt 6420 cagcaaagtt gtgaatgagt tatatcgaaa ttcaaattcc aaacaatatc ctattgctat 6480 tataaatttt attgtgccta ctaaatcata tgatgttaat gtaacacctg acaaaagaaa 6540 ggttttcttt tccgatgaag gcactcttgt gctttcatta agagaagcta tagaaaagat 6600 ctactctcca aatcaacgca gttattctat aaatggggta aagaaagtca atgaggaaac 6660 atatgagtgt gacatagacg atgccaatga aaatttgaca ctaactagat atgatagttt 6720 atgtgaagta aagaaggttg ttaatggtga tgaaatgtca ccatcaaagg atttgtttgt 6780 caatgcgcct gtggagcagc acggcagttt ttctacctgc agatataaag cttcaactag 6840 tttttataca ccaaaaagta ttactgattg taggagccct attcaatccc tggacatttt 6900 atcaagtaaa gatagccctt caaactcaaa atttgtgcaa tcttctctta caaagtttgt 6960 aacattaaat aagaggaagc atgaaaacgg ttccgatgtt ttatctgaaa tgcctatact 7020 gagatctgaa atgccatctt gtaaaattag caaaacttgt aaagaaacac atggtttggt 7080 ttcgagatcc atatcttctg aaatttcaag agattatgca cctaaagcca ccgcagaaag 7140 tctacctgga gtatcatgtg gacaggaaaa tgcctttttg gaaggtcacc aggtggatag 7200 agaagatatt gataaggtat aataattgtc aagtgcacaa cactgtacat tgttttcact 7260 tctaaaagaa accaatcctg atggtactaa atgtactgaa taactcttct tagatttaga 7320 tatttgattg cttgctagca tattatgaca ccttattgtt ttgcattgcc gacatgcaca 7380 ctacctaatt tgatattttt ttttcaaaac ataaaataaa ataaaaacct taatctccat 7440 tttggatgca gctcaatgat tgtatgtgga attttttagc taacacgtgt agtattttca 7500 tatttatatt tggtgactat tacatttagg tagaactcaa tccttgttaa gtgtctggat 7560 aaaaccaata atttatcaac cttattaccc tgatttaatg catttctagc attccttaga 7620 ctgataaatg atcctttttt tttgaattga tgagttgcca gttctgtaga cgtcatactt 7680 ccatttaatt attctattct ggagcacttc ttttcaaata ttttattctg aatattctgt 7740 acttttcagg gcaatttgga agtacctgaa acttctttga ccgatgatac aaaattgata 7800 ggtctgtctg gagaagttca tggaactata tccatagaac cacctagatc atgttctcta 7860 atggaaccat gtgatgtact tgctctcaaa ccttgttctg ctaataaaac atattctact 7920 tatcaattta aaatcgaaga tattagaaaa cggaagaaaa tgatctctag ctatttacag 7980 ttcgattcca catacaatgg aacaaacatt caaaggtagc ttagtagctt aagttgattt 8040 ttttttaaac taatggtaga atgctatact gcaagggacc tgctaacttt tgagaaacca 8100 tcctgcatgc ttgctctttg gttctacttt tctgatggat aattgtagca aggtagaatc 8160 atgtatactg actactgagc actgaaaagt attttatgaa ggataaatat ggagggaaag 8220 taatttacgt gcaaaatatc atgatcttaa gcgcgggatg gtttctaatg agcaactaaa 8280 ttgatggggt tggcctgcaa tgtagctaaa atagaccgct ctgctatttt atgatctgtc 8340 aattttgaca tattatttca ttttaaaggc gctataatgc tgcaaccctt gagaattctc 8400 agccagaaaa tgatgaagga aaatcacaag ctttagctgc tgctacaaga gagctggaga 8460 gattatttag aaaagaagat tttggaagaa tgcaggtaat tatgtaattt tttttataat 8520 tcttgtaacg caattgttgc gatgaatacc caaaatttat tgtgtcgacc gctgagcata 8580 tttaacaccc atgcactttt gaagtatttg ttatgtggca tctgcttacg aaaaataagg 8640 aatgtaccaa atagctgaaa gttctattaa catatgaaca catacattta ccgaacacgg 8700 ctagtttcaa actttaaata tttttctatg attttggtta agcctaatag tttccacttg 8760 cttctttact aatttctttt ttcatgctag gatgctttat aaaaaaaatc caaatatgct 8820 tattactgtt gttgatatta ttgatatgtt ccacttttat ctgtttattt tggtacaact 8880 ctcggcctca aatgctttac agcgtaccca tcttatcgtt ctcatttgta cttgagtggg 8940 ttatgccgag gctagtttta taccaaattt cagcatctta gccttttagt catttttgga 9000 agctttgtct ttagattatt ctctgctact gatattatga aagtatgatg gaacatgctc 9060 tcttcttctt ttcgtcgtgc aacagttgaa actgaaaatt gtattttttt gagaacttaa 9120 atttccttgt actgttttgt cgatataaaa gatctgcaga tcagaaattc atcatttttt 9180 gtatgagtag tttggatgtt actattatga tctttcatct ataagcatca ccagtggata 9240 tattattagt ttaaaactca ttatattttg tttggttaga agacgtaccc agatgctgca 9300 tcaaaagctt tctgggcaac catattactt ctcttggtga ccttttgcat ttactggtat 9360 caggttattg ggcagttcaa tcttggtttt atcattggaa aactcagtga agatttattt 9420 gtggtagatc aggttagtgt ttatatgttt tttcccatca ggctgcgctt acacattgtt 9480 agtaattcat ttgttcatct gacaatgtta ttgtgcacac cacagttatt gaaggcgcct 9540 acaagagcaa atccaaaaga ttttccaaag ggtcatagac taaaaaagaa tcaaaatcaa 9600 ctaaagatta tccttataaa gcttgttaca gtgatatatc aatttcagtt acaattgttt 9660 ctgtcaaaat ttcattaatt aaaattgtta aataatagct tatttgttgt ctatttcttt 9720 aagcacatct tcttgaatat tgtaatcaaa cgataattga gtcaatcata ttccacacag 9780 ttgattcttg tcaatcttca accaataggc agtaagacag actcttaatc tttgattaga 9840 tctcaactaa ccaatttgac gatctaacgt ataaatcctt taaccaatac caatttcaat 9900 tttaaatgca agcatttttt ttgtgacgat gatatcaatt atcaacatac atgtattgat 9960 tttctgaatc agcgcttaat catatttaca cgtttaacaa acgcaacatt acaaagataa 10020 attatatttg tcaatcagcc atggttcgta gaaaaataaa aacaaataaa tattacgttt 10080 attgacagtt tccctgaatt taaaacggtt aaggttttga caattttaca tctctttaca 10140 tctcaactgt taataaaagg aattaatttc ataacgttca acagcgtctt tctgaaacaa 10200 taaaattacc ctttatactt ctattacttt accatatttt gtttttacaa ttttaacttg 10260 agtttcaact tccattaagt tatttgcact tcactcatta ttatttactt ttttaatatg 10320 atatgatgtt ctatttttgt ttttgttttt taaaaactat ataatgatta tttttttgta 10380 ttaaaaaata tatcattttt tataacaagt cttctgtttg aaataaaaat tatatttatg 10440 aaccaaaatt agttaactaa aaatatatta gatattatat ttttgttagt gtcttgttaa 10500 cacatattat gatctaaaaa gttgatttta attgcatcca gtaagtgatg taagtttatg 10560 cacatcatgc atgtcgattt actagtatat tgaagaatag tgaaaaataa cctaatagag 10620 aagaaaataa gcgcaaagct agcactacaa tttcaaggag caacatagga tgtttcaaaa 10680 tcaatttata gaaggcgaag tcatagagca gtagcttggg tgagattgat gaatagaata 10740 aaaaaaaaga gtgaacggat ttgtgtatca attcttgaat ttggcaagaa aaattgtggg 10800 tttcaatttt ttttaatgct cgctagcgaa gaattaggat gagcaaaaaa atatactacc 10860 atagataccc catacgttga cttcctaaca ccatttcaaa aaatgaattc caaacatgtt 10920 tattttttta agacctagag gaccaaggtt atgcttgtga actccgagga attatgcagt 10980 gtacgcacaa aacatactct aagtaaatca tataaattaa atgtagatag actgaacctc 11040 tccaatgaac gaactacaaa ctacactttc tgtgagtaga aaatgtatca tttcaaccat 11100 ttatgtaata ttttgtgata cattaatatt tgaagatttc acgaatctaa taagttgaat 11160 ttacgtttga ctgcatgatt aacttcgagt ttgtgtatat atgcaatttg tttttaccaa 11220 aacaatttca tatttaaccg ttccatttct tgatttgaat ggtagcttac accgtggctg 11280 taagactgag tggatatata acaaaagtaa tatttgggtc ggcctttaaa aaaaaaggtg 11340 caagtggagg gtggatgcaa aaggagtttt atttttcttg tgagtgtaga ggtgagcgag 11400 tggagattaa atgtgggtgt atacaattac accctaaata tatatgatgt gatgagaaat 11460 actctgttgt ctttttcata atttcaaagg ttgacatttt ataaattatt agtcctagct 11520 aatttagaaa tttcgtaatt tttctctcgt aatttcaaag ggtgactttt tataaattat 11580 acgtaaattt gctattgaat gcaatgcatg gcacacaaat atgcaaaaaa aatttctgaa 11640 gcctagacct gttgcacata ccttttaatt gagatttacc ataaaaatta ctataccaca 11700 ttaattattc atcaaattat ccagatatta acatgtttgg aaaaaaaata tagttttttt 11760 cttcagaaaa caacaataat attcatagtc tcattcatct agatcccata tggtagctta 11820 tctgatctga gatgctaatg agaagtagaa gaattgaatc ttagtgatga caattagctc 11880 cttttaatta ccaagagcat gcattgtggg ttggtacttg gtgcatattt acttaggagt 11940 ttacatacaa tgttcaattt cgttagatac ccaatgaaaa ataataacac cgatataact 12000 atttttcttt accttttttt ctttctctgt ctctctctct ctctctctct ctctctctct 12060 ctctgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgttacaca 12120 catgtatgta catacatata ataacatcca tcaaaactac aagagaatta gttgcttctt 12180 gcaaataaac tatgtcttta aaatatgatt ataagagcat atgatatgat aatctcattt 12240 atgtttttaa aatgatacaa aaccatataa taattagtca aaaaatgaaa aaggcacacc 12300 ttttattgca gtacgtcatt tttaagccta atacggttca agttgtgcct tgagtgaaaa 12360 tgacacgtat gccttgccgt accttgccgt agcctgttgt ttacaacaat ggtctacata 12420 tcctagatag tttataaaac aaaactgata aaattcagtt ctgacacttt tttcattgtt 12480 tgttgttgtg caatactagc atgccgcaga tgagaaacac aacttcgaac aactttcaga 12540 ctcaacagtc ctgcatgtgc agcctctact ccagtaagaa tttgaatgaa tttttcatta 12600 ctgtaactgt agttttgtta ataatctttt atcaagtgca agacaaaatc tggtcattga 12660 acaatttaaa ggaatgtgta gcatgtgcca ccccagcatg gcaacataca tagttgtacc 12720 gattatgagt tgatttgaat ctcttttggt ctcttatatg tcaatttttg cttatggatc 12780 gctatttgtt cttttatgaa ttcttccaac tccaagttat gttgtatgct agtgatgcag 12840 aggctatccc ttaaacatgt tcgatgattg tcatgattgc ttatcaataa gttttagtct 12900 tcagttcaat gcaaaattta ctggtcttat cttaaataat atcttttgct gtcctttacg 12960 tatgagttca tactattgct ttctacggat gttatatcta ccgtttgtac gaaaacatgc 13020 agttttgttg gctatacatg cttatgttac ttttgaattc ttaggccagt tagattggaa 13080 ttatcaccag aggaagaagt aatggtatct ctgcatatgg agatcgttag gtaaggtttg 13140 tttatatagt gttcatgcaa ctcaaatact gaaattatcc agtgctagta ttttgtatgt 13200 agagtttgca gccaagtttt gtacaagttc aaattgaaaa ggaagctcgt agttgtgcca 13260 aaattgtgat tagaggggaa taatgataga aattaaagag aagcattatt agaagggaaa 13320 ctgtaaagta aggtctaaag gtttgtgcgg atgagatgaa gtacaccaat tgggtggtat 13380 gatataagag ttaacaaaga gaccccactt ttgaatatga gagggtcaaa aagtttggtt 13440 gtccccatcg gaaataaaat aaaatttaca ctactcacgg tgatcccata ccgaaaaaca 13500 catctaaatt tggcgacttc cctacagaaa accttctcgt atccaaaagt agagtctctt 13560 taacaatgtc ctttttttat agatcaataa gtacacatgt aaaagattaa taatccaaat 13620 agagtttaca aattacaatt cgaccggtta tgattattgt aagaaaacct gtttaacaat 13680 acactaaaaa aaaaatgtat gcatggacac atagatttgg gttacaaaaa ttcatagatt 13740 aaagagtaat aaatacccaa gaggaaaatg tttagtcatc acttaggagt ttgtgtggat 13800 gctaaacccc aatacaacca tataattttg tctgctcaca ccacaggtaa cagtagagat 13860 gaattctctc tttctttttc tctttcttgt caacgtgaag acttgggtac tgaagtatat 13920 aaaggcataa tcatctcatt tactacgaca acaatttctc catgcacaac ctgtcagatc 13980 ccaaaaaatc taaacaatat tcttcaattt tatcgtatct ttgattaaaa aaacatgttt 14040 aagctaggaa attagcacat tgttttaggt agcttgccat agattttgat gcttcttttt 14100 taattgctta attctggaca atgatgacat taataaaaca tataatctag gcgtgtttct 14160 gttatgtcag taccaatgtt cacatttata aggataaggt aatatagttc acgtctgtct 14220 ccccatacta tgatccatat tctttctttt tactagacag caggcggtcc cattttgcat 14280 agtaaattca tcctattagt ttctatgctg tgatctcaat tatacgtact ctctttctgc 14340 aaggaaaaat ggttttgctt taacagagga tatgcttgct cctcctggtc aacgttatct 14400 gctcaaagct gtaccattca gtaaaaaaat tacttttggg gttgaaggta aactgtatgt 14460 ttgaattctg tacctgaacc attttccacc cctgaagctt ttatatcacg aatcaaagaa 14520 aagtcaagtg tacgcatttc ttgcagattt aaaggacctt atttcaactc tctctgatag 14580 tcaaggagag tgttcgatcg ttagcagcta cagatctaac acttgtgatt ctctctgccc 14640 atcaagggtc cgtgctatgt tagcttctcg tgcatgccaa gcttctgtta tggttggtga 14700 tgcgctcgca aaaaatgaaa tgcagaatat actacgcaac ttggcaggtc tcaagtcccc 14760 ttggaattgc cctcatggta gaccaacaat gcgccacctg gttgatttga gtactcttaa 14820 acatcaaagc aacccagtag actgattcgt caagcttagg acagccactg tcaccaagca 14880 aaattttgca acttccactg 14900 <210> 3 <211> 1507 <212> DNA <213> Onion Acepa28839 Male-fertile <400> 3 ttactgtact aaacagattt caaactttgg taatggggag tgaaacagag cacgttgcca 60 tggatcgaaa gggtgtctac agccttatag gttcggtgca gaactacgat tgggggatca 120 gcggagcgtc gggttctaaa gtcgcgaggt tgtacgagaa gaatacggga ttggttgcgg 180 agcaggagaa gaagtacgca gagttttgga tgggcacgca cccctctgga ccttcatttg 240 tcctccgtga ggaggggaaa gtcaagttga aggaattcat tgaggagaat agttgtaagg 300 ttttggggca gaaggttttt gatacgtggg ggaatgatct tcctttcctg tttaaggttt 360 tatctatagc caaggcattg tcaatacaag cacatccaga taaggaatta gcaacagagt 420 tacacaagat gcacccaaat atatataaag actcaaatca taagccagag atggcagttg 480 ccatcagtga gtttgaagct ctttgtggtt ttgtcagcac caaggagctt aaggtggttt 540 tagaccgtgt tcctgaaatc aaagagttgg ttggtgaagt ggaagtaaac aaatacatgg 600 aaattaacga gcagtgcagt gtcgacgaag caaaagctca tctgcagtca atcttcacag 660 agcttatgtc agctgataaa gaaattgttt ccaaattcgt attaaaacta aaaaaccgct 720 taatcgaaga aagcaagatt aggctactga gtgagaaaga aaagcttgca ttgctattag 780 aagaacaata cccaggcgac attggggttt tatcgtcgtt tttctttaac gatgtaaagc 840 taaaaccagg cgaagcatta tatctggatg ctaatgagcc ccatgcatat atatctggag 900 aatgcatcga atgtatggcg acatccgata acgttgttcg agctggatta acctctaagt 960 acagagatgt cgagactctt tgtaggatgc tcacatacaa acagggctac cctgatattt 1020 taagaggaat ggccttaagc aagtatgtgt tgaggtacac acctccattt tatgaatttg 1080 aagttgatac tgtatcactt ccaaccggag aatctatgca attcgatgct atttctggcc 1140 cttcgatttt tgttgtcacg tctggtaatg gaaaattaag tgaaaatgta gaaataaatg 1200 agggttcggt gtttcttctt cgagcaaaca ccgagcttat aattagtgct catggtgatg 1260 gaacgttaca gttatacaga gctggggtga acagcaagtt cttggattag ctacacttat 1320 atggtatata acatataata tatatactat agagaaataa atgaagtgtg gccgtgtggg 1380 ttcttttccc atgttgggta tatcatgaaa ccaagtttgg tatatgaaat agatgtaaaa 1440 gtaaatttgc aacaataacg taagaagaaa aattttgtct aaaatgatta aaaaaaaaaa 1500 aaaaaaa 1507 <210> 4 <211> 1435 <212> DNA <213> Onion Acepa28839 Male-sterile <400> 4 caccgcttac agtactaaac agatttcaag cattggtaat ggggagtgaa acagagcacg 60 ttgccatgga tcgcaagggt gtttacagcc ttataggttc ggtgcagaac tacgattggg 120 ggatcagcgg agcgtcgggt tctaaagtcg cgaggttgta cgagaagaat acggggttgg 180 ttgtggagca ggagaagaag tacgcagagt tttggatggg cacgcacccc tctggacctt 240 catttgtcct ccgtgaggag gggaaagtca tgttgaagga attcattgag gagaatagtt 300 gtaaggtttt ggggcagaag gtttttgata cgtgggggaa tgatcttcct ttcctgttta 360 aggttttatc tgtagccaag acattgtcaa tacaagcgca tccagataag gaattagcaa 420 cagagttaca caagatgcac ccaaatatat ataaagacgc aaatcataag ccagagatgg 480 cagttgccat cagcgagttt gaagctcttt gtggttttgt cagcaccaag gagcttaagg 540 tggttttaga ctgtgttcct gaaatcagag agttggttgg tgaagtggaa gtaaacaaat 600 acatggaaat taacgagcag tgcagtttcg acgaagcaaa agctcatctg cagtcaatct 660 tcacagagct tatgtcagct gacaaagaaa ttgtttccaa attcgtatta aaactaaaaa 720 accgcttaat cgaagaaagc aagattaggc tactgagtga gaaagaaaag cttgcattgc 780 tattagaaga gcaataccca ggcgacattg gggttttatc gtcgtttttc tttaacgacg 840 taaaactaaa accaggcgaa gcattatatc tggatgctaa tgagccccat gcatatatat 900 ctggagaatg catcgaatgt atggcgacat ctgataacgt tgttcgagct ggattgactt 960 ctaagtacag agatgtcgaa actctctgta ggatgctcac atacaaacag ggctgccctg 1020 atattttaag aggaacgccc ttaagcaagt atgtgttgag gtacacacct ccattttatg 1080 aatttgaagt tgatactata tctcttccaa ccggagaatc catgcaattc gatgctattt 1140 ctggcccttc gatttttgtt gtcacgtctg gtaatggaaa attaagtgaa aatgtagaaa 1200 taaatgaggg ttcggtgttt cttcttcgag caaacaccga gcttataatt agtgctcata 1260 gtgatggaac gttacagtta tacagagctg gggtgaacag caagttcttg gattagctac 1320 acttatatgg tatataacat ataatatatg tactatagag aaataaatga agtgtggccg 1380 tgtgggttct attcccatgt tgggtatacc atgaaacaga gttcgatata tgaaa 1435 <210> 5 <211> 1306 <212> DNA <213> Onion Acepa26780 Male-fertile <400> 5 atggctgctt catcctcgtc actaaaaata gacgaatgta cggcactgga aatggtgaaa 60 aaaggagcga cccttcttct tttaaacgtt cctcagttta ctctatttgg catcgataca 120 cagatatttt ccgtgggtcc aaatttcaaa ggactgaaga tggtaccacc tgggactcat 180 tttatctact atagtgcctc aaacaaagaa ggaaatcact tttcaccaac ggttggcttc 240 ttcattacca ctcaccctgc ggaggtaata attcgtaagt ggaatccaaa ggaggaacgg 300 ttggtcaaag tttcagaaga agaggaagcc agtttcagtg atgcagtgaa gaaatttgag 360 tttgataacc agttagggcc ttaccctcta aaccactatg gagaatggaa gcagctgtcc 420 aattatattg ccgaagatgt tatagcaaaa attgaaccaa tagggggaga gatttcggtt 480 gtacatgaat cttggcttat tgacaaagtg ccattaacga ccatggagat gcaattagtg 540 gagcaattaa aggatagtaa gttctcaaag ccagtttctg aaaatattga aaagcgaaga 600 tgctattact catctattcc tcatattgtc aaggagcgct ctcaattcgg tgaagatctt 660 acttcaatga accttgataa aactagatta cttgaaacaa tcctgatgaa agaatacaag 720 ggtgaagagg atcttctcct aggagaactg cagttttcct ttattgcatt tatgatgggg 780 caatcactag aagcatttct acaatggaaa gcattggtta gtctattatt cagctgcatt 840 gaggctcccc tcaaaacacg aagtcggtta tttataaagg tcataagagt tgtctattct 900 cagctaaaat atggttttca caaagaaaac acgaataaag agaatatgga caaagggttc 960 tctctcttgc ttgatgatga atggattgca aaagatgttt ttttatttcg cctttgcaag 1020 gaatttattc ctttagtcct tgaagcacaa gtcgttgatg gtgatcttct cttatggacg 1080 agaaaactta aggggctgct tgagactacc tttgggtggg acttcaatca tagtctagct 1140 gatgcgatgg atgaagatga tgagtttgca cctgttattg tacttccagg tgatgcagta 1200 cccagcgaag atcggacgag tcagtagttc ttttatactc cagatgtatc aaagttaaaa 1260 tgcatgattg tacttgtaga tgtaagctgt atttcccaag cgtatg 1306 <210> 6 <211> 1275 <212> DNA <213> Onion Acepa26780 Male-sterile <400> 6 atggctgcat catcctcgtc actaaaaata gacgaatcta cggcactgga aatggtgaaa 60 aaaggagcga cccttcttct tttaaacgtt cctcagttta ctctatttgg catcgataca 120 caggtatttt ccgtgggtcc aaatttcaaa ggactgaaga tggtaccacc tgggactcat 180 tttatctact atagtgcctc aaacaaagaa ggaaatcact tttcaccaac ggttggcttc 240 tttattacca ctcaccctgc ggaggtaata attcgtaagt ggaatccaaa ggaggaacgg 300 ttggtcaaag tttcagaaga agaggaagcc agcttcagtg atgcagtgaa gaaatttgag 360 tttgataacc agttagggcc ttaccctcta aaccactatg gagaatggag gcagctgtcc 420 aattatattg ccgaagatat tatagcgaaa attgaaccaa tagggggaga gatttcagtt 480 gtacatgaat cttggcttat tgacaaagtg ccattaacaa ccatggagat gcaattagtg 540 gagcaattaa aggatagtaa gttctcaaag ccagtttctg aaaatattga aaagcgaaga 600 tgctattact catctattcc tcatattgtc aaggagcgct ctcgatccgg tgaagatctt 660 acttcaatga accttgataa aactagatta cttgaaacaa tcctgatgaa agaatacaag 720 ggtgaagagg atcttctcct aggagaactg cagttttcct ttattgcatt tatgatgggg 780 caatcactgg aagcatttct acaatggaaa gcattggtta gtctattatt cagctgcatt 840 gaggctcccc tcaaaacacg aagtcggtta tttataaagg tcataagaat tgtctattct 900 cagctaaaat atggttttca caacgaaaac acgaataaag agaatatgga caaagggttc 960 tctctcttgc ttgacgatga atggattgca aaagatgttt ttttatttcg cctttgcaag 1020 gaatttattc ctttagtgct tgaagcacaa gtcgttgatg gtgatcttct cttatggacg 1080 agaaaactta aggggctgct tgagactacc tttgggtggg acttcaatca tagtctagct 1140 gatgcgatgg atgaagatga tgagtttgca cctgttattg tacttccaga tgatgcagta 1200 cccagcgaag atcgaatgag tcagtagttc ttttatactc caagatgtat cgaaagttaa 1260 aatgcttgca ttgtt 1275 <210> 7 <211> 1756 <212> DNA <213> Onion Acepa27528 Male-fertile <400> 7 atgtcctcct cacaacttga aatcgatggc aacacgctaa ctgctcttgc cctaaaacgg 60 ttcaatgtgt cgcacatgtt cggagtggtc ggaatccccg taacatccct cgcgacgcgc 120 gccgtcgctc tgggtatccg atttatcgct ttccataacg aacaggcagc agggtacgct 180 gcctcagcgt atggatacct aacgaaatca gctggcgttt tcttgaccgt gtctggcccc 240 ggatgcgtgc acgggattgc tggtctggcc aacgcgcagg ccaacgcttg gcctgctgtt 300 atgatctctg gcagctgcga tcaggctgat tttgggaagg gagattttca ggagctagat 360 caaattgctg cagtgaagcc ctttgtgaaa ttttctgcaa aagccactga catttctcaa 420 attccacaat tggttcttga agttataaac actgccatat ctggccgtcc tggtggttgc 480 tatcttgaca ttccatctga tgtcctacgc cagaaaatac ccgaatcata tgcttctgaa 540 attctaaatg aggtgcaata ccctgaaata catgacctcc aaatttcagg aacccaagat 600 atccaaacag ctgtttcttt gcttagaaat gcagagaggc cattaattgt atttggaaaa 660 ggtgccgcgt tttcacgagc agaaagttcc ttgaaaaagt taattgatat cactggcata 720 ccttttcttc caactccaat gggaaaaggg ttggtgcctg atagtcatga actttctgca 780 acagctgctc gatctttagc cattggtaaa tctgatgttg ctttaatcgt aggggctcga 840 cttaattggc ttcttcattt tggcgagtct ccaaaatggt caaaagatgt caagttcata 900 ctaattgata tttcaaaaga agaaatcgag cttcgaaaac cccatttagg tttagttggt 960 gatgcaaaaa tgattcttga tttgatcaat gctgaaatca aagataatcc attctctttt 1020 gggaaatcac atccttgggt tgagacaatt tcaaaaaaag ttaaagaaaa tgtgttgaaa 1080 atggaagcac aattgtcaaa agaagtggtg ccattcaatt tttttacgcc tatgaagatt 1140 ataagggatg ctattcttga ggagggtagt ccagcaccta ttttagtttc agaaggagct 1200 aacacaatgg atgttgggag ggcagttttg gtacagaatg aaccaaggac aaggctggat 1260 gcaggaacat ggggaacaat gggggttggc ttaggatact gcattgcagc agcagttgct 1320 tcaccagata gacttgtagt cgcagtcgag ggagattcag gtttcgggtt tagtgctatt 1380 gaagtcgaga cgttggtgag atatcagctt ccagttatcg tgatcgtttt taacaacgga 1440 ggtgtatatg gtggtgatcg tagaagccct gaagaaataa ccggacccta caaaagcgac 1500 cctgccccta cttcatttgt ccctgatgct gcatatcaca agttaataga agcctttgga 1560 ggtaaaggtt acattgctag cactcctcaa gaactaaaat cagctctcaa agactctttc 1620 tctgccaaaa aacctgctgt ggttaatgtt attatcgatc cttatgctgg agcggagagt 1680 gggaggttac agcacaaaaa ctgatgcatt catgaaatgg atccgggtac tgtactcttt 1740 tccttgttga ataaat 1756 <210> 8 <211> 1863 <212> DNA <213> Onion Acepa27528 Male-sterile <400> 8 atggcattca actacacaca acttacctgc cacactgccg aaaatatccc acgaaatcaa 60 cttaagtcac tgtctttaac gacctcgatc ttgaagcaaa tttaaataat gtcctcctca 120 caacttgaaa tcgatggcaa cacgctaact gctcttgccc taaaacggtt caatgtgtcg 180 cacatgttcg gagtggtcgg aatccccgta acatcccttg cgacgcgcgc cgtcgctctg 240 ggtatccgat ttatcgcttt ccataacgaa caggcggcag ggtacgctgc ctcagcgtat 300 ggatacctaa cgaaatcagc tggcgttttc ttgaccgtgt ctggccccgg atgcgtgcac 360 gggattgctg gtctggccaa cgcgcaggcc aacgcttggc ctgctgttat gatctctggc 420 agctgcgatc aggctgattt tgggaaggga gattttcagg agctagatca aattgctgca 480 gtgaagccct ttgtgaaatt ttctgcaaaa gccactgaca tttctcaaat tccacaattg 540 gttcttgaag ttataaacac tgccatagct ggccgtcctg gtggttgcta tcttgacatt 600 ccatctgatg tcctacgaca gaaaataccc gaatcatatg cttctgaaat tttaaatgag 660 gtgcaatacc ctaaaataca tgacctccga atttcaggaa cccaagatat ccaaacagct 720 gtttctttgc ttagaaatgc agagaggcca ttaattgtat ttggaaaagg tgccgcgttt 780 tcacgagcag aaagttcctt gaaaaagtta attgatatta ctggcatacc ttttcttcca 840 actccaatgg gaaaagggtt ggtgcctgat agtcatgaac tttctgcaac agctgctcga 900 tctttagcca ttggtaaatc tgatgttgct ttaatcgtag gggctcgact taattggctt 960 cttcattttg gcgagtctcc aaaatggtca aaagatgtca agttcatact aattgatatt 1020 tcaaaagaag aaatcgagct tcgaaaaccc catttaggtt tagttggtga tgcaaaaacg 1080 attcttgatt tgatcaatgc tgaaatcaaa gataatccgt tctcttttgg gaaatcacat 1140 ccttgggttg agacaatttc aaaaaaagtt aaagaaaatg tgttgaaaat ggaagcacaa 1200 ttgtcaaaag aagtggtacc attcaatttt tttacgccta tgaagattat aagggatgct 1260 attcttgagg agggtagtcc agcacctatt ttagtttcag aaggagctaa cacaatggat 1320 gttgggaggg cagttttggt acagaatgaa ccaaggacga ggctggatgc aggaacatgg 1380 ggaacaatgg gggttggctt aggatactgc attgcagcag cagttgcttc accagataga 1440 cttgtagtcg cagtcgaggg agattcaggt ttcgggttta gtgctattga agtcgagacg 1500 ttggtgagat atcagcttcc agttatcgtg atcgttttta acaacggagg tgtatatggt 1560 ggtgatcgta gaagccctga agaaataacc ggaccctaca aaagcgaccc tgcccctact 1620 tcatttgtcc ctgatgctgc atatcacaag ttaatagaag cctttggagg taaaggttac 1680 attgcaagca ctactcaaga actaaaatca gctctcaaag actctttttc tgccaaaaaa 1740 cctgctgtgg ttaatgttat tatcgatcct tatgctggag cagagagtgg gaggttacag 1800 cacaaaaact gacgcattca tgaaatggat ccaggtactg tactcttttc cttgttgaat 1860 aaa 1863 <210> 9 <211> 871 <212> DNA <213> Onion Acepa23881 Male-fertile <400> 9 tcatcaatcc attgataaat tgataaagca ctacaaggaa attaatgtca gacaacattt 60 caaatcaatc caaatcccta attctcaaaa tcgcccaatc tttcaaccgc aaaatctctc 120 aatttttctt cattctcatc aatcaaaaga acgcgggatc tattggagct ctcgctggat 180 tagcgattgc tttgatattc atcttgaagt tattgagttt gccacgtgga aggcccagaa 240 gatccaatag agtcaaacga agagaactga gctctagaaa tgaggttgac tcgaggtcaa 300 agaatttgaa tttcactgaa tcgccagctg agcttgatct tgggaaaatt gtgaaaatga 360 agctgaatgg gggacgaaag atgactatcc aattgcttgg agcaatttta gaggagacga 420 gcatagaaga gcttcagaaa caagctacca ttaagctttc atctttaaaa gtgcttgtgg 480 aaatctccaa aacttgtgat gtctatctaa tggaaacggt gcttgatgat gaaagtgagg 540 aaaggatcct catggctttg gagaacgctg ggcttttcct caccgggagt ctgagcaagg 600 agaaggttct attctgtagc actgacattg gcagatcatc tttcgttcga caactggagt 660 cagattggca tgtagattca aatttagaag tagtttctca actagctaga tttatcagga 720 accaacttca catatcgcaa atcgacacag gttcaattgg accaaacgtg ttcacttccc 780 caagcttgga gcagtatttt tcctgcctct aatttaccac acacaattta tattgtaagt 840 ccctgcataa tatactagaa gagtagagtc a 871 <210> 10 <211> 865 <212> DNA <213> Onion Acepa23881 Male-sterile <400> 10 cttactccat tataatcgac aaagcactgc aaggaaatta atgtcagaca acatttcaaa 60 tcaatccaaa tccctaattc tcaaaatcgc ccaatctttc aaccgcaaaa tctctcagtt 120 tttcttcatt ctcatcaatc aaaagaacgc gggatctatt ggagctctcg ctggattagc 180 gattgctttg atattcatcg tgaagttatt gagtttgcca cgtggaaggc ccagaagatc 240 cgatagagtc aaacgaagag aactgagctc tagaaatgag gttgactcga ggtcaaagaa 300 tttgaatttc actgaatcgc cagctgagct tgatcttggg aaaattgtga aaatgaagct 360 gaatggggga cgaaagatga ctatccaatt gcttggagca attttagagg agacgagcat 420 agaagagctt cagaaacaag ctaccattaa gctttcatct ttaaaagcgc ttgtggaaat 480 ctcccaaact tgtgatgtct acctaatgga aacggtgctt gatgacgaaa gtgaggaaag 540 gatcctcatg gctttggaga acgctggcct tttcctcacc aggagtctga gcaaggagaa 600 ggttctattc tgtagcactg acattggcag atcatctttt gttcgacaac tggagccaga 660 ttggcatgta gattcaaatt tagaagtagt ttcgcaacta gctagattta tcaggaacca 720 acttcacata tcgcaaatcg acacaggttc aattggacca aacgtgttca cttccccaag 780 cttggagcag tatttttcct gcctgtagtt tacgacatac aatttatatt gtaagtccct 840 gcataatata ctagagagta attcg 865 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AcPMS1 forward primer <400> 11 ggctaaaggg gttcggttac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AcPMS1 reverse primer <400> 12 aagagtgcct tcatcggaaa 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Acepa28839 forward primer <400> 13 ttacacaaga tgcacccaaa 20 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Acepa28839 reverse primer <400> 14 ggggctcatt agcatccag 19 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Acepa26780 forward primer <400> 15 gggccttacc ctctaaacca 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Acepa26780 reverse primer <400> 16 cgctccttga caatatgagg a 21 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Acepa27528 forward primer <400> 17 gcttggcctg ctgttatgat 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Acepa27528 reverse primer <400> 18 ggagactcgc caaaatgaag 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Acepa23881 forward primer <400> 19 gatcctcatg gctttggaga 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Acepa23881 reverse primer <400> 20 gggaagtgaa cacgtttggt 20

Claims (7)

A nucleic acid molecule for onion male fertility screening consisting of the nucleotide sequence of Sequence Listing 3.
An onion male sterility screening nucleic acid molecule consisting of the nucleotide sequence of Sequence Listing 4.
Method of male-fertile or male-infertile discrimination of onion comprising the following steps:
(a) obtaining an onion gDNA (genomic DNA);
(b) amplifying the third sequence of Sequence Listing or the fourth sequence of Sequence Listing of the gDNA, wherein the primer set hybridizes to the 1-439 and 864-1507 regions of the Sequence Listing, Amplification using primer sets hybridized to the 1-445 and 870-1435 sites of the primer set; And
(c) analyzing the amplification product of step (b), wherein (i) the amplification product is judged to be male-infertile when the product having an Indels size of 13 bp is present, and (ii) And if the product of 13 bp is not present, it is judged male-fertile.
4. The method of claim 3, wherein the analysis of step (c) comprises: cleaved amplified polymorphic sequence (CAPS), intron length polymorphism (ILP), polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) , Sequence Characterized Amplified Regions (SCAR), DNA sequencing or Southern blot.
4. The method of claim 3, wherein step (b) is performed using a primer set of SEQ ID NO: 13 and SEQ ID NO: 14.
(a) a forward primer hybridizing to a nucleotide 1-439 region of the sequence listing third sequence or to a sequence complementary to the nucleotide 1-439 sequence of the third sequence listing; And (b) a reverse primer hybridizing to a nucleotide 864-1507 region of the third sequence or a nucleotide complementary to the nucleotide 864-1507 region of the third sequence of SEQ ID NO: 3, Analysis of the product amplified by the primer revealed that (i) the presence of a product with an Indels size of 13 bp of the amplification product is male-infertile, and (ii) there is no product with an Indels size of 13 bp If not, male - male onion judged to be fertile.
(a) a forward primer hybridizing to a nucleotide 1-445 region of Sequence Listing 4 or a sequence complementary to a nucleotide 1-445 region of Sequence Listing 4; And (b) a reverse primer hybridizing to a nucleotide sequence complementary to the nucleotide 870-1435 region of the fourth sequence or the nucleotide sequence of the nucleotide 870-1435 of the fourth sequence of SEQ ID NO: 4, Analysis of the product amplified by the primer revealed that (i) the presence of a product with an Indels size of 13 bp of the amplification product is male-infertile, and (ii) there is no product with an Indels size of 13 bp If not, male - male onion judged to be fertile.

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