Method for selecting or producing malus plant exhibiting self-compatibility, self-compatible malus plant, and primer set

Abstract

To provide a self-compatible Malus plant, a method for selecting or creating the same, and a primer set that can be utilized in the selection or creation.SOLUTION: Provided is a method for selecting a self-compatible Malus plant, the method comprising selecting a Malus plant having a predetermined S-RNase gene.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for selecting or producing a self-compatible plant of the genus Malus, and a method for selecting a self-compatible plant of the genus Malus or a primer set. [Background technology]

[0002] In angiosperms, the inability to produce normal seeds through self-pollination, for example due to a lack of fertilization, is called self-incompatibility. Conversely, the ability to produce normal fruit through self-pollination is called self-compatibility. Many Rosaceae fruit trees exhibit self-incompatibility, but there is a need to develop self-compatible varieties from the perspectives of reducing the labor required for pollination, stabilizing fruit set, reducing pollinating insects, and halting the import of pollen due to the occurrence of fire blight. For example, self-compatible breeding materials have been discovered for Japanese pears, plums, and cherries, and self-compatible varieties have already been developed. However, there have been limited reports of self-compatible varieties in the genus Malus.

[0003] Furthermore, Non-Patent Document 1 discloses a self-compatible mutant obtained by γ-ray irradiation of a diploid apple, but the mechanism of self-compatibility in the mutant and the method for producing or selecting self-compatible progeny have not been elucidated. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Abe K etal (2023). Characterization of a pollen-part self-compatible apple (Malus × domestica Borkh.) mutant induced by γ-raymutagenesis. Sci Horticul 312,111867. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the above circumstances, an object of the present invention is to provide a self-compatible plant of the genus Malus, a method for selecting or producing the plant, and a primer set that can be used for the selection or production. [Means for solving the problem]

[0006] While investigating self-fruitful plants of the genus Malus, the present inventors first discovered that the apple cultivar "Vered" is self-compatible. By analyzing the S-RNase allele of the apple cultivar "Vered," the present inventors demonstrated that the S39-RNase allele of the apple cultivar "Vered" is mutated as described below. Furthermore, they found that the self-compatibility of the apple cultivar "Vered" is due to this mutation.

[0007] The present disclosure provides, for example, the inventions described in the following [1] to

[12] . [1] A method for selecting a self-compatible plant of the genus Malus, comprising selecting a plant of the genus Malus that possesses an S-RNase allele that is any one of (1), (2), (3), (4), (5), or (6) below. (1) A gene in which the CDS sequence has a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1, and in which 200 or more consecutive bases are deleted; (2) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, in which 200 or more consecutive bases of the CDS sequence are deleted; (3) A gene whose CDS sequence has 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 3; (4) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4. (5) A gene whose CDS sequence has 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 5, or (6) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6. [2] The method according to [1], wherein in the gene (1) or (2), the deleted 200 or more consecutive bases are present in a base sequence corresponding to positions 253 to 690 of the base sequence represented by SEQ ID NO: 1. [3] The method according to [1] or [2], further comprising determining whether or not a plant of the genus Malus possesses the gene (1) or (2) by a PCR method using a primer set for detecting the presence or absence of the deletion in the gene (1) or (2). [4] The primer set is a nucleotide sequence of genomic DNA or a nucleotide sequence of cDNA containing the CDS nucleotide sequence of the gene (1) or (2) above, a first target base sequence that is different from the base sequence containing the 3'-most base in the deletion portion having the same number of bases as the base sequence containing the 3'-most base in the deletion portion, the first target base sequence being the 5'-most base of the deletion portion having the same number of bases; and a primer containing a second target base sequence that is different from the base sequence containing the 5'-most base in the deletion portion, which has the same number of bases as the base sequence containing the 5'-most base in the deletion portion, or a complementary sequence thereof; or The method according to [3], comprising primers that hybridize with DNA consisting of a first target base sequence and a second target base sequence, or their complementary sequences. [5] The primer is a reverse primer containing complementary sequences of the first and second target base sequences, the first target base sequence has 5 to 14 bases, and the second target base sequence has 1 to 32 bases; or The method according to [4], wherein the primer is a forward primer containing the first and second target base sequences, the number of bases in the first target base sequence is 5 to 35, and the number of bases in the second target base sequence is 1 to 10. [6] A method for producing a self-compatible Apple plant, comprising selecting a self-compatible Apple plant from progeny plants of the genus Apple, using a plant of the genus Apple carrying the S-RNase allele as at least one parent line, by the method described in any one of [1] to [5]. A self-compatible Malus plant produced using the method described in [7][6]. [8] (1) A gene in which the CDS sequence has a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1, and in which 200 or more consecutive bases are deleted; (2) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, in which 200 or more consecutive bases of the CDS sequence are deleted; (3) A gene whose CDS sequence has 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 3; (4) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4. (5) A gene whose CDS sequence has 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 5, or (6) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6. Self-compatible plants of the genus Malus (excluding the apple cultivar "Vered") that possess one of the following S-RNase alleles: [9] A primer set for detecting the presence or absence of a deletion of bases corresponding to positions 365 to 569 of SEQ ID NO: 1.

[10] In the base sequence represented by SEQ ID NO: 13, a third target base sequence including bases 380 to 384 of the base sequence on the 5'-terminal side of the 385th base; and a primer containing a complementary sequence of a fourth target base sequence including the 385th base in the base sequence 3'-terminal side of the 384th base, or The primer set according to [9], comprising primers that hybridize with DNA consisting of a third target base sequence and a fourth target base sequence, or their complementary sequences.

[11] The primer is a reverse primer containing a complementary sequence of the third and fourth target base sequences, the third target base sequence has 5 to 14 bases, and the fourth target base sequence has 1 to 32 bases; or The primer set according to

[10] , wherein the primer is a forward primer containing the third and fourth target base sequences, the number of bases in the third target base sequence is 5 to 35, and the number of bases in the fourth target base sequence is 1 to 10.

[12] A primer set according to any one of [9] to

[12] , wherein the primer set includes a reverse primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity to the base sequence represented by SEQ ID NO: 8. [Effects of the Invention]

[0008] According to the present invention, there are provided a self-compatible plant of the genus Malus, a method for selecting or producing the plant, and a primer set that can be used for the selection or production. [Brief explanation of the drawings]

[0009] [Figure 1A] FIG. 1 is the first half of a diagram showing the alignment of the nucleotide sequences of S39-RNase_cDNA (MG598508 and MH281944), S39 sm-RNase1 cDNA, and S39 sm-RNase2 cDNA. [Figure 1B] 1 is the second half of a diagram showing the alignment of the nucleotide sequences of S39-RNase_cDNA (MG598508 and MH281944), S39 sm-RNase1 cDNA, and S39 sm-RNase2 cDNA. [Figure 2] FIG. 1 shows an alignment of the deduced amino acid sequences of S39-RNase (MG598508), S39sm-RNase1, and S39sm-RNase2. [Figure 3] FIG. 1 shows the results of agarose gel electrophoresis of DNA fragments amplified by multiplex genomic PCR using DNA obtained from a first-generation hybrid of the apple cultivar "Vered" and the apple cultivar "Fuji" as a template, with a primer set including a forward primer consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 7 and a reverse primer consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 8, and a primer set including Normal-F (forward primer) and Normal-R (reverse primer). DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the present disclosure will be described in detail.

[0011] [Method for selecting self-compatible Malus plants] The method for selecting a self-compatible Malus plant according to this embodiment includes selecting a Malus plant that possesses an S-RNase allele that is any one of the following (1), (2), (3), (4), (5), or (6): (1) A gene in which the CDS sequence has a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1, and in which 200 or more consecutive bases are deleted; (2) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, in which 200 or more consecutive bases of the CDS sequence are deleted; (3) A gene whose CDS sequence has 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 3; (4) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4. (5) A gene whose CDS sequence has 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 5, or (6) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6.

[0012] Self-incompatibility in plants of the genus Malus is usually controlled by the S haplotype, which is a set of S genes expressed in the pistil located at the S locus (hereinafter also referred to as "pistillate S genes") and S genes expressed in pollen (hereinafter also referred to as "pollen S genes"). For example, when pollen attaches to the pistil, if the pistil S gene and pollen S gene are of the same S haplotype, fertilization is prevented by inhibiting pollen tube elongation, etc. Examples of pistil S genes include the S-RNase gene, which encodes an RNA-degrading enzyme. RNA-degrading enzymes, which are translation products of the S-RNase gene, are thought to inhibit fertilization by degrading pollen tube RNA in pollen expressing pollen S genes of the same S haplotype as the S-RNase gene. Note that pistil S genes and pollen S genes of the same haplotype are genetically linked and are usually inherited as a single set.

[0013] There are no particular limitations on the Malus plant, so long as it is a plant belonging to the genus Malus. Examples of Malus plants include European apple (Malus × domestica, Malus pumila), Siberian hawkweed (Malus baccata var. mandshurica), Malus floribunda, Japanese bean (Malus prunifolia), Japanese bean (Malus asiatica), Japanese laurel (Malus prunifolia var. ringo), Japanese bean (Malus spontanea), Japanese bean (Malus halliana), Japanese bean (Malus toringo), and Malus tschonoskii.

[0014] Whether or not a plant of the genus Apple is self-compatible can be determined, for example, according to the criteria described in Komori, Sadao, et al., "Determination of Cross-Incompatibility in Apples by Seed Number and Fruit Set Rate," (1999):97-112. That is, when a plant of the genus Apple is self-pollinated, if the fruit set rate, which is the percentage of the number of flowers that set fruit (number of fruits) to the number of self-pollinated flowers (number of cross-pollinated flowers), is 30% or higher and if the number of seeds per fruit is greater than three, the plant can be determined to be self-compatible. The fruit set rate and number of seeds per fruit may be evaluated, for example, 80 to 100 days after self-pollination. Specifically, whether or not a plant of the genus Apple is self-compatible may be determined by the method described in the Examples.

[0015] As used herein, unless otherwise specified, the term "plant" includes whole plants, plant cells, plant protoplasts, plant callus, or plant parts such as styles, embryos, pollen, ovules, gametes, seeds, leaves, flowers, branches, fruits, stems, roots, anthers, etc.

[0016] As used herein, "deletion" of a base means that a portion of a nucleotide base is missing in a gene (DNA), resulting in a change (reduction) in the number of bases. "Deletion" of an amino acid residue means that a portion of an amino acid residue is deleted in a protein, resulting in a change (reduction) in the number of amino acids.

[0017] As used herein, unless otherwise specified, the term "allele" refers to an individual gene when there are multiple types of genes occupying homologous loci.

[0018] As used herein, "sequence identity" refers to the percentage (%) of identical bases or amino acid residues in the entire overlapping DNA base sequence or entire amino acid sequence in optimal alignment of two DNA base sequences or amino acid sequences, which are achieved using a mathematical algorithm known in the art. EMBOSS Needle (provided by EMBL-EBI, URL: https: / / www.ebi.ac.uk / tools / psa / emboss_needle / ) can be used, for example, to create the alignment and calculate the sequence identity.

[0019] SEQ ID NO: 1 shows the nucleotide sequence of the CDS of the S39-RNase gene, which is a pistil-side S gene, and SEQ ID NO: 2 shows the amino acid sequence of the protein translated from the S39-RNase gene. In SEQ ID NO: 1, the region between bases 252 and 253 corresponds to the boundary between two exons. As shown in Figure 2, in SEQ ID NO: 2 ("S39-RNase (MG598508)" in Figure 2), bases 1 to 27 represent a signal peptide region, bases 29 to 42 represent a conserved region C1, bases 55 to 59 represent a conserved region C2, bases 119 to 121 represent a conserved region C3, bases 143 to 157 represent a conserved region C4, bases 192 to 201 represent a conserved region C5, and bases 76 to 91 represent a hypervariable region RHV. In addition, in SEQ ID NO: 2, the cysteine ​​residues at positions 42, 49, 75, 120, 184, 199, 210, and 222 are important for stabilizing the three-dimensional structure. In addition, in SEQ ID NO: 2, the histidine residues at positions 60 and 117 are important for RNase activity. In addition, in SEQ ID NO: 2, the amino acid residues at positions 84 and 85 correspond to the boundary between two exons.

[0020] The base sequence of SEQ ID NO: 1 is as follows: ATGGGGATTATGGGGATGATAGATAATGGTTACGATGGTATTTTCCTTAATGGTATTAATGTTTTCTTCGTCTGCGGTGAAATACGATTATTTGCATTTACGCAGCAATACCAGCCGGCTGCCTGCAACTCTAACCCTACTCCTTGTAACGATCCTACTGACAAGTTGTTTA CGGTTCACGGATTGTGGCCTTCAAAGGTTAAAGGTCCTGACCCACACGACTGCAAGACCAAAACCAACAAGTCTCAGACGATAGATATATTACTCAAACCCCAGTTGGAAATTATTTGGCCGAACGTATTCAATCGAGCCGATAATGAAAGCTTCTGGGAAACACAGTGGGAA AAACATGGCACCTGTGGATCTCCCACAATAAAAAGACAAGAACCATTACTTTGAAACAGTAATCAGAATGTACATAACCGAGAAAAAAACGTCTCTCATCACCTATCCAAGGCCAACATCAATCCGGATGGTATAGCCAGGACACGGAAGGATATTGAAATTGCCATACGCA ATAGTACTAACGATAAGGAACCAAAACTCAAGTGCCATAAGAAGAATGGGATAACTGAATTGGTGGAGGTCACTCTTTGCAGCAATTACTTCGGAAAACAATTCATAAATTGCCCTAACAAAATTCCAGAAAAATCACGATATTTCTGTCCCATCAAAGATATCCGGTATTAA

[0021] Amino acid sequence number 2. MGIMGMIDMVTMVFSLMVLMFSSSAVKYDYLQFTQQYQPAACNSNPTPCNDPTDKLFTVHGLWPSKVKGGPDPHDCKTKTNKSQTIDILLKPQLEIIWPNVFNRADNESFWETQWEKHGTCGSPTIKDKNHYFETVIRMYITEKQNVSHYLSKANINPDGIARTRKDIEIAIRNSTNDKEPKLCHKKNGITELVEVTLCSNYFGKQFINCPNKIPEKSRYFCPIKDIRY

[0022] SEQ ID NOs: 3 and 5 are the nucleotide sequences of the CDS of the mutated S39-RNase allele carried by the apple cultivar "Vered." The mutated S39-RNase allele exhibits two CDSs (SEQ ID NOs: 3 and 5) due to alternative splicing. These CDSs (SEQ ID NOs: 3 and 5) lack the nucleotide sequence corresponding to positions 365 to 569 of SEQ ID NO: 1. Accordingly, in the CDS consisting of the nucleotide sequence of SEQ ID NO: 3, the bases corresponding to thymine at position 364, adenine at position 570, and adenine at position 571 of SEQ ID NO: 1 form a stop codon. In the CDS consisting of the nucleotide sequence of SEQ ID NO: 5, the nucleotide sequence corresponding to positions 253 to 256 of SEQ ID NO: 1 (i.e., the nucleotide sequence corresponding to positions 253 to 256 of SEQ ID NO: 3) has been truncated by alternative splicing, resulting in a frameshift mutation of bases 257 and beyond relative to the nucleotide sequence of SEQ ID NO: 1. Furthermore, in the CDS consisting of the base sequence of SEQ ID NO: 5, due to the deletion of the base sequence corresponding to positions 365 to 569 of SEQ ID NO: 1 and the truncation of the base sequence corresponding to positions 253 to 256 of SEQ ID NO: 1, the bases corresponding to thymine at position 574, guanine at position 575, and adenine at position 576 of SEQ ID NO: 1 form a stop codon. SEQ ID NOs: 4 and 6 show the amino acid sequences of the proteins translated from the mutated S39-RNase gene, with the amino acid sequence of SEQ ID NO: 4 corresponding to the base sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 6 corresponding to the base sequence of SEQ ID NO: 5. As shown in Figure 2, in SEQ ID NO: 4 ("S39sm-RNase1" in Figure 2) and SEQ ID NO: 6 ("S39sm-RNase2" in Figure 2), the conserved regions C4 and C5, and the cysteine ​​residues at positions 184, 199, 210, and 222 are deleted due to the deletion of the nucleotide sequence corresponding to positions 365 to 569 of SEQ ID NO: 1 and the resulting formation of a stop codon. In addition, in SEQ ID NO: 6, the amino acid sequence from position 86 onwards causes a frameshift mutation relative to the amino acid sequence of SEQ ID NO: 2, resulting in mutations in the hypervariable region RHV, the conserved region C3, the histidine residue at position 117, and the cysteine ​​residue at position 120.Because of the above, and in particular the deletion of the nucleotide sequence corresponding to positions 365 to 569 of SEQ ID NO: 1, an S-RNase allele whose CDS sequence is SEQ ID NO: 3 or 5 lacks RNase activity. Therefore, plants of the genus Malus that possess an S-RNase allele whose CDS sequence is the nucleotide sequence of SEQ ID NO: 3 or 5 are self-compatible.

[0023] The base sequence of SEQ ID NO: 3 is as follows: ATGGGGATTATGGGGATGATAGATATGGTTACGATGGTATTTTCCTTAATTGTATTAATGTTTTCTTCGTCTGCGGTGAAATACGATTATTTGCAATTTACGCAGCATATCAGCCGGCTGCCTGCAACTCTAACCCTACTCCTTGTAACGATCCTACTGACAAGTTGTTTACGGTTCACGGA TTGTGGCCTTCAAAAGTTAAAGGTCCTGACCCACACGACTGCAAGACCAAAACCAACAAGTCTCAGACGATAGATATATTACTCAAACCCCAGTTGGAAATTATTTGGCCGAACGTATTCAATCGAGCTGATAATGAAAGCTTCTGGGAAACACAGTGGGAAAAACATGGCACCTGTGGATAA

[0024] The amino acid sequence of SEQ ID NO: 4 is as follows: MGIMGMIDMVTMVFSLIVLMFSSSAVKYDYLQFTQQYQPAACNSNPTPCNDPTDKLFTVHGGLWPSKVKGPDPHDCKTKTNKSQTIDILLKPQLEIIWPNVFNRADNESFETQWEKHGTCG

[0025] The base sequence of SEQ ID NO: 5 is as follows: ATGGGGATTATGGGGATGATAGATATGGTTACGATGGTATTTTCCTTAATTGTATTAATGTTTTCTTCGTCTGCGGTGAAATACGATTATTTGCAATTTACGCAGCATATCAGCCGGCTGCCTGCAACTCTAACCCTACTCCTTGTAACGATCCTACTGACAAGTTGTTTACGGTTCACGGA TTGTGGCCTTCAAAAGTTAAAGGTCCTGACCCACACGACTGCAAGACCAAAACCAACAAGTCTCAGACGATATATTACTCAAACCCCAGTTGGAAATTATTTGGCCGAACGTATTCAATCGAGCTGATAATGAAAGCTTCTGGGAAACACAGTGGGAAAAACATGGCACCTGTGGATAACTGA

[0026] The amino acid sequence of SEQ ID NO: 6 is as follows: MGIMGMIDMVTMVFSLIVLMFSSSAVKYDYLQFTQQYQPAACNSNPTPCNDPTDKLFTVHGGLWPSKVKGPDPHDCKTKTNKSQTIYYSNPSWKLFGRTYSIELIMKASGKHSGKNMAPVDN

[0027] As used herein, "corresponding bases" or "corresponding base sequences" can be determined by aligning a base sequence with a reference sequence in multiple alignment, based on whether the base sequence is aligned in the same row. For example, "a base sequence corresponding to positions 253 to 690 of the base sequence represented by SEQ ID NO: 1" can be determined to mean the base sequence of a target allele aligned at positions 253 to 690 of SEQ ID NO: 1 when the base sequence of the target allele is aligned with SEQ ID NO: 1 in multiple alignment.

[0028] In the S-RNase alleles (1) to (6), the sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0029] In the S-RNase allele of (1) or (2) above, the deleted 200 or more consecutive bases are preferably present in the base sequence corresponding to bases 253 to 690 of the base sequence represented by SEQ ID NO: 1, more preferably in the base sequence corresponding to bases 300 to 630, even more preferably in the base sequence corresponding to bases 330 to 600, and particularly preferably in the base sequence corresponding to bases 365 to 569.

[0030] In the S-RNase allele (1) or (2) above, the number of deleted consecutive bases of 200 or more may be 200 to 438, 200 to 300, 200 to 250, 200 to 225, 200 to 210, or 205.

[0031] In the S-RNase allele of (1) or (2), it is preferable that the nucleotide sequence corresponding to positions 253 to 256 of SEQ ID NO: 1 is further deleted. However, this does not apply if the deleted 200 or more consecutive nucleotides are present in the nucleotide sequence corresponding to positions 253 to 256 of SEQ ID NO: 1. If the nucleotide sequence corresponding to positions 253 to 256 of SEQ ID NO: 1 is further deleted, the nucleotide sequence from the nucleotide corresponding to position 256 of SEQ ID NO: 1 onwards in the nucleotide sequence of the S-RNase allele of (1) or (2) above can cause a frameshift mutation relative to the nucleotide sequence of SEQ ID NO: 1.

[0032] As described above, the S-RNase alleles (1) and (2) lack RNase activity due to the deletion of 200 or more consecutive bases. Therefore, plants of the genus Malus that possess the S-RNase alleles (1) and (2) are self-compatible.

[0033] As described above, the S-RNase allele (3) or (4) lacks the RNase activity function because the nucleotide sequence corresponding to positions 365 to 569 of SEQ ID NO: 1 is deleted in SEQ ID NO: 3. Therefore, plants of the genus Malus carrying the S-RNase allele (3) or (4) are self-compatible.

[0034] As described above, the S-RNase allele (5) or (6) lacks the RNase activity function because the nucleotide sequence corresponding to positions 365 to 569 of SEQ ID NO: 1 is deleted in SEQ ID NO: 5. Therefore, plants of the genus Malus carrying the S-RNase allele (5) or (6) are self-compatible.

[0035] A plant of the genus Malus that possesses an S-RNase allele that is any of (1), (2), (3), (4), (5), or (6) typically possesses the S-RNase allele at the S locus.

[0036] Determination of whether a plant of the genus Malus possesses any of the S-RNase alleles (1) to (6) may be performed, for example, by PCR methods such as genomic PCR and RT-PCR, nucleic acid amplification methods such as LAMP, DNA sequence analysis, or the like. When the S-RNase allele is the S-RNase allele (1) or (2), determination of whether a plant of the genus Malus possesses the S-RNase allele (1) or (2) is preferably performed by PCR using a primer set for detecting the presence or absence of the deletion of 200 or more consecutive bases in the S-RNase allele (1) or (2). That is, the method for selecting a self-compatible plant of the genus Malus according to this embodiment preferably further comprises determining whether a plant of the genus Malus possesses any of the S-RNase alleles (1) or (2) by PCR using a primer set for detecting the presence or absence of the deletion of 200 or more consecutive bases in the S-RNase allele (1) or (2).

[0037] Examples of the PCR method include genomic PCR, RT-PCR, etc. RT-PCR uses cDNA as a template DNA, and the cDNA is not particularly limited as long as it contains a part or the entire nucleotide sequence of the CDS of the S-RNase allele, and may contain a 5' untranslated region and / or a 3' untranslated region.

[0038] The primer set for detecting the presence or absence of the deletion of 200 or more consecutive bases in the S-RNase allele of (1) or (2) is a primer set comprising: a first target base sequence comprising the 3'-most base of the base sequence 5'-terminal from the deletion of 200 or more consecutive bases in the base sequence of genomic DNA or the base sequence of cDNA comprising the base sequence of the CDS of the S-RNase allele of (1) or (2); a first target base sequence different from the base sequence comprising the 3'-most base of the deletion, which has the same number of bases; and a second target base sequence comprising the 5'-most base of the base sequence 3'-terminal from the deletion, or a complementary sequence thereof; are a first target base sequence and a second target base sequence, and examples thereof include a primer set (primer set 1) containing primers that hybridize with DNA consisting of the second target base sequence, which is different from the base sequence containing the 5'-most base in the deletion portion, which has the same number of bases, or its complementary sequence; a primer set (primer set 2) designed to sandwich the deletion of 200 or more consecutive bases; and a primer set (primer set 3) containing forward and reverse primers that respectively hybridize with DNA consisting of the base sequences of two non-overlapping regions in the base sequence consisting of 200 or more consecutive bases, with primer set 1 being preferred.

[0039] As used herein, hybridization may refer to hybridization under stringent conditions. Also, as used herein, "stringent conditions" refers to conditions under which a complementary strand of a nucleotide strand having homology to a sequence in a target region preferentially hybridizes to the sequence in the target region, and a complementary strand of a non-homologous nucleotide strand does not substantially hybridize. Stringent conditions are sequence-dependent and vary in various circumstances. Longer sequences hybridize specifically at higher temperatures. Examples of stringent conditions include overnight incubation at 42°C in 50% formamide, 5x SSC (150 mM sodium chloride, 15 mM trisodium citrate, 10 mM sodium phosphate, 1 mM ethylenediaminetetraacetic acid, pH 7.2), 5x Denhardt's solution, 0.1% SDS, 10% dextran sulfate, and 100 μg / mL denatured salmon sperm DNA, followed by washing the filter in 0.2x SSC at 42°C (or 65°C for more stringent conditions).

[0040] Examples of the hybridizable DNA in primer set 1 include DNA consisting of a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, or 98% or more sequence identity to a target base sequence or its complementary sequence represented by any one of the consecutive base sequences including bases 380 to 384 and 385 in SEQ ID NO: 13 and the consecutive base sequences including bases 376 to 380 and 381 in SEQ ID NO: 14. The length of the target base sequence may be, for example, 18 to 50 bp, 18 to 35 bp, or 18 to 27 bp.

[0041] Primer set 1 can specifically amplify DNA fragments from DNA lacking 200 or more consecutive bases because the primers contained in primer set 1 can specifically hybridize to the base sequence formed by the deletion of 200 or more consecutive bases in the S-RNase allele (1) or (2). Therefore, if amplification of a DNA fragment is confirmed in a PCR using primer set 1, it can be determined that the plant in the genus Malus possesses the S-RNase allele (1) or (2). If amplification of a DNA fragment is not confirmed, it can be determined that the plant in the genus Malus does not possess the S-RNase allele (1) or (2). The presence or absence of amplification of a DNA fragment in a PCR can be confirmed by a standard method, for example, by subjecting the amplified product to agarose gel electrophoresis.

[0042] When the primers included in primer set 1 are reverse primers, the reverse primers contain complementary sequences to the first and second target base sequences. Here, "complementary sequence" refers to the reverse sequence of the inverted sequence of the target base sequence. For example, the complementary sequence of "5'-ATGC-3'" is "5'-GCAT-3'." In this case, the number of bases in the first target base sequence may be 5 to 14, and the number of bases in the second target base sequence may be 1 to 32. Furthermore, when the primers included in primer set 1 are reverse primers, the forward primer included in primer set 1 can be appropriately designed from a region of the base sequence of the template DNA that is closer to the 5' end than the region to which the reverse primer hybridizes.

[0043] When the primers included in primer set 1 are forward primers, the forward primers include the first and second target base sequences. In this case, the number of bases in the first target base sequence may be 5 to 35, and the number of bases in the second target base sequence may be 1 to 10. When the primers included in primer set 1 are forward primers, the reverse primers included in primer set 1 can be appropriately designed from a region on the 3'-end side of the region in the base sequence of the template DNA to which the forward primers hybridize.

[0044] Primer set 1 may be designed to amplify a DNA fragment of a region including 28 to 100 bp, 28 to 200 bp, 28 to 300 bp, 28 to 400 bp, 28 to 500 bp, 28 to 1000 bp, 28 to 3000 bp, 28 to 5000 bp, 28 to 10,000 bp, 28 to 50,000 bp, 100 to 500 bp, or 100 to 200 bp in a base sequence including the base sequence of the S-RNase allele (1) or (2) or the base sequence of the cDNA of said gene.

[0045] Primer set 2 may be designed in a genomic region containing a cDNA or gene containing the nucleotide sequence of the CDS of the S-RNase allele (1) or (2), or may be designed in a genomic region outside of the above. Primer set 2 may be, for example, a primer set containing a forward primer that hybridizes with DNA consisting of a portion of the complementary sequence of the nucleotide sequence located 5'-terminally closer to the deletion of 200 or more consecutive bases in a cDNA containing the nucleotide sequence of the genomic region containing the S-RNase allele (1) or (2) or the CDS of the above gene, and a reverse primer that hybridizes with DNA consisting of a portion of the nucleotide sequence located 3'-terminally closer to the deletion of 200 or more consecutive bases in a genomic DNA or a cDNA containing the nucleotide sequence of the CDS of the S-RNase allele (1) or (2).

[0046] Primer set 2 can amplify a DNA fragment consisting of the nucleotide sequence of a region containing the deletion of 200 or more consecutive bases in the nucleotide sequence of the S-RNase allele (1) or (2). The length of the DNA fragment is shorter by 200 or more bases than the DNA fragment amplified when the nucleotide sequence of the S-RNase allele (1) or (2) does not contain the deletion of 200 or more consecutive bases. Therefore, by evaluating the length of the DNA fragment amplified in a PCR method using primer set 2, it is possible to determine whether a plant of the genus Malus possesses the S-RNase allele (1) or (2). The length of the DNA fragment in a PCR method can be confirmed by a standard method, for example, by subjecting the amplified product to agarose gel electrophoresis.

[0047] Primer set 2 may be designed to amplify a DNA fragment of a region including 28 to 100 bp, 28 to 200 bp, 28 to 300 bp, 28 to 400 bp, 28 to 500 bp, 28 to 1000 bp, 28 to 3000 bp, 28 to 5000 bp, 28 to 10,000 bp, 28 to 50,000 bp, 100 to 500 bp, or 100 to 200 bp in the base sequence of a genomic region including the S-RNase allele (1) or (2), or in the base sequence of the cDNA of the above gene. Furthermore, primer set 2 may be designed to amplify a DNA fragment of a region including, for example, 233 to 305 bp, 233 to 405 bp, 233 to 505 bp, 233 to 605 bp, 233 to 1205 bp, 233 to 3205 bp, 233 to 5205 bp, 233 to 10205 bp, 233 to 50205 bp, 305 to 705 bp, or 305 to 405 bp in the base sequence of a genomic region including the S39-RNase gene or in the base sequence of the cDNA of the S39-RNase gene.

[0048] Primer set 3 can specifically amplify a DNA fragment consisting of the 200 or more consecutive bases that are deleted in the nucleotide sequence of the S-RNase allele (1) or (2). Therefore, if no amplification of a DNA fragment is confirmed in a PCR method using primer set 3, it can be determined that the plant of the genus Malus possesses the S-RNase allele (1) or (2). Conversely, if amplification of a DNA fragment is confirmed, it can be determined that the plant of the genus Malus does not possess the S-RNase allele (1) or (2). The presence or absence of amplification of a DNA fragment in the PCR method can be confirmed by standard methods, for example, by subjecting the amplified product to agarose gel electrophoresis.

[0049] Primer set 3 may be designed to amplify a DNA fragment of a region including 28 to 100 bp, 28 to 205 bp, 50 to 200 bp, or 100 to 200 bp in the base sequence of the gene or the cDNA sequence of the gene in the case where 200 or more consecutive bases are not deleted in the S-RNase allele of (1) or (2).

[0050] The length of the forward primer and the reverse primer included in the primer set may be, for example, 14 to 50 bp, 18 to 35 bp, or 18 to 27 bp. The lengths of the forward primer and the reverse primer may be the same or different.

[0051] PCR methods such as genomic PCR and RT-PCR using a primer set can be carried out by standard methods. The composition of the reaction solution, temperature and reaction time under temperature cycle conditions during DNA amplification can be appropriately determined by those skilled in the art, taking into account the Tm values ​​of the primers, the specifications of the equipment used, and other factors. When the PCR method is RT-PCR, cDNA can be obtained by reverse transcription of total RNA extracted from the style of a plant of the genus Malus, and the cDNA can then be subjected to a nucleic acid amplification reaction.

[0052] The primer set used in the method for selecting self-compatible Malus plants according to this embodiment is not limited to the embodiments specifically described above, and the primer set described below can also be used.

[0053] [Method for producing self-compatible Malus plants] The method for producing (producing) a self-compatible Apple plant according to this embodiment includes selecting a self-compatible Apple plant from progeny Apple plants obtained by crossbreeding at least one parent plant that has an Apple plant carrying any one of the S-RNase alleles (1) to (6) using the method for selecting a self-compatible Apple plant according to this embodiment described above.

[0054] A plant of the genus Malus carrying an S-RNase allele of any one of (1) to (6) may be produced, for example, by incorporating any one of the genes (1) to (6) into a plant of the genus Malus, or by converting an existing S-RNase allele to any one of the genes (1) to (6). Methods for incorporating any one of the genes (1) to (6) into a plant of the genus Malus and methods for converting an existing S-RNase allele to any one of the genes (1) to (6) can be performed using known methods, including, for example, genome editing techniques such as transcription activator-like nuclease (TALEN), zinc finger nuclease (ZFN), and CRISPR-Cas9.

[0055] A plant of the genus Malus carrying an S-RNase allele that is any one of (1) to (6) may carry the S-RNase allele in a homozygous or heterozygous state. When a plant of the genus Malus carries an S-RNase allele that is any one of (1) to (6) in a heterozygous state, the S-RNase allele on the other chromosome of the homologous chromosome is not particularly limited and may be, for example, the S1-RNase gene, the S9-RNase gene, the S24-RNase gene, or the like. An example of a plant of the genus Malus carrying an S-RNase allele that is any one of (1) to (6) in a heterozygous state is the apple cultivar "Vered." The apple cultivar "Vered" carries the above-mentioned mutated S39-RNase (corresponding to an S-RNase allele that is any one of (1) to (6)) and the S24-RNase gene. Therefore, a plant of the genus Malus carrying an S-RNase allele that is any one of (1) to (6) may be the apple cultivar "Vered" or a plant derived therefrom. Examples of plants derived from "Vered" include hybrid progeny plants produced by crossbreeding using "Vered," plants obtained by doubling haploids produced by anther culture using "Vered," and plants produced by genetic recombination or the like using "Vered," which carry an S-RNase allele that is any one of (1) to (6).

[0056] One of the parent lines does not need to have any of the S-RNase alleles (1) to (6). Such parent lines can be selected appropriately depending on the purpose, and examples include apple varieties such as "Fuji," "Tsugaru," "Orin," "Shinano Sweet," "Shinano Gold," and "Akiakari."

[0057] The progeny of the genus Malus may be a first generation hybrid (F1) obtained by crossing a plant of the genus Malus carrying any one of the S-RNase alleles (1) to (6) as one parent, or may be a second generation hybrid (F2) or later plant obtained by further crossing, such as an F3 or F4. For example, the progeny may be a first generation (BC1) obtained by backcrossing an F1 with a plant of the genus Malus that does not carry any one of the S-RNase alleles (1) to (6), or may be a second generation (BC2) or later plant obtained by further backcrossing.

[0058] [Self-compatible Malus species] A self-compatible Malus plant according to one aspect of this embodiment is a Malus plant produced by the method for producing a self-compatible Malus plant according to this embodiment.

[0059] In another aspect of this embodiment, a self-compatible plant of the genus Malus is a plant of the genus Malus that has an S-RNase allele that is any one of (1) to (6) (excluding the apple cultivar "Vered").

[0060] Self-compatible Malus plants are advantageous in that they reduce the labor required for pollination, stabilize fruit set, do not require insect pollinators for pollination, and do not require pollen from other varieties.

[0061] [Primer set] The primer set according to this embodiment is a primer set for detecting the presence or absence of a deletion of bases corresponding to positions 365 to 569 of SEQ ID NO:1.

[0062] Because plants of the genus Malus carrying an S-RNase allele lacking the bases corresponding to positions 365 to 569 of SEQ ID NO: 1 are self-compatible, the primer set can be used to select self-compatible plants of the genus Malus, particularly those carrying an S-RNase allele whose CDS sequence is the base sequence of SEQ ID NO: 3 or 5. In other words, the primer set can be a primer set for selecting self-compatible plants of the genus Malus.

[0063] SEQ ID NOs: 13 and 14 are the nucleotide sequences of cDNA containing the nucleotide sequence of the CDS of the mutated S39-RNase gene possessed by the apple cultivar "Vered." Specifically, cDNA obtained by reverse transcribing total RNA extracted from the apple cultivar "Vered" was used as a template, and a forward primer (hereinafter also referred to as "S39-F3") consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 9 and a reverse primer (Not Id(T) 18 This is the nucleotide sequence of the amplified product obtained by nested-PCR in which a first PCR is performed using a polynucleotide having the nucleotide sequence of SEQ ID NO: 10 (hereinafter also referred to as "S39-F4"), manufactured by GE Healthcare, and a second PCR is performed using the resulting PCR product as a template and a forward primer (hereinafter also referred to as "S39-F4") consisting of a polynucleotide having the nucleotide sequence of SEQ ID NO: 10 and a reverse primer (Not I-dT) consisting of a polynucleotide having the nucleotide sequence of SEQ ID NO: 12.

[0064] The base sequence of SEQ ID NO: 13 is as follows: TCTTGAACAAATATATTTCCAATGGGATTATGGGGATGATAGATATGGTTACGATGGTATTTTCCTTAATTGTATTAATGTTTCTCTGCTGCGGTGAAATACGATTATTTGCATTTACGCAGCAATATCAGCCGGCTGCCTGCAACTCTAACCCTACTCCTTGTAACGATCCTACTGACAAGTTGTTTACGGTTCACGGATTGTGGCCTTCAAAAGTTAAAGGTCCTGACCCACACGACTGCAAGACCAAAACCAACAAGTTCCAGACGATATACTCAAACCCCAGTTG GAAATTATTTGGCCGAACGTATTCAATCGAGCTGATAATGAAAGCTTCTGGGAAACACAGTGGGAAAAACATGGCACCTGTGGATAACTGAATTGGTGGAGGTCACTCTTTGCAGCAATTACTTCGGAAAACATTTCATAAATTGCCCTAACAAATTCCAGAAAAATCACGATATTCTGTCCCATCAAAGATATCCGGTATTAAGAGCGCGGCTACCCTAGTATGTATGTACGTGTTATATATATAGAAGAATACAGTTATGTGATTGTATTTCTAATGAATAAATTAGCTTAATTAA

[0065] The sequence number 14 of the base sequence is the same as shown in the following sequence. TCTTGAACAAATATATTTCAATGGGGATTATGGGGATGATAGATATGGTTACGATGGTATTTTCCTTAATTGTATTAATGTTTTCTTCGTCTGCGGTGAAATACGATTATTTGCAATTTACGCAGCAATATCAGCCGGCTGCCTGCAACTCTAACCC TACTCCTTGTAACGATCCTACTGACAAGTTGTTTACGGTTCACGGATTGTGGCCTTCAAAAGTTAAAGGTCCTGACCCACACGACTGCAAGACCAAAACCAACAAGTCTCAGACGATATATTACTCAAACCCCAGTTGGAAATTATTTGGCCGAACG TATTCAATCGAGCTGATAATGAAAGCTTCTGGGAAACACAGTGGGAAAAACATGGCACCTGTGGATAACTGAATTGGTGGAGGTCACTCTTTGCAGCAATTACTTCGGAAAACATTTCATAAATTGCCCTAACAAAATTCCAGAAAAATCACGATAT TTCTGTCCCATCAAAGATATCCGGTATTAAGAGCGCGGCTACCTAGTATGTATGTACGTGTTATATATATTAGAAGAATACAGTTATGTGATTGTATTTCTAATGAATAAATTAGCTTAATTAATTAAAATTCATGCGAATATGTGAATTTTCCTTTC

[0066] The primer set may be one used in a PCR method such as genomic PCR or RT-PCR, or a nucleic acid amplification method such as LAMP.

[0067] Primer sets used in PCR include primers containing a third target base sequence comprising bases 380 to 384 of the base sequence 5'-terminal of the 385th base in the base sequence represented by SEQ ID NO: 13, and a fourth target base sequence comprising base 385 of the base sequence 3'-terminal of the 384th base, or a complementary sequence thereof; or a primer set (primer set A) containing primers hybridizing with DNA consisting of the third target base sequence and the fourth target base sequence, or a complementary sequence thereof; a primer set (primer set B) designed to sandwich a deletion of bases corresponding to bases 365 to 569 of SEQ ID NO: 1; and a primer set (primer set C) containing forward and reverse primers that hybridize with DNA consisting of two non-overlapping regions of the base sequence consisting of bases corresponding to bases 365 to 569 of SEQ ID NO: 1, with primer set A being preferred.

[0068] 1A and 1B show the nucleotide sequences of the S39-RNase gene excluding introns ("S39-RNase_cDNA (MG598508)" and "S39b-RNase_cDNA (MH281944)") and two types of cDNAs containing the CDS of the "Vered" S39-RNase allele ("S 39 sm -RNase1', and 'S 39 sm For example, the primer sets are "S39-RNase_cDNA (MG598508)" and "S39b-RNase_cDNA (MH281944)" shown in Figures 1A and 1B, and "S 39 sm -RNase1', and 'S 39 sm It may also be designed based on the base sequence of "-RNase2".

[0069] Primer set A can specifically amplify a DNA fragment from DNA lacking bases corresponding to positions 365 to 569 of SEQ ID NO: 1 because the primers contained in primer set A can specifically hybridize to the base sequence of SEQ ID NO: 13 or 14 formed by the deletion of bases corresponding to positions 365 to 569 of SEQ ID NO: 1 (i.e., a base sequence including consecutive bases corresponding to bases 360 to 364 and 570 of SEQ ID NO: 1). Therefore, in a PCR method using primer set A, if amplification of a DNA fragment is confirmed, it can be determined that bases corresponding to positions 365 to 569 of SEQ ID NO: 1 have been deleted, and if amplification of a DNA fragment is not confirmed, it can be determined that bases corresponding to positions 365 to 569 of SEQ ID NO: 1 have not been deleted. The presence or absence of amplification of a DNA fragment in a PCR method can be confirmed by a standard method, for example, by subjecting the amplified product to agarose gel electrophoresis.

[0070] When the primers contained in primer set A are reverse primers, the reverse primers contain sequences complementary to the third and fourth target base sequences. In this case, it is preferable that the number of bases in the third target base sequence is 5 to 14, and the number of bases in the fourth target base sequence is 1 to 32. When the primers contained in primer set A are reverse primers, the forward primers contained in primer set A can be appropriately designed from a region on the 5'-end side of the region to which the reverse primers hybridize.

[0071] When the primers contained in primer set A are forward primers, the forward primers contain the third and fourth target base sequences. In this case, it is preferable that the number of bases in the third target base sequence is 5 to 35, and the number of bases in the fourth target base sequence is 1 to 10. Furthermore, when the primers contained in primer set A are forward primers, the reverse primers contained in primer set A can be appropriately designed from a region on the 3'-terminal side of the region to which the forward primers hybridize.

[0072] Primer set A may be designed to amplify a DNA fragment of a region including 28 to 100 bp, 28 to 200 bp, 28 to 300 bp, 28 to 400 bp, 28 to 500 bp, 28 to 1000 bp, 28 to 3000 bp, 28 to 5000 bp, 28 to 10,000 bp, 28 to 50,000 bp, 100 to 500 bp, or 100 to 200 bp in a base sequence including the base sequence of SEQ ID NO: 13 or 14.

[0073] It is preferable that primer set A includes a reverse primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity to the base sequence represented by SEQ ID NO: 8, and in this case it is more preferable that the forward primer is a primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity to the base sequence represented by SEQ ID NO: 7.

[0074] The base sequence of SEQ ID NO: 7 is as follows: ATTACTCAAACCCCAGTTGG

[0075] The base sequence of SEQ ID NO: 8 is as follows: CACCAATTCAGTTATCCACAGG

[0076] When a reverse primer consisting of a polynucleotide containing the base sequence represented by SEQ ID NO: 8 hybridizes to the base sequence of SEQ ID NO: 13 or 14, the 13th base of SEQ ID NO: 8 forms a base pair with the 385th base of SEQ ID NO: 13 or the 381st base of SEQ ID NO: 14, and the 14th base of SEQ ID NO: 8 forms a base pair with the 384th base of SEQ ID NO: 13 or the 380th base of SEQ ID NO: 14. In addition, in SEQ ID NO: 13, the position corresponding to the deletion of bases corresponding to positions 365 to 569 of SEQ ID NO: 1 is between the 384th and 385th bases, and in SEQ ID NO: 14, the position corresponding to the deletion of bases corresponding to positions 365 to 569 of SEQ ID NO: 1 is between the 380th and 381st bases.

[0077] The reverse primer consisting of a polynucleotide containing a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence represented by SEQ ID NO:8 preferably contains the nucleotide sequence TCAGTTATCCAC. TCAGTT hybridizes to the nucleotide sequence in SEQ ID NO:13 or SEQ ID NO:14, which is closer to the 5' end than the position corresponding to the deleted part of nucleotides corresponding to positions 365 to 569 in SEQ ID NO:1, and ATCCAC hybridizes to the nucleotide sequence in SEQ ID NO:13 or SEQ ID NO:14, which is closer to the 3' end than the position corresponding to the deleted part of nucleotides corresponding to positions 365 to 569 in SEQ ID NO:1.

[0078] In the above reverse primer and forward primer, the sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0079] When the bases corresponding to positions 365 to 569 of SEQ ID NO: 1 are deleted in the template DNA, primer set B can amplify a DNA fragment consisting of the base sequence of the region containing the deletion. Primer set B may be set on SEQ ID NO: 13 or 14, or may be set in a genomic region outside of these. The DNA fragment amplified with this primer set is 205 bases shorter than the DNA fragment amplified when DNA containing the base sequence of SEQ ID NO: 1 is used as a template. Therefore, by evaluating the length of the DNA fragment amplified in PCR using primer set B, it is possible to determine whether the bases corresponding to positions 365 to 569 of SEQ ID NO: 1 are deleted. The length of the DNA fragment in PCR can be confirmed by a conventional method, for example, by subjecting the amplified product to agarose gel electrophoresis.

[0080] Examples of primer set B include a primer set (primer set B-1) containing a forward primer that hybridizes with DNA consisting of a portion of the complementary sequence of the 1st to 384th bases in the base sequence represented by SEQ ID NO: 13, and a reverse primer that hybridizes with DNA consisting of a portion of the complementary sequence of the 385th to 599th bases in the base sequence represented by SEQ ID NO: 13; and a primer set (primer set B-2) containing a forward primer that hybridizes with DNA consisting of a portion of the complementary sequence of the 1st to 380th bases in the base sequence represented by SEQ ID NO: 14, and a reverse primer that hybridizes with DNA consisting of a portion of the complementary sequence of the 381st to 629th bases in the base sequence represented by SEQ ID NO: 14.

[0081] Primer set B-1 can amplify a DNA fragment consisting of the nucleotide sequence of a region of SEQ ID NO: 13 that contains a deletion of nucleotides corresponding to positions 365 to 569 of SEQ ID NO: 1. The length of the DNA fragment is 205 nucleotides shorter than the DNA fragment amplified when DNA containing the nucleotide sequence of SEQ ID NO: 1 is used as a template. Therefore, by evaluating the length of the DNA fragment amplified in a PCR method using primer set B-1, it is possible to determine whether or not the nucleotides corresponding to positions 365 to 569 of SEQ ID NO: 1 are deleted. The length of the DNA fragment in the PCR method can be confirmed by a conventional method, for example, by subjecting the amplified product to agarose gel electrophoresis.

[0082] Primer set B-2 can amplify a DNA fragment consisting of the nucleotide sequence of SEQ ID NO: 14, which contains a deletion of nucleotides corresponding to positions 365 to 569 of SEQ ID NO: 1. The length of the DNA fragment is 205 to 209 nucleotides shorter than the DNA fragment amplified when DNA containing the nucleotide sequence of SEQ ID NO: 1 is used as a template. Therefore, by evaluating the length of the DNA fragment amplified in PCR using primer set B-2, it is possible to determine whether or not the nucleotides corresponding to positions 365 to 569 of SEQ ID NO: 1 are deleted. The length of the DNA fragment in PCR can be confirmed by a standard method, for example, by subjecting the amplified product to agarose gel electrophoresis.

[0083] Primer sets B-1 and B-2 may be designed to amplify DNA fragments in regions including 28 to 100 bp, 28 to 200 bp, 28 to 300 bp, 28 to 400 bp, 28 to 500 bp, 28 to 1000 bp, 28 to 3000 bp, 28 to 5000 bp, 28 to 10,000 bp, 28 to 50,000 bp, 100 to 500 bp, or 100 to 200 bp in the base sequence of SEQ ID NO: 13 or 14, respectively. Furthermore, primer sets B-1 and B-2 may be designed to amplify a DNA fragment of a region including, for example, 233 to 305 bp, 233 to 405 bp, 233 to 505 bp, 233 to 605 bp, 233 to 1205 bp, 233 to 3205 bp, 233 to 5205 bp, 233 to 10205 bp, 233 to 50205 bp, 305 to 705 bp, or 305 to 405 bp in the base sequence of a genomic region including the S39-RNase gene or in the base sequence of the cDNA of the S39-RNase gene.

[0084] Primer set C can specifically amplify a DNA fragment consisting of bases corresponding to positions 365 to 569 of SEQ ID NO: 1. Therefore, if amplification of a DNA fragment is not confirmed in a PCR method using primer set C, it can be determined that the bases corresponding to positions 365 to 569 of SEQ ID NO: 1 have been deleted, and if amplification of a DNA fragment is confirmed, it can be determined that the bases corresponding to positions 365 to 569 of SEQ ID NO: 1 have not been deleted. Whether or not a DNA fragment has been amplified in the PCR method can be confirmed by a standard method, and for example, this can be done by subjecting the amplified product to agarose gel electrophoresis.

[0085] Primer set C may be designed to amplify a DNA fragment of a region containing 28 to 100 bp, 28 to 205 bp, 50 to 200 bp, or 100 to 200 bp in the base sequence consisting of bases corresponding to positions 365 to 569 of SEQ ID NO:1.

[0086] The length of the forward primer and reverse primer included in the primer set according to this embodiment (e.g., primer sets A to C) may be, for example, 14 to 50 bp, 18 to 35 bp, or 18 to 27 bp. The lengths of the forward primer and reverse primer may be the same or different.

[0087] Genomic PCR using the primer set according to this embodiment (for example, primer sets A to C) may be performed by amplifying a DNA fragment using, for example, genomic DNA extracted from a plant of the genus Malus as a template.

[0088] Genomic DNA may be extracted from plants of the genus Malus, for example, from the style, embryo, pollen, ovule, gamete, seed, leaf, flower, branch, fruit, stem, root, anther, etc., of the plant, and is preferably extracted from the leaf. Genomic DNA extraction may be performed by a conventional method.

[0089] In the genomic PCR method, the composition of the reaction solution, the temperature and reaction time of the temperature cycle conditions, etc. when carrying out the DNA amplification reaction may be appropriately determined by a person skilled in the art taking into consideration the Tm values ​​of the primers, the specifications of the equipment used, etc.

[0090] The reaction solution composition may contain, for example, in addition to the primer set according to this embodiment and the genomic DNA extracted by the above-described method, DNA polymerase, deoxynucleoside triphosphates (dNTPs: dATP, dTTP, dCTP, and dGTP), a buffer solution, salts such as magnesium chloride, etc. Alternatively, the reaction solution can be prepared using the primer set according to this embodiment, genomic DNA extracted from a plant of the genus Malus, and a commercially available kit such as GoTaq G2 Hot Start Colorless Master Mix (Promega) according to the manufacturer's instructions.

[0091] For example, when using GoTaq G2 Hot Start Colorless Master Mix (Promega), the temperature cycling conditions may be as follows: an initial denaturation step at 90°C to 98°C for 1 to 3 minutes, followed by a denaturation step at 90°C to 98°C for 15 seconds to 1 minute, an annealing step at 55°C to 65°C for 15 seconds to 1 minute, and an extension step at 65°C to 80°C for 30 seconds to 2 minutes, with each cycle consisting of these steps being repeated 20 to 50 times, followed by a final extension step at 65°C to 75°C for 3 to 8 minutes. [Example]

[0092] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0093] The inventors investigated self-fruitful plants of the genus Malus and selected the apple cultivar "Vered." The following tests were carried out on the apple cultivar "Vered."

[0094] [Self-pollination test of 'Vered'] We investigated whether the self-fruitfulness of the apple cultivar "Vered" is due to self-compatibility, i.e., whether the apple cultivar "Vered" exhibits self-compatibility. Self-pollination tests were conducted annually for five years from 2018 to 2022 using the following procedure. Anthers were collected from "Vered" flowers 1–2 days before flowering and allowed to open at 20°C to prepare pollen. One day before flowering, petals were removed from "Vered" flowers, and self-pollination was performed using the prepared pollen. Afterward, the plants were covered with a bag to block out other pollen. Fruit set and seed number per fruit were investigated 84–91 days after self-pollination. Fruit set was calculated as the percentage of the number of flowers that set fruit (number of fruits) compared to the number of self-pollinated flowers (number of hybrid flowers). According to the criteria of Komori Sadao et al., "Determining Cross-Incompatibility in Apples by Seed Number and Fruit Set Rate," (1999): 97-112, "Vered" was determined to be self-compatible if the fruit set rate was 30% or higher and the number of seeds per fruit was greater than three. Table 1 shows the number of hybrid flowers, number of fruits, fruit set rate (%), number of seeds per fruit, and whether or not the fruit was self-compatible for each year. In Table 1, the number of seeds per fruit is shown as the mean ± standard deviation.

[0095] [Table 1]

[0096] As shown in Table 1, "Vered" was determined to be self-compatible every year for the five years from 2018 to 2022. Therefore, "Vered" was shown to be self-compatible.

[0097] [Determination of the nucleotide sequence of the S39-RNase gene of "Vered"] The S genotype (S-RNase genotype) of the apple cultivar "Vered" was unknown. Therefore, we analyzed the S genotype (S-RNase genotype) of "Vered." Specifically, two S-RNases from "Vered" were amplified by genomic PCR using an S-RNase gene-specific primer set, including a forward primer (hereinafter referred to as "FTQQYQ") consisting of the polynucleotide sequence of SEQ ID NO: 17 and a reverse primer (hereinafter referred to as "anti-(I / M)IWPNV") consisting of the polynucleotide sequence of SEQ ID NO: 18. The PCR products were then electrophoresed, extracted from the gel, cloned, and sequenced. A BLAST search of these sequences revealed 100% identity with S24-RNase and 95.06-99.87% identity with S39-RNase of the genus Malus. Therefore, it was shown that "Vered" carries the S24-RNase gene and the mutated S39-RNase allele in a heterozygous state.

[0098] Next, the base sequence of the S39-RNase allele was determined. Total RNA was extracted from the style of "Vered" by the usual method, and cDNA was synthesized. 39 -RNase (International Nucleotide Sequence Database accession number: MG598508) and S 24 Based on the differences in the nucleotide sequence between this S-RNase and the S-RNase (International Nucleotide Sequence Database accession number: MG598502) (De Franceschi, Paolo, et al. "Characterization of 25 full-length S-RNase alleles, including flanking regions, from a pool of resequenced apple cultivars." Plant molecular biology 97 (2018): 279-296.), 39Two RNase-specific primers were designed: a forward primer (hereinafter also referred to as "S39-F3") consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 9, and a forward primer (hereinafter also referred to as "S39-F4") consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 10. A reverse primer (Not Id(T)) consisting of S39-F3 and a polynucleotide with the nucleotide sequence of SEQ ID NO: 11 was also designed. 18 PCR was performed using a PCR primer set (GE Healthcare), and nested PCR was then performed using the amplified PCR product as a template and a reverse primer (Not I-dT) consisting of S39-F4 and a polynucleotide having the nucleotide sequence of SEQ ID NO: 12. The nested PCR product was electrophoresed on a 1.5% agarose gel, extracted from the gel, cloned into a vector, and then sequenced.

[0099] The nucleotide sequences determined as described above are shown in Figures 1A and 1B. In Figures 1A and 1B, "S39-RNase_cDNA (MG598508)" and "S39b-RNase_cDNA (MH281944)" are the nucleotide sequences obtained by excluding introns from the genomic sequences of the S39-RNase gene (De Franceschi, Paolo, et al. "Characterization of 25 full-length S-RNase alleles, including flanking regions, from a pool of resequenced apple cultivars." Plant molecular biology 97 (2018): 279-296.) and S39b-RNase (Sheick, Ryan, et al. "Characterization of a novel S-RNase allele and genotyping of new apple cultivars." Scientia Horticulturae 273 (2020): 109630.), respectively. In addition, in Figure 1B, the black downward triangles indicate the positions of introns in "S39-RNase_cDNA (MG598508)" and "S39b-RNase_cDNA (MH281944)". Two types of cDNAs (hereinafter, the two types of cDNAs are referred to as "S") were isolated from the total RNA of "Vered". 39 sm -RNase1', and 'S 39 sm The two cDNAs were synthesized by alternative splicing. 39 sm -RNase2, S 39 sm This is because the base sequence corresponding to positions 253 to 256 in the base sequence from the start codon to the stop codon of RNase 1 cDNA (SEQ ID NO: 3) was truncated (the diagonal hatching in Figure 1B indicates the truncated portion). As shown in Figure 1B, 39 sm -RNase 1 and S 39 sm-RNase2 was missing the base sequence corresponding to positions 365 to 569 of the base sequence (sequence number 1) from the start codon to the stop codon of S39-RNase_cDNA and S39b-RNase_cDNA (in Figure 1B, dotted hatching indicates the deleted site).

[0100] Next, S 39 sm -RNase 1 and S 39 sm Figure 2 shows the alignment of the deduced amino acid sequences obtained by translating S-RNase2 (SEQ ID NOs: 4 and 6, respectively) with the amino acid sequence of "S39-RNase (MG598508)" (SEQ ID NO: 2, De Franceschi, Paolo, et al. "Characterization of 25 full-length S-RNase alleles, including flanking regions, from a pool of resequenced apple cultivars." Plant Molecular Biology 97 (2018): 279-296.). In Figure 2, "Signal peptide" indicates the signal peptide region of S39-RNase, "C1" to "C5" indicate conserved regions of S-RNase, and "RHV" indicates the hypervariable region of S-RNase. The conserved regions C1 to C5 are important for the formation of the three-dimensional structure of S39-RNase. In Figure 2, asterisks indicate cysteine ​​residues important for stabilizing the three-dimensional structure of S39-RNase, and "#" indicates a histidine residue essential for RNase activity. The black downward triangle indicates the position of the intron in "S39-RNase (MG598508)".

[0101] As shown in Figure 2, S 39 sm -RNase 1 and S 39 smIn the deduced amino acid sequence obtained by translating S-RNase2, the amino acid sequence from position 122 onwards in SEQ ID NO: 2 was deleted. The amino acid sequence from position 122 onwards in SEQ ID NO: 2 contains the conserved regions C4 and C5 and four cysteine ​​residues that are important for stabilizing the three-dimensional structure. 39 sm -RNase 1 and S 39 sm The protein consisting of the predicted amino acid sequence obtained by translating S39-RNase2 cannot form the normal three-dimensional structure of S39-RNase, and therefore lacks RNase activity. 39 sm -RNase2, S 39 sm The base sequence corresponding to positions 253 to 256 in the base sequence of the CDS of RNase 1 (SEQ ID NO: 3) was truncated by alternative splicing, and therefore, the amino acid sequence from position 86 onwards in SEQ ID NO: 6 caused a frameshift mutation relative to the amino acid sequence of SEQ ID NO: 2. 39 sm -RNase2 had mutations in the hypervariable region and the conserved region C3.

[0102] [Primer set design] S 39 sm -RNase 1 and S 39 sm A primer set was designed to detect the presence or absence of a deletion of bases corresponding to positions 365 to 569 of SEQ ID NO: 1 in S-RNase2. 39 sm -RNase 1 and S 39 sm A primer set was designed that included a forward primer (SEQ ID NO: 7) consisting of a polynucleotide with the base sequence indicated by the right-pointing arrow in Figure 1B for the base sequence of RNase 2, and a reverse primer (SEQ ID NO: 8) consisting of a polynucleotide with the complementary sequence to the base sequence indicated by the left-pointing arrow.

[0103] [Selection of self-compatible individuals from the cross between "Vered" and "Fuji"] DNA was extracted from leaves of the first generation hybrid obtained by crossing "Vered" with "Fuji" (genotype S1S9). Using this DNA as a template, multiplex genomic PCR was performed using the primer set described above in "Primer Set Design," a co-specific primer set containing Normal-F (forward primer) and Normal-R (reverse primer) as described in Okada, Kazuma, et al., "Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (Malus × domestica)," Journal of Plant Research 129 (2016): 1109-1126, as a positive control, and GoTaq G2 Hot Start Colorless Master Mix (Promega). The nucleotide sequences of Normal-F and Normal-R are set forth in SEQ ID NOs: 15 and 16, respectively. The multiplex genomic PCR was performed using a program consisting of one cycle of 94°C for 2 minutes, followed by 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute, followed by one cycle of 72°C for 5 minutes. The PCR products were electrophoresed on a 2% agarose gel and then stained with gel green. The results are shown in Figure 3. In Figure 3, "M" indicates the marker, "2" indicates "Fuji," "1" indicates "Vered," and "FV1" to "FV30" indicate the first generation hybrids. The genotypes of "FV1" to "FV30" in Figure 3 were determined by electrophoresis after amplification with an S-RNase gene-specific primer set including the forward primer FTQQYQ and the reverse primer anti-(I / M)IWPNV. The "S" in the above genotypes was determined by electrophoresis after amplification with an S-RNase gene specific primer set including the forward primer FTQQYQ and the reverse primer anti-(I / M)IWPNV. 39 sm " means the allele of the S39-RNase gene possessed by "Vered".

[0104] As shown in Figure 3, S39 sm In the "Vered" strain and the first generation hybrid, PCR products were confirmed using the primer set designed in the above [Primer set design]. Therefore, PCR using the above primer set can be used to detect S, which lacks the nucleotide sequence corresponding to positions 365 to 569 of SEQ ID NO: 1. 39 It was shown that the RNase gene can be specifically amplified.

Claims

1. A method for selecting a plant of the genus Malus that exhibits self-compatibility, comprising selecting a plant of the genus Malus that possesses any one of the following S-RNase alleles (1), (2), (3), (4), (5), or (6): (1) A gene in which 200 or more consecutive bases are deleted in a base sequence in which the CDS sequence has 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1; (2) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, in which 200 or more consecutive bases in the CDS sequence are deleted; (3) a gene whose CDS sequence has 90% or more sequence identity with the base sequence represented by SEQ ID NO: 3; (4) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4; (5) A gene whose CDS sequence has 90% or more sequence identity with the base sequence represented by SEQ ID NO: 5, or (6) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

6.

2. The method according to claim 1, wherein in the gene (1) or (2), the deleted 200 or more consecutive bases are present in a base sequence corresponding to bases 253 to 690 of the base sequence represented by SEQ ID NO:

1.

3. The method according to claim 1, further comprising determining whether or not a plant of the genus Malus possesses the gene (1) or (2) by a PCR method using a primer set for detecting the presence or absence of the deletion in the gene (1) or (2).

4. The primer set is a nucleotide sequence of a genomic DNA or a nucleotide sequence of a cDNA containing the nucleotide sequence of the CDS of the gene (1) or (2), a first target base sequence that includes the base closest to the 3' end among the base sequences located 5'-terminally from the deletion portion of 200 or more consecutive bases, and that is different from a base sequence including the base closest to the 3' end in the deletion portion that has the same number of bases; and a primer containing a second target base sequence that is different from the base sequence containing the 5'-most base in the deletion portion, which has the same number of bases as the base sequence containing the 5'-most base in the deletion portion, or a complementary sequence thereof; or The method according to claim 3, further comprising a primer that hybridizes with DNA consisting of the first target base sequence and the second target base sequence, or a complementary sequence thereof.

5. the primer is a reverse primer containing complementary sequences of the first and second target base sequences, the first target base sequence having 5 to 14 bases, and the second target base sequence having 1 to 32 bases; or The method according to claim 4, wherein the primer is a forward primer comprising the first and second target base sequences, the number of bases in the first target base sequence is 5 to 35, and the number of bases in the second target base sequence is 1 to 10.

6. A method for producing a self-compatible plant of the genus Malus, comprising selecting a self-compatible plant of the genus Malus from progeny plants of Malus obtained by crossbreeding at least one parent plant with a plant of the genus Malus carrying the S-RNase allele by the method according to any one of claims 1 to 5.

7. A self-compatible plant of the genus Malus produced by the method of claim 6.

8. (1) A gene in which 200 or more consecutive bases are deleted in a base sequence in which the CDS sequence has 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1; (2) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2, in which 200 or more consecutive bases in the CDS sequence are deleted; (3) a gene whose CDS sequence has 90% or more sequence identity with the base sequence represented by SEQ ID NO: 3; (4) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4; (5) A gene whose CDS sequence has 90% or more sequence identity with the base sequence represented by SEQ ID NO: 5, or (6) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

6. A self-compatible plant of the genus Malus (excluding the apple cultivar "Vered") that possesses any one of the S-RNase alleles.

9. A primer set for detecting the presence or absence of a deletion of bases corresponding to positions 365 to 569 of SEQ ID NO:

1.

10. In the base sequence represented by SEQ ID NO: 13, a third target base sequence including bases 380 to 384 of the base sequence on the 5'-terminal side of the 385th base; and a primer containing a complementary sequence of a fourth target base sequence including the 385th base in the base sequence 3'-terminal side of the 384th base, or The primer set according to claim 9 , comprising primers that hybridize with DNA consisting of the third target base sequence and the fourth target base sequence, or sequences complementary thereto.

11. the primer is a reverse primer containing complementary sequences of the third and fourth target base sequences, the third target base sequence having 5 to 14 bases, and the fourth target base sequence having 1 to 32 bases; or The primer set according to claim 10, wherein the primer is a forward primer comprising the third and fourth target base sequences, the number of bases in the third target base sequence is 5 to 35, and the number of bases in the fourth target base sequence is 1 to 10.

12. The primer set according to claim 9, comprising a reverse primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity to the base sequence represented by SEQ ID NO:8.

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