NZ762905B2

NZ762905B2 - Determination method for onion

Info

Publication number: NZ762905B2
Application number: NZ762905A
Authority: NZ
Inventors: Masahiro Kato; Noriya Masamura; Masae UEYAMA
Original assignee: House Foods Group Inc
Priority date: 2017-09-25
Filing date: 2018-09-21
Publication date: 2021-11-30

Abstract

The present invention relates to a method of discriminating traits of an onion comprising: a first determination step of determining presence of a first mutation site comprising one or more nucleotides selected from nucleotides at positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 in a nucleic acid derived from the onion, wherein the onion is discriminated to be an onion with no pungent taste and/or tear-inducing property if the presence of the first mutation site is not determined in the first determination step. The method can further comprise a second determination step as an internal control by determining presence of the nucleotide sequence of SEQ ID NO:2 in the nucleic acid derived from the onion, or determining presence of a second mutation site comprising one or more nucleotides selected from nucleotides at positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 in the nucleic acid. ide sequence of SEQ ID NO:1 in a nucleic acid derived from the onion, wherein the onion is discriminated to be an onion with no pungent taste and/or tear-inducing property if the presence of the first mutation site is not determined in the first determination step. The method can further comprise a second determination step as an internal control by determining presence of the nucleotide sequence of SEQ ID NO:2 in the nucleic acid derived from the onion, or determining presence of a second mutation site comprising one or more nucleotides selected from nucleotides at positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 in the nucleic acid.

Description

(12) Granted patent speciﬁcaon (19) NZ (11) 762905 (13) B2 (47) Publicaon date: 2021.12.24 (54) DETERMINATION METHOD FOR ONION (51) Internaonal Patent Classiﬁcaon(s): C12Q 1/6895 C12N 15/09 C12N 9/88 C12N 15/60 (22) Filing date: (73) Owner(s): 2018.09.21 HOUSE FOODS GROUP INC. (23) Complete caon ﬁling date: (74) Contact: 2018.09.21 Spruson & Ferguson Pty Ltd (30) Internaonal Priority Data: (72) Inventor(s): JP 2017-184019 2017.09.25 KATO Masahiro MASAMURA Noriya (86) Internaonal Applicaon No.: UEYAMA Masae (87) aonal aon number: WO/2019/059350 (57) Abstract: The present invenon relates to a method of discriminang traits of an onion comprising: a ﬁrst determinaon step of determining presence of a ﬁrst mutaon site comprising one or more nucleodes selected from nucleodes at posions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleode sequence of SEQ ID NO:1 in a nucleic acid derived from the onion, wherein the onion is discriminated to be an onion with no pungent taste and/or tear-inducing property if the presence of the ﬁrst mutaon site is not determined in the ﬁrst determinaon step. The method can further comprise a second determinaon step as an internal control by determining presence of the de sequence of SEQ ID NO:2 in the nucleic acid derived from the onion, or determining presence of a second n site comprising one or more nucleodes selected from des at ns 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleode sequence of SEQ ID NO:2 in the nucleic acid. 762905 B2 Description Title of Invention: INATION METHOD FOR ONION Technical Field The present invention relates to a method of discriminating whether an onion is an onion with no pungent taste and/or tear-inducing property, a primer, a primer set, and a kit used for the method, and a method of ng an onion using the method.

Background Art Patent ture 1 discloses that 29 types of alliinases, which are enzymes involved in production of lachrymatory factor (LF), a component causing a pungent taste, are expressed in an onion; that, among them, a specific alliinase consisting of an amino acid sequence as set forth in SEQ ID NO:5 in Patent Literature 1 is the primary ase associated with a pungent taste and a nducing property of an onion; and that two lines of tearless and non-pungent onions were produced in which the activity of the specific alliinase was suppressed.

Non Patent Literature 1 also discloses that tearless and non-pungent onions were produced in which the activity of the alliinase was suppressed, as in Patent Literature 1.

Non Patent Literature 2 discloses an amino acid sequence of an alliinase that has a high sequence identity to the amino acid ce of the specific alliinase disclosed in Patent Literature 1. However, Non Patent Literature 2 does not show the magnitude of contribution of this alliinase to a pungent taste or a tear-inducing property or does not suggest the presence of other alliinases.

Non Patent Literatures 3 to 6 suggest the presence of onion alliinases other than the specific alliinase disclosed in Non Patent Literature 2. However, these literatures disclose neither sequence information of these other alliinases nor explanation of whether they are associated with a pungent taste and a tear-inducing property.

Citation List Patent ture Patent Literature 1: JP Patent No. 5671657 Non Patent Literatures Non Patent Literature 1: Kato et al. "Production and characterization of tearless and nonpungent onion" DOI: 10.1038/srep23779 Non Patent Literature 2: GenBank Accession No. AAA32639.1 Non Patent Literature 3: King et al. "A nsity genetic map of onion reveals a role for tandem duplication in the evolution of an extremely large d genome." Theor Appl Genet 96, 52-62 (1998) Non Patent Literature 4: Van Heusden, A. W., Shigyo, M., Tashiro, Y., Vrielink-van Ginkel, R. & Kik, C. AFLP linkage group assignment to the somes of Allium cepa L. via monosomic addition lines. Theor Appl Genet 100, 480-486 (2000) Non Patent Literature 5: Martin, W. J. et al. c mapping of expressed sequences in onion and in silico comparisons with rice show scant colinearity. Mol Genet Genom 274, 197-204 (2005) Non Patent ture 6: Khrustaleva, L. et al. The some organization of genes and some types of extragenic DNA in Allium. Acta Hort 969, 43-52 (2012) Summary of Invention [0007a] In a first aspect, the present invention provides a method of discriminating traits of an onion, comprising: a first determination step comprising determining the presence of a first mutation site in a c acid derived from the onion, said mutation site comprising one or more nucleotides selected from nucleotides at positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the tide sequence of SEQ ID NO:1, wherein the onion is discriminated to be an onion with no pungent taste and/or tear-inducing property if the presence of said first mutation site is not determined in the first determination step. [0007b] In a second aspect, the present invention provides a method of breeding an onion with no pungent taste and/or tear-inducing property, comprising: 1) discriminating whether an onion is an onion with no pungent taste and/or tearinducing property by a method according to the first aspect; 2) using the onion discriminated to be an onion with no pungent taste and/or tearinducing property to breed an onion; and 3) discriminating whether the onion bred in 2) above is an onion with no pungent taste and/or tear-inducing property by a method according to the first aspect. [0007c] In a third aspect, the present invention provides a primer set when used in a method of the invention, comprising: a primer comprising a first polynucleotide ting of a first nucleotide ce or consisting of the first nucleotide sequence at the 3' end and a further tide sequence having or fewer nucleotides linked to the first nucleotide sequence at the 5' end thereof, n the first nucleotide sequence is i) identical to, or ii) identical in a region of 3 nucleotides at the 3' end and homologous in the remaining region on the 5’ end, to a first partial nucleotide sequence of 17 to 50 consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:1, the first partial nucleotide ce comprising a first nucleotide selected from positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 within 2 nucleotides from the 3' end, n the gous nucleotide sequence of the remaining region in ii) above is obtained by deleting, substituting, adding, and/or inserting one or two nucleotides in the first partial nucleotide sequence; and a primer sing a second polynucleotide consisting of a second tide sequence or consisting of the second nucleotide sequence at the 3' end and a further nucleotide sequence having 20 or fewer nucleotides linked to the second nucleotide sequence at the 5' end thereof, wherein the second nucleotide sequence is iii) identical to, or iv) identical in a region of 3 nucleotides at the 3' end and homologous in the ing region on the 5' end, to a second partial tide ce of 17 to 50 consecutive nucleotides comprised in a complementary nucleotide sequence to the tide sequence of SEQ ID NO:1, the second partial nucleotide sequence sing a nucleotide complementary to a second nucleotide selected from positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 within 2 nucleotides from the 3' end, wherein the homologous nucleotide sequence of the remaining region in iv) above is obtained by deleting, substituting, adding, and/or inserting one or two nucleotides in the second partial nucleotide sequence, and wherein the first nucleotide is positioned closer to the 5' end than the second nucleotide in the nucleotide sequence of SEQ ID NO:1. [0007d] In a fourth aspect, the present invention provides primer set when used in a method of the invention, comprising: a primer comprising a first polynucleotide consisting of a first nucleotide sequence or consisting of the a first nucleotide ce at the 3' end and a further nucleotide sequence having 20 or fewer nucleotides linked to the first tide sequence at the 5' end thereof, wherein the first nucleotide sequence is v) identical to, or vi) identical in a region of 3 nucleotides at the 3' end and homologous in the remaining region on the 5' end, to a first partial nucleotide sequence of 17 to 50 utive nucleotides comprised in the nucleotide sequence of SEQ ID NO:2, the first partial nucleotide sequence sing a first nucleotide selected from positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 within 2 nucleotides from the 3' end, wherein the homologous nucleotide sequence of the remaining region in vi) above is obtained by deleting, substituting, adding, and/or inserting one or two tides in the first partial nucleotide sequence; a primer comprising a second polynucleotide consisting of a second nucleotide sequence or consisting of the second nucleotide sequence at the 3' end and a r nucleotide ce having 20 or fewer nucleotides linked to the second nucleotide sequence at the 5' end thereof, wherein the second nucleotide sequence is vii) identical to, or viii) identical in a region of 3 nucleotides at the 3' end and homologous in the remaining region on the 5' end, to a second partial nucleotide sequence of 17 to 50 utive nucleotides comprised in a complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:2, the second partial nucleotide ce comprising a nucleotide complementary to a second nucleotide selected from positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the tide sequence of SEQ ID NO:2 within 2 nucleotides from the 3' end, wherein the homologous nucleotide sequence of the remaining region in viii) above is obtained by deleting, substituting, adding, and/or inserting one or two nucleotides in the second partial nucleotide sequence; and wherein the first tide is positioned closer to the 5' end than the second nucleotide in the tide sequence of SEQ ID NO:2. [0007e] In a fifth aspect, the present invention provides a kit for discriminating traits of an onion when used in a method of the invention, comprising: the primer set according to the third aspect; and the primer set according to the fourth .

Technical Problem 2d followed by page 3 The onion with suppressed expression of the ic alliinase ting of the amino acid ce as set forth in SEQ ID NO:5 in Patent Literature 1, which is disclosed in Patent ture 1, does not have a pungent taste or a tear-inducing property. However, Patent Literature 1 does not disclose the gene sequence of the ic alliinase or cause of not being expressed (e.g., destruction of regulatory region, transcription factor, or the gene itself).

Additionally, Patent Literature 1 does not disclose information on the alliinase gene that was not expressed. Non Patent Literature 1 also discloses that tearless and non-pungent onions were produced in which the activity of the alliinase was suppressed, as in Patent Literature 1, but does not se the complete sequence of the alliinase with suppressed expression or cause of the suppressed sion.

The onion alliinase gene is a multicopy gene. As sed in Patent Literature 1, there are 29 alliinase genes that are expressed, and even more alliinase genes are present in an onion if genes that are not expressed are included. Additionally, Non Patent Literatures 2 to 6 also indicate the presence of alliinase genes other than the specific alliinase genes disclosed in Patent Literature 1. Thus, other alliinase genes might be detected in error, and it was therefore difficult to discriminate whether the onion is an onion with no pungent taste by determining the presence or absence of the gene encoding the specific alliinase sed in Patent Literature 1.

The present invention provides a means for discriminating whether an onion is an onion with no pungent taste and/or tear-inducing property by specifically determining the presence of an alliinase gene associated with a pungent taste and a tear-inducing property in the onion in distinction from other genes.

Solution to Problem The ication discloses the following inventions as solutions to the above-described problem: (1) A method of discriminating traits of an onion, comprising: a first determination step of determining presence of the nucleotide sequence of SEQ ID NO:1 in a nucleic acid derived from the onion; and a second determination step of determining presence of the nucleotide sequence of SEQ ID NO:2 in the c acid derived from the onion, wherein the onion is discriminated to be an onion with no pungent taste and/or nducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is not determined in the first determination step and the presence of the nucleotide sequence of SEQ ID NO:2 is determined in the second determination step, and the onion is discriminated to be an onion with a pungent taste and/or a tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is determined in the first determination step, and the presence of the nucleotide sequence of SEQ ID NO:2 is determined in the second determination step. (2) The method according to (1), wherein the first determination step comprises ining presence of a first mutation site comprising one or more nucleotides selected from nucleotides at positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide ce of SEQ ID NO:1 in the nucleic acid, and the second determination step comprises determining presence of a second mutation site comprising one or more nucleotides selected from nucleotides at positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 in the nucleic acid. (3) The method according to (2), wherein the first ination step comprises: performing a first nucleic acid ication reaction on a c DNA or a cDNA of the onion as a template using a first primer set comprising: a first primer comprising a polynucleotide comprising a nucleotide sequence which is identical or homologous to a partial nucleotide sequence sing the first mutation site in the nucleotide sequence of SEQ ID NO:1; and/or a second primer comprising a polynucleotide comprising a nucleotide sequence which is cal or homologous to a complementary tide sequence to the partial nucleotide sequence sing the first mutation site in the nucleotide sequence of SEQ ID NO:1; and confirming an amplification product of the first nucleic acid amplification reaction, the second determination step comprises: performing a second nucleic acid amplification reaction on a c DNA of the onion as a template using a second primer set comprising: a third primer comprising a polynucleotide comprising a tide sequence which is identical or homologous to a partial nucleotide ce comprising the second mutation site in the nucleotide sequence of SEQ ID NO:2; and/or a fourth primer comprising a polynucleotide comprising a nucleotide sequence which is identical or homologous to a complementary nucleotide sequence to the partial nucleotide sequence sing the second on site in the nucleotide sequence of SEQ ID NO:2; and confirming an amplification product of the second nucleic acid amplification reaction. (4) A method of discriminating traits of an onion, comprising a first determination step of determining presence of the nucleotide sequence of SEQ ID NO:1 in a nucleic acid derived from the onion, the first determination step comprising determining presence of a first mutation site comprising one or more nucleotides selected from nucleotides at ons 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 in the nucleic acid, wherein the onion is discriminated to be an onion with no pungent taste and/or tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is not determined in the first determination step, and the onion is discriminated to be an onion with a pungent taste and/or a tear-inducing property if the ce of the nucleotide ce of SEQ ID NO:1 is determined in the first determination step. (5) The method according to (4), wherein the first determination step comprises: performing a first nucleic acid amplification reaction on a genomic DNA or a cDNA of the onion as template using a first primer set comprising: a first primer comprising a polynucleotide comprising a nucleotide ce which is identical or homologous to a partial nucleotide sequence comprising a first mutation site in the nucleotide ce of SEQ ID NO:1; and/or a second primer comprising a polynucleotide comprising a nucleotide sequence which is identical or homologous to a complementary nucleotide sequence to the partial tide sequence comprising the first mutation site in the nucleotide sequence of SEQ ID NO:1; and confirming an amplification product of the first nucleic acid amplification reaction. (6) A method of discriminating traits of an onion, sing: minating r the onion is an onion with no pungent taste and/or tear-inducing property according to any of the methods according to (1) to (5); and discriminating whether the onion is an onion with no pungent taste and/or tear-inducing property based on one or more of the following traits as indicator(s): a) production of pyruvic acid at disruption of onion cells is reduced compared with conventional varieties; b) the quantity of PRENCSO remaining after tion of onion cells is greater compared with conventional varieties; and c) production of lachrymatory factor (LF) at disruption of onion cells is reduced compared with conventional varieties. (7) A method of breeding an onion with no pungent taste and/or nducing property, comprising: discriminating whether an onion is an onion with no pungent taste and/or tear-inducing property according to any of the methods ing to (1) to (6); and using the onion discriminated to be an onion with no pungent taste and/or tear-inducing property to breed an onion. (8) A primer comprising a first cleotide comprising a first nucleotide sequence at the 3' end, wherein the first nucleotide ce is identical or homologous to a first partial nucleotide sequence of 10 or more consecutive nucleotides sed in the nucleotide sequence of SEQ ID NO:1, the first partial nucleotide sequence comprising a first nucleotide selected from positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the tide sequence of SEQ ID NO:1 within 2 nucleotides from the 3' end. (9) A primer comprising a second polynucleotide comprising a second tide sequence at the 3' end, wherein the second nucleotide sequence is identical or homologous to a second partial nucleotide sequence of 10 or more consecutive nucleotides comprised in a complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:1, the second partial nucleotide sequence comprising a nucleotide complementary to a second nucleotide selected from positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide ce of SEQ ID NO:1 within 2 nucleotides from the 3' end. (10) A primer set comprising: the primer according to (8); and the primer according to (9), wherein the first nucleotide and the second nucleotide are identical to each other, or the first nucleotide is positioned more closely to the 5' endthan the second nucleotide in the tide sequence of SEQ ID NO:1. (11) A primer comprising a third polynucleotide comprising a third nucleotide sequence at the 3' end, wherein the third nucleotide sequence is identical or homologous to a third partial nucleotide sequence of 10 or more consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:2, the third partial nucleotide sequence comprising a third tide selected from positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the tide sequence of SEQ ID NO:2 within 2 tides from the 3' end. (12) A primer comprising a fourth polynucleotide comprising a fourth nucleotide ce at the 3' end, wherein the fourth nucleotide sequence is identical or homologous to a fourth partial nucleotide sequence of 10 or more consecutive tides comprised in a complementary tide sequence to the nucleotide sequence of SEQ ID NO:2, the fourth partial nucleotide sequence comprising a nucleotide complementary to a fourth nucleotide selected from positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 within 2 nucleotides from the 3' end. (13) A primer set, sing: the primer according to (11); and the primer according to (12), wherein the third nucleotide and the fourth nucleotide are identical to each other, or the third nucleotide is positioned more closely to the 5' end than the fourth nucleotide in the nucleotide sequence of SEQ ID NO:2. (14) A kit for discriminating traits of an onion, comprising: one selected from the primer ing to (8), the primer according to (9), and the primer set ing to (10); and one selected from the primer according to (11), the primer according to (12), and the primer set according to (13). (15) A marker gene for minating a pungent taste and/or a tear-inducing property of an onion, for which the cDNA nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:1.

The specification encompasses the disclosure of JP Patent Application No. 2017- 184019, on which the priority of the present application is based. ageous Effects of Invention According to the onion discrimination method of the present invention, an onion with no pungent taste and/or tear-inducing property, which cannot be discriminated in appearance, can be discriminated accurately. ing to the breeding method of the present invention, a target onion with no pungent taste and/or tear-inducing property can be ed accurately in a setting of breeding a new variety using an onion with no pungent taste and/or tear-inducing property as one of materials.

The primers, the primer sets, and the kit of the present invention can be used to amplify alliinase gene 1 or alliinase gene 2 specifically.

The marker gene of the present invention can be used to detect a pungent taste and/or a tear-inducing property of an onion.

Brief Description of gs e 1-1] Figure 1-1 shows the alignment results for the nucleotide sequence of SEQ ID NO:1, the nucleotide sequence of SEQ ID NO:2, and the nucleotide sequence of the alliinase gene of GenBank ion No. AAA32639.1 and the position of each primer. e 1-2] Continued from Figure 1-1 [Figure 1-3] Continued from Figure 1-1 [Figure 1-4] Continued from Figure 1-1 [Figure 2] Figure 2 shows the detection results for amplification products of PCR on the genomic DNAs of onion #6 with no pungent taste and a control variety as templates using a primer set of the nucleotide sequences of SEQ ID NOS: 3 and 4.

[Figure 3-1] Figure 3-1 shows the detection s for alliinase gene 1 and alliinase gene 2 in amplification products of PCR on the genomic DNAs of onion #6 with no pungent taste and a control variety as templates using various primer sets.

[Figure 3-2] Continued from Figure 3-1 [Figure 4] Figure 4 shows the detection results for alliinase gene 1 and alliinase gene 2 in ication products of PCR on the c DNAs of 14 varieties of cially available onions, a control y, and onion #6 as templates using various primer sets.

[Figure 5] Figure 5 shows the detection results for alliinase gene 1 in amplification products of PCR on the genomic DNAs of leaves of a plurality of F2 onions derived from onion #6 with no pungent taste as templates using SF2-SR1 as a primer set.

[Figure 6] Figure 6 shows the measurement results for the quantity of pyruvic acid produced in onion bulbs in which alliinase gene 1 was detected in the genomic DNA (selected bulbs) and onion bulbs in which alliinase gene 1 was not ed elected bulbs), among F2 onions derived from onion #6 with no pungent taste. e 7-1] Figure 7-1 shows the positions of primers in the nucleotide sequence of SEQ ID NO:1.

[Figure 7-2] Continued from Figure 7-1 [Figure 8-1] Figure 8-1 shows the positions of primers in the nucleotide ce of SEQ ID NO:2.

[Figure 8-2] Continued from Figure 8-1.

Description of Embodiments <1. Terms> In the ication, "gene 1" refers to a gene encoding an alliinase comprised in the genomic DNA which is transcribed to an mRNA corresponding to a cDNA comprising the nucleotide sequence of SEQ ID NO:1.

Additionally, in the specification, "gene 2" refers to a gene encoding an ase, the genomic DNA nucleotide sequence of which comprises the tide sequence of SEQ ID NO:2.

The "gene for which the cDNA nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:1" as used herein refers to a gene for which the nucleotide sequence of the cDNA corresponding to mRNA transcribed from the gene comprises the nucleotide sequence of SEQ ID NO:1.

Messenger RNA (mRNA) as used herein refers to a mature mRNA consisting of exon regions ligated by splicing of a precursor mRNA biosynthesized by RNA polymerase from the genomic DNA as a template in the cell of an onion. The process of production of mRNA (mature mRNA) from genomic DNA is "transcription." Complementary DNA (cDNA) as used herein refers to a double-stranded DNA consisting of a first DNA complementary to mRNA, which is sized by reverse riptase using mRNA as a te, and a second DNA mentary to the first DNA (i.e., consisting of a nucleotide sequence obtained by substituting u with t in the nucleotide sequence of mRNA), the single-stranded first DNA, or the single-stranded second DNA. The "nucleotide sequence of the cDNA corresponding to mDNA" as used herein refers to the nucleotide sequence of the second DNA.

In the present invention, "polynucleotide" refers to a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA), typically a DNA. In RNA, thymine (T) can be substituted with uracil (U). A DNA comprising U, which is synthesized by substituting T with U at one or more positions, can also be used as a DNA. The polynucleotide may comprise modified nucleotides, such as inosine (I), as part f.

The polynucleotide may be present as a single-stranded chain or a double-stranded chain. If the polynucleotide is present as a -stranded chain, it is sufficient that at least one strand is a polynucleotide having characteristics detailed in this specification.

The method of manufacturing a polynucleotide is not particularly limited, and the polynucleotide can be manufactured using a polynucleotide synthesizer.

In the present invention, the "nucleic acid derived from an onion" refers to a genomic DNA, an mRNA, or a cDNA derived from an onion of which traits are to be discriminated (target onion). The genomic DNA, the mRNA, and the cDNA also e amplified nts of the genomic DNA, amplified fragments of the mRNA, and amplified fragments of the cDNA, respectively.

In the present invention, the expression "a nucleotide sequence Y homologous to a nucleotide sequence X" or "a nucleotide sequence X and a nucleotide sequence Y are homologous to each other" refers to a combination of a DNA chain consisting of the complementary sequence to the nucleotide sequence X and a DNA chain consisting of the nucleotide ce Y that can form hydrogen bonds enough to hybridize these DNA chains under the annealing condition of a nucleic acid amplification on to form a stable stranded chain, and the nucleotide sequences X and Y may be partially different. For example, a polynucleotide consisting of the complementary sequence to the nucleotide sequence X and a polynucleotide consisting of the nucleotide sequence Y may comprise some mismatches, such as one mismatch in 10 nucleotides, one mismatch in 20 nucleotides, or one mismatch in 30 nucleotides. Typically, the expression a nucleotide sequence Y "homologous" to a nucleotide sequence X means that the nucleotide sequences X and Y satisfy any of the following relationships: (A) the nucleotide sequence Y is a nucleotide sequence obtained by ng, tuting, adding, and/or inserting one or several nucleotides in the nucleotide sequence X; (B) the nucleotide sequence Y is a nucleotide sequence having a 90% or higher identity to the nucleotide sequence X; (C) a polynucleotide consisting of the nucleotide ce Y can be hybridized with a polynucleotide consisting of the nucleotide sequence mentary to SEQ ID NO:X under stringent conditions; and (D) e (T) at an arbitrary position in either the nucleotide sequence X or the nucleotide ce Y is substituted with uracil (U) in the other nucleotide sequence.

In the (A), "one or l" means preferably one or two, and most ably one.

In the (B), the value of ty indicates a value obtained using a software to calculate the identity between a plurality of nucleotide sequences (e.g., FASTA, DNASIS, and BLAST) under the default setting. The value of nucleotide sequence identity is obtained by counting the number of coincident tides when a pair of nucleotide sequences are aligned at the maximum degree of coincidence and calculating the proportion of the number of coincident nucleotides to the total number of nucleotides in the ed nucleotide sequence. For details of the method of determining identity, refer to, for example, ul et al., Nuc Acids.

Res. 25, 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215, 403-410, 1990.

In the (B), the identity is more preferably 95% or higher, more preferably 96% or higher, more preferably 97% or higher, more ably 98% or higher, and more preferably 99% or higher.

In the (C), the term "stringent conditions" means conditions under which a so-called specific hybrid is formed, and a nonspecific hybrid is not formed. For example, the ent conditions can be appropriately determined with reference to Green and Sambrook, Molecular Cloning, 4th Ed. (2012), Cold Spring Harbor Laboratory Press. Specifically, stringent conditions can be set for temperature and a salt concentration in a solution for rn hybridization and temperature and a salt concentration in a solution for the g s in southern hybridization. <2. First discrimination method of the present invention> Firstly, the method of discriminating traits of an onion of the present ion is a method comprising a first determination step of determining presence of the nucleotide sequence of SEQ ID NO:1 in a nucleic acid derived from the onion, and a second determination step of determining presence of the nucleotide sequence of SEQ ID NO:2 in the c acid derived from the onion, wherein the onion is discriminated to be an onion with no pungent taste and/or tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is not determined in the first determination step and the presence of the nucleotide sequence of SEQ ID NO:2 is determined in the second determination step, and the onion is discriminated to be an onion with a pungent taste and/or a tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is determined in the first determination step and the presence of the nucleotide sequence of SEQ ID NO:2 is ined in the second ination step. This method is the "first discrimination method" of the t invention.

The present inventors found that, as the nucleotide sequence of the gene encoding alliinase, both the nucleotide sequence of gene 1 and the nucleotide sequence of gene 2 were present in the c DNA of a usual onion, whereas the nucleotide ce of gene 2 was present but the nucleotide sequence of gene 1 was not present in the genomic DNA of an onion with no pungent taste and/or tear-inducing property. The alliinase encoded by the nucleotide sequence of gene 1 is a specific ase consisting of the amino acid sequence as set forth in SEQ ID NO:5 disclosed in Patent Literature 1. Although Patent Literature 1 discloses that an onion with suppressed expression of the specific alliinase does not have a pungent taste and/or a tear-inducing property, the cause of not being expressed (e.g., regulatory region, transcription factor, destruction of gene itself) is not disclosed. The t inventors surprisingly found that the nucleotide sequence of gene 1 encoding the specific alliinase was not present in the genomic DNA of an onion with no pungent taste and/or tear-inducing property, and that the nucleotide sequence of gene 2 with a high homology to the nucleotide sequence of gene 1, which is not associated with a pungent taste, was present in the c DNA. Here, representative examples of the "onion with no pungent taste and/or tear-inducing property" include, but are not limited to, an onion of which seed has been ted in an International Depository ity under Accession No. NCIMB 42219, its progenies, and onions with no t taste and/or tear-inducing property bred using these onions as one of materials.

The nucleotide sequence of SEQ ID NO:1 is the cDNA nucleotide sequence of alliinase gene 1, and the tide ce of SEQ ID NO:2 is the genomic DNA nucleotide sequence of alliinase gene 2.

The "determining presence of the nucleotide ce of SEQ ID NO:1" and "determining presence of the first mutation site (in the nucleotide sequence of SEQ ID NO:1)" in the first determination step mean mining presence of a nucleotide sequence corresponding to the nucleotide ce of SEQ ID NO:1" and "determining presence of a nucleotide sequence corresponding to the first mutation site (in the nucleotide sequence of SEQ ID NO:1)," respectively. Similarly, "determining presence of the nucleotide sequence of SEQ ID NO:2" and "determining ce of the second mutation site (in the nucleotide sequence of SEQ ID NO:2)" in the second determination step mean "determining presence of a nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO:2" and "determining presence of a nucleotide sequence corresponding to the second on site (in the nucleotide sequence of SEQ ID " respectively.

The "nucleotide ce corresponding to the nucleotide sequence of SEQ ID NO:1" refers to the genomic DNA nucleotide ce of an alliinase gene for which the cDNA nucleotide sequence is the nucleotide sequence of SEQ ID NO:1 or the complementary nucleotide sequence thereto if the nucleic acid derived from an onion is a genomic DNA; a nucleotide sequence derived from SEQ ID NO:1 by substitution of T with U if the nucleic acid derived from an onion is an mRNA; or the nucleotide sequence of SEQ ID NO:1 or the complementary nucleotide ce thereto if the nucleic acid derived from an onion is a cDNA.

The "nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO:2" refers to the nucleotide sequence of SEQ ID NO:2 or the complementary nucleotide sequence thereto if the nucleic acid derived from an onion is a genomic DNA; a nucleotide sequence d from the nucleotide sequence of SEQ ID NO:2 by deletion of the intron regions and substitution of T with U if the c acid derived from an onion is an mRNA; or a nucleotide sequence in which intron regions are removed from the nucleotide sequence of SEQ ID NO:2 or the complementary nucleotide sequence thereto if the nucleic acid derived from an onion is a cDNA.

The "nucleotide sequence corresponding to the first mutation site (in the nucleotide sequence of SEQ ID NO:1)" refers to the nucleotide sequence of a site corresponding to the first mutation site in the nucleotide sequence of the genomic DNA of an alliinase gene for which the cDNA nucleotide ce is the nucleotide sequence of SEQ ID NO:1 or the complementary nucleotide ce thereto if the nucleic acid derived from an onion is a c DNA; the nucleotide sequence of a site corresponding to the first mutation site in a nucleotide sequence derived from SEQ ID NO:1 by substitution of T with U if the nucleic acid derived from an onion is an mRNA; or the first mutation site in the nucleotide sequence of SEQ ID NO:1 or the complementary nucleotide ce thereto if the nucleic acid derived from an onion is a cDNA.

The "nucleotide sequence corresponding to the second on site (in the nucleotide sequence of SEQ ID NO:2)" refers to the second mutation site in the nucleotide sequence of SEQ ID NO:2 or the complementary nucleotide sequence thereto if the nucleic acid derived from an onion is a genomic DNA; the nucleotide sequence of a site corresponding to the second mutation site in a nucleotide sequence derived from the tide sequence of SEQ ID NO:2 by deletion of the intron regions and substitution of T with U if the nucleic acid derived from an onion is an mRNA; or the nucleotide sequence of a site corresponding to the second mutation site in the nucleotide sequence derived from the nucleotide sequence of SEQ ID NO:2 by deletion of the intron regions, or the mentary nucleotide sequence o if the nucleic acid derived from an onion is a cDNA.

It is preferable to analyze a genomic DNA as a nucleic acid derived from an onion because the quantity does not change depending on the growth stage of the target onion or the site thereof. Furthermore, a genomic DNA is preferable because r an analysis is being performed under appropriate conditions can be confirmed by ming not only the presence or absence of the nucleotide sequence of gene 1, but also the presence of the nucleotide sequence of gene 2, which is present regardless of the presence of a pungent taste of an onion in the first mination method of the present invention.

The "first determination step" will be explained in detail below.

The first determination step is a step of determining the presence of the nucleotide sequence of SEQ ID NO:1 in a nucleic acid derived from an onion. In the step, preferably, a nucleotide sequence sing at least a partial nucleotide sequence of the nucleotide sequence of SEQ ID NO:1 or of a nucleotide sequence derived from SEQ ID NO:1 by substitution of T with U or the mentary nucleotide sequence to the partial nucleotide ce is detected in the nucleic acid derived from the onion.

When the nucleotide sequence of SEQ ID NO:1, which is the cDNA nucleotide sequence of gene 1, and the nucleotide sequence of SEQ ID NO:2, which is the genomic DNA nucleotide sequence of gene 2, were d to obtain the maximum matching, some mismatching nucleotides were found in regions with a high homology as shown in s 1-1, 1-2, 1-3, and 1-4. ically, positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 and positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 are mismatching nucleotides comprised in the regions with a high homology.

Accordingly, in the first determination step, it is preferable to determine the ce of a first mutation site comprising one or more nucleotides selected from nucleotides at positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1.

This enables to specifically detect the nucleotide sequence of SEQ ID NO:1 comprised in an nucleic acid derived from an onion, without detecting the nucleotide sequence of SEQ ID NO:2 in error.

As a specific means for determining the presence of the first mutation site in the nucleotide sequence of SEQ ID NO:1 in the nucleic acid derived from the onion, it is preferable to perform a first nucleic acid amplification on using the following first primer set and a nucleic acid derived from an onion, in particular, a genomic DNA or a cDNA as a te and to confirm an amplification product of the first nucleic acid amplification reaction.

A first primer set preferably comprises at least either one of a first primer comprising a polynucleotide comprising a nucleotide ce which is identical or homologous to a partial tide sequence comprising the first mutation site in the nucleotide sequence of SEQ ID NO:1, and a second primer comprising a polynucleotide comprising a nucleotide ce which is identical or homologous to the complementary tide sequence to a partial nucleotide sequence comprising the first mutation site in the nucleotide sequence of SEQ ID NO:1, and particularly preferably both the first and second primers.

Examples of a preferred embodiment of the first primer include a first primer comprising a first polynucleotide comprising a first nucleotide sequence at the 3' end which is identical or homologous to a first partial tide ce of 10 or more consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:1, the first partial nucleotide ce sing a first nucleotide selected from positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide ce of SEQ ID NO:1 within 2 nucleotides from the 3' end.

Examples of a preferred embodiment of the second primer include a second primer comprising a second polynucleotide comprising a second nucleotide sequence at the 3' end which is identical or homologous to a second partial nucleotide sequence of 10 or more consecutive nucleotides comprised in the complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:1, the second partial tide sequence comprising a nucleotide complementary to a second nucleotide ed from positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide ce of SEQ ID NO:1 within 2 tides from the 3' end.

The preferred ment of the first primer will be explained below.

The first partial nucleotide sequence described in association with the first primer is a partial nucleotide sequence of 10 or more consecutive nucleotides, preferably 15 or more utive nucleotides, more preferably 17 or more consecutive nucleotides, more preferably or more consecutive nucleotides, and more preferably 23 or more consecutive nucleotides comprised in the tide sequence of SEQ ID NO:1. Of note, to prevent a nonspecific hybrid formation between the primer and a region other than the end region of the target nucleic acid to be amplified, it is nown that designing a primer with the number of primer nucleotides within the above-described ranges, in particular 17 or more tides, is desirable (for example, refer to http://www.takara-bio.co.jp/prt/pdfs/prt3-1.pdf). The upper limit of the number of nucleotides in the first partial nucleotide sequence is not particularly limited, and is preferably 50 or fewer nucleotides, more preferably 40 or fewer nucleotides, more preferably 35 or fewer nucleotides, and more preferably 30 or fewer nucleotides. When the first l nucleotide sequence comprises the first nucleotide as a tide within 2 nucleotides from the 3' end, preferably as a nucleotide at the 3' end, a region on the 3' end side from a nucleotide corresponding to the first nucleotide in the nucleotide sequence on the sense chain of gene 1 in the template nucleic acid is specifically elongated from the 3' end of the first primer in the first nucleic acid amplification reaction.

The first polynucleotide comprises the first nucleotide sequence which is identical or homologous to the first partial nucleotide sequence at the 3' end. Here, the term "homologous" is defined as described above. More preferably, the first partial nucleotide sequence and the first nucleotide sequence are identical to each other preferably in a region of 3 nucleotides at the 3' end, more preferably in a r egion of 5 nucleotides at the 3' end, more ably a region of 8 nucleotides at the 3' end, and more preferably in a region of 10 nucleotides at the 3' end (further preferably in a region of 15 nucleotides at the 3' end if the length of the first partial nucleotide sequence is 15 or more nucleotides, further preferably in a region of 17 nucleotides at the 3' end if the length of the first partial nucleotide sequence is 17 or more nucleotides, and further preferably in a region of 20 nucleotides at the 3' end if the length of the first partial nucleotide sequence is 20 or more nucleotides) and are homologous to each other in the ing region on the 5' end side. Specific examples of the first nucleotide sequence e the nucleotide sequence as set forth in SEQ ID NO:5, 6, 7, or 8 and a partial nucleotide sequence comprising preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, and more preferably 20 or more consecutive tides from the 3' end of the nucl eotide sequence as set forth in SEQ ID NO:5, 6, 7, or 8. The expression that the first polynucleotide "comprises the first nucleotide sequence at the 3' end" encompasses both a case where the whole nucleotide sequence of the first cleotide consists only of the first nucleotide sequence and a case where the first polynucleotide ses the first nucleotide sequence and a further nucleotide sequence linked to the first nucleotide sequence at the 5' e nd f. It is sufficient that the further nucleotide sequence does not substantially inhibit the first nucleic acid amplification on, and the number of nucleotides thereof is, for example, 20 or fewer nucleotides, preferably 10 or fewer nucleotides, more preferably 5 or fewer nucleotides, and more preferably 1 or 2 nucleotides.

In the specification, the first primers comprising the first polynucleotide consisting of the nucleotide sequences of SEQ ID NOS: 5, 6, 7, and 8 are referred to as SF1, SF2, SF3, and SF4, respectively, and their positions in the tide sequence of SEQ ID NO:1 are shown in Figures 7-1 and 7-2.

The first primer may consist of the first polynucleotide or may comprise the first polynucleotide and useful chemical ures such as a labeling tag, a labeling substance, and lization tag added to the first polynucleotide. Such useful chemical structures can be detected.

If the first primer set comprises the first primer, the other primer is preferably a primer comprising a polynucleotide comprising a nucleotide sequence at the 3' end which is identical or homologous to a partial nucleotide sequence of 10 to 50 consecutive nucleotides, preferably to 30 consecutive nucleotides comprised in the complementary nucleotide sequence to the tide sequence of SEQ ID NO:1, which is mentary to a region positioned more closely to the 3' end than the first tide in the nucleotide sequence of SEQ ID NO:1, and more preferably the second primer.

The preferred embodiment of the second primer will be explained below.

The second partial nucleotide sequence described in association with the second primer is a l nucleotide sequence of 10 or more consecutive nucleotides, preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, more preferably or more consecutive nucleotides, and more preferably 23 or more consecutive nucleotides comprised in the complementary nucleotide sequence to the nucleotide ce of SEQ ID NO:1. The upper limit of the number of nucleotides in the second partial nucleotide sequence is not particularly limited, and is preferably 50 or fewer tides, more ably 40 or fewer nucleotides, more preferably 35 or fewer nucleotides, and more preferably 30 or fewer nucleotides. When the second partial nucleotide ce comprises the complementary nucleotide of the second nucleotide as a nucleotide within 2 nucleotides from the 3' end, preferably as a nucleotide at the 3' end, a region on the 3' end side from a nucleotide corresponding to the complementary nucleotide of the second nucleotide in the nucleotide sequence on the antisense chain of gene 1 in the template c acid is specifically elongated from the 3' end of the second primer in the first nucleic acid amplification reaction.

The second polynucleotide ses the second nucleotide sequence which is identical or homologous to the second partial nucleotide sequence at the 3' end. Here, the term "homologous" is defined as described above. More preferably, the second partial nucleotide sequence and the second nucleotide sequence are identical to each other, preferably in a region of 3 nucleotides at the 3' end, more preferably in a region of 5 nucleotides at the 3' end, more preferably in a region of 8 nucleotides at the 3' end, and more preferably in a region of 10 tides at the 3' end (further preferably in a region of 15 nucleotides at the 3' end if the length of the second l nucleotide sequence is 15 or more nucleotides, further preferably in a region of 17 nucleotides at the 3' end if the length of the second partial tide ce is 17 or more nucleotides, and further preferably in a region of 20 nucleotides at the 3' end if the length of the second partial nucleotide sequence is 20 or more nucleotides) and are homologous to each other in the remaining region on the 5' end side. ic examples of the second tide sequence include the nucleotide sequence as set forth in SEQ ID NO:9, 10, 11, or 12 and a partial nucleotide sequence comprising preferably or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, and more preferably 20 or more consecutive nucleotides from the 3' end of the nucleotide sequence as set forth in SEQ ID NO:9, 10, 11, or 12. The expression that the second polynucleotide ises the second nucleotide ce at the 3' end" encompasses both a case where the whole nucleotide sequence of the second polynucleotide consists only of the second nucleotide sequence and a case where the second polynucleotide comprises the second nucleotide sequence and a further tide ce linked to the second nucleotide sequence at the 5' end thereof. It is sufficient that the further nucleotide sequence does not ntially inhibit the first nucleic acid amplification reaction, and the number of nucleotides thereof is, for example, 20 or fewer nucleotides, preferably 10 or fewer nucleotides, more preferably 5 or fewer nucleotides, and more preferably 1 or 2 nucleotides.

In the specification, the second primers comprising the second polynucleotides consisting of the nucleotide sequences of SEQ ID NOS: 9, 10, 11, and 12 are referred to as SR1, SR2, SR3, and SR4, respectively, and their positions in the nucleotide sequence of SEQ ID NO:1 are shown in Figures 7-1 and 7-2.

The second primer may t of the second polynucleotide or may comprise the second cleotide and useful chemical structures such as a labeling tag, a labeling substance, and immobilization tag added to the second polynucleotide. Such useful chemical structures can be detected.

If the first primer set comprises the second primer, the other primer is preferably a primer comprising a polynucleotide comprising a nucleotide sequence at the 3' end which is identical or homologous to a l nucleotide sequence of 10 to 50 consecutive nucleotides, preferably 15 to 30 utive nucleotides comprised in the nucleotide sequence of SEQ ID NO:1, which is a region positioned more closely to the 5' end than the second nucleotide in the nucleotide sequence of SEQ ID NO:1, and more preferably the first .

If the first primer set is a combination of the first primer and the second primer, it is sufficient that the first primer and the second primer are selected so that the first nucleotide and the second nucleotide are identical to each other, or the first nucleotide is positioned more closely to the 5' end than the second nucleotide in the nucleotide sequence of SEQ ID NO:1.

In a preferred embodiment in which the first primer set is a combination of the first primer and the second primer, the first tide for the first primer is at position 94, 130, 1312, or 1348 in the nucleotide sequence of SEQ ID NO:1, and the second nucleotide for the second primer is at position 187, 358, 791, or 1467 in SEQ ID NO:1, provided that the first nucleotide is identical to the second nucleotide or is oned more closely to the 5' end than the second nucleotide in the nucleotide sequence of SEQ ID NO:1.

The d determination step" will be explained in detail below.

The second determination step is a step of determining the presence of the nucleotide sequence of SEQ ID NO:2 in a nucleic acid derived from an onion. In the step, preferably, a nucleotide sequence comprising at least a partial nucleotide sequence of the nucleotide sequence of SEQ ID NO:2 or of a nucleotide sequence derived from SEQ ID NO:2 substitution of T with U or the complementary tide sequence to the partial nucleotide sequence is detected in the nucleic acid derived from the onion.

In the second ination step, it is preferable to determine the presence of a second mutation site comprising one or more nucleotides selected from nucleotides at positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2. This enables to specifically detect the nucleotide sequence of SEQ ID NO:2 comprised in a nucleic acid derived from an onion, without detecting the nucleotide sequence of SEQ ID NO:1 in error.

As a specific means for determining the ce of the second on site in the nucleotide sequence of SEQ ID NO:2 in the nucleic acid derived from the onion, it is preferable to perform a second nucleic acid ication reaction using the following second primer set and a nucleic acid derived from an onion, in particular, a c DNA as a te and to confirm an amplification product of the second nucleic acid amplification reaction.

A second primer set preferably comprises at least either one of a third primer comprising a polynucleotide comprising a nucleotide sequence which is identical or homologous to a partial tide sequence comprising the second mutation site in the nucleotide sequence of SEQ ID NO:2, and a fourth primer comprising a polynucleotide sing a nucleotide sequence which is cal or homologous to the complementary nucleotide sequence to the partial nucleotide sequence comprising the second mutation site in the nucleotide sequence of SEQ ID NO:2, and particularly preferably both the third and fourth primers.

Examples of a preferred embodiment of the third primer include a third primer comprising a third polynucleotide comprising a third nucleotide sequence at the 3' end which is identical or homologous to a third partial nucleotide sequence of 10 or more consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:2 comprising a third nucleotide selected from positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 within 2 nucleotides from the 3' end.

Examples of a preferred embodiment of the fourth primer e a fourth primer comprising a fourth polynucleotide comprising a fourth nucleotide sequence at the 3' end which is identical or gous to a fourth partial nucleotide sequence of 10 or more consecutive nucleotides sed in the complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:2 comprising a nucleotide complementary to a fourth nucleotide ed from positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 within 2 nucleotides from the 3' end.

The preferred embodiment of the third primer will be explained below.

The third l nucleotide sequence described in ation with the third primer is a l nucleotide sequence of 10 or more consecutive nucleotides, preferably 15 or more consecutive tides, more preferably 17 or more consecutive tides, more preferably or more consecutive tides, and more preferably 23 or more consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:2. The upper limit of the number of nucleotides in the third partial nucleotide sequence is not particularly limited, and is preferably 50 or fewer nucleotides, more preferably 40 or fewer nucleotides, more preferably 35 or fewer nucleotides, and more preferably 30 or fewer nucleotides. When the third partial nucleotide sequence comprises the third tide as a nucleotide within 2 nucleotides from the 3' end, preferably as a nucleotide at the 3' end, a region on the 3' end side from a nucleotide corresponding to the third nucleotide in the nucleotide sequence on the sense chain of gene 2 in the template nucleic acid is specifically elongated from the 3' end of the third primer in the second nucleic acid amplification reaction.

The third polynucleotide comprises the third nucleotide sequence which is identical or homologous to the third partial nucleotide sequence at the 3' end. Here, the term "homologous" is defined as described above. More preferably, the third partial nucleotide sequence and the third nucleotide sequence are identical to each other, preferably in a region of 3 nucleotides at the 3' end, more preferably in a region of 5 nucleotides at the 3' end, more preferably in a region of 8 nucleotides at the 3' e nd, and more preferably in a region of 10 nucleotides at the 3' end (further preferably in a region of 15 nucleotides at the 3' end if the length of the third partial nucleotide sequence is 15 or more nucleotides, further ably in a region of 17 nucleotides at the 3' end if the length of the third partial nucleotide sequence is 17 or more nucleotides, and further preferably in a region of 20 nucleotides at the 3' end if the length of the third partial nucleotide sequence is 20 or more nucleotides) and are homologous to each other in the remaining region on the 5' end side. ic examples of the third nucleotide sequence include the tide sequence as set forth in SEQ ID NO:13, 14, 15, or 16 and a partial nucleotide sequence comprising preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, and more preferably 20 or more consecutive nucleotides from the 3' end of the nucl eotide sequence as set forth in SEQ ID NO:13, 14, 15, or 16. The expression that the third cleotide ises the third tide sequence at the 3' end" encompasses both a case where the whole nucleotide sequence of the third polynucleotide consists only of the third nucleotide sequence and a case where the third polynucleotide comprises the third nucleotide sequence and a further nucleotide sequence linked to the third tide sequence at the 5' end thereof. It is sufficient that the further nucleotide sequence does not substantially inhibit the second nucleic acid amplification reaction, and the number of nucleotides thereof is, for example, 20 or fewer tides, preferably 10 or fewer tides, more ably 5 or fewer nucleotides, and more preferably 1 or 2 nucleotides.

In the ication, the third primers comprising the third polynucleotide consisting of the nucleotide sequences of SEQ ID NOS: 13, 14, 15, and 16 are referred to as UF1, UF2, UF3, and UF4, respectively, and their positions in the nucleotide sequence of SEQ ID NO:2 are shown in Figures 8-1 and 8-2.

The third primer may consist of the third polynucleotide or may comprise the third polynucleotide and useful al structures such as a labeling tag, a labeling substance, and immobilization tag added to the third cleotide. Such useful chemical structures can be detected.

If the second primer set comprises the third , the other primer is preferably a primer comprising a cleotide comprising a nucleotide sequence at the 3' end which is identical or homologous to a partial nucleotide sequence of 10 to 50 consecutive nucleotides, preferably 15 to 30 consecutive nucleotides comprised in the complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:2 which is complementary to a region positioned more closely to the 3' end than the thir d nucleotide in the nucleotide sequence of SEQ ID NO:2, and more preferably the fourth primer.

The preferred embodiment of the fourth primer will be explained below.

The fourth partial nucleotide sequence described in association with the fourth primer is a partial tide sequence of 10 or more consecutive tides, preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, more preferably or more consecutive nucleotides, and more preferably 23 or more consecutive nucleotides comprised in the complementary tide sequence to the nucleotide sequence of SEQ ID NO:2. The upper limit of the number of nucleotides in the fourth partial nucleotide sequence is not particularly limited, and is preferably 50 or fewer nucleotides, more preferably 40 or fewer tides, more preferably 35 or fewer nucleotides, and more ably 30 or fewer nucleotides. When the fourth partial nucleotide sequence comprises the complementary nucleotide of the fourth nucleotide as a nucleotide within 2 nucleotides from the 3' end, preferably as a nucleotide at the 3' end, and a reg ion on the 3' end side from a nucleotide corresponding to the complementary nucleotide of the fourth tide in the nucleotide sequence on the nse chain of gene 2 in the template nucleic acid is specifically elongated from the 3' end of the fourth primer in the secondnucleic acid amplification reaction.

The fourth polynucleotide ses the fourth nucleotide sequence which is identical or homologous to the fourth partial tide sequence at the 3' end. Here, the term "homologous" is defined as described above. More preferably, the fourth partial tide sequence and the fourth nucleotide sequence are identical to each other, preferably in a region of 3 tides at the 3' end, more preferably in a region of 5 nucleotides at the 3' end, more preferably in a region of 8 nucleotides at the 3' e nd, and more preferably in a region of 10 nucleotides at the 3' end (further preferably in a region of 15 nucleotides at the 3' end if the length of the fourth l nucleotide sequence is 15 or more nucleotides, further preferably in a region of 17 nucleotides at the 3' end if the length of the fourth partial nucleotide sequence is 17 or more nucleotides, and further preferably in a region of 20 nucleotides at the 3' end if the length of the fourth l nucleotide sequence is 20 or more nucleotides) and are homologous to each other in the remaining region on the 5' end side. Specific examples of the fourth nucleotide sequence include the nucleotide ce as set forth in SEQ ID NO:17, 18, 19, or and a partial nucleotide sequence comprising preferably 15 or more consecutive nucleotides, more preferably 17 or more consecutive nucleotides, and more preferably 20 or more consecutive tides from the 3' end of the nucl eotide sequence as set forth in SEQ ID NO:17, 18, 19, or 20. The expression that the fourth polynucleotide "comprises the fourth nucleotide sequence at the 3' end" encompasses both a case where the whole nucleotide ce of the fourth polynucleotide consists only of the fourth nucleotide sequence and a case where the fourth cleotide ses the fourth nucleotide sequence and a further nucleotide sequence linked to the fourth nucleotide sequence at the 5' end thereof. It is sufficient that the further nucleotide sequence does not substantially t the second nucleic acid amplification reaction, and the number of nucleotides thereof is, for example, 20 or fewer nucleotides, preferably 10 or fewer nucleotides, more preferably 5 or fewer nucleotides, and more preferably 1 or 2 nucleotides.

In the specification, the fourth primers comprising the fourth polynucleotide consisting of the nucleotide sequence of SEQ ID NOS: 17, 18, 19, and 20 are referred to as UR1, UR2, UR3, and UR4, respectively, and their positions in the nucleotide sequence of SEQ ID NO:2 are shown in Figures 8-1 and 8-2.

The fourth primer may consist of the fourth polynucleotide or may comprise the fourth polynucleotide and useful chemical structures such as a labeling tag, a ng substance, and immobilization tag added to the fourth polynucleotide. Such useful chemical structures can be ed.

If the second primer set comprises the fourth primer, the other primer is preferably a primer comprising a polynucleotide comprising a nucleotide sequence at the 3' end which is identical or homologous to a partial nucleotide sequence of 10 to 50 consecutive nucleotides, preferably 15 to 30 consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:2, which is a region positioned more y to the 5' end than the fourth nucleotide in the nucleotide sequence of SEQ ID NO:2, and more preferably the third primer.

If the second primer set is a combination of the third primer and the fourth primer, it is sufficient that the third primer and the fourth primer are selected so that the third nucleotide and the fourth nucleotide are identical to each other, or the third nucleotide is positioned more closely to the 5' end than the fourth nucleotide in the nucleotide sequence of SEQ ID NO:2.

In a preferred embodiment in which the second primer set is a ation of the third primer and the fourth primer, the third nucleotide for the third primer is at position 34, 70, 1667, or 1703 in the nucleotide sequence of SEQ ID NO:2, and the fourth nucleotide for the fourth primer is at on 127, 409, 943, or 1822 in SEQ ID NO:2, provided that the third nucleotide is identical to the fourth nucleotide or is positioned more closely to the 5' end than the fourth nucleotide in the tide sequence of SEQ ID NO:2.

In the present invention, both the first c acid amplification reaction and the second nucleic acid ication reaction (hereinafter referred collectively to as "nucleic acid amplification reaction") can be performed according to a usual polymerase chain reaction (PCR) .

A nucleic acid, such as a genomic DNA or a cDNA, used as a te can be prepared from an onion according to a usual method.

It is sufficient that a DNA polymerase used for PCR is a thermostable DNA polymerase, and DNA polymerases are not particularly limited. In the present invention, cially available DNA polymerases can be used. Additionally, primer concentration, number of cycles, temperature, time, compositions of buffer solutions, and other ions can be appropriately selected depending on the DNA polymerase used, the concentration of each primer, and other ions.

In the nucleic acid amplification reaction, a polynucleotide fragment comprising a predetermined target region is produced as an ication product when the nucleic acid derived from the target onion contains a tide sequence to be detected.

The method of confirming the amplification product of the nucleic acid amplification reaction is not particularly limited, and examples thereof include a method in which, after completion of the nucleic acid ication reaction, the reaction mixture of the c acid amplification reaction is fractioned by gel electrophoresis, and the presence of a band of the size corresponding to the polynucleotide fragment comprising the predetermined target region is confirmed and a method in which the ication product is labelled and detected.

Additionally, the amplification product can also be detected by a real-time PCR method while performing the nucleic acid amplification reaction. <3. Second discrimination method of the present invention> Secondly, the method of discriminating traits of an onion of the present invention is a method comprising a first determination step of determining presence of the nucleotide sequence of SEQ ID NO:1 in a nucleic acid derived from the onion, the first determination step comprising determining presence of a first mutation site comprising one or more nucleotides selected from tides at positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 in the nucleic acid, wherein the onion is discriminated to be an onion with no pungent taste and/or tear-inducing property if the presence of the nucleotide sequence of SEQ ID NO:1 is not determined in the first determination step; and the onion is discriminated to be an onion with a pungent taste and/or a tear-inducing property if the presence of the tide sequence of SEQ ID NO:1 is determined in the first determination step. This method is the "second discrimination method" of the present invention.

The first determination step in the second discrimination method is the same as the first determination step in the first discrimination method. Specific embodiments of the first determination step in the second discrimination method are the same as the specific embodiments of the first determination step in the first mination method.

The first determination step in the second discrimination method preferably comprises performing a first nucleic acid amplification reaction using a first primer set comprising at least either one of a first primer and a second primer and a nucleic acid of an onion as a template and confirming an amplification product of the first nucleic acid ication reaction. The first , the second primer, the first primer set, the first nucleic acid amplification reaction, and the method of detecting the amplification product in this ment of the second discrimination method are as explained for the first discrimination <4. Combination with further discrimination methods> The first or second discrimination method of the present invention may be used in combination with further methods for discriminating an onion with no pungent taste and/or tear-inducing property. By using a ity of different discrimination methods in combination, an onion with no pungent taste and/or tear-inducing property can be discriminated tely.

Examples of further s for discriminating an onion with no pungent taste and/or tear-inducing ty include discriminating whether the onion is an onion with no pungent taste and/or tear-inducing property based on one or more of the following traits as indicator(s): a) tion of pyruvic acid at disruption of onion cells is reduced compared with conventional varieties; b) the quantity of PRENCSO remaining after disruption of onion cells is greater compared with conventional varieties; and c) production of lachrymatory factor (LF) at disruption of onion cells is reduced compared with tional varieties.

An onion having one or more of the above traits can be discriminated to be an onion with no pungent taste and/or tear-inducing ty. <5. ng method> The present invention also provides a method of breeding an onion with no pungent taste and/or tear-inducing property, comprising discriminating whether an onion is an onion with no pungent taste and/or tear-inducing property by the first or second discrimination method of the present invention, and using the onion discriminated to be an onion with no pungent taste and/or tear-inducing property to breed onions.

In a setting of breeding a new variety using an onion with no pungent taste and/or tearinducing property as one of materials, an onion with no pungent taste and/or tear-inducing property can be accurately ed among onions produced by crossing using the first or second discrimination method of the present invention. The selected onion with no pungent taste and/or tear-inducing property can be used for further breeding. <6. Primers, primer sets, kit> Both the first primer and the second primer are useful as a primer to specifically amplify a part comprising a nucleotide sequence corresponding to the nucleotide ce of SEQ ID NO:1 in a c acid derived from an onion.

The first primer set comprising the first primer and the second primer in which the first nucleotide and the second nucleotide are identical to each other, or the first tide is positioned more closely to the 5' end than the second nucleotide in the nucleotide sequence of SEQ ID NO:1 is useful as a primer set to specifically amplify a part comprising a nucleotide ce corresponding to the nucleotide sequence of SEQ ID NO:1 in the nucleic acid derived from the onion.

Both the third primer and the fourth primer are useful as a primer to specifically amplify a part comprising a nucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO:2 in a nucleic acid derived from an onion.

The second primer set comprising the third primer and the fourth primer in which the third nucleotide and the fourth nucleotide are identical to each other, or the third nucleotide is positioned more closely to the 5' end than the fourth nucleotide in the nucleotide sequence of SEQ ID NO:2 is useful as a primer set to specifically y a part comprising a nucleotide sequence corresponding to the nucleotide ce of SEQ ID NO:2 in a nucleic acid derived from an onion.

A kit comprising one or more of these primers or primer sets is useful as a kit to discriminate traits of an onion. The kit can r comprise various components (e.g., DNA polymerase, various buffer solutions, dNTPs) used to perform a nucleic acid ication reaction and m an amplification product. <7. Marker gene> Alliinase gene 1, for which the cDNA nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:1, is not present in an onion with no pungent taste and/or ducing ty and is present in a usual onion with a pungent taste and/or a tear-inducing ty. Therefore, alliinase gene 1 is useful as a marker gene to detect a t taste and/or a tear-inducing property of an onion. If alliinase gene 1 is detected in a nucleic acid derived from an onion, the onion can be discriminated to be an onion with a pungent taste and/or a tear-inducing property. If alliinase gene 1 is not detected in a nucleic acid derived from an onion, the onion is discriminated to be an onion with no pungent taste and/or tearinducing property. <8. Further ions> The present invention further includes the following inventions A to C: A. A plant body of an onion with no pungent taste and/or tear-inducing property discriminated to be an onion with no pungent taste and/or tear-inducing property by the first or second discrimination method of the present invention, a y thereof, or a part thereof.

B. A method of manufacturing a plant body of an onion with no pungent taste and/or tearinducing property, a progeny thereof, or a part thereof, comprising the following steps: (i) a step of inducing a mutation in an onion seed; (ii) a step of cultivating the onion seed with the induced mutation to obtain a plant body of the onion or a part thereof, and (iii) a step of discriminating and ing the plant body of an onion with no pungent taste and/or tear-inducing property or a part thereof by the first or second discrimination method of the t invention from plant bodies or parts thereof of the obtained onions.

C. A method of manufacturing a plant body of an onion with no pungent taste and/or tearinducing property, a progeny thereof, or a part thereof, comprising crossing a first onion discriminated to be an onion with no t taste and/or tear-inducing property by the first or second discrimination method of the present invention and a second onion.

The description in Patent Literature 1 may be referred to for specific aspects of the ions A to C.

Example 1: ing the gene sequence of alliinase associated with a pungent taste and/or a tear-inducing property of an onion Three bulbs of a tearless and non-pungent onion #6 disclosed in Patent Literature 1 (the seed of onion #6 has been deposited in an International Depository Authority under Accession No. NCIMB 42219. Hereinafter, the onion is ed to as "onion #6") and three bulbs of an onion having a pungent taste obtained by repeating the same number of times of selfreproduction of the same variety as onion #6 (hereinafter referred to as "control variety") as l bulbs were rapidly frozen with liquid nitrogen and then cryopreserved at -80°C. The skin of each frozen onion bulb was peeled, 100 mg of onion tissue was measured and taken out, and a total RNA was collected using the RNeasy Plant Mini kit (manufactured by Qiagen) according to the ed manual. Further, the collected total RNA was subjected to DNase ent using the RNeasy Mini Kit (manufactured by Qiagen) and the RNase-Free DNase Set (manufactured by Qiagen). The treated total RNA was subjected to concentration measurement and quality confirmation using the Nanodrop (manufactured by Nanodrop Technologies) and the Agilent 2100 Bioanalyzer actured by Agilent Technologies).

In the quality confirmation, it was confirmed that samples had A260/A280 absorbance ratios of 1.8 or higher and RNA Integrity Numbers of 8.0 or higher, and a sequence y was prepared using the TruSeq RNA Sample Prep Kit (manufactured by Illumina) according to the attached manual. The prepared sequence library was subjected to sequence analysis using a next-generation sequencer HiSeq (manufactured by Illumina) under the following conditions: (Sequence conditions) Analytical method, paired end sequencing; number of specimens, 6; number of lanes, 3; read nucleotide length, 100 nucleotides/read (Data processing) The obtained data were subjected to the ing information processing: - Data with a low fluorescence purity were removed using a formula called Chastity.

"Chastity" is ented by a formula of "I1/(I1 + I2)," where the largest value of signals from 4 different tides is I1 and the second largest value is I2. In this example, data were selected under a condition of "I1/(I1 + I2) > 0.6." - The selected data were sorted out by specimen acc ording to the specimen unique index information.

- Reads comprising an adaptor ce were removed , and read pairs in which the Quality Value was 20 or higher for 90% or more of the comprised nucleotides were further extracted.

De novo assembly was performed for data of all these specimens using Trinity (URL: http://trinityrnaseq.sourceforge.net/index.html version 201325).

- The assembly data (estimated ript sequence) were annotated by BLAST . The BLASTX was used as the annotation program, and the amino acid sequences in RefSeq-Fungi, RefSeq-Microbial, RefSeq-Plant of NCBI and the amino acid sequences and the gene sequences (converted to amino acid sequence) registered under Allieae in the NCBI classification were integrated and used as the database. The BLASTX parameter used was evalue 1E-5/num_alignments tfmt "6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue re qlen slen stitle qcovs p"/, and default conditions were used for others.

As a result, a cDNA nucleotide sequence encoding alliinase comprising "amino acid sequence 5" disclosed in Patent Literature 1 was acquired. The cDNA nucleotide sequence of "amino acid sequence 5" encoding alliinase is shown as SEQ ID NO:1 in the sequence listing.

Example 2: ition of sequences of other alliinases with a high homology to the ase encoded by the nucleotide sequence of SEQ ID NO:1 A d primer corresponding to the end part of the nucleotide sequence of SEQ ID NO:1 encoding alliinase that comprises the start codon and a reverse primer corresponding to the end part of the nucleotide sequence that comprises the stop codon were prepared, 100 mg each of samples were ted from both onion #6 and the control variety used in Example 1, the genomic DNA was prepared using the DNeasy Plant Mini Kit (manufactured by Qiagen) according to the ed manual, and a polymerase chain reaction (PCR) was performed using the prepared genomic DNA as a template. Amplification products were analyzed using the MultiNA Electrophoresis System (manufactured by Shimadzu Corporation). The tide sequences of the s are as shown below, and the positions of the primers are shown in Figures 1-1, 1-2, 1-3, and 1-4.

Forward primer 1: GAGTCTTACCACAAAGTTGGCAGT-3' (SEQ ID NO:3) Reverse primer 2: GCCCATACATGATCACAAACATGAAC-3' (SEQ ID NO:4) The results of the analysis of the amplification products by electrophoresis are shown in Figure 2. Amplification products of the same size were confirmed for both onion #6 and the control variety (Figure 2). The result of directly sequencing the amplification products confirmed that the product of amplification of the genomic DNA of onion #6 as a template had the genomic DNA nucleotide sequence as set forth in SEQ ID NO:2 exhibiting an approximately 95% ty to the nucleotide sequence of SEQ ID NO:1 in the exon parts (Table 1). Of note, in the nucleotide sequence of SEQ ID NO:1, the first exon is at positions 50 to 339, the second exon is at positions 340 to 651, the third exon is at positions 652 to 935, the fourth exon is at positions 936 to 1235, and the fifth exon is at positions 1236 to 1621. In the nucleotide sequence of SEQ ID NO:2, the part corresponding to the first exon in SEQ ID NO:1 is positions 1 to 279, the part corresponding to the second exon in SEQ ID NO:1 is positions 391 to 702, the part corresponding to the third exon in SEQ ID NO:1 is positions 804 to 1087, the part corresponding to the fourth exon in SEQ ID NO:1 is positions 1188 to 1487, and the part corresponding to the fifth exon in SEQ ID NO:1 is positions 1591 to 1975.

However, the product of amplification of the c DNA of the control variety onion as a template comprised a mixture of the amplification product comprising of genomic DNA fragments of the gene for which the cDNA nucleotide sequence is the nucleotide sequence of SEQ ID NO:1 and the amplification t sing genomic DNA fragments comprising the nucleotide ce of SEQ ID NO:2, and clear results could not be obtained by direct sequencing. This result indicated that the control variety onion had, as the alliinase gene, both the alliinase gene for which the cDNA nucleotide sequence comprised the nucleotide sequence of SEQ ID NO:1 nafter referred to as "alliinase gene 1") and the alliinase gene for which the genomic DNA nucleotide sequence comprised the nucleotide sequence of SEQ ID NO:2 (hereinafter referred to as nase gene 2") and that onion #6 had alliinase gene 2 but did not have alliinase gene 1 as alliinase genes. Of note, alliinase gene 2 appears to be a pseudogene, which is not expressed, because RNA corresponding to this gene was not detected in the RNA-seq analysis of Example 1.

[Table 1] Example 3: Preparation of primers that can distinguish a tearless and non-pungent onion and a usual onion To prepare primers to detect alliinase gene 1 and alliinase gene 2 specifically, the nucleotide sequences of the alliinase genes of SEQ ID NO:1, SEQ ID NO:2, and k Accession No. AAA32639.1 were compared, and primers that specifically amplified only either one of alliinase gene 1 and alliinase gene 2 were prepared, wherein one or more tides at the 3' end of each primer are identical to only either one of the nucleotide sequence of SEQ ID NO:1 and the nucleotide sequence of SEQ ID NO:2 at the corresponding positions. As control primers, primers were ed for a part common to the nucleotide sequence of SEQ ID NO:1 and the nucleotide sequence of SEQ ID NO:2. The nucleotide sequence of each primer is shown in Table 2, and the ons of the primers are shown in Figures 7-1, 7-2, 8-1, and 8-2.

[Table 2] A mixture of UF3 primers (SEQ ID NO:15) that comprised T or C at the position of Y were used.

PCR was performed on the genomic DNAs of onion #6 and the control variety prepared in Example 2 as templates using the prepared primers, and amplification products were analyzed using the MultiNA Electrophoresis System (manufactured by Shimadzu Corporation). The results are shown in Figures 3-1 and 3-2. Combinations of the forward primer and the reverse primer used are shown in Figures 3-1 and 3-2.

When the genomic DNA of onion #6 was used as a template, ication products corresponding to the nucleotide ce of SEQ ID NO:2 were obtained, and when the genomic DNA of the control y was used as a template, both amplification products corresponding to the nucleotide sequence of SEQ ID NO:1 and amplification products corresponding to the nucleotide sequence of SEQ ID NO:2 were ed. From these s, it was ded that whether an onion was a tearless onion or a control variety could be discriminated by whether alliinase gene 1 comprising the nucleotide sequence of SEQ ID NO:1 could be detected, and that primers achieving the discrimination could be prepared.

Furthermore, to m the versatility of the above-described primers, the effectiveness of the above-described primers was investigated using the genomic DNAs of 14 commercially available onion varieties shown in Table 2, the above-described control variety, and onion #6 as templates. The conditions for PCR were the same as the above-described conditions.

The results are shown in Figure 4. The lane numbers in Figure 4 correspond to the onion variety numbers in Table 3. Lanes 1 to 14 show the analysis results for commercially available varieties, lane 15 is the analysis results for the above-described control variety, and lane 16 is the analysis results for onion #6. The results shown in Figure 4 med that the 14 commercially available varieties and the control variety had alliinase gene 1 and ase gene 2, and that onion #6 had alliinase gene 2 but did not have alliinase gene 1. These results support that the method of determining onion #6 using the above-described primers can be applied widely independently of the variety.

[Table 3] Example 4: Use of the discrimination method The above-described primers can be used to select a tearless and ngent onion which is incorporated with useful traits from progenies obtained by crossing the tearless and non-pungent onion #6 and a second onion having the useful traits by a common crossing method.

A long-day onion having a good bulb size was crossed with onion #6. Specifically, onion #6 and a male sterile long-day onion having a trait of a good bulb size (HTA that is not a commercially available variety) were d by a tional method in Hokkaido to obtain F1 seeds. The F1 seeds were seeded, and bulbs of the F1 onions (F1 bulbs) were harvested by the conventional planting method in Hokkaido. The harvested F1 bulbs were selfreproduced and cultivated to obtain F2 seeds. The obtained F2 seeds were cultivated by the conventional method to obtain bulbs of the F2 onions (F2 bulbs). Onion selection was performed by sampling leaves of 4249 bulbs in the s of cultivating the F2 bulbs, extracting genomic DNAs, and performing PCR using the genomic DNAs as templates and three primer sets of SF2-SR1, SF1-SR1, and 1 among the above-described s.

Then, F2 bulbs were selected for which amplification ts corresponding to alliinase gene 2 were obtained, and amplification products corresponding to alliinase gene 1 were not obtained. The F2 bulbs selected according to this ion are presumed to be tearless and non-pungent onion bulbs. As a , 866 onion bulbs were selected from 4249 bulbs.

Figure 5 shows one example of the detection results for the amplification products of PCR using genomic DNAs from leaves of a plurality of F2 onions as templates and SF2-SR1 as a primer set. The upper column of Figure 5 shows the names of the F2 onion samples.

To confirm the identity between the selection results by PCR and the phenotypes, sensory evaluation was med, and the ty of pyruvic acid, which is an indicator of a tear-inducing property and a pungent taste, was measured for 94 F2 bulbs of the F2 onions for which the amplification product of alliinase gene 2 was obtained in the described detection, and the amplification product of alliinase gene 1 was not obtained (referred to as "selected bulbs") and for 30 F2 bulbs of the F2 onions for which both the amplification products were obtained (referred to as "non-selected bulbs"). The quantity of pyruvic acid produced was measured using the method disclosed in Patent Literature 1. The measurement results for the quantity of pyruvic acid produced are shown in Figure 6. The results of sensory evaluation showed that a tear-inducing property and a pungent taste were not sensed in any of the selected bulbs. Additionally, the quantity of pyruvic acid ed in the selected bulbs was approximately 1 mmol/g, which was comparable with the ty of pyruvic acid produced in the tearless and non-pungent onion disclosed in JP Patent No. 5671657.

However, a tear-inducing property and a pungent taste were sensed in the non-selected bulbs in sensory evaluation, and the quantity of c acid produced were more than 6 mmol/g.

The above results t that the discrimination method of the present invention can accurately select traits of not inducing tears and having no pungent taste.

Industrial Applicability The present invention can be used to discriminate an onion with no pungent taste in the fields such as agriculture and food manufacturing.

All publications, patents, and patent applications cited in this specification are incorporated fully herein by reference.

The present invention has been ned in detail, but it will become nt to those skilled in the art that various modifications can be made without departing from the scope of the present ion, and the present invention is not limited to the description in this specification.

Claims [Claim 1] A method of discriminating traits of an onion, comprising: a first determination step comprising determining the presence of a first mutation site in a nucleic acid derived from the onion, said mutation site comprising one or more nucleotides selected from nucleotides at positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1, n the onion is discriminated to be an onion with no t taste and/or tear-inducing property if the presence of said first mutation site is not determined in the first determination step.

[Claim 2] The method ing to claim 1, wherein the first determination step comprises: performing a first nucleic acid amplification reaction on a genomic DNA or a cDNA of the onion as a template using a first primer set comprising a first primer comprising a polynucleotide consisting of a first nucleotide sequence or consisting of the first nucleotide sequence at the 3' end and a further nucleotide sequence having 20 or fewer nucleotides linked to the first nucleotide sequence at the 5' end f, wherein the first tide sequence is i) identical to, or ii) identical in a region of 3 nucleotides at the 3' end and homologous in the remaining region on the 5' end, to a first partial nucleotide sequence of 17 to 50 utive nucleotides comprised in the nucleotide sequence of SEQ ID NO:1, the first partial nucleotide sequence comprising one or more first nucleotides selected from nucleotides at ons 94, 130, 1312, 1348, 187, 358, 791, and 1467 within 2 nucleotides from the 3' end, wherein the homologous nucleotide sequence of the remaining region in ii) above is obtained by ng, tuting, adding, and/or inserting one or two nucleotides in the first partial nucleotide sequence; and a second primer comprising a polynucleotide consisting of a second nucleotide sequence or consisting of the second nucleotide sequence at the 3' end and a further nucleotide sequence having 20 or fewer tides linked to the second nucleotide sequence at the 5' end thereof, wherein the second nucleotide sequence is iii) cal to, or iv) identical in a region of 3 nucleotides at the 3' end and homologous in the remaining region on the 5' end, to a second partial nucleotide sequence of 17 to 50 consecutive nucleotides comprised in a complementary nucleotide ce to the nucleotide sequence of SEQ ID NO:1, the second partial nucleotide sequence comprising one or more nucleotides mentary to one or more second nucleotides selected from nucleotides at positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 within 2 nucleotides from the 3' end, wherein the homologous nucleotide sequence of the remaining region in iv) above is obtained by deleting, substituting, , and/or inserting one or two nucleotides in the second l nucleotide sequence; and confirming an amplification t of the first nucleic acid amplification reaction, wherein said first nucleotide is positioned closer to the 5' end than said second nucleotide in the nucleotide sequence of SEQ ID NO:1.

[Claim 3] The method according to claim 1 or 2, further comprising a second determination step comprising determining the presence of a second on site in a nucleic acid derived from the onion, said mutation site comprising one or more nucleotides selected from nucleotides at positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2, wherein the onion is discriminated to be an onion with no t taste and/or tear-inducing property if the presence of said first mutation site is not determined in the first determination step and the presence of said second mutation site is determined in the second determination step.

[Claim 4] The method according to claim 3, wherein the second determination step comprises: performing a second nucleic acid amplification reaction on a c DNA of the onion as a template using a second primer set comprising: a third primer comprising a polynucleotide consisting of a third nucleotide sequence or consisting of the third nucleotide sequence at the 3' end and a further tide sequence having 20 or fewer nucleotides linked to the third nucleotide sequence at the 5' end thereof, wherein the third nucleotide ce is v) identical to, or vi) identical in a region of 3 nucleotides at the 3' end and homologous in the remaining region on the 5' end, to a third partial nucleotide sequence of 17 to 50 consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:2, the third partial tide ce comprising one or more third nucleotides selected from nucleotides at positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 within 2 nucleotides from the 3' end, wherein the homologous nucleotide sequence of the remaining region in vi) above is obtained by deleting, substituting, adding, and/or inserting one or two nucleotides in the third partial nucleotide sequence; and a fourth primer comprising a polynucleotide consisting of a fourth nucleotide sequence or consisting of the fourth nucleotide sequence at the 3' end and a r nucleotide sequence having 20 or fewer nucleotides linked to the fourth tide sequence at the 5' end thereof, wherein the fourth nucleotide ce is vii) identical to, or viii) identical in a region of 3 nucleotides at the 3' end and gous in the remaining region on the 5' end, to a fourth partial nucleotide sequence of 17 to 50 consecutive nucleotides comprised in a complementary nucleotide ce to the nucleotide sequence of SEQ ID NO:2, the fourth partial nucleotide sequence comprising one or more nucleotides mentary to one or more fourth nucleotides selected from nucleotides at positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide ce of SEQ ID NO:2 within 2 nucleotides from the 3' end, wherein the homologous nucleotide sequence of the remaining region in viii) above is obtained by deleting, substituting, adding, and/or inserting one or two nucleotides in the fourth partial nucleotide sequence; and confirming an amplification product of the second nucleic acid amplification reaction, wherein said third nucleotide is positioned closer to the 5' end than said fourth nucleotide in the nucleotide ce of SEQ ID NO:2.

[Claim 5] A method of breeding an onion with no pungent taste and/or tear-inducing property, comprising: 1) discriminating whether an onion is an onion with no t taste and/or tearinducing property by a method according to any one of claims 1 to 4; 2) using the onion discriminated to be an onion with no pungent taste and/or tear-inducing property to breed an onion; and 3) discriminating whether the onion bred in 2) above is an onion with no pungent taste and/or tear-inducing property by a method according to any one of claims 1 to 4.

[Claim 6] A primer set when used in a method of any one of claims 1 to 5, comprising: a primer comprising a first polynucleotide consisting of a first tide sequence or consisting of the first nucleotide sequence at the 3' end and a further nucleotide sequence having 20 or fewer nucleotides linked to the first tide ce at the 5' end thereof, wherein the first nucleotide sequence is i) identical to, or ii) identical in a region of 3 nucleotides at the 3' end and homologous in the remaining region on the 5' end, to a first l nucleotide sequence of 17 to 50 consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:1, the first partial nucleotide sequence comprising a first nucleotide selected from positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 within 2 nucleotides from the 3' end, wherein the homologous tide sequence of the remaining region in ii) above is obtained by ng, substituting, adding, and/or inserting one or two nucleotides in the first partial nucleotide sequence; and a primer comprising a second polynucleotide consisting of a second nucleotide sequence or consisting of the second nucleotide sequence at the 3' end and a further nucleotide ce having 20 or fewer tides linked to the second nucleotide sequence at the 5' end f, wherein the second nucleotide sequence is iii) identical to, or iv) identical in a region of 3 nucleotides at the 3' end and homologous in the remaining region on the 5' end, to a second l nucleotide sequence of 17 to 50 consecutive nucleotides comprised in a complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:1, the second partial nucleotide sequence comprising a nucleotide complementary to a second nucleotide selected from positions 94, 130, 1312, 1348, 187, 358, 791, and 1467 in the nucleotide sequence of SEQ ID NO:1 within 2 nucleotides from the 3' end, wherein the homologous nucleotide sequence of the remaining region in iv) above is obtained by deleting, substituting, adding, and/or inserting one or two nucleotides in the second partial nucleotide ce, and wherein the first nucleotide is positioned closer to the 5' end than the second nucleotide in the tide sequence of SEQ ID NO:1.

[Claim 7] A primer set when used in a method of claim 3 or 4, sing: a primer comprising a first polynucleotide consisting of a first tide sequence or ting of the a first nucleotide sequence at the 3' end and a further nucleotide sequence having 20 or fewer nucleotides linked to the first nucleotide sequence at the 5' end thereof, wherein the first nucleotide sequence is v) identical to, or vi) identical in a region of 3 nucleotides at the 3' end and homologous in the remaining region on the 5' end, to a first partial nucleotide sequence of 17 to 50 consecutive nucleotides comprised in the nucleotide sequence of SEQ ID NO:2, the first l nucleotide sequence comprising a first nucleotide selected from ons 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 within 2 nucleotides from the 3' end, wherein the homologous nucleotide sequence of the remaining region in vi) above is obtained by deleting, substituting, adding, and/or inserting one or two nucleotides in the first partial tide sequence; a primer comprising a second polynucleotide consisting of a second nucleotide sequence or consisting of the second nucleotide sequence at the 3' end and a further tide ce having 20 or fewer tides linked to the second nucleotide sequence at the 5' end thereof, wherein the second tide sequence is vii) identical to, or viii) identical in a region of 3 nucleotides at the 3' end and homologous in the remaining region on the 5' end, to a second partial nucleotide sequence of 17 to 50 consecutive nucleotides comprised in a complementary nucleotide sequence to the nucleotide sequence of SEQ ID NO:2, the second partial nucleotide sequence comprising a tide complementary to a second nucleotide selected from positions 34, 70, 1667, 1703, 127, 409, 943, and 1822 in the nucleotide sequence of SEQ ID NO:2 within 2 nucleotides from the 3' end, wherein the homologous nucleotide sequence of the remaining region in viii) above is obtained by deleting, substituting, adding, and/or ing one or two nucleotides in the second partial nucleotide sequence; and wherein the first nucleotide is positioned closer to the 5' end than the second nucleotide in the nucleotide sequence of SEQ ID NO:2.

[Claim 8] A kit for discriminating traits of an onion when used in a method of claim 3 or 4, comprising: the primer set according to claim 6; and the primer set according to claim 7.

SEQUENCE LISTING <110> House Foods Group Inc. <120> A method for distinguishing onions <130> PHPCT <150> JP 2017-184019 <151> 201725 <160> 22 <170> PatentIn version 3.5 <210> 1 <211> 1663 <212> DNA <213> Allium cepa <400> 1 ccgagattac aagtggagca ttaaatatcc atagcagagc taattagcta tggagtctta 60 ccacaaagtt ggcagtaata aaatgccaag ccttcttatt tgca taatcatgtc 120 tgtt aacaataata tagctcaagc gaaggtgaca tggagtttga aggcagcaga 180 agaggcagaa gcagtggcta actg gcat gggagagctt ttttggacgg 240 aattctttca tctc ctaaatgcga gtgcaatact tgctacactg gtgcagactg 300 ctctgaaaag attacaggtt gctctgctga tgttgccagt ggtgatggac tgtttctaga 360 agaatactgg cagcagcaca aggaaaacag tgcagtgctg gtttcaggat ggcacagaat 420 cttt ttcaacccag ttagcaattt catatctttc gagcttgaaa aaacaattaa 480 agaactacat gagatagtcg gaaatgctgc tgcaaaggac aggtacattg tgtttggagt 540 aggggtgact caactcatcc atggattggt tctt tcaccaaata tgactgccac 600 tccttgtgca ccacaatcta aagttgttgc tcatgcccct tattatccgg tgtttagaga 660 1/10 acaaacaaag tactttgaca agaaagggta cgagtggaaa ggaaatgcag cggattacgt 720 ttca actccagagc aattcattga gatggttact aata acccagaagg 780 tcgc catgaagtaa taaagggatg caaatccatc tacgatatgg tttactactg 840 gcctcattac accccaatca agtacaaagc cgatgaagat atcatgttgt ttacaatgtc 900 taaatacact ggacactctg gtagtcgatt tgggtgggca ctgataaagg atgaaactgt 960 gtataataaa ttgttgaatt acatgacaaa gaacacggag ggcacttccc caca 1020 gctacgatcg ctcaaaattc taaaagaagt tatagcaatg gttaaaacac agaaaggcac 1080 catgcgcgac ctcaacacat ttca gaaactaaga gagaggtggg taaatatcac 1140 ttcattgctc gataaatccg acagattctc ctatcaaaag cttccacaaa gtgaatattg 1200 caattacttc aggagaatga gacctccatc ttat gcatgggtga agtgtgaatg 1260 ggaagaagac aaagattgct accagacatt tcaaaatggg cgtatcaata cgcaaagtgg 1320 agagggtttc ggta gtcgttatgt gcgtttgagt ttgatcaaga caaaagatga 1380 ttttgatcaa ctaatgtact atttgaagaa tatggttgaa gcaaagagga agactcctct 1440 catcaaacaa ctttccaatg atcagatctc ccgccgtcct ttcatttaag tactcatgtt 1500 atgtattgct ctgctgtttt gtat gactatgttc atacatccta tggt 1560 agtaaggagt atctttctat gcaataaata aagttcatgt ttgtgatcat gtatgggcta 1620 ctatgatttt ataataaaat caattttcat ataaaaaaaa aaa 1663 <210> 2 <211> 1975 <212> DNA <213> Allium cepa <400> 2 2/10 ccacaaagtt ggcagtagta aaatgccaag cctacttatt ttgatatgca taatcatgtc 60 ttcatttgtc aacaataata tagctcaagg gaaggtgaca tggagtttga aggcagcaga 120 agaggcggag gcagtggcca atataaactg ttcagggcat ggaagagctt ttttggatgg 180 aattctttca gatggctctc ctaaatgcga tact tgctacactg gtgcagattg 240 ctctcaaaag attacaggtt gctctgcgga tgttgccagg ttaatatttc tctgttcttc 300 acaatacatg gtagtttaac tcaa acacactgga caatatttaa tgacatgctt 360 aaggaattga atgatatatt gtatacacag tgstgatgga ctgwttcttg aggaatattg 420 gcagcascac maggaaaaca gtgcagtgct cgtttcagga tggcacagaa tgagctactt 480 tttcaaccca gytagcaatt tcatatcgtt cgagcttgaa aaaacaatta aggaactaca 540 tgagatagtc ggaaatgctg ctgcaaagga caggtacatt gtgtttggag tgac 600 tcaactcatc catggattgg tcatctytct ttcaccaaat atgactgcca ctccttgtgc 660 accacaatct aaagttgttg ctcatgcccc tttttatccg gtaactctac gcatgtttct 720 aact acctagagat aactgtttat ttcttatgta tgac tgacttaatt 780 tgaacaaatt aaatgataca ggtgttcaga gaacaaacaa aatattttga aggg 840 tacgagtgga aaggaaatgc agcgaattac gtgaacactt caactcctga gcagttcatt 900 gagatggtta ctaa agaa ggtctgcttc aagt aatcaaggga 960 tgcaaatcca tttacgatat ggtttactac tggcctcatt acaccccaat caagtacaaa 1020 gccgacgaag tgtt gtttacaatg tctaaataca ctggacactc tggtagtcga 1080 tttgggtatg tccacatatt attacctcac ctct acctataatt aacatattta 1140 agttggttag ttagtaactc atactttaat atcttattaa attaggtggg cgttgataaa 1200 ggatgaaacc gtgtataata waaa ttacatgaca aaaaacacgg aaggcactcc 1260 3/10 tcgggaaaca cagctacgat cgctcaaaat tmtaaaagaa gtcatagcaa tggttaaaac 1320 aggc accatgcgtg acctcaacac atttggtttt aagaaactaa gagagaggtg 1380 ggtaaatatc actgcattgc tggataaatc agacagattc tcctatcaaa caca 1440 aagtgaatat tgcaattact tccgaagaat gagacctcca tccccatgta ccag 1500 atca tttattgaaa gatataatat tatagattat aaacataaca atgyagcatt 1560 aatcaatgat atatatatac acgatgacag cttatgcatg ggtgaagtgt gaatgggaag 1620 aagacaaaga ttgctaccag acatttcaaa atggacgyat caacacacaa agtggagagg 1680 gtttcgaagc gggcagtcgt cgty tgagtttgat caagacaaag gatgattttg 1740 atcaactcat gtactatttg aagactatgg ttgaagcaaa gaggaagact cctctcatca 1800 aacaactttc caatgatcag acatcccgcc gtcctttcat ttaagtactc atgttatgta 1860 tcgctcgctg ttttgttagt gtatgactat acat cctaatgcta tggtcgtaag 1920 gagttcctat ctttgtaata aataaagttc atgtttgtga tcatgtatgg rctac 1975 <210> 3 <211> 27 <212> DNA <213> Artificial <220> <223> Primer <400> 3 atggagtctt aagt tggcagt 27 <210> 4 <211> 29 <212> DNA <213> Artificial 4/10 <220> <223> Primer <400> 4 gtagcccata catgatcaca aacatgaac 29 <210> 5 <211> 25 <212> DNA <213> Artificial <220> <223> Primer <400> 5 tggcagtaat aaaatgccaa gcctt 25 <210> 6 <211> 25 <212> DNA <213> Artificial <220> <223> Primer <400> 6 atgcataatc atgtcttcat ttgtt 25 <210> 7 <211> 30 <212> DNA <213> Artificial <220> <223> Primer <400> 7 tttc aaaatgggcg tatcaatacg 30 /10 <210> 8 <211> 29 <212> DNA <213> Artificial <220> <223> Primer <400> 8 gagagggttt cgaagcaggt agtcgttat 29 <210> 9 <211> 25 <212> DNA <213> Artificial <220> <223> Primer <400> 9 tata ttagccactg cttct 25 <210> 10 <211> 23 <212> DNA <213> Artificial <220> <223> Primer <400> 10 tgtgctgctg ccagtattct tct 23 <210> 11 <211> 25 <212> DNA <213> Artificial 6/10 <220> <223> Primer <400> 11 atttgcatcc ctttattact tcatg 25 <210> 12 <211> 27 <212> DNA <213> Artificial <220> <223> Primer <400> 12 aaat gaaaggacgg cgggaga 27 <210> 13 <211> 25 <212> DNA <213> Artificial <220> <223> Primer <400> 13 tggcagtagt aaaatgccaa gccta 25 <210> 14 <211> 25 <212> DNA <213> Artificial <220> <223> Primer <400> 14 atgcataatc atgtcttcat ttgtc 25 7/10 <210> 15 <211> 30 <212> DNA <213> cial <220> <223> Primer <400> 15 cagacatttc aaaatggacg yatcaacaca 30 <210> 16 <211> 29 <212> DNA <213> Artificial <220> <223> Primer <400> 16 gagagggttt cgaagcgggcag tcgttac 29 <210> 17 <211> 25 <212> DNA <213> Artificial <220> <223> Primer <400> 17 acagtttata ttggccactg cctcc 25 <210> 18 <211> 23 <212> DNA <213> Artificial 8/10 <220> <223> Primer <400> 18 ggtggtgctg ccaatattcc tca 23 <210> 19 <211> 29 <212> DNA <213> Artificial <220> <223> Primer <400> 19 atggatttgc atcccttgat tacttcctt 29 <210> 20 <211> 27 <212> DNA <213> Artificial <220> <223> Primer <400> 20 gtacttaaat gaaaggacgg cgggatg 27 <210> 21 <211> 28 <212> DNA <213> cial <220> <223> Primer <400> 21 atatggttta ctactggcct cattacac 28 9/10 <210> 22 <211> 26 <212> DNA <213> cial <220> <223> Primer <400> 22 ccattcacac ttcacccatg cataag 26 /10 Forward primer 1 1 Fig. 1－ 39.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 1/14 2 Fig. 1－ AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 39.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 2/14 3 Fig. 1－ 39.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 3/14 e primer 2 4 Fig. 1－ AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 AAA32639.1 SEQ ID NO:1 SEQ ID NO:2 4/14 Fig. 2 (UM) 2500 1600 1200 1000 800 #6 l variety /14 Control #6 F3-R3 F3-R3 Control #6 SF2-SR3 UF2-UR3 Control #6 2 UF2-UR2 Control #6 SF2-SR1 UF2-UR1 Control #6 SF1-SR3 UF1-UR3 Control #6 SF1-SR2 UF1-UR2 Control 1 R -S 1 S F UF1-UR1 1 M Fig. 3－ 1500 800 600 500 400 300 200 100 (LM) 1500 800 600 500 400 300 200 100 (LM) 6/14 Fig. 3-2 M l #6 Control #6 (UM) SF3-SR4 SF4-SR4 (LM) (UM) UF3-UR4 UF4-UR4 (LM) 7/14 #6 16 7 8 9101112131415 6 UF2-UR3 4 5 3 2 #6 1 8 9101112131415 16 6 7 UF1-UR1 ication specific to alliinase gene 2 4 5 3 2 #6 1 8 9101112131415 16 7 6 SF2-SR3 4 3 #6 2 1 8 9101112131415 16 7 SF1-SR1 Amplification specific to alliinase gene 1 5 6 4 3 1 2 Fig. 4 1500 800 600 500 400 300 200 100 (LM) 8/14 (40) D5:36 (39) C5:35 (38) B5:34 (37) A5:33 (36) H4:32 (35) G4:31 (34) F4:30 (33) E4:29 (32) D4:28 (31) C4:27 (30) B4:26 (29) A4:25 (28) H3:24 (27) G3:23 (26) F3:22 (25) E3:21 (24) D3:20 (23) C3:19 (22) B3:18 (21) A3:17 (20) H2:16 (19) G2:15 (18) F2:14 (17) E2:13 (16) D2:12 (15) C2:11 (14) B2:10 (13) A2:9 (12) H1:8 (11) G1:7 (10) F1:6 (9) E1:5 (8) D1:4 (7) C1:3 (6) B1:2 (5) A1:1 Fig. 5 (1) 5bp [bp] (UM) 450 350 275 225 175 150 125 100 75 50 25 (LM) 9/14 Fig. 6 ed bulb Non-selected bulb /14 Fig. 7-1 SEQ ID 番号1 ccgagattac aagtggagca ttaaatatcc atagcagagc taattagct a tggagtctta 60 ccacaaagtt ggcagt aata aaatgccaag ccttcttatt ttgatatgca taatcatgtc 120 Forward primer ﾞﾌﾟﾗｲﾏｰ1 SF1 ttcatttgtt aacaataata tagctcaagc gaaggtgaca tggagtttga aggcagcaga 180 agaggcagaa gcagtggcta atataaactg gcat gggagagctt acgg 240 aattctttca gatggatctc ctaaatgcga gtgcaatact actg gtgcagactg 300 aaag attacaggtt gctctgctga tgttgccagt ggtgatggac tgtttctaga 360 agaatactgg cagcagcaca aggaaaacag tgcagtgctg gtttcaggat ggcacagaat 420 gagctacttt ttcaacccag ttagcaattt catatctttc gagcttgaaa aaacaattaa 480 acat gagatagtcg gaaatgctgc tgcaaaggac aggtacattg gagt 540 aggggtgact caactcatcc atggattggt catctctctt tcaccaaata tgactgccac 600 tccttgtgca ccacaatcta aagttgttgc tcatgcccct tattatccgg gaga 660 acaaacaaag tactttgaca agaaagggta cgagtggaaa gcag cggattacgt 720 gaacacttca actccagagc aattcattga gatggttact tcacctaata acccagaagg 780 tctgcttcgc catgaagtaa taaagggatg caaatccatc tacg atatgg tttactactg 840 gcctcattac ac cccaatca agtacaaagc cgatgaagat atcatgttgt ttacaatgtc 900 taaatacact ggacactctg gtagtcgatt tgggtgggca ctgataaagg atgaaactgt 960 gtataataaa aatt acatgacaaa gaacacggag ggcacttccc gagaaacaca 1020 gctacgatcg ctcaaaattc taaaagaagt tatagcaatg gttaaaacac agaaaggcac 1080 catgcgcgac ctcaacacat ttggttttca gaaactaaga gagaggtggg taaatatcac 1140 ttcattgctc gataaatccg acagattctc ctatcaaaag caaa gtgaatattg 1200 11/14 Fig. 7-2 SEQ ID NO:1配列番号1 caattacttc aggagaatga catc cccat cttat gcatgggtga agtgtgaatg 1260 ggaagaagac aaagattgct accagacatt tcaaaatggg cgtatcaata cgcaaagt gg 1320 agagggtttc gaagcaggta gtcgttatgt gcgtttgagt ttgatcaaga caaaagatga 1380 tcaa ctaatgtact atttgaagaa tatggttgaa gcaaagagga agactcctct 1440 catcaaacaa ctttccaatg atcagatctc ccgccgtcct ttcatttaag tactcatgtt 1500 tgct ctgctgtttt gttagtgtat gactatgttc atacatccta atgctatggt 1560 agtaaggagt atctttctat gcaataaata aa gttcatgt ttgtgatcat gcta 1620 ﾘﾊﾞｰｽﾌﾟﾗｲﾏｰ2Reverse primer 2 ctatgatttt ataataaaat caattttcat ataaaaaaaa aaa 1663 12/14 Fig. 8-1 SEQ ID NO:2配列番号2 ccacaaagtt ggcagt agta aaatgccaag cctacttatt ttgatatgca taatcatgtc 60 Forward primer 1ﾌｫﾜｰﾄﾞﾌﾟﾗｲﾏｰ1 UF1 ttcatttgtc aacaataata tagctcaagg gaaggtgaca tggagtttga aggcagcaga 120 agaggcggag gcagtggcca atataaactg ttcagggcat ggaagagctt ttttggatgg 180 aattctttca gatggctctc ctaaatgcga gtgcaatact tgctacactg gtgcagattg 240 ctctcaaaag attacaggtt cgga cagg tttc tctgttcttc 300 acaatacatg gtagtttaac tttatatcaa acacactgga caatatttaa tgacatgctt 360 aaggaattga atgatatatt gtatacacag tgga ctgwttcttg aggaatattg 420 gcagcascac maggaaaaca tgct cgtttcagga tggcacagaa tgagctactt 480 tttcaaccca gytagcaatt tcatatcgtt cgagcttgaa aaaacaatta aggaactaca 540 tgagatagtc ggaaatgctg ctgcaaagga caggtacatt gtgtttggag tcggggtgac 600 tcaactcatc catggattgg tcatctytct ttcaccaaat atgactgcca ctccttgtgc 660 accacaatct aaagttgttg ctcatgcccc tttttatccg gtaactctac gcatgtttct 720 aagttgaact acctagagat aactgtttat ttcttatgta ctcgtgtgac tgacttaatt 780 tgaacaaatt aaatgataca caga gaacaaacaa aatattttga caagaaaggg 840 tacgagtgga aaggaaatgc agcgaattac gtgaacactt caactcctga gcagttcatt 900 gagatggtta cttcacctaa taacccagaa ggtctgcttc gcaaggaagt aatcaaggga 960 tgcaaatcca tttacg atat ggtttactac tggcctcatt acac cccaat caagtacaaa 1020 gccgacgaag atatcatgtt gtttacaatg taca actc tggtagtcga 1080 tatg tatt attacctcac atctttctct acctataatt ttta 1140 agttggttag ttagtaactc atactttaat atcttattaa attaggtggg cgttgataaa 1200 13/14 Fig. 8-2 SEQ ID NO:2配列番号2 ggatgaaacc gtgtataata agttgtwaaa ttacatgaca aaaaacacgg aaggcactcc 1260 tcgggaaaca cagctacgat cgctcaaaat tmtaaaagaa gtcatagcaa tggttaaaac 1320 acagaaaggc accatgcgtg acctcaacac atttggtttt aagaaactaa gagagaggtg 1380 ggtaaatatc ttgc tggataaatc agacagattc tcctatcaaa agcttccaca 1440 atat tgcaattact gaat gagacctcca tccccatgta tgtataccag 1500 tatattatca tttattgaaa gatataatat tatagattat aaacataaca atgyagcatt 1560 aatcaatgat atac acgatgacag cttatgcatg ggtgaagtgt gaatgg gaag 1620 aagacaaaga ttgctaccag acatttcaaa atggacgyat acaa agtggagagg 1680 gtttcgaagc gggcagtcgt tacgtgcgty tgagtttgat caagacaaag gatgattttg 1740 atcaactcat gtactatttg aagactatgg ttgaagcaaa gaggaagact cctctcatca 1800 aacaactttc caatgatcag acatcccgcc tcat ttaagtactc atgttatgta 1860 tcgctcgctg ttttgttagt gtatgactat gttcatacat cctaatgcta tggtcgtaag 1920 gagttcctat ctttgtaata aataaa gttc gtga tcatgtatgg rctac 1975 ﾘﾊﾞｰｽﾌﾟﾗｲﾏｰ2Reverse primer 2 14/14