Hexaploid wheat with low polyphenol oxidase activity and resistance to wheat yellow mosaic disease

Abstract

To provide hexaploid wheat with low polyphenol oxidase activity and preserving quantitative trait locus Q.Ymym for wheat yellow mosaic disease resistance, and to provide a primer set that enables determination of whether or not a Triticum plant possesses a loss-of-function Ppo-D1 gene.SOLUTION: Provided is a hexaploid wheat having a homozygous type gene which is a Ppo-D1 gene with defective or reduced function of polyphenol oxidase activity and a quantitative trait locus Q.Ymym for resistance to wheat yellow mosaic disease, on chromosome 2D.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to hexaploid wheat having low polyphenol oxidase activity and resistance to wheat yellow mosaic disease. [Background technology]

[0002] It is known that the color of dough (kneaded wheat flour) becomes dull (discolored) during the addition of water and kneading process during wheat flour processing. This phenomenon is caused by polyphenol oxidase (PPO) contained in wheat flour, and therefore there has been a demand for the development of varieties with low PPO activity. It has been reported that two genes, the Ppo-A1 gene and the Ppo-D1 gene, are the major PPO activity genes in hexaploid wheat. The Ppo-A1 gene in hexaploid wheat is broadly classified into the Ppo-A1a gene (high activity), the Ppo-A1b gene (low activity), and the Ppo-A1i gene (functionally defective) depending on the genotype. However, the Ppo-A1i gene is a recently discovered genotype and has not yet been used in breeding (Non-Patent Document 1). The Ppo-D1 gene in hexaploid wheat is broadly classified into the Ppo-D1a gene (low activity) and the Ppo-D1b gene (high activity) depending on the genotype. However, no hexaploid wheat carrying a functionally defective Ppo-D1 gene has been reported, at least in Japan. Until now, the goal for reducing PPO activity has been to combine the Ppo-A1b gene (low activity) and the Ppo-D1a gene (low activity).

[0003] Wheat yellow mosaic virus (WYMV), which is transmitted by the fungus Polymyxa graminis, is known to be one of the major diseases of hexaploid wheat. Wheat yellow mosaic virus in Japan is classified into three pathotypes, I, II, and III. Q.Ymym has been reported as a quantitative trait locus for wheat yellow mosaic disease resistance that confers resistance to all three pathotypes (Non-Patent Document 2). Because of its potent effect, Q.Ymym is one of the most valuable quantitative trait loci for wheat yellow mosaic disease resistance breeding.

[0004] The Q.Ymym and Ppo-D1b genes (high activity) are tightly linked, and it was known that prioritizing reduction of PPO activity in hexaploid wheat would result in hexaploid wheat that lacks resistance to wheat yellow mosaic disease, while prioritizing resistance to wheat yellow mosaic disease would result in hexaploid wheat with high PPO activity. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Nakamaru et al. (2023) A null alleleof the polyphenol oxidase gene Ppo-A1 in hexaploidwheat originates from tetraploid wheat., Crop Science, 63:2844-2855 DOI:10.1002 / csc2.21075 [Non-patent document 2] Kobayashi et al. (2019)Characterization of the Q. Ymym region on wheat chromosome 2D associated with wheatyellow mosaic virus resistance., Plant Breeding, 139: 93-106DOI:10.1002 / csc2.21075 Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above circumstances, the present invention aims to provide a primer set that enables determination of whether hexaploid wheat, which has lower polyphenol oxidase activity than hexaploid wheat homozygously carrying the Ppo-D1a gene and which carries the quantitative trait locus Q.Ymym for resistance to wheat yellow mosaic disease, and Triticum plants, carry a functionally defective Ppo-D1 gene. [Means for solving the problem]

[0007] The present inventors selected hexaploid wheats with low polyphenol oxidase activity and performed genetic analysis. They found that the Ppo-D1 gene had a mutation, rendering it functionally defective. They also found that crossing wheat carrying the functionally defective Ppo-D1 gene with wheat carrying the Q.Ymym quantitative trait locus for wheat yellow mosaic disease resistance could produce lines carrying both the homozygous functionally defective Ppo-D1 gene and the Q.Ymym quantitative trait locus for wheat yellow mosaic disease resistance.

[0008] The present disclosure provides, for example, the inventions described in the following [1] to [6]. [1] A homozygous Ppo-D1 gene in which the function of polyphenol oxidase activity is deficient or reduced, and which is one of the following (1), (2), or (3): Hexaploid wheat carrying the wheat yellow mosaic disease resistance quantitative trait locus Q.Ymym and on chromosome 2D. (1) A gene consisting of a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1, in which 20 or more consecutive bases are deleted, and the amino acid sequence of the encoded protein is shorter than the amino acid sequence represented by SEQ ID NO: 2; (2) A gene consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 3, or (3) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4. [2] The hexaploid wheat described in [1], wherein in the gene (1) above, the deletion of bases includes a deletion of 20 or more consecutive bases in the base sequence corresponding to positions 1576 to 1648 of SEQ ID NO: 1, and the base sequence corresponding to positions 1737 to 1739 of SEQ ID NO: 1 is a stop codon. [3] The hexaploid wheat according to [1] or [2], wherein the gene is a gene consisting of the base sequence of SEQ ID NO: 3. [4] A hexaploid wheat according to any one of [1] to [3], which has a Ppo-A1 gene homozygously on chromosome 2A that is deficient in the polyphenol oxidase activity function of any one of (6), (7), (10), or (11) below. (6) A gene consisting of the base sequence represented by SEQ ID NO: 8. (7) A gene consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 8. (10) A gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 23, or (11) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 23. [5] A primer set designed to sandwich the 1536th and 1537th bases in the base sequence represented by SEQ ID NO: 3. [6] A primer set according to [5], comprising a primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity to the base sequence of SEQ ID NO: 9, and a primer consisting of a polynucleotide containing a base sequence having 90% or more sequence identity to the base sequence of SEQ ID NO: 10. [6] A method for producing hexaploid wheat according to any one of [1] to [3]. [Effects of the Invention]

[0009] The present invention provides a primer set that enables determination of whether a hexaploid wheat possesses a Q.Ymym quantitative trait locus conferring resistance to wheat yellow mosaic disease (Wheat YMMD), which has lower polyphenol oxidase activity than hexaploid wheat homozygously possessing the Ppo-D1a gene, and whether a Triticum possesses a loss-of-function Ppo-D1 gene. Furthermore, the primer set can identify the genotype of the Ppo-D1 gene in Triticum plants. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows a portion of the alignment of the nucleotide sequences of the Ppo-D1a gene, the Ppo-D1b gene, and the Ppo-D1d gene, including the nucleotide sequence corresponding to positions 1537 to 1611 of SEQ ID NO: 5 (the nucleotide sequence of the Ppo-D1a gene). [Figure 2] FIG. 1 is a schematic diagram showing the exons and introns of the Ppo-D1a gene, the Ppo-D1b gene, and the Ppo-D1d gene, and a diagram showing a portion of the alignment of the amino acid sequences of the proteins encoded by these three genes, including the amino acid sequence corresponding to positions 435 to 467 of SEQ ID NO: 6 (the amino acid sequence of the protein encoded by the Ppo-D1a gene). [Figure 3] FIG. 1 shows the annealing positions of primers comprising the primer set according to the present embodiment in the Ppo-D1a gene, the Ppo-D1b gene, and the Ppo-D1d gene. [Figure 4] This figure shows the results of electrophoresis of amplification products obtained by PCR using DNA containing the Ppo-D1a gene, the Ppo-D1b gene, or the Ppo-D1d gene as a template and the co-dominant marker for the Ppo-D1 gene, the co-dominant marker TNAC3152CD of Q.Ymym, or the co-dominant marker for the Ppo-A1 gene. [Figure 5] 1 is a graph showing the results of measuring the PPO activity of Fukuhonoka, Fukuhonoka PPO-deficient line, Ppo-D1d-Q.Ymym recombinant line, Ppo-D1a-Q.Ymym NIL, and Ppo-D1b-Q.Ymym NIL. [Figure 6] FIG. 1 shows the annealing positions of the primers for the co-dominant marker PPO18Plus of the Ppo-A1 gene in the Ppo-A1a gene, the Ppo-A1b gene, and the Ppo-A1i gene. [Figure 7] This figure shows the results of electrophoresis of amplification products obtained by PCR using genomic DNA extracted from Ppo-A1b / Ppo-D1a-Q.Ymym NIL, Ppo-A1b / Ppo-D1b-Q.Ymym NIL, Ppo-A1b / Ppo-D1d-Q.Ymym NIL, Ppo-A1i / Ppo-D1a-Q.Ymym NIL, Ppo-A1i / Ppo-D1b-Q.Ymym NIL, and Ppo-A1i / Ppo-D1d-Q.Ymym NIL as a template, the co-dominant marker for the Ppo-D1 gene, the co-dominant marker for Q.Ymym TNAC3152CD, or the co-dominant marker for the Ppo-A1 gene PPO18Plus. [Figure 8] Graph showing the results of measuring the PPO activity of Fukuhonoka, Fukuhonoka PPO-deficient line, Ppo-A1b / Ppo-D1a-Q.Ymym NIL, Ppo-A1b / Ppo-D1b-Q.Ymym NIL, Ppo-A1b / Ppo-D1d-Q.Ymym NIL, Ppo-A1i / Ppo-D1a-Q.Ymym NIL, Ppo-A1i / Ppo-D1b-Q.Ymym NIL and Ppo-A1i / Ppo-D1d-Q.Ymym NIL. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, embodiments of the present disclosure will be described in detail.

[0012] [Hexaploid wheat carrying a homozygous Ppo-D1 gene with a null or reduced polyphenol oxidase function and a quantitative trait locus Q.Ymym for wheat yellow mosaic disease resistance on chromosome 2D] The hexaploid wheat of this embodiment has a homozygous Ppo-D1 gene that lacks or has reduced polyphenol oxidase activity (hereinafter also referred to as "PPO activity"), and a quantitative trait locus Q.Ymym (hereinafter also referred to simply as "Q.Ymym") for resistance to wheat yellow mosaic disease on chromosome 2D.

[0013] Hexaploid wheat is a plant belonging to the genus Triticum in the family Poaceae. Hexaploid wheat is an allohexaploid with a genome structure represented by AABBDD, and has a total of 21 pairs of chromosomes represented by 1A-7A, 1B-7B, and 1D-7D. Hexaploid wheat may possess the Ppo-D1 gene and the wheat yellow mosaic disease resistance quantitative trait locus Q.Ymym (hereinafter simply referred to as "Q.Ymym") on chromosome 2D, and the Ppo-A1 gene on chromosome 2A.

[0014] Examples of hexaploid wheat species include bread wheat (scientific name: Triticum aestivum, also known as common wheat) and spelt wheat (scientific name: Triticum spelta). Examples of hexaploid wheat varieties include "Fukuhonoka," "Yumechikara," and "Tamaizumi." The hexaploid wheat of this embodiment may be, for example, a line derived from these species or varieties. "Lines derived from these species or varieties" may also be the progeny of the species or varieties.

[0015] Known Ppo-D1 genes in hexaploid wheat include the Ppo-D1b gene (SEQ ID NO: 1), which exhibits high PPO activity, and the Ppo-D1a gene (SEQ ID NO: 5), which exhibits lower PPO activity compared to the Ppo-D1b gene. Figure 3 shows a conceptual diagram of the Ppo-D1b and Ppo-D1a genes. In Figure 3, rectangles in the nucleotide sequence of each gene represent exons, and solid lines represent introns. The 5' end of each gene sequence is on the left. As shown in Figure 3, the Ppo-D1b and Ppo-D1a genes have exon 1 consisting of 595 bases, exon 2 consisting of 261 bases, and exon 3 consisting of 875 bases. The Ppo-D1b and Ppo-D1a genes encode proteins consisting of 577 amino acid residues.

[0016] Known Ppo-A1 genes in hexaploid wheat include the Ppo-A1a gene (SEQ ID NO: 20), which exhibits high PPO activity; the Ppo-A1b gene (SEQ ID NO: 7), which exhibits low PPO activity; and the Ppo-A1i gene (SEQ ID NO: 8), which lacks PPO activity. Figure 6 shows conceptual diagrams of the Ppo-A1a, Ppo-A1b, and Ppo-A1i genes. In Figure 6, rectangles in the nucleotide sequence of each gene represent exons, and solid lines represent introns. The 5' end of each gene's nucleotide sequence is on the left. As shown in Figure 6, the Ppo-A1a gene has, from the 5' end, an exon consisting of 596 bases, an intron consisting of 102 bases, an exon consisting of 262 bases, an intron consisting of 125 bases, and an exon consisting of 876 bases. The Ppo-A1a gene encodes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 21. As shown in Figure 6, the Ppo-A1b gene has, from the 5' end, an exon consisting of 581 bases, an intron consisting of 293 bases, an exon consisting of 262 bases, an intron consisting of 125 bases, and an exon consisting of 876 bases. The Ppo-A1b gene encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 20. As shown in Figure 6, the Ppo-A1i gene has, from the 5' end, an exon consisting of 596 bases, an intron consisting of 102 bases, an exon consisting of 262 bases, an intron consisting of 3241 bases, and an exon consisting of 876 bases. The Ppo-A1i gene encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 23.

[0017] The Ppo-D1 gene in which the PPO activity function is deficient or reduced according to this embodiment is any one of the following genes (1), (2), and (3): (1) A gene consisting of a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1, in which 20 or more consecutive bases are deleted, and the amino acid sequence of the encoded protein is shorter than the amino acid sequence represented by SEQ ID NO: 2; (2) a gene consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 3; (3) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 4.

[0018] As used herein, a "Ppo-D1 gene lacking PPO activity" means that the protein encoded by the Ppo-D1 gene does not have PPO activity. Furthermore, as used herein, a "Ppo-D1 gene with reduced PPO activity" means that the protein encoded by the Ppo-D1 gene has lower PPO activity than the protein encoded by the Ppo-D1b gene.

[0019] As used herein, "sequence identity" refers to the percentage (%) of identical bases or amino acid residues in the entire overlapping DNA base sequence or entire amino acid sequence in optimal alignment of two DNA base sequences or amino acid sequences, which are achieved using a mathematical algorithm known in the art. EMBOSS Needle (provided by EMBL-EBI, URL: https: / / www.ebi.ac.uk / tools / psa / emboss_needle / ) can be used, for example, to create the alignment and calculate the sequence identity.

[0020] In the genes (1) to (3), the sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0021] The number of bases in the base sequences of the genes (1) to (3) may be 1900 or less, 1800 or less, 1700 or less, or 1650 or less, and the lower limit of these may be 1000, 1200, 1400, or 1600. Furthermore, the genes (1) to (3) may consist of three exons and two introns, in which case the number of bases in exon 3 may be 800 or less, 700 or less, 600 or less, or 550 or less, and the lower limit of these may be 200, 300, 400, or 500.

[0022] In the gene (1), the deletion of bases means that some nucleotide bases are missing from the gene (DNA), resulting in a change (decrease) in the number of bases. In the gene (1), 20 or more consecutive bases are deleted, but preferably 25 or more, 30 or more, 40 or more, 50 or more, 60 or more, or 70 or more consecutive bases are deleted. The upper limit of these deletions may be 300, 200, 150, 100, or 73.

[0023] The location of the nucleotide deletion is not particularly limited as long as the PPO activity of the gene (1) is lost or reduced; however, in the gene (1), the nucleotide deletion preferably includes deletion of 20 or more consecutive nucleotides in the nucleotide sequence corresponding to positions 1576 to 1648 of SEQ ID NO: 1, and more preferably deletion of all nucleotides in the nucleotide sequence. In these preferred embodiments, the nucleotide deletion may include deletion of nucleotides upstream of the nucleotide corresponding to position 1576 of SEQ ID NO: 1, deletion of nucleotides downstream of the nucleotide corresponding to position 1648 of SEQ ID NO: 1, or deletion of all nucleotides from the nucleotide corresponding to position 1649 of SEQ ID NO: 1 to the termination codon. Note that in the nucleotide sequence of the Ppo-D1b gene (SEQ ID NO: 1), bases 1576 to 1648 are present in exon 3.

[0024] In the gene (1), the deletion of bases is preferably a mutation that causes a frameshift mutation, more preferably a mutation that causes a frameshift mutation that generates a new stop codon (thymine-adenine-adenine, thymine-adenine-guanine, or thymine-guanine-adenine), and even more preferably a mutation that causes a frameshift mutation in which the base sequence corresponding to bases 1737 to 1739 of SEQ ID NO: 1 becomes the stop codon. That is, in the gene (1), an embodiment in which a stop codon generated by a frameshift mutation exists is more preferred, and an embodiment in which the base sequence corresponding to bases 1737 to 1739 of SEQ ID NO: 1 becomes the stop codon is even more preferred. Note that in the base sequence of the Ppo-D1b gene (SEQ ID NO: 1), bases 1737 to 1739 are present in exon 3.

[0025] The amino acid sequence of the protein encoded by the gene (1) is shorter than the amino acid sequence of the protein encoded by the gene consisting of the base sequence represented by SEQ ID NO: 1 (SEQ ID NO: 2, 577 amino acid residues) and the amino acid sequence of the protein encoded by the gene consisting of the base sequence represented by SEQ ID NO: 5 (SEQ ID NO: 6, 577 amino acid residues), but it is preferable that the amino acid sequence is 50 or more amino acid residues shorter than these amino acid sequences, or 100 or more amino acid residues shorter than these amino acid sequences.

[0026] As described above, the gene (1) lacks at least 20 consecutive bases, and the amino acid sequence of the protein it encodes is shorter than the amino acid sequences represented by SEQ ID NOs: 2 and 6. Therefore, the PPO activity is either absent or reduced compared to the Ppo-D1a gene.

[0027] The nucleotide sequence shown in SEQ ID NO: 3 is the nucleotide sequence of the Ppo-D1 gene of a hexaploid wheat line with low PPO activity derived from "Fukuhonoka," as described in the Examples below. FIG. 1 shows a portion of an alignment of the nucleotide sequences of the Ppo-D1 gene (SEQ ID NO: 3, designated "Ppo-D1d" in FIG. 1), the Ppo-D1a gene (SEQ ID NO: 5), and the Ppo-D1b gene (SEQ ID NO: 1) of the hexaploid wheat with low PPO activity, including the nucleotide sequence corresponding to positions 1537 to 1611 of SEQ ID NO: 5. In FIG. 1, "1537" and "1611" refer to the nucleotide numbers in SEQ ID NO: 5. The left side of each nucleotide sequence is the 5' end. As shown in FIG. 1, the Ppo-D1 gene (SEQ ID NO: 3) of the hexaploid wheat with low PPO activity lacks 73 bases, corresponding to positions 1538 to 1610 of SEQ ID NO: 5 and positions 1576 to 1648 of SEQ ID NO: 1.

[0028] The deletion of 73 bases in SEQ ID NO: 3 causes a frameshift mutation relative to the base sequences represented by SEQ ID NOs: 1 and 5. Due to this frameshift mutation, the reading frame of the base sequence from base 1537 onwards in SEQ ID NO: 3 is shifted relative to the base sequences represented by SEQ ID NOs: 1 and 5, and the thymine-guanine-adenine sequences corresponding to bases 1737 to 1739 of SEQ ID NO: 1 and bases 1699 to 1701 of SEQ ID NO: 5 become stop codons.

[0029] As described above, the nucleotide sequence of SEQ ID NO: 3 lacks 73 nucleotides, the reading frame of the nucleotide sequence from position 1537 onwards is shifted relative to the nucleotide sequences of SEQ ID NOs: 1 and 5, and the stop codon is also present. Therefore, the gene consisting of the nucleotide sequence of SEQ ID NO: 3 lacks PPO activity. Therefore, the gene (2) consisting of a nucleotide sequence that shares 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 3 lacks PPO activity or has reduced PPO activity compared to the Ppo-D1a gene.

[0030] The amino acid sequence shown in SEQ ID NO: 4 is the amino acid sequence of a protein encoded by the gene consisting of the nucleotide sequence shown in SEQ ID NO: 3, as deduced from the nucleotide sequence shown in SEQ ID NO: 3 and the positions of exons and introns in the nucleotide sequences of the Ppo-D1b gene and the Ppo-D1a gene. In SEQ ID NO: 4, the above-mentioned deletion of 73 nucleotides results in deletion of amino acid residues corresponding to amino acid residues 437 to 461 in SEQ ID NO: 2 and 437 to 461 in SEQ ID NO: 6. Furthermore, the amino acid residues from position 437 onward in SEQ ID NO: 4 correspond to the nucleotide sequence of SEQ ID NO: 3, the reading frame of which has been shifted by the frameshift mutation. Furthermore, the above-mentioned termination codon results in deletion of amino acid residues corresponding to amino acid residues 467 and onward in SEQ ID NO: 2 and SEQ ID NO: 6. Therefore, the amino acid sequence shown in SEQ ID NO: 4 has 466 amino acid residues, which is shorter than the amino acid sequences shown in SEQ ID NOs: 2 and 6, each of which has 577 amino acid residues.

[0031] Furthermore, as described above, the amino acid sequence of SEQ ID NO: 4 lacks amino acid residues due to the deletion of the above-mentioned 73 bases, and the amino acid residues from position 437 onwards are amino acid residues corresponding to the base sequence of SEQ ID NO: 3, the reading frame of which has been shifted by the frameshift mutation, and furthermore, the amino acid residues corresponding to the amino acid residues from position 467 onwards in SEQ ID NO: 2 and SEQ ID NO: 6 have been deleted by the above-mentioned stop codon, so the gene encoding the protein consisting of the amino acid sequence of SEQ ID NO: 4 lacks PPO activity. Therefore, the gene (3) encoding the protein consisting of an amino acid sequence sharing 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 4 lacks PPO activity or has reduced PPO activity compared to the Ppo-D1a gene.

[0032] If the base sequence of any of the genes (1) to (3) lacks 50 or more, 60 or more, or 70 or more consecutive bases relative to the base sequence of SEQ ID NO: 1, if the reading frame of the base sequence corresponding to the base sequence from position 565 onwards, 1160 or more, 1223 or more, or 1537 or more of SEQ ID NO: 1 is shifted relative to the base sequence of SEQ ID NO: 1, or if a stop codon occurs in the base sequence corresponding to the base sequence before position 728, before position 1198, before position 1352, or before position 1739 of SEQ ID NO: 1, it can be assumed that the PPO activity function of the genes (1) to (3) is lost.

[0033] In the PpO-D1 gene, the nucleotide sequences corresponding to positions 565 to 728 and positions 1223 to 1352 of SEQ ID NO: 1 are conserved regions, and therefore these nucleotide sequences are significantly involved in PPO activity (He et al. (2007) Allelic variation of polyphenol oxidase (PPO) genes located on chromosomes 2A and 2D and development of functional markers for the PPO genes in common wheat., Theoretical and Applied Genetics 115:47-58, DOI:10.1007 / s00122-007-0539-8). Therefore, if the base sequences of the genes (1) to (3) lack at least a portion of the base sequence (conserved sequence) corresponding to bases 565 to 728 or 1223 to 1352 of SEQ ID NO: 1, if the reading frame of at least a portion of the base sequence of the conserved sequence is shifted relative to the base sequence of SEQ ID NO: 1, or if a stop codon occurs in the conserved sequence or upstream of the conserved sequence, it can be assumed that the PPO activity function of the genes (1) to (3) is lost.

[0034] As described above, the gene consisting of the base sequence shown in SEQ ID NO: 3 causes a frameshift mutation relative to the base sequences shown in SEQ ID NOs: 1 and 5. Therefore, the gene (2) consisting of a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 3 lacks the PPO activity function.

[0035] Furthermore, as described above, the amino acid sequence shown in SEQ ID NO: 4 has amino acid residues from position 437 onwards that correspond to the base sequence whose reading frame has been shifted due to the frameshift mutation. Therefore, the gene (3) that encodes a protein consisting of an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4 lacks PPO activity.

[0036] The hexaploid wheat according to the present embodiment has low PPO activity due to the loss or reduction of the PPO activity function of the Ppo-D1 gene, which it possesses homozygously. For example, if the PPO activity of the hexaploid wheat according to the present embodiment is lower than that of a control carrying the Ppo-D1b gene, the PPO activity of the hexaploid wheat according to the present embodiment can be determined to be low. For example, if the PPO activity of the hexaploid wheat according to the present embodiment is 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.8-fold or less, 0.7-fold or less, 0.6-fold or less, 0.5-fold or less, or 0.25-fold or less compared to that of the control carrying the Ppo-D1b gene, the PPO activity of the hexaploid wheat according to the present embodiment can be determined to be lower than that of the control carrying the Ppo-D1b gene.

[0037] The "control carrying the Ppo-D1b gene" is a hexaploid wheat carrying the Ppo-D1b gene in a heterozygous or homozygous state. The "control carrying the Ppo-D1b gene" may be the same hexaploid wheat as the hexaploid wheat of this embodiment, except that it carries the Ppo-D1b gene in a heterozygous or homozygous state.

[0038] The PPO activity may be evaluated by the L-DOPA method (Ito, M. et al., "Development of a Simple Method for Evaluating Polyphenol Oxidase Activity in Wheat Flour," Journal of the Crop Science Society of Japan, 77.2 (2008): 159-166). The L-DOPA method may be performed, for example, by grinding wheat seeds in a grinder or the like and passing them through a 0.4-0.6 mm sieve to obtain whole wheat flour. The whole wheat flour is then mixed with an aqueous solution containing 5-15 mM 3,4-dihydroxy-L-phenylalanine and 25-100 mM 3-morpholinopropanesulfonic acid for 5-20 minutes, followed by centrifugation to collect the supernatant, and measuring the optical density (OD value) at 475 nm of the supernatant. Specifically, PPO activity may be evaluated by the method described in the Examples below.

[0039] Hexaploid wheat carrying any one of the genes (1) to (3) on its 2D chromosome may be produced by, for example, incorporating any one of the genes (1) to (3) into the 2D chromosome of hexaploid wheat, or by converting an existing Ppo-D1 gene to any one of the genes (1) to (3). Methods for incorporating any one of the genes (1) to (3) into the 2D chromosome of hexaploid wheat and converting an existing Ppo-D1 gene to any one of the genes (1) to (3) can be performed using known methods, such as transcription activator-like nuclease (TALEN), zinc finger nuclease (ZFN), and CRISPR-Cas9 genome editing techniques. Whether or not a hexaploid wheat has any one of the genes (1) to (3) on its 2D chromosome can be confirmed using the primer set described below or by DNA sequence analysis. DNA sequence analysis may be performed, for example, by amplifying a region of hexaploid wheat genomic DNA containing the ORF of the Ppo-D1 gene by PCR. An example of a primer set capable of amplifying a region of hexaploid wheat genomic DNA containing the ORF of the Ppo-D1 gene is a primer set containing a forward primer consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 18 and a reverse primer consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 19.

[0040] Wheat yellow mosaic virus in Japan is classified into three pathotypes, types I to III, and the wheat yellow mosaic virus resistance quantitative trait locus Q.Ymym is a quantitative trait locus that confers resistance to all three pathotypes (Non-Patent Document 2). The hexaploid wheat of this embodiment possesses Q.Ymym and therefore can be resistant to wheat yellow mosaic virus types I to III. The hexaploid wheat of this embodiment preferably possesses Q.Ymym in a homozygous state.

[0041] Examples of hexaploid wheat that has Q.Ymym on chromosome 2D include wheat varieties such as "Yumechikara" and "Seto no Hohoemi," and wheat lines such as "KS831957." "Yumechikara" has Q.Ymym homozygously on chromosome 2D.

[0042] Whether or not hexaploid wheat has Q.Ymym on chromosome 2D can be confirmed using the Q.Ymym codominant marker TNAC3152CD, described in Kobayashi et al. (2019) Characterization of the Q.Ymym region on wheat chromosome 2D associated with wheat yellow mosaic virus resistance., Plant Breeding 139: 93-106, DOI: 10.1002 / csc2.21075. Confirmation using TNAC3152CD is highly reliable.

[0043] TNAC3152CD consists of four primers: a primer consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 11, a primer consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 12, a primer consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 13, and a primer consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 14. TNAC3152CD can be used to determine whether a hexaploid wheat possesses Q.Ymym based on the length of the DNA fragment amplified by PCR using TNAC3152CD. The length of the Q.Ymym-derived DNA fragment amplified by PCR is typically 720 to 780 bp, particularly 749 bp. On the other hand, when wheat varieties that do not possess Q.Ymym, such as "Fukuhonoka" and "Tamaizumi," are subjected to PCR using TNAC3152CD, DNA fragments with a length of 550 to 600 bp, particularly 579 bp, are typically amplified. When hexaploid wheat heterozygous for Q.Ymym is subjected to the above PCR method, two DNA fragments of different lengths are amplified.

[0044] The PCR method may be carried out by using genomic DNA extracted from hexaploid wheat as a template and amplifying a DNA fragment using TNAC3152CD.

[0045] Genomic DNA may be extracted from hexaploid wheat, for example, from embryos, pollen, ovules, gametes, seeds, leaves, flowers, branches, fruits, stems, roots, anthers, etc., preferably from hexaploid wheat leaves, and more preferably from hexaploid wheat leaves 1 to 3 weeks after sowing. Genomic DNA may be extracted by conventional methods, for example, the potassium acetate method (Dellaporta et al. (1983) "A plant DNA minipreparation: version II," Plant molecular biology reporter 1., 19-21, DOI: 10.1007 / BF02712670).

[0046] In PCR, the composition of the reaction solution, the temperature and reaction time of the temperature cycle conditions, etc. when carrying out the DNA amplification reaction may be appropriately determined by a person skilled in the art taking into consideration the Tm values ​​of the primers, the specifications of the equipment used, etc.

[0047] The reaction solution may contain, for example, genomic DNA extracted from TNAC3152CD and hexaploid wheat, as well as DNA polymerase, deoxynucleoside triphosphates (dNTPs: dATP, dTTP, dCTP, and dGTP), a buffer solution, and salts such as magnesium chloride. Alternatively, the reaction solution may be prepared using genomic DNA extracted from TNAC3152CD and hexaploid wheat and a commercially available kit such as Hotstar Taq Plus (Qiagen), TaKaRa Ex Taq Hot Start Version (TaKaRa Bio), or Quick Taq HS DyeMix (Toyobo) according to the manufacturer's instructions. The concentration of the forward primer or reverse primer in the reaction solution may be 0.1 to 0.3 μM. The concentration of genomic DNA extracted from hexaploid wheat in the reaction solution may be 1 to 3 ng / μL.

[0048] For example, when Quick Taq HS DyeMix (Toyobo) is used, the temperature cycle conditions may be such that 20 to 50 cycles are performed, each cycle consisting of an initial denaturation step at 90°C to 98°C for 1 to 4 minutes, a denaturation step at 90°C to 98°C for 15 seconds to 1 minute, an annealing step at 55°C to 65°C for 15 seconds to 1 minute, and an extension step at 60°C to 75°C for 30 seconds to 2 minutes.

[0049] The length of the DNA fragment amplified by PCR can be confirmed, for example, by subjecting the DNA fragment to agarose gel electrophoresis and comparing it with a molecular weight marker.

[0050] The hexaploid wheat of this embodiment may have a Ppo-A1 gene with a defective or reduced PPO activity function on chromosome 2A in a homozygous state. In this case, the PPO activity can be further reduced. Examples of a Ppo-A1 gene with a defective or reduced PPO activity function include a Ppo-A1 gene with a defective or reduced PPO activity function that falls under any of the following (4) to (7), or a Ppo-A1 gene with a defective or reduced PPO activity function that falls under any of (4) to (11). A Ppo-A1 gene with a defective PPO activity function that falls under any of (6), (7), (10), or (11) is preferred, and a Ppo-A1 gene with a defective PPO activity function that falls under any of (6) or (10) is more preferred. (4) Ppo-A1b gene (SEQ ID NO: 7), i.e., a gene consisting of the base sequence represented by SEQ ID NO: 7; (5) A gene consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 7. (6) Ppo-A1i gene (SEQ ID NO: 8), i.e., a gene consisting of the base sequence represented by SEQ ID NO: 8; (7) A gene consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 8. (8) A gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 22. (9) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22. (10) A gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 23, or (11) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 23.

[0051] In the genes of (5), (7), (9), and (11), the sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0052] Examples of hexaploid wheat that homozygously carries the Ppo-A1b gene on chromosome 2A include wheat varieties such as "Fukuhonoka" and "Ginga no Chikara."

[0053] Examples of hexaploid wheat that homozygously carries the Ppo-A1i gene on chromosome 2A include wheat varieties such as "Norin 27," "Norin 53," and "Nanbukomugi."

[0054] Hexaploid wheat carrying any one of the genes (4) to (11) on chromosome 2A may be produced, for example, by incorporating any one of the genes (4) to (11) into chromosome 2A of hexaploid wheat, or by modifying an existing Ppo-A1 gene to any one of the genes (4) to (11). Methods for incorporating any one of the genes (4) to (11) into chromosome 2A of hexaploid wheat and methods for modifying an existing Ppo-D1 gene to any one of the genes (4) to (11) can be performed using known methods, including, for example, genome editing techniques such as transcription activator-like nuclease (TALEN), zinc finger nuclease (ZFN), and CRISPR-Cas9.

[0055] Whether hexaploid wheat has any of the genes (4) to (11) on chromosome 2A can be confirmed using the codominant marker PPO18Plus for the Ppo-A1 gene described in Nakamaru et al. (2023) "A null allele of the polyphenol oxidase gene Ppo-A1 in hexaploid wheat originates from tetraploid wheat." Crop Science, 63:2844-2855, DOI: 10.1002 / csc2.21075, or by DNA sequence analysis. The codominant marker PPO18Plus for the Ppo-A1 gene consists of a forward primer (PPO18F) consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 15, a reverse primer (PPO18R) consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 16, and a reverse primer (PPO18R3) consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 17. A schematic diagram of the annealing positions of each primer of the co-dominant marker PPO18Plus to the Ppo-A1a gene, Ppo-A1b gene, and Ppo-A1i gene is shown in Figure 6. As shown in Figure 6, the reverse primer (PPO18R3) consisting of the polynucleotide having the nucleotide sequence of SEQ ID NO: 17 anneals to the Ppo-A1i gene but does not anneal to the Ppo-A1a gene or Ppo-A1b gene. Using these co-dominant markers, the genotype of the Ppo-A1 gene in hexaploid wheat can be identified based on the length of the DNA fragment amplified by PCR using the co-dominant markers. The length of the DNA fragment derived from the Ppo-A1a gene amplified by the PCR method is usually 670 to 700 bp, particularly 685 bp, the length of the DNA fragment derived from the Ppo-A1b gene is usually 860 to 900 bp, particularly 876 bp, and the length of the DNA fragment derived from the Ppo-A1i gene is usually 520 to 550 bp, particularly 536 bp. When hexaploid wheat heterozygously carrying the Ppo-A1 gene is subjected to the PCR method, two DNA fragments of different lengths may be amplified.

[0056] The PCR method may be performed by using genomic DNA extracted from hexaploid wheat as a template and amplifying a DNA fragment using the co-dominant marker for the Ppo-A1 gene. The template may be the same as the template used in the PCR method using TNAC3152CD described above. The composition of the reaction solution used in the DNA amplification reaction may be the same as that used in the PCR method using TNAC3152CD described above, except that the co-dominant marker for the Ppo-A1 gene is used instead of TNAC3152CD. The content of the forward primer or reverse primer for the co-dominant marker for the Ppo-A1 gene in the reaction solution may be 0.1 to 0.3 μM. For example, when using TaKaRa Ex Taq Hot Start Version (TaKaRa Bio), the temperature cycling conditions for the DNA amplification reaction may be 20 to 50 cycles, each cycle consisting of a denaturation step at 90 to 99°C for 5 to 20 seconds, an annealing step at 55 to 75°C (at a temperature 0.3°C lower than the previous cycle) for 15 seconds to 1 minute, and an extension step at 65 to 80°C for 30 seconds to 2 minutes. The length of the amplified DNA fragment can be confirmed, for example, by subjecting the DNA fragment to agarose gel electrophoresis and comparing it with a molecular weight marker.

[0057] The hexaploid wheat of this embodiment may be produced (production method 1), for example, by crossing a hexaploid wheat having any of the genes (1) to (3) on chromosome 2D with a hexaploid wheat having Q.Ymym on chromosome 2D.

[0058] Production method 1 may be performed by repeatedly crossing, backcrossing, or successive backcrossing two hexaploid wheats as the P generation, and selecting hexaploid wheats that have any one of the genes (1) to (3) or Q.Ymym in a heterozygous or homozygous state on chromosome 2D. For example, the hexaploid wheats can be produced by crossing two hexaploid wheats to obtain an F1 generation, selecting wheats that have any one of the genes (1) to (3) and Q.Ymym in a heterozygous state, and then crossing these wheats to obtain an F2 generation, and selecting wheats that have any one of the genes (1) to (3) and Q.Ymym in a homozygous state.

[0059] In production method 1, the hexaploid wheat carrying Q.Ymym on chromosome 2D may be a hexaploid wheat carrying Q.Ymym and Ppo-D1a genes on chromosome 2D in a homozygous state, from the viewpoint of facilitating the production of the hexaploid wheat of this embodiment. The hexaploid wheat may be produced by crossing a hexaploid wheat carrying Q.Ymym on chromosome 2D with a hexaploid wheat carrying the Ppo-D1a gene on chromosome 2D. The production may be performed by repeatedly crossing, backcrossing, or successive backcrossing the two hexaploid wheats as the P generation, and selecting for a hexaploid wheat carrying any of the genes (1) to (3) or Q.Ymym on chromosome 2D in a heterozygous or homozygous state.

[0060] In the production method 1, a hexaploid wheat further carrying any one of the genes (4) to (11) on chromosome 2A may be used as the former or latter hexaploid wheat. In this case, it is possible to produce a hexaploid wheat according to this embodiment that further carries a Ppo-A1 gene with a defective or reduced function on chromosome 2A in a homozygous state.

[0061] The hexaploid wheat of this embodiment can be produced by incorporating any of the genes (1) to (3) into the 2D chromosome of a hexaploid wheat that homozygously carries Q.Ymym on the 2D chromosome, or by replacing the existing Ppo-D1 gene in the hexaploid wheat with any of the genes (1) to (3) (Production Method 2-1). If the hexaploid wheat thus produced also heterozygously carries any of the genes (1) to (3) on the 2D chromosome, the hexaploid wheat can also be produced by crossbreeding the hexaploid wheat (Production Method 2-2). The methods for incorporating any of the genes (1) to (3) into the 2D chromosome of a hexaploid wheat and for replacing the existing Ppo-D1 gene with any of the genes (1) to (3) may be the same as the above-described methods for producing a hexaploid wheat that has any of the genes (1) to (3) on the 2D chromosome.

[0062] Furthermore, in the production methods 2-1 and 2-2, the hexaploid wheat homozygously carrying Q.Ymym on chromosome 2D may be a hexaploid wheat further carrying any one of the genes (4) to (11) on chromosome 2A. This hexaploid wheat may be produced by incorporating any one of the genes (4) to (11) into chromosome 2A of the hexaploid wheat homozygously carrying Q.Ymym on chromosome 2D, or by replacing the existing Ppo-A1 gene in the hexaploid wheat with any one of the genes (4) to (11). The methods for incorporating any one of the genes (4) to (11) into chromosome 2A of the hexaploid wheat and for replacing the existing Ppo-D1 gene with any one of the genes (4) to (11) may be the same as the above-described methods for producing a hexaploid wheat homozygously carrying Q.Ymym on chromosome 2A. In this way, it is possible to produce the hexaploid wheat according to this embodiment that further has a Ppo-A1 gene with a defective or reduced function on chromosome 2A in a homozygous state.

[0063] The hexaploid wheat of this embodiment may be produced by successively backcrossing and crossing the hexaploid wheat having any of the genes (1), (2), or (3) and Q.Ymym homozygously on chromosome 2D obtained as described above with a hexaploid wheat of a desired species or variety, thereby introducing a homozygous form of any of the genes (1), (2), or (3) and Q.Ymym into the hexaploid wheat of the desired species or variety. In the successive backcrossing, the hexaploid wheat having any of the genes (1), (2), or (3) and Q.Ymym homozygously on chromosome 2D is the monophasic parent, and the hexaploid wheat of the desired species or variety is the recurrent parent. The above-mentioned successive backcrossing and crossbreeding may be carried out, for example, by selecting individuals that possess either gene (1), (2), or (3) and Q.Ymym in a heterozygous state from the BC1F1 to BC5F1 generations, and then selecting individuals that possess either gene (1), (2), or (3) and Q.Ymym in a homozygous state in the BC5F2 generation.

[0064] Furthermore, if the hexaploid wheat obtained as described above has any one of the genes (1), (2), or (3) and Q.Ymym homozygously on chromosome 2D, but does not have any one of the genes (4) to (11) homozygously on chromosome 2A, the hexaploid wheat of this embodiment having any one of the genes (4) to (11) homozygously on chromosome 2A can be produced by crossing the hexaploid wheat with another hexaploid wheat having any one of the genes (4) to (11) homozygously on chromosome 2A.

[0065] In the method for producing hexaploid wheat according to the present embodiment described above, selection of hexaploid wheats carrying any one of the genes (1) to (3) in a homozygous or heterozygous state may be performed using the primer set described below or by DNA sequence analysis, and selection of hexaploid wheats carrying Q.Ymym in a homozygous or heterozygous state may be confirmed using the co-dominant marker TNAC3152CD described above, and selection of hexaploid wheats carrying any one of the genes (4) to (11) in a homozygous or heterozygous state may be performed using the co-dominant marker for the Ppo-A1 gene described above or by DNA sequence analysis.

[0066] [Primer set] The primer set according to this embodiment is designed to sandwich the 1536th and 1537th bases in the base sequence represented by SEQ ID NO: 3. The 1536th base in SEQ ID NO: 3 corresponds to the 1575th base in SEQ ID NO: 1 and the 1537th base in SEQ ID NO: 5, and the 1537th base in SEQ ID NO: 3 corresponds to the 1649th base in SEQ ID NO: 1 and the 1611th base in SEQ ID NO: 5.

[0067] The primer set allows for determining whether or not a Triticum plant possesses a functionally defective Ppo-D1 gene based on the length of the DNA fragment amplified by PCR using the primer set. Thus, the primer set may be a primer set for determining whether or not a Triticum plant possesses a functionally defective Ppo-D1 gene.

[0068] The Triticum plant is not particularly limited as long as it is a Triticum plant that can carry the Ppo-D1 gene, and may be a Triticum plant that carries the D genome or the above-mentioned hexaploid wheat.

[0069] An example of the functionally defective Ppo-D1 gene is a gene consisting of the nucleotide sequence of SEQ ID NO: 3. The nucleotide sequence of SEQ ID NO: 3 lacks bases corresponding to positions 1576 to 1648 of SEQ ID NO: 1 (i.e., bases corresponding to positions 1538 to 1610 of SEQ ID NO: 5), and therefore the DNA fragment derived from the gene consisting of the nucleotide sequence of SEQ ID NO: 3 is shorter than the DNA fragment that can be amplified from the Ppo-D1b gene or the Ppo-D1a gene.

[0070] The primer set may be any primer that can determine whether or not a Triticum plant possesses a functionally defective Ppo-D1 gene, and may be based on the nucleotide sequence of SEQ ID NO: 3. The primer set is designed to sandwich the 1536th and 1537th bases in the nucleotide sequence of SEQ ID NO: 3, but may also be designed to amplify a DNA fragment containing, for example, a region of 36 to 100 bp, 36 to 200 bp, 36 to 300 bp, 36 to 400 bp, 36 to 500 bp, 36 to 1000 bp, 36 to 2000 bp, 36 to 3000 bp, 200 to 2000 bp, or 500 to 1000 bp in the genome of a Triticum plant possessing the functionally defective Ppo-D1 gene.

[0071] The primer set may include a forward primer and a reverse primer, and the forward primer and reverse primer may be designed to anneal to the 5'-terminal nucleotide sequence and the 3'-terminal nucleotide sequence of the 1536th and 1537th bases, respectively, in the nucleotide sequence represented by SEQ ID NO: 3. The length of the forward primer and the reverse primer may be, for example, 18 to 50 bp, 18 to 35 bp, or 18 to 27 bp. The length of the forward primer and the reverse primer may be the same or different.

[0072] The primer set according to this embodiment is preferably a primer set designed to sandwich the 911th and 1615th bases in the base sequence represented by SEQ ID NO: 3, and more preferably includes a primer (hereinafter also referred to as a "first primer") consisting of a polynucleotide containing a base sequence having 90% or more sequence identity to the base sequence of SEQ ID NO: 9, and a primer (hereinafter also referred to as a "second primer") consisting of a polynucleotide containing a base sequence having 90% or more sequence identity to the base sequence of SEQ ID NO: 10. For example, it is preferable that the forward primer is the first primer and the reverse primer is the second primer.

[0073] The sequence identity between the first primer and the second primer may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. When the first primer consists of a polynucleotide consisting of the base sequence of SEQ ID NO: 9, self-complementarity is unlikely to occur. Furthermore, when the second primer consists of a polynucleotide consisting of the base sequence of SEQ ID NO: 10, self-complementarity is unlikely to occur.

[0074] A primer set designed to sandwich the 911th and 1615th bases in the base sequence represented by SEQ ID NO: 3, or a primer set including a first primer and a second primer, can be used to determine whether a Triticum plant possesses the loss-of-function Ppo-D1 gene based on the length of the DNA fragment amplified by PCR using the primer set. Furthermore, the genotype of the Ppo-D1 gene in a Triticum plant can be identified. Therefore, the primer set may be a primer set for identifying the genotype of the Ppo-D1 gene in a Triticum plant. For example, the primer set can identify whether a plant possesses a loss-of-function Ppo-D1 gene, Ppo-D1a gene, or Ppo-D1b gene in a homozygous or heterozygous state.

[0075] The primer set including the first and second primers was designed from a common nucleotide sequence among the nucleotide sequences of SEQ ID NO: 3, SEQ ID NO: 1, and SEQ ID NO: 5. Figure 3 shows the annealing positions of the primers in the primer set according to this embodiment in the nucleotide sequences of these three Ppo-D1 genes. In Figure 3, "Ppo-D1d" refers to the gene consisting of the nucleotide sequence represented by SEQ ID NO: 3, and "Primer F" and "Primer R" refer to the first and second primers, respectively. As shown in Figure 3, the first primer anneals to exon 2 of the three Ppo-D1 genes, and the second primer anneals to exon 3 of the three Ppo-D1 genes. Because intron 2 in SEQ ID NO: 1 is longer than intron 2 in SEQ ID NO: 3 and SEQ ID NO: 5, the DNA fragments amplified by PCR using the primer set derived from the nucleotide sequence of SEQ ID NO: 1 are longer than the DNA fragments derived from the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 5. Furthermore, because the first and second primers anneal to the nucleotide sequence of SEQ ID NO: 3 so as to sandwich the 73-nucleotide deletion site as described above, the DNA fragment amplified by PCR using the primer set derived from the nucleotide sequence of SEQ ID NO: 3 is shorter than the DNA fragments derived from the nucleotide sequences of SEQ ID NOs: 1 and 5. The length of the DNA fragment amplified by PCR using the primer set derived from the nucleotide sequence of SEQ ID NO: 1 is typically 794 to 830 bp, particularly 813 bp. The length of the DNA fragment derived from the nucleotide sequence of SEQ ID NO: 3 is typically 695 to 715 bp, particularly 705 bp. The length of the DNA fragment derived from the nucleotide sequence of SEQ ID NO: 5 is typically 768 to 788 bp, particularly 778 bp.

[0076] The PCR method may be carried out by using genomic DNA extracted from a Triticum plant as a template and amplifying a DNA fragment using the primer set according to this embodiment.

[0077] Genomic DNA may be extracted from Triticum plants, for example, from embryos, pollen, ovules, gametes, seeds, leaves, flowers, branches, fruits, stems, roots, anthers, etc., preferably from leaves of Triticum plants, and more preferably from leaves of Triticum plants 1 to 3 weeks after sowing. Genomic DNA extraction may be carried out by a conventional method, for example, the potassium acetate method (Dellaporta et al. (1983) "A plant DNA minipreparation: version II." Plant molecular biology reporter 1., 19-21.).

[0078] In PCR, the composition of the reaction solution, the temperature and reaction time of the temperature cycle conditions, etc. when carrying out the DNA amplification reaction may be appropriately determined by a person skilled in the art taking into consideration the Tm values ​​of the primers, the specifications of the equipment used, etc.

[0079] The reaction solution composition may contain, for example, in addition to the primer set according to this embodiment and genomic DNA extracted from a Triticum plant, DNA polymerase, deoxynucleoside triphosphates (dNTPs: dATP, dTTP, dCTP, and dGTP), a buffer solution, salts such as magnesium chloride, and the like. Alternatively, the reaction solution may be prepared using the primer set according to this embodiment, genomic DNA extracted from a Triticum plant, and a commercially available kit such as Hotstar Taq Plus (Qiagen), TaKaRa Ex Taq Hot Start Version (TaKaRa Bio), or Quick Taq HS DyeMix (Toyobo) according to the manufacturer's instructions. The content of each primer in the primer set according to this embodiment in the reaction solution may be 0.1 to 0.3 μM. The content of genomic DNA extracted from a Triticum plant in the reaction solution may be 1 to 3 ng / μL.

[0080] For example, when using Hotstar taq Plus (Qiagen), the temperature cycle conditions may be as follows: an initial denaturation step at 90°C to 98°C for 5 to 20 minutes, followed by 20 to 50 cycles of a denaturation step at 90°C to 98°C for 30 seconds to 2 minutes, an annealing step at 55°C to 65°C for 30 seconds to 2 minutes, and an extension step at 60°C to 75°C for 30 seconds to 2 minutes, and the cycle is repeated until a final extension step at 65°C to 75°C for 5 to 20 minutes.

[0081] The DNA fragments amplified by PCR can be detected by standard methods, such as agarose gel electrophoresis, fluorescent DNA probes, or fluorescent intercalators. Real-time PCR may also be used as the PCR method. [Example]

[0082] The present invention will be described in more detail below with reference to examples, but is not limited to these examples. In the following examples, the quantitative trait locus Q.Ymym for yellow mosaic disease resistance may be referred to simply as Q.Ymym, and the quantitative trait locus for yellow mosaic disease susceptibility may be referred to simply as q.ymym. The Ppo-D1a gene and the Ppo-D1b gene may be referred to simply as Ppo-D1a and Ppo-D1b, respectively. Similarly, the Ppo-A1i gene, the Ppo-A1a gene, and the Ppo-A1b gene may be referred to simply as Ppo-A1i, Ppo-A1a, and Ppo-A1b, respectively. Furthermore, the notation Ppo-D1b-Q.Ymym means that the Ppo-D1b gene and Q.Ymym are homozygous. This also applies to other Ppo genes and q.ymym. Furthermore, the notation Ppo-A1i / Ppo-D1d-Q.Ymym means that the Ppo-A1i gene, Ppo-D1d gene, and Q.Ymym gene are homozygous. This also applies to other Ppo genes and q.ymym. Wheat varieties are written in quotation marks. Furthermore, "line" refers to an experimentally produced line. A line that has undergone five consecutive backcrosses is considered a near-isogenic line (hereinafter also referred to as "NIL").

[0083] <Analysis of the Ppo-D1 gene in wheat with low PPO activity> When the inventors evaluated the polyphenol oxidase activity of synthetic wheat they possessed (hereinafter also referred to as "PPO activity"), they discovered wheat with low PPO activity in the Fukuhonoka strain (hereinafter also referred to as "Fukuhonoka Ppo-deficient strain"). The Fukuhonoka Ppo-deficient strain was created by crossing 'Fukuhonoka' with synthetic wheat 'SYN-1' having low PPO activity, performing 5 consecutive backcrosses using 'Fukuhonoka' as the recurrent parent, self-propagating in each generation of backcross (e.g., BC1F1, BC2F1, etc.) to create the F3 generation (e.g., BC1F3, BC2F3, etc.), and selecting strains that do not brown in the phenol reaction (immersed in 1% phenol solution).

[0084] To analyze the Ppo-D1 gene of the Fukuhonoka Ppo-deficient strain, genomic DNA was extracted from leaves about 2 weeks after sowing of the Fukuhonoka Ppo-deficient strain by the potassium acetate method (Dellaporta et al. (1983) A plant DNA minipreparation: version II. "Plant molecular biology reporter 1., 19-21, DOI: 10.1007 / BF02712670), diluted to 100 ng / μL, and used for PCR. PCR was performed using a Veriti 96 well thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). PCR was performed using a forward primer consisting of the nucleotide sequence of SEQ ID NO: 18, a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 19, which amplify the region containing the Open Reading Frame (ORF) of the Ppo-D1 gene, and LA taq (TaKaRa Bio, Shiga, Japan). The reaction conditions were 1 cycle of 94°C for 1 minute, 35 cycles of 94°C for 20 seconds followed by 68°C for 3 minutes, and 1 cycle of 72°C for 10 minutes. PCR amplification products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide. The target amplification products were excised under UV irradiation and recovered using AMpure (Beckman Coulter, Brea, CA, USA). The recovered target amplification products were subjected to Sanger sequencing to determine the nucleotide sequence of the Ppo-D1 gene.

[0085] The results showed that the Fukuhonoka Ppo deletion strain homozygously possesses the Ppo-D1 gene consisting of the nucleotide sequence shown in SEQ ID NO: 3. As shown in Figure 1, the nucleotide sequence of the Ppo-D1 gene possessed by the Fukuhonoka Ppo deletion strain was deleted, consisting of 73 bases corresponding to positions 1576 to 1648 of SEQ ID NO: 1, which shows the nucleotide sequence of the Ppo-D1b gene.

[0086] Furthermore, based on the nucleotide sequence shown in SEQ ID NO: 3 and the positions of exons and introns in the nucleotide sequences of the Ppo-D1b and Ppo-D1a genes, the amino acid sequence of the protein encoded by the Ppo-D1 gene carried by the Fukuhonoka Ppo-deficient strain was predicted to be the amino acid sequence shown in SEQ ID NO: 4. Figure 2 shows an alignment of the amino acid sequences of the proteins encoded by the Ppo-D1, Ppo-D1a, and Ppo-D1b genes carried by the Fukuhonoka Ppo-deficient strain (SEQ ID NOs: 4, 2, and 6, respectively), including the amino acid sequence corresponding to positions 435 to 467 of SEQ ID NO: 6. In Figure 2, "435" and "467" refer to the amino acid residue numbers in SEQ ID NO: 6. In Figure 2, an "*" indicates that a corresponding amino acid residue does not exist. Figure 2 shows that the amino acid sequence (SEQ ID NO: 4) of the protein encoded by the Ppo-D1 gene in the Fukuhonoka Ppo-deficient strain contains a frameshift mutation due to the deletion of the 73 bases, resulting in the creation of a new stop codon. Furthermore, the Ppo-D1 gene in the Fukuhonoka Ppo-deficient strain encodes 466 amino acids, which is shorter than Ppo-D1a and Ppo-D1b, which encode 577 amino acids.

[0087] These mutations indicated that the Ppo-D1 gene of the Fukuhonoka Ppo-deficient line was a loss-of-function type, and it was also shown that the loss of function of the Ppo-D1 gene contributed to the low PPO activity of the Fukuhonoka Ppo-deficient line. When Blast search was performed on NCBI (https: / / www.ncbi.nlm.nih.gov / ), the nucleotide sequence shown in SEQ ID NO: 3 matched the Ppo-D1d gene (EU371657) of Aegilops tauschii (scientific name) and had a 73-bp deletion that was the cause of the frameshift mutation, and the ORF sequences also mostly matched. Therefore, hereinafter, the Ppo-D1 gene possessed by the Fukuhonoka Ppo-deficient line is referred to as the Ppo-D1d gene (hereinafter, also simply referred to as "Ppo-D1d"). There has been no report on hexaploid wheat possessing the Ppo-D1d gene, at least in Japan so far. Also, as described later, the Fukuhonoka Ppo-deficient line was homozygous for q.ymym.

[0088] <Creation of a co-dominant marker for the Ppo-D1 gene> From the common sequences of the Ppo-D1d gene (SEQ ID NO: 3), the Ppo-D1a gene (SEQ ID NO: 5, EF070149), and the Ppo-D1b gene (SEQ ID NO: 1, EF070150) obtained by Blast search on NCBI (https: / / www.ncbi.nlm.nih.gov / ), a co-dominant marker was created so as to sandwich the deletion site of the above 73 bases in SEQ ID NO: 3. The created co-dominant marker is a primer set consisting of a forward primer (hereinafter, also referred to as "Primer F") consisting of a polynucleotide having the nucleotide sequence of SEQ ID NO: 9 and a reverse primer (hereinafter, also referred to as "Primer R") consisting of a polynucleotide having the nucleotide sequence of SEQ ID NO: 10. A schematic diagram of the annealing positions of the primer set with the Ppo-D1a gene, the Ppo-D1b gene, and the Ppo-D1d gene is shown in Figure 3.

[0089] Genomic DNA was extracted from leaves of the Fukuhonoka Ppo-deficient line (homozygous for the Ppo-D1d gene), Fukuhonoka (homozygous for the Ppo-D1a gene), and Yumechikara (homozygous for the Ppo-D1b gene) approximately 2 weeks after sowing using the codominant markers described above by the potassium acetate method (Dellaporta et al. 1983). The DNA was diluted to 100 ng / μL and used as template DNA. PCR was performed using a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). The PCR reaction mixture was prepared by combining template DNA, the codominant markers, and Hotstar Taq Plus (Qiagen) as shown in Table 1. The PCR conditions were 95°C for 5 minutes (1 cycle), 94°C for 1 minute, followed by 60°C for 1 minute, followed by 72°C for 1 minute (33 cycles), and 72°C for 10 minutes (1 cycle).

[0090] [Table 1]

[0091] Next, the PCR amplification products were separated by 2% agarose gel electrophoresis and stained with Gel Red stain (Biotium, Fremont, CA, USA). The results are shown in Figure 4. As shown in Figure 4, DNA fragments of 705 bp, 778 bp, and 813 bp were confirmed to be amplified in the Fukuhonoka Ppo deletion strains, Fukuhonoka, and Yumechikara, respectively. Therefore, by using the above codominant markers, it was possible to identify the genotype of the Ppo-D1 gene based on the length of the DNA fragment amplified by PCR.

[0092] <Confirmation of the Ppo-D1 gene, Ppo-A1 gene, and Q.Ymym genotype in wheat> In the following examples, the genotypes of the Ppo-D1 gene, Ppo-A1 gene, and Q.Ymym in wheat were confirmed by the following method. First, genomic DNA was extracted from the leaves of wheat about 2 weeks after sowing by the potassium acetate method (Dellaporta et al. 1983), diluted to 100 ng / μL, and used for PCR. PCR was performed using a Veriti 96 well thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA).

[0093] For the Ppo-D1 gene, PCR was performed in the same manner as in <Creation of a co-dominant marker for the Ppo-D1 gene>, except that the genomic DNA diluted to 100 ng / μL was used instead of the template DNA.

[0094] For the Ppo-A1 gene, PCR was performed using the codominant marker PPO18Plus for the Ppo-A1 gene described in Nakamaru et al. (2023) A null allele of the polyphenol oxidase gene Ppo-A1 in hexaploid wheat originates from tetraploid wheat., Crop Science, 63:2844-2855 DOI:10.1002 / csc2.21075, and TaKaRa Ex Taq Hot Start Version (TaKaRa Bio). The reaction conditions were 95°C for 5 minutes (1 cycle), 94°C for 30 seconds, followed by 66°C for 30 seconds (with a 0.3°C decrease each cycle), followed by 72°C for 1 minute (35 cycles), followed by 72°C for 5 minutes (1 cycle). The co-dominant marker for the Ppo-A1 gene is a primer set consisting of a forward primer (PPO18F) consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 15, a reverse primer (PPO18R) consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 16, and a reverse primer (PPO18R3) consisting of a polynucleotide with the nucleotide sequence of SEQ ID NO: 17. A schematic diagram of the annealing positions of this primer set with the Ppo-A1a gene, Ppo-A1b gene, and Ppo-A1i gene is shown in Figure 6.

[0095] For Q.Ymym, PCR was performed using the co-dominant marker TNAC3152CD of Q.Ymym described in Kobayashi et al. (2019) Characterization of the Q. Ymym region on wheat chromosome 2D associated with wheat yellow mosaic virus resistance., Plant Breeding, 139: 93-106 DOI:10.1002 / csc2.21075 and Quick Taq HS DyeMix (Toyobo), with the reaction conditions being 1 cycle of 95°C for 2 minutes, 30 seconds at 94°C, followed by 30 seconds at 60°C, and further followed by 1 minute at 68°C for 30 cycles. TNAC3152CD is a primer set consisting of four primers, namely, a primer consisting of a polynucleotide having the nucleotide sequence of SEQ ID NO: 11, a primer consisting of a polynucleotide having the nucleotide sequence of SEQ ID NO: 12, a primer consisting of a polynucleotide having the nucleotide sequence of SEQ ID NO: 13, and a primer consisting of a polynucleotide having the nucleotide sequence of SEQ ID NO: 14.

[0096] Next, the PCR amplification products were separated by 2% agarose gel electrophoresis and stained with Gel Red stain (Biotium, Fremont, CA, USA). As illustrated in Figure 4, the genotypes of the Ppo-D1 gene, Ppo-A1 gene, and Q.Ymym can be determined based on the length of the DNA fragments that are the amplification products.

[0097] <Creation of <Ppo-D1d-Q.Ymym> recombinant lines> To produce the Ppo-D1d-Q.Ymym recombinant line, we crossed the TY714-10 line (Ppo-D1a-Q.Ymym) with the Fukuhonoka Ppo-deficient line (Ppo-D1d-q.ymym). In the F2 generation, we selected individuals (Ppo-D1d-Q.Ymym) that were homozygous for Ppo-D1d and Q.Ymym. To unify the genetic background of the two NILs described below with that of Fukuhonoka, we subjected these individuals (Ppo-D1d-Q.Ymym) to repeated backcrossing with Fukuhonoka (Ppo-D1a-q.ymym) as the recurrent parent. From the BC1F1 to BC3F1 generations, individuals carrying Ppo-D1d and Q.Ymym heterozygously were selected. Three individuals carrying Ppo-D1d and Q.Ymym homozygously were selected in the BC3F2 generation, and these were designated Ppo-D1d-Q.Ymym recombinant lines. Selection of individuals carrying Ppo-D1d and Q.Ymym heterozygously or homozygously was performed according to the method described above in <Confirmation of the Ppo-D1 gene, Ppo-A1 gene, and Q.Ymym genotype in wheat>. The TY714-10 line was developed by crossing "Yumechikara" (Ppo-D1b-Q.Ymym) with "Tamaizumi" (Ppo-D1a-q.ymym).

[0098] The genotypes of the Ppo-A1 gene and Q.Ymym in the Fukuhonoka Ppo-deficient line, Fukuhonoka, and Yumechikara were confirmed using the method described above in <Confirmation of the genotypes of the Ppo-D1 gene, Ppo-A1 gene, and Q.Ymym in wheat>, and the results are shown in Figure 4. As shown in Figure 4, the Fukuhonoka Ppo-deficient line and Fukuhonoka were confirmed to homozygously carry q.ymym, and Yumechikara was confirmed to homozygously carry Q.Ymym. Furthermore, the Fukuhonoka Ppo-deficient line was confirmed to homozygously carry Ppo-A1i, Fukuhonoka was confirmed to homozygously carry Ppo-A1b, and Yumechikara was confirmed to homozygously carry Ppo-A1a.

[0099] The results of confirming the genotypes of the Ppo-D1 gene, Ppo-A1 gene, and Q.Ymym in three Ppo-D1d-Q.Ymym recombinant lines were obtained by the method described in <Confirmation of the genotypes of the Ppo-D1 gene, Ppo-A1 gene, and Q.Ymym in wheat> and are shown in Figure 4. As shown in Figure 4, all three Ppo-D1d-Q.Ymym recombinant lines were homozygous for the Ppo-D1d gene, Ppo-A1a gene, and Q.Ymym, respectively.

[0100] <Preparation of Ppo-D1a-Q.Ymym lines and Ppo-D1b-Q.Ymym lines> To create Ppo-D1a-Q.Ymym lines and Ppo-D1b-Q.Ymym lines with a unified genetic background of 'Fukuhonoka', TY714-10 line (Ppo-D1a-Q.Ymym) or 'Yumechikara' (Ppo-D1b-Q.Ymym) was subjected to successive backcrossing with 'Fukuhonoka' (Ppo-D1a-q.ymym) as the recurrent parent. In the creation of the Ppo-D1a-Q.Ymym line, individuals carrying Q.Ymym in heterozygous form were selected from the BC1F1 to BC5F1 generations, and individuals carrying Q.Ymym in homozygous form were selected in the BC5F2 generation, and these individuals were designated as Ppo-D1a-Q.Ymym NIL. In the creation of the Ppo-D1b-Q.Ymym line, individuals carrying Ppo-D1b and Q.Ymym in heterozygous form were selected from the BC1F1 to BC5F1 generations, and individuals carrying Ppo-D1b and Q.Ymym in homozygous form were selected in the BC5F2 generation, and these individuals were designated as Ppo-D1b-Q.Ymym NIL. The selection of individuals carrying Ppo-D1b and / or Q.Ymym in heterozygous or homozygous form was carried out based on the method described in <Confirmation of the genotypes of the Ppo-D1 gene, Ppo-A1 gene, and Q.Ymym in wheat>.

[0101] The genotypes of the Ppo-D1 gene, Ppo-A1 gene, and Q.Ymym gene in the Ppo-D1a-Q.Ymym NILs and Ppo-D1b-Q.Ymym NILs were confirmed by the method described above in <Confirmation of the Genotypes of the Ppo-D1 Gene, Ppo-A1 Gene, and Q.Ymym Gene in Wheat>, and the results are shown in Figure 4. As shown in Figure 4, the Ppo-D1a-Q.Ymym NILs possessed the Ppo-D1a gene, Ppo-A1a gene, and Q.Ymym gene in homozygous form, while the Ppo-D1b-Q.Ymym NILs possessed the Ppo-D1b gene, Ppo-A1a gene, and Q.Ymym gene in homozygous form.

[0102] <Measurement of polyphenol oxidase activity> To measure PPO activity, a scaled-down version of the L-DOPA method (Ito, M. et al., "Development of a Simple Method for Assessing Polyphenol Oxidase Activity in Wheat Flour," Journal of the Crop Science Society of Japan, 77.2 (2008): 159-166) was used. Specifically, 10 wheat seeds were air-milled using a Cyclotec CT293 (Foss, Hilleroed, Denmark) equipped with a 0.5 mm sieve to obtain whole wheat flour. 1 mL of L-DOPA solution (10 mM 3,4-dihydroxy-L-phenylalanine, 50 mM 3-morpholinopropanesulfonic acid) was added to 100 mg of whole wheat flour, mixed for 10 minutes, and then centrifuged to collect the supernatant. The OD value at 475 nm was measured using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific). Statistical analysis was performed using R version 4.3.0, with multiple comparisons performed using Tukey's HSD at a 5% significance level.

[0103] The results of analyzing the PPO activities of "Fukuhonoka", the Fukuhonoka Ppo-deficient line, the Ppo-D1d-Q.Ymym recombinant line, the Ppo-D1a-Q.Ymym NIL, and the Ppo-D1b-Q.Ymym NIL by the L-DOPA method are shown in Fig. 5. In Fig. 5, "a", "b", "c", "cd", "e", and "f" are labels in the Tukey's HSD test, and the significance level is 5% or less between different labels. As a result, there were significant differences in the PPO activities among the Ppo-D1d-Q.Ymym recombinant line, the Ppo-D1a-Q.Ymym NIL, and the Ppo-D1b-Q.Ymym NIL, and from the highest PPO activity, it was Ppo-D1b-Q.Ymym NIL > Ppo-D1a-Q.Ymym NIL > Ppo-D1d-Q.Ymym line. In addition, the 475 nm OD value is not 0 even in the Fukuhonoka Ppo-deficient line having homozygous Ppo-D1d and Ppo-A1i because in hexaploid wheat, at least four genes other than the Ppo-D1 gene and the Ppo-A1 gene contribute to the PPO activity.

[0104] <Creation of Ppo-D1d-Q.Ymym Recombinant Line 2> To create the Ppo-D1d-Q.Ymym recombinant line, the TY714-10 line (Ppo-D1a-Q.Ymym) and the Fukuhonoka Ppo-deficient line (Ppo-D1d-q.ymym) were crossed. In the F2 generation, individuals (Ppo-D1d-Q.Ymym) carrying homozygous Ppo-D1d and Q.Ymym were selected. To make the genetic background "Fukuhonoka", the above individuals (Ppo-D1d-Q.Ymym) were subjected to continuous backcrossing with "Fukuhonoka" (Ppo-D1a-q.ymym) as the recurrent parent. Individuals carrying heterozygous Q.Ymym were selected from the BC1F1 to BC5F1 generations, and individuals carrying homozygous Q.Ymym were selected in the BC5F2 generation, and these individuals were designated as Ppo-D1d-Q.Ymym NIL. The selection of individuals carrying heterozygous or homozygous Ppo-D1d and / or Q.Ymym was performed based on the method described in the above <Confirmation of Genotypes of Ppo-D1 Gene, Ppo-A1 Gene, and Q.Ymym in Wheat>.

[0105] <Development of Ppo-A1i / Ppo-D1a-Q.Ymym NIL, Ppo-A1i / Ppo-D1b-Q.Ymym NIL, and Ppo-A1i / Ppo-D1d-Q.Ymym NIL> As shown in FIG. 7 described below, the Ppo-D1a-Q.Ymym NIL and Ppo-D1b-Q.Ymym NIL created in the <Preparation of Ppo-D1a-Q.Ymym line and Ppo-D1b-Q.Ymym line>, and the Ppo-D1d-Q.Ymym NIL created in the <Creation 2 of Ppo-D1d-Q.Ymym recombinant line> carried Ppo-A1b because they were in the BC5F2 generation obtained by backcrossing five times with 'Fukuhonoka' (Ppo-A1b / Ppo-D1a-q-ymym). Therefore, these three lines were designated as Ppo-A1b / Ppo-D1a-Q.Ymym NIL, Ppo-A1b / Ppo-D1b-Q.Ymym NIL, and Ppo-A1b / Ppo-D1d-Q.Ymym NIL in order. Seeds of the F1 generation were produced by crossing each of these three lines with the Fukuhonoka Ppo-deficient line (Ppo-A1i / Ppo-D1d-q.ymym). The seeds of each created F1 generation were sown, and seeds of the F2 generation were produced by self-pollination. The seeds of each F2 generation were sown, and DNA marker selection was performed for individuals that homozygously carried Ppo-A1i and homozygously carried Ppo-D1a-Q.Ymym, Ppo-D1b-Q.Ymym, or Ppo-D1d-Q.Ymym. The individuals selected thereby were designated as Ppo-A1i / Ppo-D1a-Q.Ymym NIL, Ppo-A1i / Ppo-D1b-Q.Ymym NIL, and Ppo-A1i / Ppo-D1d-Q.Ymym NIL, respectively. In addition, seeds of the F3 generation were obtained from each of these three types of individuals. The selection of individuals carrying Ppo-A1i, Ppo-D1d, and / or Q.Ymym in heterozygous or homozygous form was performed based on the method described in the <Confirmation of Genotypes of Ppo-D1 Gene, Ppo-A1 Gene, and Q.Ymym in Wheat>.

[0106] The genotypes of the Ppo-D1 gene, Ppo-A1 gene, and Q.Ymym in six hexaploid wheat lines, namely, Ppo-A1b / Ppo-D1a-Q.Ymym NIL, Ppo-A1b / Ppo-D1b-Q.Ymym NIL, Ppo-A1b / Ppo-D1d-Q.Ymym NIL, Ppo-A1i / Ppo-D1a-Q.Ymym NIL, Ppo-A1i / Ppo-D1b-Q.Ymym NIL, and Ppo-A1i / Ppo-D1d-Q.Ymym NIL, were confirmed using the method described above in <Confirmation of the genotypes of the Ppo-D1 gene, Ppo-A1 gene, and Q.Ymym in wheat>. The results are shown in Figure 7. As shown in Figure 7, the Ppo-A1i / Ppo-D1a-Q.Ymym NILs homozygously carried the Ppo-A1i gene, the Ppo-D1a gene, and Q.Ymym, respectively; the Ppo-A1i / Ppo-D1b-Q.Ymym NILs homozygously carried the Ppo-A1i gene, the Ppo-D1b gene, and Q.Ymym, respectively; and the Ppo-A1i / Ppo-D1d-Q.Ymym NILs homozygously carried the Ppo-A1i gene, the Ppo-D1d gene, and Q.Ymym, respectively.

[0107] <Measurement of polyphenol oxidase activity 2> PPO activity was measured and analyzed using the L-DOPA method in Fukuhonoka, the Fukuhonoka Ppo-deficient line, and the six hexaploid wheat lines described above, using the same method as described above for polyphenol oxidase activity. For the six hexaploid wheat lines, F3 seeds were subjected to the L-DOPA method. The results are shown in Figure 8. In Figure 8, "a," "b," "c," "bc," "bd," "ae," and "f" are labels used in Tukey's HSD tests, and the significance level between different labels is 5% or less. As shown in Figure 8, when comparing lines that share the Ppo-D1 gene and Q.Ymym, lines homozygously carrying the Ppo-A1i gene had significantly lower PPO activity than lines homozygously carrying the Ppo-A1b gene. Furthermore, there was a significant difference in PPO activity among the Ppo-A1i / Ppo-D1a-Q.Ymym NIL, Ppo-A1i / Ppo-D1b-Q.Ymym NIL, and Ppo-A1i / Ppo-D1d-Q.Ymym NIL, with the order of highest PPO activity being Ppo-A1i / Ppo-D1b-Q.Ymym NIL > Ppo-A1i / Ppo-D1a-Q.Ymym NIL > Ppo-A1i / Ppo-D1d-Q.Ymym NIL. Furthermore, of the six hexaploid wheat lines, Ppo-A1i / Ppo-D1d-Q.Ymym NIL had the lowest PPO activity.

Claims

1. A homozygous Ppo-D1 gene having a lack of or reduced polyphenol oxidase activity, which is any one of the following (1), (2), or (3): A hexaploid wheat having a quantitative trait locus for wheat yellow mosaic disease resistance Q. Ymym and Ymym on chromosome 2D. (1) A gene consisting of a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO: 1, in which 20 or more consecutive bases are deleted, and the amino acid sequence of the encoded protein is shorter than the amino acid sequence represented by SEQ ID NO: 2; (2) A gene consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 3, or (3) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

4.

2. 2. The hexaploid wheat according to claim 1, wherein in the gene (1), the deletion of bases comprises a deletion of 20 or more consecutive bases in the base sequence corresponding to bases 1576 to 1648 of SEQ ID NO: 1, and the base sequence corresponding to bases 1737 to 1739 of SEQ ID NO: 1 is a stop codon.

3. 2. The hexaploid wheat according to claim 1, wherein the gene is a gene consisting of the base sequence of SEQ ID NO:

3.

4. The hexaploid wheat according to claim 1, having a Ppo-A1 gene homozygously on chromosome 2A that is deficient in the function of polyphenol oxidase activity, which is any one of (6), (7), (10), or (11) below. (6) A gene consisting of the base sequence represented by SEQ ID NO:

8. (7) A gene consisting of a base sequence having 90% or more sequence identity with the base sequence represented by SEQ ID NO:

8. (10) A gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 23, or (11) A gene encoding a protein consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:

23.

5. A primer set designed to sandwich the 1536th and 1537th bases in the base sequence represented by SEQ ID NO:

3.

6. The primer set according to claim 5, comprising a primer consisting of a polynucleotide comprising a base sequence having 90% or more sequence identity to the base sequence of SEQ ID NO: 9, and a primer consisting of a polynucleotide comprising a base sequence having 90% or more sequence identity to the base sequence of SEQ ID NO: 10.

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