JP5445400B2 - Genetic polymorphism marker set, probe set, detection kit, and breed determination method - Google Patents

Description

  The present invention relates to a genetic polymorphism marker set, a probe set, a detection kit, and a breed determination method.

  In quality control of plants, for example, it is important to determine the variety and purity of seeds. However, conventionally, determination of varieties and purity has been carried out by actually cultivating seeds, observing phenotypes such as morphological characteristics and cultivation characteristics in the initial stage, and confirming differences between varieties. . However, in this way, the judgment based on the expression requires skillful judgment by the breeder and cannot be easily performed by anyone. Moreover, since cultivation is essential, there is a problem that it takes time to obtain a determination result.

  Therefore, in recent years, methods for determining the variety and purity by genetic analysis have been put into practical use, and among them, determination by analysis of single nucleotide polymorphisms (SNPs) has been attempted (Patent Document 1). However, for Brassicaceae plants, although a technique for identifying an S-type gene has been reported (Patent Document 2), an effective SNP for discriminating between related varieties has not been clarified. It was difficult to determine the variety, purity, etc. by scientific analysis.

Japanese Patent No. 4389783 Japanese Patent No. 4346933

  Therefore, an object of the present invention is to provide a genetic polymorphic marker set, a probe set, a detection kit, and a variety determination method that enable discrimination of Brassica plant varieties by genetic analysis.

The genetic polymorphic marker set of the present invention is a genetic polymorphic marker set for discriminating the varieties of Brassicaceae plants, characterized in that it comprises a marker comprising the following polynucleotides (1) to (13): .
(1) The polynucleotide of (S) or (A) below.
(S) 10 to 2 containing the 11th mutant base (G) in the DNA sequence of SEQ ID NO: 1
A polynucleotide comprising 73 consecutive DNA sequences.
(A) 10 to 2 containing the 11th mutant base (A) in the DNA sequence of SEQ ID NO: 2
A polynucleotide comprising 83 consecutive DNA sequences.
(2) The polynucleotide of (S) or (A) below.
(S) In the DNA sequence of SEQ ID NO: 3, the 175th to 176th mutant base (CT)
And 10 to 366 consecutive DNA sequences containing the 184th mutant base (C)
A polynucleotide comprising:
(A) In the DNA sequence of SEQ ID NO: 4, a mutant base between positions 165 and 166
(CT deletion) and 173 th 10-353 consecutive, including mutant base (G)
A polynucleotide comprising a DNA sequence.
(3) The polynucleotide of (S) or (A) below.
(S) 10 to 1 including the 90th mutant base (A) in the DNA sequence of SEQ ID NO: 5
A polynucleotide comprising 40 consecutive DNA sequences.
(A) 10 to 1 containing the 92nd mutant base (G) in the DNA sequence of SEQ ID NO: 6
A polynucleotide comprising 45 consecutive DNA sequences.
(4) The polynucleotide of (S) or (A) below.
(S) A mutated base between the 99th and 100th positions in the DNA sequence of SEQ ID NO: 7
(C deletion) and mutated bases between positions 100 and 101 (A deletion)
A polynucleotide comprising 10 to 349 continuous DNA sequences.
(A) The 159th mutant base (C) and 161 in the DNA sequence of SEQ ID NO: 8
A poly consisting of 10 to 406 continuous DNA sequences containing the second mutant base (A)
nucleotide.
(5) The polynucleotide of (S) or (A) below.
(S) 10-2 including the 93rd mutant base (C) in the DNA sequence of SEQ ID NO: 9
A polynucleotide comprising 63 consecutive DNA sequences.
(A) 10 containing the 108th mutant base (T) in the DNA sequence of SEQ ID NO: 10
A polynucleotide consisting of ~ 232 continuous DNA sequences.
(6) The polynucleotide of (S) or (A) below.
(S) 10 containing the 264th mutant base (C) in the DNA sequence of SEQ ID NO: 11
A polynucleotide consisting of ~ 302 continuous DNA sequences.
(A) 10 containing the 301st mutant base (T) in the DNA sequence of SEQ ID NO: 12
A polynucleotide consisting of ~ 336 consecutive DNA sequences.
(7) The polynucleotide of (S) or (A) below.
(S) The 254th to 255th mutant base (CT) in the DNA sequence of SEQ ID NO: 13
A polynucleotide comprising 10 to 439 continuous DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 14, the 261st to 262nd mutant base (TC)
A polynucleotide comprising 10 to 467 consecutive DNA sequences.
(8) The polynucleotide of (S) or (A) below.
(S) In the DNA sequence of SEQ ID NO: 15, the 197th mutant base (T) and 20
A sequence consisting of 10 to 619 continuous DNA sequences containing the 0th mutant base (G).
Renucleotide.
(A) In the DNA sequence of SEQ ID NO: 16, the 208 th mutant base (A) and 21
A sequence consisting of 10 to 620 consecutive DNA sequences containing the first mutant base (A).
Renucleotide.
(9) The polynucleotide of (S) or (A) below.
(S) Mutant salt between the 263rd and 264th positions in the DNA sequence of SEQ ID NO: 17
Group (C deletion), 265th mutant base (T) and 267th mutant base (G
A polynucleotide comprising 10 to 567 consecutive DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 18, the 265th mutant base (C), 267
10 to 563 including the mutated base of the eye (A) and the mutated base of the 269th (T)
A polynucleotide comprising a continuous DNA sequence.
(10) The polynucleotide of (S) or (A) below.
(S) 1 including the 292nd mutant base (A) in the DNA sequence of SEQ ID NO: 19
A polynucleotide comprising a continuous DNA sequence of 0 to 352.
(A) 1 containing the 292nd mutant base (G) in the DNA sequence of SEQ ID NO: 20
A polynucleotide comprising a continuous DNA sequence of 0 to 352.
(11) The polynucleotide of (S) or (A) below.
(S) 10 containing the 89th mutant base (G) in the DNA sequence of SEQ ID NO: 21
A polynucleotide consisting of ˜405 continuous DNA sequences.
(A) 10 containing the 90th mutant base (A) in the DNA sequence of SEQ ID NO: 22
A polynucleotide consisting of ˜408 consecutive DNA sequences.
(12) The polynucleotide of (S) or (A) below.
(S) 1 containing the 290th mutant base (C) in the DNA sequence of SEQ ID NO: 23
A polynucleotide consisting of 0 to 324 continuous DNA sequences.
(A) 1 containing the 300th mutant base (T) in the DNA sequence of SEQ ID NO: 24
A polynucleotide consisting of 0 to 335 continuous DNA sequences.
(13) The polynucleotide of (S) or (A) below.
(S) 1 containing the 226th mutant base (C) in the DNA sequence of SEQ ID NO: 25
A polynucleotide consisting of 0 to 329 continuous DNA sequences.
(A) 1 containing the 212th mutant base (T) in the DNA sequence of SEQ ID NO: 26
A polynucleotide consisting of 0 to 296 continuous DNA sequences.

  The probe set of the present invention is a probe set for detecting the genetic polymorphic marker set of the present invention, which is a DNA sequence containing the mutant base in the polynucleotides (1) to (13) or a complement thereof. It is characterized by including a target sequence.

  The detection kit of the present invention is a detection kit for detecting the genetic polymorphic marker set of the present invention, and includes the probe set of the present invention.

  The cultivar determination method of the present invention is a cultivar determination method for determining the varieties of cruciferous plants, characterized in that the determination is made using the genetic polymorphic marker set of the present invention in the genome of the cruciferous plant.

  As a result of intensive studies, the present inventors have clarified a plurality of types of polynucleotides containing SNPs that can discriminate Brassicaceae varieties, and have completed the present invention based on these. According to the present invention, a subject of a cruciferous plant is detected by a genetic analysis method using these polynucleotides as a genetic marker set, thereby quickly and easily having excellent reliability. It became possible to distinguish the type of specimen. This also makes it possible to perform seed purity tests, etc. quickly and easily with excellent reliability. For this reason, the present invention can be said to be an extremely useful technique in quality display and quality confirmation in the distribution of seedlings and the like, for example.

FIG. 1 is a photograph showing an example of a seed purity test result in an example of the present invention. FIG. 2 is a photograph showing another example of the seed purity test result in the example of the present invention.

<Polynucleotide>
As described above, the polynucleotide of the present invention includes at least one selected from the group consisting of the following polynucleotides (1) to (13).
(1) The polynucleotide of (S) or (A) below.
(S) 10 to 2 containing the 11th mutant base (G) in the DNA sequence of SEQ ID NO: 1
A polynucleotide comprising 73 consecutive DNA sequences.
(A) 10 to 2 containing the 11th mutant base (A) in the DNA sequence of SEQ ID NO: 2
A polynucleotide comprising 83 consecutive DNA sequences.
(2) The polynucleotide of (S) or (A) below.
(S) In the DNA sequence of SEQ ID NO: 3, the 175th to 176th mutant base (CT)
And 10 to 366 consecutive DNA sequences containing the 184th mutant base (C)
A polynucleotide comprising:
(A) In the DNA sequence of SEQ ID NO: 4, a mutant base between positions 165 and 166
(CT deletion) and 173 th 10-353 consecutive, including mutant base (G)
A polynucleotide comprising a DNA sequence.
(3) The polynucleotide of (S) or (A) below.
(S) 10 to 1 including the 90th mutant base (A) in the DNA sequence of SEQ ID NO: 5
A polynucleotide comprising 40 consecutive DNA sequences.
(A) 10 to 1 containing the 92nd mutant base (G) in the DNA sequence of SEQ ID NO: 6
A polynucleotide comprising 45 consecutive DNA sequences.
(4) The polynucleotide of (S) or (A) below.
(S) A mutated base between the 99th and 100th positions in the DNA sequence of SEQ ID NO: 7
(C deletion) and mutated bases between positions 100 and 101 (A deletion)
A polynucleotide comprising 10 to 349 continuous DNA sequences.
(A) The 159th mutant base (C) and 161 in the DNA sequence of SEQ ID NO: 8
A poly consisting of 10 to 406 continuous DNA sequences containing the second mutant base (A)
nucleotide.
(5) The polynucleotide of (S) or (A) below.
(S) 10-2 including the 93rd mutant base (C) in the DNA sequence of SEQ ID NO: 9
A polynucleotide comprising 63 consecutive DNA sequences.
(A) 10 containing the 108th mutant base (T) in the DNA sequence of SEQ ID NO: 10
A polynucleotide consisting of ~ 232 continuous DNA sequences.
(6) The polynucleotide of (S) or (A) below.
(S) 10 containing the 264th mutant base (C) in the DNA sequence of SEQ ID NO: 11
A polynucleotide consisting of ~ 302 continuous DNA sequences.
(A) 10 containing the 301st mutant base (T) in the DNA sequence of SEQ ID NO: 12
A polynucleotide consisting of ~ 336 consecutive DNA sequences.
(7) The polynucleotide of (S) or (A) below.
(S) The 254th to 255th mutant base (CT) in the DNA sequence of SEQ ID NO: 13
A polynucleotide comprising 10 to 439 continuous DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 14, the 261st to 262nd mutant base (TC)
A polynucleotide comprising 10 to 467 consecutive DNA sequences.
(8) The polynucleotide of (S) or (A) below.
(S) In the DNA sequence of SEQ ID NO: 15, the 197th mutant base (T) and 20
A sequence consisting of 10 to 619 continuous DNA sequences containing the 0th mutant base (G).
Renucleotide.
(A) In the DNA sequence of SEQ ID NO: 16, the 208 th mutant base (A) and 21
A sequence consisting of 10 to 620 consecutive DNA sequences containing the first mutant base (A).
Renucleotide.
(9) The polynucleotide of (S) or (A) below.
(S) Mutant salt between the 263rd and 264th positions in the DNA sequence of SEQ ID NO: 17
Group (C deletion), 265th mutant base (T) and 267th mutant base (G
A polynucleotide comprising 10 to 567 consecutive DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 18, the 265th mutant base (C), 267
10-563 containing the mutated base of the eye (A) and the 269th mutated base (T)
A polynucleotide comprising a continuous DNA sequence.
(10) The polynucleotide of (S) or (A) below.
(S) 1 including the 292nd mutant base (A) in the DNA sequence of SEQ ID NO: 19
A polynucleotide comprising a continuous DNA sequence of 0 to 352.
(A) 1 containing the 292nd mutant base (G) in the DNA sequence of SEQ ID NO: 20
A polynucleotide comprising a continuous DNA sequence of 0 to 352.
(11) The polynucleotide of (S) or (A) below.
(S) 10 containing the 89th mutant base (G) in the DNA sequence of SEQ ID NO: 21
A polynucleotide consisting of ˜405 continuous DNA sequences.
(A) 10 containing the 90th mutant base (A) in the DNA sequence of SEQ ID NO: 22
A polynucleotide consisting of ˜408 consecutive DNA sequences.
(12) The polynucleotide of (S) or (A) below.
(S) 1 containing the 290th mutant base (C) in the DNA sequence of SEQ ID NO: 23
A polynucleotide consisting of 0 to 324 continuous DNA sequences.
(A) 1 containing the 300th mutant base (T) in the DNA sequence of SEQ ID NO: 24
A polynucleotide consisting of 0 to 335 continuous DNA sequences.
(13) The polynucleotide of (S) or (A) below.
(S) 1 containing the 226th mutant base (C) in the DNA sequence of SEQ ID NO: 25
A polynucleotide consisting of 0 to 329 continuous DNA sequences.
(A) 1 containing the 212th mutant base (T) in the DNA sequence of SEQ ID NO: 26
A polynucleotide consisting of 0 to 296 continuous DNA sequences.

In addition, the polynucleotide of the present invention is characterized by including at least one selected from the group consisting of the following polynucleotides (14) to (23).
(14) The polynucleotide of (S) or (A) below.
(S) 10 containing the 60th mutant base (C) in the DNA sequence of SEQ ID NO: 53
A polynucleotide consisting of ˜344 continuous DNA sequences.
(A) 10 containing the 56th mutant base (T) in the DNA sequence of SEQ ID NO: 54
A polynucleotide consisting of ˜344 continuous DNA sequences.
(15) The polynucleotide of (S) or (A) below.
(S) Mutation between positions 131 and 132 in the DNA sequence of SEQ ID NO: 55
Poly consisting of 10 to 401 consecutive DNA sequences including bases (CAG deletion)
nucleotide.
(A) In the DNA sequence of SEQ ID NO: 56, the 133-135th mutant base (CA
A polynucleotide comprising 10 to 414 consecutive DNA sequences, including G).
(16) The polynucleotide of (S) or (A) below.
(S) In the DNA sequence of SEQ ID NO: 57, 10 containing the 79th mutant base (T)
A polynucleotide consisting of ˜534 consecutive DNA sequences.
(A) 10 including the 75th mutant base (C) in the DNA sequence of SEQ ID NO: 58
A polynucleotide consisting of 531 continuous DNA sequences.
(17) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 59, 10 containing the 72nd mutant base (T)
A polynucleotide consisting of ˜363 continuous DNA sequences.
(A) 10 containing the 72nd mutant base (G) in the DNA sequence of SEQ ID NO: 60
A polynucleotide consisting of ~ 350 continuous DNA sequences.
(18) The polynucleotide of (S) or (A) below:
(S) 1 containing the 176th mutant base (C) in the DNA sequence of SEQ ID NO: 61
A polynucleotide comprising 0 to 313 continuous DNA sequences.
(A) 1 containing the 175th mutant base (G) in the DNA sequence of SEQ ID NO: 62
A polynucleotide comprising 0 to 312 continuous DNA sequences.
(19) The polynucleotide of (S) or (A) below.
(S) 1 containing the 196th mutant base (G) in the DNA sequence of SEQ ID NO: 63
A polynucleotide comprising 0 to 316 consecutive DNA sequences.
(A) 1 including the 176th mutant base (C) in the DNA sequence of SEQ ID NO: 64
A polynucleotide comprising 0 to 302 consecutive DNA sequences.
(20) The polynucleotide of (S) or (A) below.
(S) 1 containing the 241st mutant base (G) in the DNA sequence of SEQ ID NO: 65
A polynucleotide consisting of 0 to 350 continuous DNA sequences.
(A) 1 containing the 243rd mutant base (T) in the DNA sequence of SEQ ID NO: 66
A polynucleotide comprising a continuous DNA sequence of 0 to 352.
(21) The polynucleotide of (S) or (A) below.
(S) In the DNA sequence of SEQ ID NO: 67, 10 containing the 53rd mutant base (C)
A polynucleotide consisting of ~ 603 continuous DNA sequences.
(A) 10 including the 86th mutant base (A) in the DNA sequence of SEQ ID NO: 68
A polynucleotide consisting of ~ 633 consecutive DNA sequences.
(22) The polynucleotide of (S) or (A) below.
(S) 1 containing the 193rd mutant base (C) in the DNA sequence of SEQ ID NO: 69
A polynucleotide comprising a continuous DNA sequence of 0 to 249.
(A) 1 containing the 172nd mutant base (T) in the DNA sequence of SEQ ID NO: 70
A polynucleotide consisting of 0 to 239 continuous DNA sequences.
(23) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 71, 1 containing the 156th mutant base (T)
A polynucleotide comprising a continuous DNA sequence of 0 to 352.
(A) 1 containing the 227th mutant base (C) in the DNA sequence of SEQ ID NO: 72
A polynucleotide comprising a continuous DNA sequence of 0 to 422.

In addition, the polynucleotide of the present invention is characterized by including at least one selected from the group consisting of the following polynucleotides (24) to (51).
(24) The polynucleotide of (S) or (A) below.
(S) 1 containing the 100th mutant base (T) in the DNA sequence of SEQ ID NO: 73
A polynucleotide consisting of 0 to 376 continuous DNA sequences.
(A) 1 containing the 119th mutant base (G) in the DNA sequence of SEQ ID NO: 74
A polynucleotide consisting of 0 to 376 continuous DNA sequences.
(25) The polynucleotide of (S) or (A) below.
(S) 1 containing the 348th mutant base (G) in the DNA sequence of SEQ ID NO: 75
A polynucleotide consisting of 0 to 358 continuous DNA sequences.
(A) 1 including the 333rd mutant base (T) in the DNA sequence of SEQ ID NO: 76
A polynucleotide comprising a continuous DNA sequence of 0 to 342.
(26) The polynucleotide of (S) or (A) below.
(S) In the DNA sequence of SEQ ID NO: 77, the 163rd to 165th mutant base (AT
A polynucleotide comprising 10 to 510 consecutive DNA sequences, including G).
(A) Mutation between positions 134 and 135 in the DNA sequence of SEQ ID NO: 78
Poly consisting of 10 to 483 continuous DNA sequences including bases (ATG deletion)
nucleotide.
(27) The polynucleotide of (S) or (A) below.
(S) 1 containing the 273rd mutant base (C) in the DNA sequence of SEQ ID NO: 79
A polynucleotide consisting of 0 to 468 continuous DNA sequences.
(A) 1 containing the 268th mutant base (T) in the DNA sequence of SEQ ID NO: 80
A polynucleotide comprising a continuous DNA sequence of 0 to 465.
(28) The polynucleotide of (S) or (A) below:
(S) 1 containing the 278th mutant base (A) in the DNA sequence of SEQ ID NO: 81
A polynucleotide consisting of 0 to 429 continuous DNA sequences.
(A) 1 including the 488th mutant base (T) in the DNA sequence of SEQ ID NO: 82
A polynucleotide consisting of 0 to 642 continuous DNA sequences.
(29) The polynucleotide of (S) or (A) below:
(S) 1 containing the 321st mutant base (G) in the DNA sequence of SEQ ID NO: 83
A polynucleotide consisting of 0 to 332 continuous DNA sequences.
(A) 1 including the 320th mutant base (A) in the DNA sequence of SEQ ID NO: 84
A polynucleotide consisting of 0 to 332 continuous DNA sequences.
(30) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 85, the 375 th to 376 th mutant base (CT
A polynucleotide consisting of 10 to 436 consecutive DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 86, the 373rd to 374th mutant bases (TC
A polynucleotide consisting of 10 to 435 consecutive DNA sequences.
(31) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 87, 1 containing the 152 th variant base (C)
A polynucleotide comprising 0 to 333 continuous DNA sequences.
(A) 1 containing the 148th mutant base (G) in the DNA sequence of SEQ ID NO: 88
A polynucleotide comprising 0 to 327 continuous DNA sequences.
(32) The polynucleotide of (S) or (A) below:
(S) 1 containing the 225th mutant base (G) in the DNA sequence of SEQ ID NO: 89
A polynucleotide comprising a continuous DNA sequence of 0 to 359.
(A) 1 containing the 227th mutant base (A) in the DNA sequence of SEQ ID NO: 90
A polynucleotide comprising 0 to 361 continuous DNA sequences.
(33) The polynucleotide of (S) or (A) below.
(S) 1 containing the 285th mutant base (G) in the DNA sequence of SEQ ID NO: 91
A polynucleotide consisting of 0 to 383 continuous DNA sequences.
(A) 1 containing the 271nd mutant base (C) in the DNA sequence of SEQ ID NO: 92
A polynucleotide consisting of 0 to 382 continuous DNA sequences.
(34) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 93, 1 containing the 124th mutant base (A)
A polynucleotide comprising a continuous DNA sequence of 0 to 247.
(A) 1 containing the 125th mutant base (G) in the DNA sequence of SEQ ID NO: 94
A polynucleotide consisting of 0 to 251 continuous DNA sequences.
(35) The polynucleotide of (S) or (A) below.
(S) 1 which includes the 121st mutant base (A) in the DNA sequence of SEQ ID NO: 95
A polynucleotide comprising 0 to 319 continuous DNA sequences.
(A) 1 containing the 120th mutant base (G) in the DNA sequence of SEQ ID NO: 96
A polynucleotide consisting of 0 to 358 continuous DNA sequences.
(36) The polynucleotide of (S) or (A) below:
(S) 1 including the 321st mutant base (A) in the DNA sequence of SEQ ID NO: 97
A polynucleotide consisting of 0 to 367 continuous DNA sequences.
(A) 1 including the 335th mutant base (G) in the DNA sequence of SEQ ID NO: 98
A polynucleotide consisting of 0 to 382 continuous DNA sequences.
(37) The polynucleotide of (S) or (A) below:
(S) 10 containing the 90th mutant base (A) in the DNA sequence of SEQ ID NO: 99
A polynucleotide consisting of ˜284 consecutive DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 100, containing the 116th mutant base (G)
A polynucleotide consisting of 10 to 318 continuous DNA sequences.
(38) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 101, the 234th mutant base (G) is included.
A polynucleotide comprising 10 to 384 continuous DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 102, the 227th mutant base (T) is included.
A polynucleotide consisting of 10 to 376 continuous DNA sequences.
(39) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 103, the 284th mutant base (C) is included.
A polynucleotide comprising 10 to 516 continuous DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 104, contains the 302nd mutant base (A)
A polynucleotide comprising 10 to 531 continuous DNA sequences.
(40) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 105, containing the 122nd mutant base (G)
A polynucleotide comprising 10 to 385 continuous DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 106, contains the 122nd mutation base (A)
A polynucleotide comprising 10 to 385 continuous DNA sequences.
(41) The polynucleotide of (S) or (A) below.
(S) In the DNA sequence of SEQ ID NO: 107, contains the 107th mutant base (C)
A polynucleotide comprising 10 to 280 continuous DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 108, the 108th mutation base (A) is included.
A polynucleotide comprising 10 to 283 continuous DNA sequences.
(42) The polynucleotide of (S) or (A) below:
(S) 1 containing the 41st mutant base (T) in the DNA sequence of SEQ ID NO: 109
A polynucleotide comprising a continuous DNA sequence of 0 to 262.
(A) 1 containing the 41st mutant base (C) in the DNA sequence of SEQ ID NO: 110
A polynucleotide comprising a continuous DNA sequence of 0 to 264.
(43) The following polynucleotide (S) or (A):
(S) In the DNA sequence of SEQ ID NO: 111, the 236th mutant base (G) is included.
A polynucleotide comprising 10 to 320 consecutive DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 112, the 231st mutant base (T) is included.
A polynucleotide comprising 10 to 305 continuous DNA sequences.
(44) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 113, the 153rd mutant base (G) is included.
A polynucleotide comprising 10 to 283 continuous DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 114, the 174th mutant base (A) is included.
A polynucleotide comprising 10 to 287 continuous DNA sequences.
(45) The polynucleotide of (S) or (A) below:
(S) 1 containing the 63rd mutant base (C) in the DNA sequence of SEQ ID NO: 115
A polynucleotide consisting of 0 to 336 continuous DNA sequences.
(A) 1 containing the 63rd mutant base (T) in the DNA sequence of SEQ ID NO: 116
A polynucleotide comprising 0 to 333 continuous DNA sequences.
(46) The polynucleotide of (S) or (A) below.
(S) In the DNA sequence of SEQ ID NO: 117, the 258th mutant base (A) is included.
A polynucleotide comprising 10 to 452 continuous DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 118, the 271nd mutant base (T) is included.
A polynucleotide comprising 10 to 465 continuous DNA sequences.
(47) The polynucleotide of (S) or (A) below.
(S) Variation between positions 226 and 227 in the DNA sequence of SEQ ID NO: 119
A polynucleotide comprising 10 to 274 consecutive DNA sequences containing different bases (T deletion)
Creotide.
(A) In the DNA sequence of SEQ ID NO: 120, the 251st mutant base (T) is included.
A polynucleotide comprising 10 to 309 continuous DNA sequences.
(48) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 121, the 340th mutant base (A)
A polynucleotide comprising 10 to 627 consecutive DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 122, the 358th mutant base (T) is included.
A polynucleotide comprising 10 to 514 continuous DNA sequences.
(49) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 123, the 107th mutation base (C) is included.
A polynucleotide consisting of 10 to 260 continuous DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 124, the 104th mutation base (T) is included.
A polynucleotide comprising 10 to 257 continuous DNA sequences.
(50) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 125, the 342nd mutant base (A) is included.
A polynucleotide comprising 10 to 627 consecutive DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 126, the 323rd mutant base (G) is included.
A polynucleotide comprising 10 to 606 consecutive DNA sequences.
(51) The polynucleotide of (S) or (A) below:
(S) In the DNA sequence of SEQ ID NO: 127, the 186th mutant base (A) is included.
A polynucleotide comprising 10 to 250 consecutive DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 128, the 155th mutant base (T) is included.
A polynucleotide comprising 10 to 221 continuous DNA sequences.

  Hereinafter, in the present invention, the polynucleotide (S) is referred to as an (S) type polynucleotide, and the polynucleotide (A) is referred to as an (A) type polynucleotide. Since diploid cruciferous plants can be classified into homozygous and heterozygous for a given locus, for example, the polynucleotides of (1) to (51) are, for example, homozygous of (S) polynucleotide It can be said that there is a hetero type having a homo type of (A) type polynucleotide and (S) type polynucleotide and (A) type polynucleotide. For each of the polynucleotides (1) to (51), the combination of the (S) type polynucleotide and the (A) type polynucleotide is referred to as a polynucleotide set. Specifically, for example, the combination of the (S) -type polynucleotide of (1) and the (A) -type polynucleotide is referred to as the polynucleotide set of (1), and the other (2) to (51) ) Is the same. Hereinafter, the polynucleotide of the present invention may be represented by the names shown in Tables 1 and 2.

  In the present invention, the mutated base is a base that is involved in variety discrimination between the (S) type polynucleotide and the (A) type polynucleotide of the polynucleotide set in the DNA sequence of each polynucleotide. Means. The mutated base is a base that is substituted, deleted, inserted, or added in each polynucleotide. Hereinafter, each base is also referred to as a single nucleotide polymorphism (SNP).

  The present inventors constructed a plurality of primer sets based on the EST information of the 20th Japanese radish published at Michigan State University, and performed nucleic acid amplification for each of the radish and blue neck radish. By comparing the sequences to be amplified, SNP candidates with different bases were searched for. As a result of further examination of these SNP candidates, a polynucleotide having a SNP capable of distinguishing the Brassicaceae plant has been determined. In other words, for the Brassicaceae plant, it is possible to discriminate the variety by detecting which type of the predetermined SNP between the Saya radish and the blue neck radish is. These polynucleotides are the polynucleotides (1) to (51). In the polynucleotides (1) to (51), the (S) -type polynucleotide is a polynucleotide having a sequence derived from Soya radish, and the (A) -type polynucleotide is a sequence having a sequence derived from blue neck radish. It is a polynucleotide. In the present invention, the use of the polynucleotides (1) to (51) as a genetic polymorphic marker set, as will be described later, makes it possible to discriminate the varieties of cruciferous plants. The determination method will be described in detail in the kind determination method of the present invention.

  The polynucleotide of the present invention may be, for example, a polynucleotide comprising a complementary sequence to any of the polynucleotides (1) to (51). The polynucleotide of the present invention may be, for example, a polynucleotide comprising an RNA sequence in which thymine is replaced with uracil in any of the polynucleotides (1) to (51).

  Although the total length of the polynucleotide of the present invention is not particularly limited, for example, it is 10 to 500 bases long, preferably 10 to 300 bases long, more preferably 10 to 200 bases long, further preferably 10 to 30 bases long, The length is particularly preferably 15 to 25 bases, and most preferably 17 bases.

  Examples of the polynucleotide of the present invention include the DNA sequences shown in SEQ ID NOs: 1-128, among which the DNA sequences shown in SEQ ID NOs: 1-26 and the DNA sequences shown in SEQ ID NOs: 27-72 are preferred, more preferably These are the DNA sequences shown in SEQ ID NOs: 1 to 26. In addition, the polynucleotide of the present invention may be, for example, a partial sequence containing the mutated base in the DNA sequence shown in SEQ ID NOs: 1-128. For example, the 17-base sequence shown in Tables 1 and 2 below or the 17 A sequence containing a base length can be exemplified. In the base sequences of Table 1 and Table 2 below, the underlined part and the * mark are the mutant bases, and the * mark indicates a deletion. In the present invention, the polynucleotides shown in both tables are examples and are not limited thereto.

<Genetic polymorphic marker set>
As described above, the genetic polymorphic marker set of the present invention is a genetic polymorphic marker set for distinguishing cruciferous plant varieties, and includes a marker comprising the polynucleotides (1) to (13) above. It is characterized by that.

  The cruciferous plants that can be cultivated by the genetic polymorphic marker set of the present invention are not particularly limited. For example, radish, Chinese cabbage, cabbage, Takana, mustard, turnip, broccoli, cauliflower, watercress, rape (rapeseed), Komatsuna , Singing rhinoceros, Sirona, Pakchoi, Kailan, Mekabetsu, Kohlrabi, Rutabaga, Japanese radish and the like. Among them, the genetic polymorphic marker of the present invention is suitable, for example, for distinguishing radish varieties.

For example, when the type of radish is discriminated by the genetic polymorphic marker of the present invention, the type of radish that can be discriminated is not particularly limited, and various radish can be discriminated. Table 3 below exemplifies radish varieties that can be identified, but the present invention is not limited to this.

Among the various radish ( Raphanus sativus L. ) shown in Table 3, for example, variety numbers 1 to 32 and 54 to 56 are from Takii Seed Co., Ltd., and variety numbers 33 to 41 are from Sakata Seed Co., Ltd. Number 42 is from Kaneko Seed Co., Ltd., Variety Nos. 43 to 49 are from Mikado Kyowa Co., Ltd., Variety No. 50 is from Tohoku Co., Ltd., Variety Nos. 51 and 52 are from Variety No. 53, Variety No. 53. Are available from Thompson & Morgan from Matsushita Seedlings Co., Ltd. Variety numbers 73 to 94 are registered in the agricultural biological resource gene bang (http://www.gene.affrc.go.jp/index_j.php) and are available.

  In the genetic polymorphic marker set of the present invention, each marker included is called a genetic polymorphic marker. Further, hereinafter, in the genetic polymorphic marker set of the present invention, the marker comprising the (S) type polynucleotide is referred to as the (S) type marker or the (S) type, and the marker comprising the (A) type polynucleotide. Is referred to as (A) type marker or (A) type. Since diploid cruciferous plants can be classified into homotypes and heterotypes for a given locus, the markers (1) to (51) are, for example, homotypes of (S) type markers, ( It can be said that there is a heterotype having a homotype of (A) type marker and (S) type marker and (A) type marker.

  As described above, the genetic polymorphic marker set of the present invention is not limited as long as it includes the marker comprising the polynucleotides (1) to (13) described above. The genetic polymorphic marker set of the present invention may further include, for example, a marker comprising the polynucleotides of (14) to (23), and a marker comprising the polynucleotides of (24) to (51). May be included. Hereinafter, in the present invention, the markers comprising the polynucleotides (1) to (51) are also referred to as markers (1) to (51), respectively. Hereinafter, each marker may be represented by the names shown in Tables 1 and 2 above.

  In the present invention, the markers (1) to (51) may be, for example, any of the (S) type marker and the (A) type marker, but preferably each of the (S) type marker. It is preferable that both of the above-mentioned (A) type markers are included. Regarding the markers (1) to (51), the (S) type marker and the (A) type marker are also referred to as (S) type and (A) type marker pairs, respectively. As a specific example, when the marker in (1) includes the (S) type marker and the (A) type marker, the marker in (1) is also referred to as the marker pair in (1). The same applies to (2) to (51). The genetic polymorphism marker set of the present invention preferably includes, for example, the marker pairs of (1) to (13) as the markers of (1) to (13). The marker pair of 23) and / or the marker pair of (24) to (51) may be included.

  Examples of the genetic polymorphic marker in the present invention include the DNA sequences shown in SEQ ID NOs: 1-128, among which the DNA sequences shown in SEQ ID NOs: 1-26 and the DNA sequences shown in SEQ ID NOs: 27-72 are preferable, More preferably, it is a DNA sequence shown to sequence number 1-26. In addition, the polynucleotide of the present invention may be, for example, a partial sequence containing the mutated base in the DNA sequence shown in SEQ ID NOs: 1-128. For example, the 17-base sequence shown in Table 1 and Table 2, or the 17 A sequence containing a base length can be exemplified. In the present invention, the genetic polymorphic markers shown in both tables are illustrative and not limiting.

<Probe>
As described above, the probe set of the present invention is a probe set for detecting the genetic polymorphic marker set of the present invention, and includes the mutant base in the polynucleotides (1) to (13). It contains a DNA sequence or its complementary sequence. The probe set of the present invention is only required to detect the genetic polymorphic marker set of the present invention, and other conditions are not limited at all.

In the probe set of the present invention, the probe containing the DNA sequence is, for example, at least one selected from the group consisting of the following probes (1) to (13).
(1) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 27 or DNA of SEQ ID NO: 27
1 to several bases other than the 9th base in the sequence are substituted, deleted, inserted, added
And the polynucleotide (1) (S) above is high.
A probe capable of bridging.
(A) Probe consisting of the DNA sequence of SEQ ID NO: 28 or DNA of SEQ ID NO: 28
1 to several bases other than the 9th base in the sequence are substituted, deleted, inserted, added
And the polynucleotide (1) (A) above is high.
A probe capable of bridging.
(2) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 29 or DNA of SEQ ID NO: 29
In the sequence, 1 to several other than the 7th to 8th bases and the 16th base
It consists of a DNA sequence in which a base is substituted, deleted, inserted or added, and (2) (
A probe capable of hybridizing to the polynucleotide of S).
(A) Probe consisting of the DNA sequence of SEQ ID NO: 30 or DNA of SEQ ID NO: 30
Mutation between the 8th and 9th in the sequence (CT deletion) and the 16th salt
It consists of a DNA sequence in which one to several bases other than the group are substituted, deleted, inserted, added,
And a probe capable of hybridizing to the polynucleotide of (2) (A)
.
(3) The probe of the following (S) or (A).
(S) a probe comprising the DNA sequence of SEQ ID NO: 31 or DNA of SEQ ID NO: 31
1 to several bases other than the 9th base in the sequence are substituted, deleted, inserted, added
And the polynucleotide (3) (S) above is high.
A probe capable of bridging.
(A) Probe consisting of the DNA sequence of SEQ ID NO: 32 or DNA of SEQ ID NO: 32
1 to several bases other than the 9th base in the sequence are substituted, deleted, inserted, added
And the polynucleotide (3) (A) above is high.
A probe capable of bridging.
(4) The following probe (S) or (A).
(S) a probe comprising the DNA sequence of SEQ ID NO: 33 or DNA of SEQ ID NO: 33
In sequence, mutation between 8th and 9th (C deletion) and 9th and 10th
One to several bases other than the mutation between the eyes (A deletion) are substituted, deleted, inserted, added
And a hive to the polynucleotide (4) (S).
A reproducible probe.
(A) Probe consisting of the DNA sequence of SEQ ID NO: 34 or DNA of SEQ ID NO: 34
In the sequence, 1 to several bases other than the 9th and 11th bases are substituted or missing.
Consisting of a deleted, inserted and added DNA sequence, and the polynucleotide of (4) (A) above
Probe capable of hybridizing to leotide.
(5) The probe of the following (S) or (A).
(S) Probe consisting of the DNA sequence of SEQ ID NO: 35 or DNA of SEQ ID NO: 35
1 to several bases other than the 9th base in the sequence are substituted, deleted, inserted, added
And the polynucleotide (5) (S) above is high.
A probe capable of bridging.
(A) Probe consisting of the DNA sequence of SEQ ID NO: 36 or DNA of SEQ ID NO: 36
1 to several bases other than the 9th base in the sequence are substituted, deleted, inserted, added
And the polynucleotide (5) (A) above is high.
A probe capable of bridging.
(6) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 37 or DNA of SEQ ID NO: 37
1 to several bases other than the 9th base in the sequence are substituted, deleted, inserted, added
And the polynucleotide (6) (S) above is high.
A probe capable of bridging.
(A) a probe comprising the DNA sequence of SEQ ID NO: 38 or DNA of SEQ ID NO: 38
1 to several bases other than the 9th base in the sequence are substituted, deleted, inserted, added
And the polynucleotide (6) (A) above is a high sequence.
A probe capable of bridging.
(7) The probe of the following (S) or (A).
(S) a probe comprising the DNA sequence of SEQ ID NO: 39 or DNA of SEQ ID NO: 39
In the sequence, 1 to several bases other than the 9th and 10th bases are substituted or missing.
Consisting of a deleted, inserted, added DNA sequence, and the polynucleotide of (7) (S) above
Probe capable of hybridizing to leotide.
(A) Probe consisting of the DNA sequence of SEQ ID NO: 40 or DNA of SEQ ID NO: 40
In the sequence, 1 to several bases other than the 9th and 10th bases are substituted or missing.
Consisting of a deleted, inserted, added DNA sequence, and the polynucleotide of (7) (A) above
Probe capable of hybridizing to leotide.
(8) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 41 or DNA of SEQ ID NO: 41
In the sequence, 1 to several bases other than the 8th and 11th bases are substituted or missing.
Consisting of a deleted, inserted, added DNA sequence, and the polynucleotide of (8) (S) above
Probe capable of hybridizing to leotide.
(A) Probe consisting of the DNA sequence of SEQ ID NO: 42 or DNA of SEQ ID NO: 42
In the sequence, 1 to several bases other than the 8th and 11th bases are substituted or missing.
Consisting of a deleted, inserted and added DNA sequence, and the polynucleotide (8) (A)
Probe capable of hybridizing to leotide.
(9) The probe according to (S) or (A) below.
(S) Probe consisting of the DNA sequence of SEQ ID NO: 43 or DNA of SEQ ID NO: 43
In sequence, mutation between 7th and 8th (C deletion), 9th and 11th
From a DNA sequence in which one to several bases other than the base of the eye are substituted, deleted, inserted or added
And hybridizing to the polynucleotide of (9) (S) above
Possible probe.
(A) Probe consisting of the DNA sequence of SEQ ID NO: 44 or DNA of SEQ ID NO: 44
1 to several bases other than the 7th, 9th and 11th bases in the sequence
Consisting of a DNA sequence with substitutions, deletions, insertions, additions, and (9) (A)
A probe capable of hybridizing to a polynucleotide.
(10) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 45 or DN of SEQ ID NO: 45
In the A sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted,
The polynucleotide of (10) (S), comprising the added DNA sequence
Probes that can hybridize to the probe.
(A) a probe comprising the DNA sequence of SEQ ID NO: 46 or DN of SEQ ID NO: 46
In the A sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted,
The polynucleotide of (10) (A), comprising an added DNA sequence
Probes that can hybridize to the probe.
(11) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 47 or DN of SEQ ID NO: 47
In the A sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted,
The polynucleotide of (11) (S), comprising the added DNA sequence
Probes that can hybridize to the probe.
(A) a probe comprising the DNA sequence of SEQ ID NO: 48 or DN of SEQ ID NO: 48
In the A sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted,
The polynucleotide of (11) (A) above, comprising an added DNA sequence
Probes that can hybridize to the probe.
(12) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 49 or DN of SEQ ID NO: 49
In the A sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted,
The polynucleotide of (12) (S), comprising the added DNA sequence
Probes that can hybridize to the probe.
(A) a probe comprising the DNA sequence of SEQ ID NO: 50 or DN of SEQ ID NO: 50
In the A sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted,
The polynucleotide of (12) (A) above, comprising an added DNA sequence
Probes that can hybridize to the probe.
(13) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 51 or DN of SEQ ID NO: 51
In the A sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted,
A polynucleotide comprising the added DNA sequence and the polynucleotide of (13) (S)
Probes that can hybridize to the probe.
(A) a probe comprising the DNA sequence of SEQ ID NO: 52 or DN of SEQ ID NO: 52
In the A sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted,
The polynucleotide of (13) (A), comprising the added DNA sequence
Probes that can hybridize to the probe.

In addition to this, the probe set of the present invention further comprises a DNA sequence containing the mutated base in at least one polynucleotide selected from the group consisting of the polynucleotides of (14) to (23) or a complementary sequence thereof. May be included. The probe containing the DNA sequence is, for example, at least one selected from the group consisting of the following probes (14) to (23).
(14) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 129, or of SEQ ID NO: 129
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (14) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 130, or of SEQ ID NO: 130
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (14) (A)
A probe that can hybridize to an tide.
(15) The probe of the following (S) or (A).
(S) a probe comprising the DNA sequence of SEQ ID NO: 131, or of SEQ ID NO: 131
1 other than mutation between 6th and 7th (CAG deletion) in DNA sequence
~ Consisting of a DNA sequence in which several bases are substituted, deleted, inserted, added, and
(15) A probe capable of hybridizing to the polynucleotide of (S).
(A) a probe comprising the DNA sequence of SEQ ID NO: 132, or of SEQ ID NO: 132
In the DNA sequence, 1 to several bases other than the 5th to 7th bases are substituted or deleted.
The inserted and added DNA sequences, and the polynucleotide of (15) (A)
A probe capable of hybridizing to a nucleotide.
(16) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 133, or of SEQ ID NO: 133
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (16) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 134, or of SEQ ID NO: 134
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (16) (A) above
A probe that can hybridize to an tide.
(17) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 135, or of SEQ ID NO: 135
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (17) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 136, or of SEQ ID NO: 136
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (17) (A) above
A probe that can hybridize to an tide.
(18) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 137, or of SEQ ID NO: 137
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (18) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 138, or of SEQ ID NO: 138
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (18) (A) above
A probe that can hybridize to an tide.
(19) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 139, or of SEQ ID NO: 139
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (19) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 140, or of SEQ ID NO: 140
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide (19) (A).
A probe that can hybridize to an tide.
(20) The probe of the following (S) or (A).
(S) a probe comprising the DNA sequence of SEQ ID NO: 141, or of SEQ ID NO: 141
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (20) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 142, or of SEQ ID NO: 142
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide (20) (A).
A probe that can hybridize to an tide.
(21) The probe of the following (S) or (A).
(S) a probe comprising the DNA sequence of SEQ ID NO: 143, or of SEQ ID NO: 143
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (21) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 144, or of SEQ ID NO: 144
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (21) (A)
A probe that can hybridize to an tide.
(22) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 145, or of SEQ ID NO: 145
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (22) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 146, or of SEQ ID NO: 146
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (22) (A) above
A probe that can hybridize to an tide.
(23) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 147, or of SEQ ID NO: 147
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (23) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 148, or of SEQ ID NO: 148
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Consisting of an inserted and added DNA sequence, and the polynucleotide of (23) (A) above
A probe that can hybridize to an tide.

In addition to this, the probe of the present invention further comprises a DNA sequence containing the mutant base in at least one polynucleotide selected from the group consisting of the polynucleotides of (24) to (51) or a complementary sequence thereof. But you can. The probe containing the DNA sequence is, for example, at least one selected from the group consisting of the following probes (24) to (51).
(24) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 149, or of SEQ ID NO: 149
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (24) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 150, or of SEQ ID NO: 150
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (24) (A) above
A probe that can hybridize to an tide.
(25) The probe of the following (S) or (A).
(S) a probe comprising the DNA sequence of SEQ ID NO: 151, or of SEQ ID NO: 151
In the DNA sequence, 1 to several bases other than the 10th base are substituted, deleted,
The polynucleotide of (25) (S), comprising an inserted and added DNA sequence
Probe capable of hybridizing to leotide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 152, or of SEQ ID NO: 152
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (25) (A) above
A probe that can hybridize to an tide.
(26) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 153, or of SEQ ID NO: 153
In the DNA sequence, 1 to several bases other than the 8th to 10th bases are substituted or missing.
Consisting of a deleted, inserted and added DNA sequence, and the poly (26) (S)
A probe capable of hybridizing to a nucleotide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 154, or of SEQ ID NO: 154
1 other than the mutation between the eighth and ninth positions (ATG deletion) in the DNA sequence
~ Consisting of a DNA sequence in which several bases are substituted, deleted, inserted, added, and
(26) A probe capable of hybridizing to the polynucleotide of (A).
(27) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 155, or of SEQ ID NO: 155
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence added and added, and the polynucleotide of (27) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 156, or of SEQ ID NO: 156
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (27) (A) above
A probe that can hybridize to an tide.
(28) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 157, or of SEQ ID NO: 157
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (28) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 158, or of SEQ ID NO: 158
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (28) (A)
A probe that can hybridize to an tide.
(29) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 159, or of SEQ ID NO: 159
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (29) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 160, or of SEQ ID NO: 160
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (29) (A) above
A probe that can hybridize to an tide.
(30) The probe of the following (S) or (A).
(S) a probe comprising the DNA sequence of SEQ ID NO: 161, or of SEQ ID NO: 161
1 to several bases other than the 8th to 9th bases are substituted or deleted in the DNA sequence
A DNA sequence inserted, added, and the polynucleotide of (30) (S)
A probe capable of hybridizing to a nucleotide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 162, or of SEQ ID NO: 162
1 to several bases other than the 8th to 9th bases are substituted or deleted in the DNA sequence
A DNA sequence inserted, added, and the polynucleotide of (30) (A)
A probe capable of hybridizing to a nucleotide.
(31) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 163, or of SEQ ID NO: 163
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (31) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 164, or of SEQ ID NO: 164
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (31) (A) above
A probe that can hybridize to an tide.
(32) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 165, or of SEQ ID NO: 165
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (32) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 166, or of SEQ ID NO: 166
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (32) (A) above
A probe that can hybridize to an tide.
(33) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 167, or of SEQ ID NO: 167
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (33) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 168, or of SEQ ID NO: 168
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (33) (A) above
A probe that can hybridize to an tide.
(34) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 169, or of SEQ ID NO: 169
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence added and added, and the polynucleotide of (34) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 170, or of SEQ ID NO: 170
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Consisting of an inserted and added DNA sequence, and the polynucleotide of (34) (A) above
A probe that can hybridize to an tide.
(35) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 171 or SEQ ID NO: 171
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (35) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 172, or of SEQ ID NO: 172
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (35) (A) above
A probe that can hybridize to an tide.
(36) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 173, or of SEQ ID NO: 173
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (36) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 174, or of SEQ ID NO: 174
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide (36) (A).
A probe that can hybridize to an tide.
(37) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 175, or of SEQ ID NO: 175
In the DNA sequence, 1 to several bases other than the 10th base are substituted, deleted,
The polynucleotide of (37) (S) above, comprising an inserted and added DNA sequence
Probe capable of hybridizing to leotide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 176, or of SEQ ID NO: 176
In the DNA sequence, 1 to several bases other than the 10th base are substituted, deleted,
The polynucleotide of (37) (A), comprising an inserted and added DNA sequence
Probe capable of hybridizing to leotide.
(38) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 177, or of SEQ ID NO: 177
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (38) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 178, or of SEQ ID NO: 178
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (38) (A) above
A probe that can hybridize to an tide.
(39) The probe of the following (S) or (A).
(S) a probe comprising the DNA sequence of SEQ ID NO: 179, or of SEQ ID NO: 179
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (39) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 180, or of SEQ ID NO: 180
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Consisting of an inserted and added DNA sequence, and the polynucleotide of (39) (A) above
A probe that can hybridize to an tide.
(40) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 181 or SEQ ID NO: 181
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (40) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 182 or SEQ ID NO: 182
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (40) (A) above
A probe that can hybridize to an tide.
(41) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 183, or of SEQ ID NO: 183
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (41) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 184, or of SEQ ID NO: 184
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (41) (A)
A probe that can hybridize to an tide.
(42) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 185, or of SEQ ID NO: 185
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (42) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 186, or of SEQ ID NO: 186
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (42) (A)
A probe that can hybridize to an tide.
(43) The probe of the following (S) or (A).
(S) a probe comprising the DNA sequence of SEQ ID NO: 187, or of SEQ ID NO: 187
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (43) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 188, or of SEQ ID NO: 188
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (43) (A)
A probe that can hybridize to an tide.
(44) The probe of the following (S) or (A).
(S) a probe comprising the DNA sequence of SEQ ID NO: 189, or of SEQ ID NO: 189
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (44) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 190, or of SEQ ID NO: 190
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (44) (A).
A probe that can hybridize to an tide.
(45) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 191 or SEQ ID NO: 191
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (45) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 192, or of SEQ ID NO: 192
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (45) (A) above
A probe that can hybridize to an tide.
(46) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 193, or of SEQ ID NO: 193
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (46) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 194, or of SEQ ID NO: 194
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (46) (A)
A probe that can hybridize to an tide.
(47) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 195, or of SEQ ID NO: 195
1 to number other than mutation between 8th and 9th (T deletion) in DNA sequence
Consisting of a DNA sequence in which one base is substituted, deleted, inserted or added, and said (
47) A probe capable of hybridizing to the polynucleotide of (S).
(A) a probe comprising the DNA sequence of SEQ ID NO: 196, or of SEQ ID NO: 196
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (47) (A)
A probe that can hybridize to an tide.
(48) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 197 or SEQ ID NO: 197
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (48) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 198, or of SEQ ID NO: 198
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (48) (A).
A probe that can hybridize to an tide.
(49) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 199, or of SEQ ID NO: 199
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (49) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 200, or of SEQ ID NO: 200
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (49) (A) above
A probe that can hybridize to an tide.
(50) The probe of the following (S) or (A).
(S) a probe comprising the DNA sequence of SEQ ID NO: 201, or of SEQ ID NO: 201
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Consisting of an inserted and added DNA sequence, and the polynucleotide of (50) (S) above
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 202, or of SEQ ID NO: 202
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (50) (A) above
A probe that can hybridize to an tide.
(51) The probe according to (S) or (A) below.
(S) a probe comprising the DNA sequence of SEQ ID NO: 203, or of SEQ ID NO: 203
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of the above (51) (S)
A probe that can hybridize to an tide.
(A) a probe comprising the DNA sequence of SEQ ID NO: 204, or of SEQ ID NO: 204
In the DNA sequence, 1 to several bases other than the 9th base are substituted, deleted, inserted
Comprising the DNA sequence inserted and added, and the polynucleotide of (51) (A) above
A probe that can hybridize to an tide.

  Hereinafter, in the present invention, the probe of (S) is also referred to as a probe for (S) type for detecting the (S) type polynucleotide or the (S) type marker, and the probe of (A) is: It is also referred to as a (A) type probe for detecting the (A) type polynucleotide or the (A) type marker.

  In the present invention, the probes of (1) to (51) may be, for example, either the (S) type probe or the (A) type probe, but preferably each of the (S) type probe. It is preferable that both the type probe and the (A) type probe are included. Regarding the probes (1) to (51), the (S) type probe and the (A) type probe are also referred to as (S) type and (A) type probe pairs, respectively. As a specific example, when the probe of (1) includes the (S) type probe and the (A) type probe, the probe of (1) may be referred to as the probe pair of (1). The same applies to the other (2) to (51). The probe set of the present invention preferably includes, for example, the probe pairs (1) to (13) as the probes (1) to (13), and the probes (14) to (23). A pair and / or a probe pair of (24) to (51) may be included.

  The probe in the present invention may be, for example, a probe that can hybridize to the antisense strand or a probe that can hybridize to the sense strand. The probes (1) to (51) are probes that can hybridize to the antisense strand. The probe of the present invention may be, for example, a probe composed of a complementary sequence to the probes (1) to (51), and these are probes that can hybridize to the sense strand. Probes having a complementary sequence to the (S) type probe of (1) to (51) are also referred to as (S) type probes for detecting the sense strand, and (A) of (1) to (51) above. A probe having a complementary sequence to the type probe is also referred to as a (A) type probe for detecting the sense strand. The probe in the present invention may be, for example, a probe comprising an RNA sequence in which thymine is replaced with uracil in any of the probes (1) to (51).

  The total length of the probe in the present invention is not particularly limited, and is, for example, 10 to 100 bases long, preferably 10 to 50 bases long, more preferably 15 to 25 bases long, and particularly preferably 17 bases. It is long.

  As a probe in this invention, the sequence | arrangement of 17 base length shown in Table 8 and Table 9 mentioned later can be illustrated, for example. In the present invention, the probes shown in both tables are examples and are not limited thereto. Each probe in the present invention can be appropriately designed by those skilled in the art from the sequences of various cruciferous plants registered in a database or the like based on the sequence of the polynucleotide or marker of the present invention.

  The probe in the present invention may be, for example, capable of hybridizing to the polynucleotide or genetic polymorphic marker of the present invention, but is preferably capable of specifically hybridizing. In the present invention, “specifically hybridizable” means, for example, significantly hybridizing to the target polynucleotide as compared to other polynucleotides under general hybridization conditions. Preferably, under stringent hybridization conditions (eg, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition 1989). It means that it can hybridize significantly to a polynucleotide compared to other polynucleotides.

The probe in the present invention may be, for example, a labeled probe having a labeling substance. The labeling substance is not particularly limited, and for example, a known labeling substance such as a fluorescent substance, a quenching substance for the fluorescent substance, a coloring substance, a radioactive substance such as ³² P, an antibody such as digoxigenin (DIG), avidin or biotin, etc. can give. These labeling substances can be appropriately set according to, for example, the method for detecting a polynucleotide or genetic polymorphic marker of the present invention.

  In the probe of the present invention, the labeling site by the labeling substance is not particularly limited, and examples thereof include at least one of 5 'end and 3' end, but 5 'end is preferable.

<Primer>
As described above, the primer of the present invention is a primer for detecting the polynucleotide of the present invention or the genetic polymorphism marker of the present invention by a gene amplification method, and the primer of the above (1) to (13) It comprises a DNA sequence containing the mutated base in at least one polynucleotide selected from the group consisting of polynucleotides or a complementary sequence thereof. The probe of the present invention only needs to be able to amplify, for example, a region comprising the polynucleotide of the present invention or a genetic polymorphism marker or a region containing them in the gene of the subject, and other conditions are not limited at all.

  In addition to this, the primer of the present invention comprises a DNA sequence containing the mutated base in at least one polynucleotide selected from the group consisting of the polynucleotides of (14) to (23) or a complementary sequence thereof. Features.

  In addition to this, the primer of the present invention comprises a DNA sequence containing the mutated base in at least one polynucleotide selected from the group consisting of the polynucleotides (24) to (51) or a complementary sequence thereof. Features.

  The primer of the present invention may be, for example, a forward primer or a reverse primer. In addition, a primer set including the primer of the present invention can be used, for example, in the method for determining the type of the present invention described later.

  The full length of the primer of this invention is not restrict | limited in particular, For example, it is 10-50 base length, Preferably it is 10-30 base length, More preferably, it is 20-25 base length. The length of the region to be amplified according to the present invention is not particularly limited, but is, for example, 100 to 800 bases long, preferably 120 to 650 bases long, more preferably 120 to 600 bases long.

<Detection kit>
As described above, the detection kit of the present invention is a detection kit for detecting the polynucleotide of the present invention or the genetic polymorphic marker set of the present invention, and includes the probe set of the present invention. Features.

  The detection kit of the present invention preferably includes, for example, an (S) type probe and an (A) type probe for each marker as the probe. In this case, depending on the polymorphism to be detected, it is preferable that either one is the labeled probe and the other is an unlabeled probe. Specifically, when the marker for detection is of the (S) type, for example, it is preferable to include a labeled probe for the (S) type and an unlabeled probe for the (A) type. Thus, when a probe for the (A) type marker different from the detection purpose is used in combination as an unlabeled probe, the labeled probe for the (S) type can be prevented from binding to the (A) type marker due to competition. Therefore, the detection of the (S) type marker can be performed with higher accuracy. For the same reason, when the marker for detection is of type (A), for example, it is preferable to include a labeled probe for type (A) and an unlabeled probe for type (S).

  When the detection kit of the present invention has the labeled probe and the unlabeled probe, the ratio of both is not particularly limited, but for example, the molar ratio of the labeled probe (L) and the unlabeled probe (N) (L: N) is preferably 1: 5 to 1: 100, more preferably 1:10 to 1:50, still more preferably 1:10 to 1:20, and particularly preferably 1:10. is there.

  The detection kit of the present invention is only required to include the probes (1) to (13), and other configurations are not particularly limited. The detection kit of the present invention may further contain, for example, the probes (14) to (23) or the probes (24) to (51). Since cruciferous plants are diploid, for example, for the markers (1) to (51), (S) type homo, (A) type homo, (S) type and (A) type It is preferable to determine which is hetero. For this reason, it is preferable to have a probe pair of the (S) type probe and the (A) type probe with respect to the genetic polymorphic marker for detection purposes.

  The detection kit of the present invention may further include, for example, a primer for amplifying a region to which the probe is hybridized. The primer may include a primer set consisting of a forward primer and a reverse primer.

  The detection kit of the present invention may optionally further contain other reagents depending on, for example, the method for detecting the polynucleotide or genetic polymorphic marker of the present invention. Examples of the other reagent include an enzyme, a substrate of the enzyme, a buffer solution, and the like. In addition, the detection kit of the present invention may further include a container such as a plate or a tube. The detection kit of the present invention may further include instructions for use, for example.

<Product type determination method>
As described above, the cultivar determination method of the present invention is a cultivar determination method for determining the varieties of cruciferous plants, and is performed using the genetic polymorphic marker set of the present invention in the genome of the cruciferous plant. It is characterized by. The present invention is characterized by using the genetic polymorphism marker set of the present invention, and other conditions are not limited at all.

  The breed determination method of the present invention can be performed, for example, by detecting the genetic polymorphic marker set of the present invention in the genome of a subject. Specifically, for example, the genetic polymorphic marker set is detected for the genome of the Brassicaceae plant, and it can be detected by detecting whether each has the (S) type or (A) type. Since cruciferous plants are diploid, for example, each genetic polymorphism marker is classified into (S) type homo, (A) type homo, (S) type, and (A) type hetero. can do. Therefore, the type of the subject is determined by comparing the detection result of the genetic polymorphic marker set for the subject with the respective types of genetic polymorphic markers of various cruciferous plants identified in advance. can do.

  The cruciferous plant is not particularly limited, and examples thereof include those mentioned above. Among them, radish is preferable. Further, the type of radish to be determined is not particularly limited, and examples thereof include 96 types of radish shown in Table 3 as described above.

In the type determination method of the present invention, when determining the type of radish, for example, the radish is 96 types shown in Table 3, and the determination is performed using the determination table shown in Table 4 or Table 5 below. This can be implemented by determining which of the 96 types corresponds. Table 5 below is a table showing the contents of Table 4 below in a tree diagram.

  The determination using the determination table shown in Table 4 or Table 5 can be performed as follows, for example. For example, when determining which of two varieties is any of the specimens, detection of genetic polymorphic markers having different mutated bases between the two varieties and the other varieties and A genetic polymorphic marker having a mutated base that differs between the two types of varieties is detected. As an example, in the case of determining whether the subject is a variety number 95 (blue neck radish) or a variety number 64 (Zlata), a genetic having a different mutated base between both varieties and other varieties. Detection of (3) and (7) is performed as a genetic polymorphic marker, and detection of (8) is performed as a genetic polymorphic marker having a mutated base different between both varieties. Specifically, first, after confirming that the genetic polymorphic markers (3) and (7) are of the (S) type, the genetic polymorphic marker (8) is transformed into the (S) type marker and the (A) ) Detect which type marker. When the genetic polymorphic marker (8) of the subject is the (S) type marker, it can be determined as the breed number 95, and when it is the (A) type marker, it is determined as the breed number 64. it can. In addition, when the breed for determination is not specified, for example, the genetic polymorphic markers (1) to (13) are detected, and (S) type homozygous and (A) type for each marker. It is only necessary to detect the homozygous or heterozygous and to determine the variety based on Table 4 or Table 5 above. In the present invention, the 96 types of Japanese radish shown in Table 3 can be determined by detecting, for example, the genetic polymorphism markers (1) to (13).

  The method for detecting the genetic polymorphic marker in the genome is not particularly limited, and those skilled in the art can appropriately perform a conventional known method based on the sequence of the genetic polymorphic marker of the present invention. .

  The detection of the genetic polymorphic marker in the Brassicaceae plant genome is, for example, because the genetic polymorphic marker has a different mutated base (SNP) between the (S) type and the (A) type. It is only necessary to determine which mutated base in each genetic polymorphic marker.

  The method for determining the mutant base in the Brassicaceae genome is not particularly limited, and a conventionally known method can be employed. Examples of the determination method include a direct sequencing method, a RFLP (Restriction Fragment Length Polymorphism) analysis method, a PCR-RFLP method, a PCR-SSCP (single-strand conformation polymorphism: single chain) Higher order structure polymorphism) method, TaqMan ™ PCR method using reporter substance and quencher substance, Invader method, Pyrosequencing method, Acyclo Prime method, SNuPe method, MALDI-TOF MS method, DNA array method, allele specific Oligonucleotide (Allele Specific Oligonucleotide / ASO) hybridization method, Ttoburotto method, and the like. In the case of the dot blot method, for example, the nucleic acid of the analyte immobilized on the membrane is reacted with the probe for the target genetic polymorphic marker, and the presence or absence of binding to the probe is determined to determine the mutant base. it can. At this time, it is preferable to confirm the presence or absence of binding for each genetic polymorphic marker using the probe for type (S) and the probe for type (A). Thus, it is possible to determine whether the subject is the (S) type, the (A) type, or the heterogeneous for each genetic polymorphic marker. In addition, for example, when confirming whether the genetic polymorphism marker is of the (S) type, a labeled (S) type probe is used for detection, and unlabeled for competition. (A) type probe is preferably used in combination. If the unlabeled (A) type probe is allowed to coexist, the (S) type probe can be prevented from binding to the analyte having the sequence of the (A) type marker. Can be improved.

  For detection of the genetic polymorphism marker in the genome, for example, depending on the method for determining the mutant base, genomic DNA or RNA derived from a cruciferous subject may be used as it is, or the genomic DNA or RNA An amplification product such as DNA or RNA amplified by a nucleic acid amplification method using RNA as a template may be used. When performing the nucleic acid amplification, for example, it is preferable to amplify a region containing a genetic polymorphic marker for detection purposes using a primer. The genomic DNA derived from the subject can be prepared from, for example, leaves, roots, seeds, callus, cultured cells, and the like. The method for preparing the genomic DNA is not particularly limited, and a conventionally known method can be adopted. As a specific example, when the seed is used, for example, the seed can be ground in an alkaline aqueous solvent, and genomic DNA can be extracted from the ground product. The nucleic acid amplification method is not limited in any way, and examples thereof include a PCR (Polymerase Chain Reaction) method, a reverse transcription (RT) -PCR method, an ICAN (Isothermal and chiral primer-initiated amplification of nucleic acid) nuclease acid (NABA) Known nucleic acid amplification methods such as a base amplification (TMA) method, a TMA (Transscription-Mediated Amplification) method, and an SDA (Strand Displacement Amplification) method can be employed.

<Method for testing similarity between varieties>
As described above, the inter-variety similarity test method of the present invention is an inter-variety similarity test method for testing the similarity between two or more varieties of Brassicaceae plants, and the present invention in the genome of a Brassicaceae plant. It is characterized by testing using the genetic polymorphism marker set of. The present invention is characterized by using the genetic polymorphism marker set of the present invention, and other conditions are not limited at all.

  The cruciferous plant is not particularly limited, and examples thereof include those mentioned above. Among them, radish is preferable. In addition, the varieties of radish to be tested are not particularly limited. For example, as described above, the radish of 96 varieties shown in Table 3 can be mentioned.

  In the inter-variety similarity test method of the present invention, when testing the similarity between radish varieties, for example, the radish is 96 varieties shown in Table 3, and the test uses the test table shown in Table 5 above. This can be carried out by testing the similarity in the 96 varieties.

<Seed purity test method>
As described above, the seed purity test method of the present invention is a seed purity test method for testing the purity of the seeds of cruciferous plants, and uses the genetic polymorphic marker set of the present invention in the genome of the cruciferous plant. The method is characterized in that a ratio of a target variety among a plurality of seeds is obtained. The present invention is characterized by using the genetic polymorphism marker set of the present invention, and other conditions are not limited at all.

  Generally, seeds are distributed in units of several tens to several hundreds. However, for example, there is a possibility that seeds of other varieties may be mixed with the seeds of the target varieties, or self-propagated seeds of the parent line may be mixed with the target F1 seeds. Therefore, the purity test is important from the viewpoint of quality control. . According to the present invention, as described above, the use of the genetic polymorphism marker of the present invention makes it possible to discriminate varieties, so that the purity test can be performed quickly and easily with excellent accuracy. It is.

  The cruciferous plant is not particularly limited, and examples thereof include those mentioned above. Among them, radish is preferable. In addition, the varieties of radish to be tested are not particularly limited. For example, as described above, the radish of 96 varieties shown in Table 3 can be mentioned.

  In the seed purity test method of the present invention, when testing the seed purity of radish, for example, the radish is 96 varieties shown in Table 3, and the test uses the determination table shown in Table 4 or Table 5, This is performed by determining the varieties of the plurality of seeds.

The seed purity test method of the present invention can be performed, for example, as follows. A predetermined number of seeds are collected from a sample containing a plurality of seeds, and genomic DNA derived from various offspring is extracted. Then, for each genomic DNA, a genetic polymorphic marker capable of determining a target variety is detected. If this detection result is different from the result of the genetic polymorphism marker of the target variety, it is determined that the seed is from another variety. If the result of the genetic polymorphism marker of the target variety is the same, the seed of the target variety is determined. Is determined. Then, from the number of seeds of other varieties in the collected seeds, the ratio of the seeds of the target variety may be obtained and calculated as the purity.

  In addition, when the purity test for the F1 seed mother line is performed, genomic DNA is extracted in the same manner, and a genetic polymorphic marker showing a different mutated base between F1 seed and the parent line seed is detected. Do. If the detection result is the same as the result of the genetic polymorphism marker of the F1 seed, it is determined as F1 seed, and if the detection result is the same as the result of the genetic polymorphism marker of the parent line seed, it is determined as the parent line seed. . And what is necessary is just to obtain | require the ratio of F1 seed | species from the number of the seeds of the parent system | strain in the extract | collected seed, and calculate this as purity.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

[Example 1]
In this example, for the 96 kinds of radish varieties shown in Table 3, 51 kinds of marker regions were amplified, and polymorphism analysis was performed by the dot blot method. And based on the analysis result of polymorphism, radish variety was discriminated.

(Primer set)
Tables 6 and 7 below show the base sequences of the primer sets used for the amplification of the 51 types of marker sets described above.

(probe)
Tables 8 and 9 below show the sequences of the probes used for the polymorphism detection. In both tables, the upper row is the sequence of the (S) type probe, and the lower row is the sequence of the (A) type probe. Each probe was a 17-base long oligonucleotide containing the polymorphic site in the marker sequence. In addition, for each of the probes, a probe labeled with biotin at the 5 ′ end and an unlabeled probe were prepared, and the former was used as a detection probe and the latter was used as a competition probe. As described later, for the detection of the (S) type marker, a detection (S) type probe and a competitive (A) type probe are used, and for the detection of the (A) type marker, A detection (A) type probe and a competitive (S) type probe were used. In both cases, the compounding ratio indicates the molar ratio (D: C) of the detection probe (D) and the competition probe (C) in the reaction solution for nucleic acid amplification.

<Product type determination>
As the subject, 96 kinds of radish seeds shown in Table 3 were used. The seeds of each of the varieties were pulverized, and an extract having the following composition was added and mixed. Then, a phenol / chloroform / isoamyl alcohol mixture was added, and the supernatant was collected by centrifugation. An equal amount of isopropanol was added to the supernatant to precipitate DNA, and centrifuged again. The obtained precipitate was dissolved in 1 × TE buffer solution having the following composition to prepare a DNA sample. Then, PCR was performed on 10 μL of a PCR reaction solution having the following composition to which the DNA sample was added. The PCR was performed at 94 ° C. for 1 minute, followed by 40 cycles of treatment at 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 72 ° C. for 30 seconds, and treatment at 72 ° C. for 1 minute. In addition, the primer set corresponding to each above-mentioned marker was used for the forward primer and reverse primer in the following PCR reaction liquid.

(Extract)
200 mmol / L Tris-HCl buffer (pH 7.5)
250 mmol / L NaCl
25 mmol / L EDTA
0.5w / v% SDS
(1 x TE buffer)
10 mmol / L Tris-HCl (pH 8.0)
1mmol / L EDTA
(PCR reaction solution: 10 μL)
10 ng DNA
10 pmol forward primer 10 pmol reverse primer 1 x ExTaq buffer ^{* 1}
2 μmol dNTP
5U Taq polymerase <sup>* 2
* 1</sup> Product name, manufactured by Takara Biomedical
^{* 2} Product name ExTaq, manufactured by Takara Biomedical

After the PCR reaction, an equal amount of an alkali-denatured solution having the following composition was added to the reaction solution. Using a multi-pin blotter (ATTO), the denatured reaction solution was dot-blotted on two nylon membranes (trade name NytranN, GE Bioscience) in the same arrangement, and UV crosslinker (manufactured by UVP). The DNA contained in the reaction solution was bound to the membrane by UV irradiation. For each reaction solution, one set of two membranes to which DNA was bound was prepared.
(Alkali modified solution)
0.8 mol / L NaOH
20 mmol / L EDTA

  Next, a prehybridization solution and a hybridization solution having the following composition were prepared. The detection probe and the competition probe in the hybridization solution are as described above. Here, for the detection of the (S) type marker, the (S) type hybridization solution containing the (S) type detection probe and the (A) type competition probe is used, and the (A) type marker is used. For the marker detection, the (A) type hybridization solution containing the (A) type detection probe and the (S) type competition probe was used.

(Prehybridization solution)
5 x SSC
0.1 w / v% sarkosyl 0.02 w / v% SDS
1w / v% Blocking Reagent (Roche)
(Hybridization solution)
Prehybridization solution 10mL
100 μmol / L detection probe 5 μL
100 μmol / L Competitive probe 50 μL

The membrane to which DNA was bound was immersed in the prehybridization solution at 50 ° C. for 1 hour or longer, and then the prehybridization solution was removed. Then, the hybridization solution containing the probe was treated in hot water for 10 minutes, quenched, and then immersed in the hybridization solution at 50 ° C. overnight. At this time, one set of membranes was immersed in the (S) type hybridization solution, and the other was immersed in the (A) type hybridization solution. Then, the membrane after immersion was washed twice for 5 minutes at room temperature using the cleaning solution 1 (0.1 w / v% SDS-containing 2 × SSC), and then the cleaning solution 2 (0.1 w / v % SDS containing 0.1 × SSC) was washed twice at 50 ° C. for 20 minutes, and further washed at room temperature for 5 minutes using TBS having the following composition. And the blocking process was performed by preparing the said TBS solution containing 1v / v% Blocking Reagent (brand name, Roche), and immersing the said membrane in this for 30 minutes or more at room temperature.
(TBS, pH 8.0)
50 mmol / L Tris-HCl buffer solution 0.9 w / v% NaCl
0.02 w / v% NaN ₃

A digoxigenin antibody-alkaline phosphatase complex solution (manufactured by Roche) was diluted 5000 times with the blocking solution, and the membrane thus blocked was immersed at 25 ° C. for 30 minutes. After soaking, the membrane was washed three times for 10 minutes at room temperature using TBS having the above composition. Further, after immersing the membrane in AP9.5 having the following composition at room temperature for 3 minutes, CSPD (Roche) was diluted 500 times with AP9.5, and the membrane was then added at 37 ° C. for 5 minutes. Soaked. Then, the membrane was exposed to an X-ray film, and a light emission signal was detected.
(AP 9.5, pH 9.5)
100 mmol / L Tris-HCl buffer 100 mmol / L NaCl
5 mmol / L MgCl ₂

  Based on the obtained signals, for the 96 types of samples, regarding the markers (1) to (23), the (S) type marker homotype, (A) type marker homotype, (S) type It was classified as a hetero type having a marker and a (A) type marker. As a result, it was found that 96 kinds of samples (variety numbers 1 to 96) can be discriminated by the markers (1) to (23).

  Table 10 below shows the types of the 23 types of markers for 96 types of samples (types 1 to 96). As shown in Table 10 below, 96 types of samples could be classified by discriminating the types of the 23 types of markers. Further, among the 23 types of markers, a dendrogram was prepared in which 96 types of samples were classified according to the S type, A type, and hetero type of 13 types of markers. This dendrogram is shown in Table 11 below. As a result, it is confirmed that 96 types of samples can be classified by discriminating the types of the 23 types of markers, as well as 96 types of samples can be classified by discriminating the types of the 13 types of markers. It was.

[Example 2]
<Seed purity test>
In this example, from the 96 varieties of radish shown in Table 3, single-cross F1 varieties of 2 lines (white skin beauty and puninosuke) seeds were used as seed purity tests as parent lines. The presence or absence of contamination of self-propagating seeds was confirmed.

  A genetic polymorphism marker for discriminating between the F1 seed and the seed of the parental line was selected from Table 1 for each of the white-skinned beauty and the Shinosuke. In the F1 seed, the gene polymorphism marker indicates a hetero type having (S) type and (A) type, and in the parent line seed of the F1 seed, (S) type homo type or (A) type Those showing the homotype of were selected. Specifically, the marker (15) in Table 1 was selected for the white skin beauty, and the marker (5) in Table 1 was selected for Shinosuke.

  First, 96 individuals of single-cross F1 varieties (white skin beautiful woman and puninosuke) were used from 96 radish seeds shown in Table 3 as subjects. The seeds of each of the varieties were pulverized, and an extract having the following composition was added and mixed. Then, a phenol / chloroform / isoamyl alcohol mixture was added, and the supernatant was collected by centrifugation. An equal amount of isopropanol was added to the supernatant to precipitate DNA, and centrifuged again. The obtained precipitate was dissolved in 1 × TE buffer solution having the following composition to prepare a DNA sample. Then, PCR was performed on 10 μL of a PCR reaction solution having the following composition to which the DNA sample (10 ng) was added. The PCR was performed at 94 ° C. for 1 minute, followed by 40 cycles of treatment at 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 72 ° C. for 30 seconds, and treatment at 72 ° C. for 1 minute. For the PCR of white-skinned beauty, the primers shown in Table 6 corresponding to the marker of (15) are used as the forward primer and the reverse primer, respectively, and for the PCR of Shinosuke, as the forward primer and the reverse primer, The primers listed in Table 6 corresponding to the marker (5) were used.

(Extract)
200 mmol / L Tris-HCl buffer (pH 7.5)
250 mmol / L NaCl
25 mmol / L EDTA
0.5w / v% SDS
(1 x TE buffer)
10 mmol / L Tris-HCl (pH 8.0)
1mmol / L EDTA
(PCR reaction solution: 10 μL)
10 ng DNA sample 10 pmol forward primer 10 pmol reverse primer 1 × ExTaq buffer ^{* 1}
2 μmol dNTP
5U Taq polymerase <sup>* 2
* 1</sup> Product name, manufactured by Takara Biomedical
^{* 2} Product name ExTaq, manufactured by Takara Biomedical

  After the PCR reaction, an equal amount of an alkali-denatured solution having the following composition was added to the reaction solution. The denatured reaction solution was dot-blotted with the same arrangement on two nylon membranes (trade name NytranN, GE Bioscience) using a multi-pin blotter (ATTO). Then, using a UV crosslinker (manufactured by UVP), the membrane was irradiated with UV to bind the DNA contained in the reaction solution to the membrane. As described above, one set of two membranes was prepared for each of the white skin beautifying PCR reaction solution and Shinnosuke's PCR reaction solution.

(Alkali modified solution)
0.8 mol / L NaOH
20 mmol / L EDTA

  Next, a prehybridization solution and a hybridization solution having the following composition were prepared. The detection probes and the competition probes in the hybridization solution are as shown in Table 8 above. Here, for the detection of the (S) type marker, the (S) type hybridization solution containing the (S) type detection probe and the (A) type competition probe is used, and the (A) type marker is used. For the marker detection, the (A) type hybridization solution containing the (A) type detection probe and the (S) type competition probe was used.

(Prehybridization solution)
5 x SSC
0.1 w / v% sarkosyl 0.02 w / v% SDS
1w / v% Blocking Reagent (Roche)
(Hybridization solution)
Prehybridization solution 10mL
100 μmol / L detection probe 5 μL
100 μmol / L Competitive probe 50 μL

  The membrane to which DNA was bound was immersed in the prehybridization solution at 50 ° C. for 1 hour or longer, and then the prehybridization solution was removed. Then, the hybridization solution containing the probe was treated in hot water for 10 minutes, quenched, and then immersed in the hybridization solution at 50 ° C. overnight. At this time, one set of membranes was immersed in the (S) type hybridization solution, and the other was immersed in the (A) type hybridization solution. Then, the membrane after immersion was washed twice for 5 minutes at room temperature using the cleaning solution 1 (0.1 w / v% SDS-containing 2 × SSC), and then the cleaning solution 2 (0.1 w / v % SDS containing 0.1 × SSC) was washed twice at 50 ° C. for 20 minutes, and further washed at room temperature for 5 minutes using TBS having the following composition. And the blocking process was performed by preparing the said TBS solution containing 1v / v% Blocking Reagent (brand name, Roche), and immersing the said membrane in this for 30 minutes or more at room temperature.

(TBS, pH 8.0)
50 mmol / L Tris-HCl buffer solution 0.9 w / v% NaCl
0.02 w / v% NaN ₃

  A digoxigenin antibody-alkaline phosphatase complex solution (Roche) was diluted 5000 times with the blocking solution. The blocked membrane was immersed in this diluted solution at 25 ° C. for 30 minutes. After soaking, the membrane was washed three times for 10 minutes at room temperature using TBS having the above composition. Further, the membrane was immersed in AP9.5 having the following composition at room temperature for 3 minutes. Then, CSPD (Roche) was diluted 500 times with AP9.5, and the membrane was immersed in this diluted solution at 37 ° C. for 5 minutes. Then, the membrane was exposed to an X-ray film, and a light emission signal was detected.

(AP 9.5, pH 9.5)
100 mmol / L Tris-HCl buffer 100 mmol / L NaCl
5 mmol / L MgCl ₂

  These results are shown in FIG. 1 and FIG. 1 and 2 are photographs showing emission signals in the X-ray film. FIG. 1 shows the results relating to a beautiful white skin, (A) shows the signal detection of the (S) type marker, and (B) shows the results of signal detection of the (A) type marker. FIG. 2 shows the results relating to Shinosuke, where (A) shows the signal detection of the (S) type marker, and (B) shows the result of signal detection of the (A) type marker.

  Based on FIG. 1 and FIG. 2, the genetic polymorphism markers were classified for the samples of each of 96 individuals, white-skinned beauties and shinnosuke. Specifically, regarding the marker of (15) and the marker of (5) above, (S) type marker homotype, (A) type marker homotype, (S) type marker and (A) type marker It was classified as a hetero type having As a result, as shown in FIG. 1, all 96 individuals with white skin beauty show signals with both (S) type marker and (A) type marker, and have (S) type marker and (A) type marker. Heterotype was determined. From this result, it was confirmed that all 96 white-skinned beauty individuals were F1 seeds, and the possibility of contamination of self-propagated seeds and other lines was extremely low. On the other hand, as shown in FIG. 2, in Shinosuke, 95 out of 96 individuals showed signals with both (S) type marker and (A) type marker, and (S) type marker and (A) type marker Heterotype having However, in one individual indicated by the arrow in FIG. 2A, the signal of the (S) type marker was not detected, and the homozygous type of the (A) type marker was determined. From this result, it was confirmed that 95 individuals of Shinosuke were F1 seeds, but 1 individual was a self-propagating seed (maternal line), and it was found that self-propagating seeds were mixed. From the above results, it was found that, according to the gene polymorphism marker of the present invention, for example, single purity F1 varieties can be tested for seed purity.

  According to the present invention, a subject of a cruciferous plant is detected by a genetic analysis method using these polynucleotides as a genetic marker set, thereby quickly and easily having excellent reliability. It became possible to distinguish the type of specimen. This also makes it possible to perform the purity test of the cruciferous plant seeds quickly and easily with excellent reliability. For this reason, the present invention can be said to be a very useful technique in, for example, quality display and quality confirmation in the distribution of seedlings and the like.

Claims (12)

A genetic polymorphic marker set for distinguishing cruciferous plant varieties, comprising a marker comprising the following polynucleotides (1) to (13):
(1) The polynucleotide of (S) or (A) below.
(S) 10 to 2 containing the 11th mutant base (G) in the DNA sequence of SEQ ID NO: 1
A polynucleotide comprising 73 consecutive DNA sequences.
(A) 10 to 2 containing the 11th mutant base (A) in the DNA sequence of SEQ ID NO: 2
A polynucleotide comprising 83 consecutive DNA sequences.
(2) The polynucleotide of (S) or (A) below.
(S) In the DNA sequence of SEQ ID NO: 3, the 175th to 176th mutant base (CT)
And 10 to 366 consecutive DNA sequences containing the 184th mutant base (C)
A polynucleotide comprising:
(A) In the DNA sequence of SEQ ID NO: 4, a mutant base between positions 165 and 166
(CT deletion) and 173 th 10-353 consecutive, including mutant base (G)
A polynucleotide comprising a DNA sequence.
(3) The polynucleotide of (S) or (A) below.
(S) 10 to 1 including the 90th mutant base (A) in the DNA sequence of SEQ ID NO: 5
A polynucleotide comprising 40 consecutive DNA sequences.
(A) 10 to 1 containing the 92nd mutant base (G) in the DNA sequence of SEQ ID NO: 6
A polynucleotide comprising 45 consecutive DNA sequences.
(4) The polynucleotide of (S) or (A) below.
(S) A mutated base between the 99th and 100th positions in the DNA sequence of SEQ ID NO: 7
(C deletion) and mutated bases between positions 100 and 101 (A deletion)
A polynucleotide comprising 10 to 349 continuous DNA sequences.
(A) The 159th mutant base (C) and 161 in the DNA sequence of SEQ ID NO: 8
A poly consisting of 10 to 406 continuous DNA sequences containing the second mutant base (A)
nucleotide.
(5) The polynucleotide of (S) or (A) below.
(S) 10-2 including the 93rd mutant base (C) in the DNA sequence of SEQ ID NO: 9
A polynucleotide comprising 63 consecutive DNA sequences.
(A) 10 containing the 108th mutant base (T) in the DNA sequence of SEQ ID NO: 10
A polynucleotide consisting of ~ 232 continuous DNA sequences.
(6) The polynucleotide of (S) or (A) below.
(S) 10 containing the 264th mutant base (C) in the DNA sequence of SEQ ID NO: 11
A polynucleotide consisting of ~ 302 continuous DNA sequences.
(A) 10 containing the 301st mutant base (T) in the DNA sequence of SEQ ID NO: 12
A polynucleotide consisting of ~ 336 consecutive DNA sequences.
(7) The polynucleotide of (S) or (A) below.
(S) The 254th to 255th mutant base (CT) in the DNA sequence of SEQ ID NO: 13
A polynucleotide comprising 10 to 439 continuous DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 14, the 261st to 262nd mutant base (TC)
A polynucleotide comprising 10 to 467 consecutive DNA sequences.
(8) The polynucleotide of (S) or (A) below.
(S) In the DNA sequence of SEQ ID NO: 15, the 197th mutant base (T) and 20
A sequence consisting of 10 to 619 continuous DNA sequences containing the 0th mutant base (G).
Renucleotide.
(A) In the DNA sequence of SEQ ID NO: 16, the 208 th mutant base (A) and 21
A sequence consisting of 10 to 620 consecutive DNA sequences containing the first mutant base (A).
Renucleotide.
(9) The polynucleotide of (S) or (A) below.
(S) Mutant salt between the 263rd and 264th positions in the DNA sequence of SEQ ID NO: 17
Group (C deletion), 265th mutant base (T) and 267th mutant base (G
A polynucleotide comprising 10 to 567 consecutive DNA sequences.
(A) In the DNA sequence of SEQ ID NO: 18, the 265th mutant base (C), 267
10 to 563 including the mutated base of the eye (A) and the mutated base of the 269th (T)
A polynucleotide comprising a continuous DNA sequence.
(10) The polynucleotide of (S) or (A) below.
(S) 1 including the 292nd mutant base (A) in the DNA sequence of SEQ ID NO: 19
A polynucleotide comprising a continuous DNA sequence of 0 to 352.
(A) 1 containing the 292nd mutant base (G) in the DNA sequence of SEQ ID NO: 20
A polynucleotide comprising a continuous DNA sequence of 0 to 352.
(11) The polynucleotide of (S) or (A) below.
(S) 10 containing the 89th mutant base (G) in the DNA sequence of SEQ ID NO: 21
A polynucleotide consisting of ˜405 continuous DNA sequences.
(A) 10 containing the 90th mutant base (A) in the DNA sequence of SEQ ID NO: 22
A polynucleotide consisting of ˜408 consecutive DNA sequences.
(12) The polynucleotide of (S) or (A) below.
(S) 1 containing the 290th mutant base (C) in the DNA sequence of SEQ ID NO: 23
A polynucleotide consisting of 0 to 324 continuous DNA sequences.
(A) 1 containing the 300th mutant base (T) in the DNA sequence of SEQ ID NO: 24
A polynucleotide consisting of 0 to 335 continuous DNA sequences.
(13) The polynucleotide of (S) or (A) below.
(S) 1 containing the 226th mutant base (C) in the DNA sequence of SEQ ID NO: 25
A polynucleotide consisting of 0 to 329 continuous DNA sequences.
(A) 1 containing the 212th mutant base (T) in the DNA sequence of SEQ ID NO: 26
A polynucleotide consisting of 0 to 296 continuous DNA sequences. The genetic polymorphic marker set according to claim 1, wherein the cruciferous plant is radish. The genetic polymorphic marker set according to claim 2, wherein the radish is at least one selected from the group consisting of varieties shown in Table 1 below.
A probe set for detecting the genetic polymorphic marker set according to any one of claims 1 to 3, wherein the DNA sequence comprises the mutant base in the polynucleotide of (1) to (13) or A probe set comprising the complementary sequence. The probe set according to claim 4, wherein the probe containing the DNA sequence is a probe of the following (1) to (13).
(1) The probe according to (S) or (A) below.
(S) probe consisting of the DNA sequence of SEQ ID NO: 27.
(A) probe comprising a DNA sequence of SEQ ID NO: 28.
(2) The probe according to (S) or (A) below.
(S) probe consisting of a DNA sequence of SEQ ID NO: 29.
(A) probe comprising a DNA sequence of SEQ ID NO: 30.
(3) The probe of the following (S) or (A).
(S) probe consisting of the DNA sequence of SEQ ID NO: 31.
(A) probe comprising a DNA sequence of SEQ ID NO: 32.
(4) The following probe (S) or (A).
(S) probe consisting of a DNA sequence of SEQ ID NO: 33.
(A) probe comprising a DNA sequence of SEQ ID NO: 34.
(5) The probe of the following (S) or (A).
(S) probe consisting of a DNA sequence of SEQ ID NO: 35.
(A) probe comprising a DNA sequence of SEQ ID NO: 36.
(6) The probe according to (S) or (A) below.
(S) probe consisting of the DNA sequence of SEQ ID NO: 37.
(A) probe comprising a DNA sequence of SEQ ID NO: 38.
(7) The probe of the following (S) or (A).
(S) probe consisting of the DNA sequence of SEQ ID NO: 39.
(A) probe comprising a DNA sequence of SEQ ID NO: 40.
(8) The probe according to (S) or (A) below.
(S) probe consisting of the DNA sequence of SEQ ID NO: 41.
(A) probe comprising a DNA sequence of SEQ ID NO: 42.
(9) The probe according to (S) or (A) below.
(S) probe consisting of a DNA sequence of SEQ ID NO: 43.
(A) probe comprising a DNA sequence of SEQ ID NO: 44.
(10) The probe according to (S) or (A) below.
(S) probe consisting of the DNA sequence of SEQ ID NO: 45.
(A) probe comprising a DNA sequence of SEQ ID NO: 46.
(11) The probe according to (S) or (A) below.
(S) probe consisting of a DNA sequence of SEQ ID NO: 47.
(A) A probe consisting of the DNA sequence of SEQ ID NO: 48.
(12) The probe according to (S) or (A) below.
(S) probe consisting of a DNA sequence of SEQ ID NO: 49.
(A) probe comprising a DNA sequence of SEQ ID NO: 50.
(13) The probe according to (S) or (A) below.
(S) probe consisting of the DNA sequence of SEQ ID NO: 51.
(A) probe comprising a DNA sequence of SEQ ID NO: 52. A detection kit for detecting the genetic polymorphic marker set according to any one of claims 1 to 3, comprising the probe set according to claim 4 or 5. A cultivar determination method for determining the varieties of cruciferous plants, wherein the varieties are determined using the genetic polymorphic marker set according to claim 1 in the genome of the cruciferous plant. The method according to claim 7, wherein the cruciferous plant is a radish. The radish is 96 varieties shown in Table 1 below, and the determination is carried out by determining which of the 96 varieties corresponds to the determination table shown in Table 2 below. The product type determination method according to claim 8, characterized in that:

A seed purity test method for testing the purity of a seed of a cruciferous plant, wherein the ratio of a target variety in a plurality of seeds is obtained using the genetic polymorphic marker set according to claim 1 in the genome of the cruciferous plant. A seed purity test method. The seed purity test method according to claim 10, wherein a ratio of seeds of the target variety is obtained from the number of seeds of the target variety and the seeds of other varieties among the plurality of seeds. The seed purity test method according to claim 10, wherein a ratio of the target F1 seed is obtained from the number of the target F1 seed and the parent line seed in the plurality of seeds.