JP6934647B2 - How to identify the variety of potatoes or tissues derived from them, or their processed products - Google Patents

Description

The present invention relates to a method for identifying potatoes or tissues derived from them, or varieties of processed products thereof (eg, Japanese varieties, US varieties).

Crop variety identification technology is an important technology used for proper food labeling and protection of breeders' rights. In potatoes, there is a need to identify varieties of processed products, and there is also a need to identify varieties in similarity tests for cases of infringement of breeders' rights.

As a variety identification technique for potatoes, a method using the RAPD (Random Amplified Polymorphic DNA) method (Non-Patent Document 1) and a database of Japanese domestic varieties using SSR (Simple Sequence Repeat) markers (Non-Patent Documents 2 and 3). (Non-Patent Document 4) has been reported.

Motoyuki MORI et al. , Jpn. J. Genet. (1993) 68, pp. 167-174 M. Ghisline et al. , Theor. Apple. Genet. , 2004, 108: 881-90 Marc Ghislain et al. , Mol Breeding (2009) 23: 377-388 Creation of a potato genotype database by DNA analysis, breeding research, 10: 63-69 (2008)

However, the RAPD marker has a problem that the reproducibility of the result is low and the genetic polymorphism information obtained is small.

On the other hand, SSR markers have good reproducibility and are rich in polymorphism, but most of the SSR markers used in the prior art are composed of short repeating units of 2 to 3 bases, so that they have stutter peaks (stutter peaks). Accompanying peaks) are likely to occur, and accurate judgment may be difficult.

Therefore, more accurate variety identification technology for potatoes is required.

As a result of diligent studies, the present inventors have succeeded in identifying eight types of highly distinguishable 4-base repeating SSR markers in potatoes. These SSR markers have few stutter peaks and can accurately detect peaks according to the variety of potatoes. By using one or more of these SSR markers, we succeeded in identifying a wide range of potato varieties of Japanese varieties and American varieties with high accuracy, and completed the present invention.

That is, the present invention is based on genetic polymorphisms of 4-base repeating SSRs (SSR ID4026 / 4027, SSR ID8242, SSR ID12002, SSR ID16410, SSR ID31924, SSR ID35584, SSR ID43016, and SSR ID46514) as described below. To provide a method for identifying varieties of potatoes or tissues derived from them, or their processed products.

The SSR marker has the advantages that the results are more reproducible than the RAPD marker and that a large amount of polymorphic information can be obtained. Further, since the 4-base repetitive SSR marker has fewer stutter peaks than the SSR marker composed of short repetitive units of 2 to 3 bases, it is possible to accurately detect the peak according to the variety of the potato. Therefore, the identification method of the present invention is excellent in the ability to identify potatoes or structures derived from them, or their processed products. In addition, the identification method of the present invention can detect a variety-specific peak corresponding to each SSR marker, and by combining these SSR markers, it is possible to identify a variety of potatoes with higher accuracy.

FIG. 1 shows the R.I. The analysis chart figure by capillary electrophoresis of the amplification fragment corresponding to 8 kinds of SSR markers using Burbank as a subject is shown. The horizontal axis shows the nucleotide length (nt) of the amplified fragment, and the vertical axis shows the signal intensity of the amplified fragment peak. The eight SSR markers were amplified by multiplex PCR using primer pairs labeled with two fluorescent dyes of four colors each, and the reactants were analyzed by capillary electrophoresis. Each analysis chart in FIG. 1 shows a diagram decomposed for each type of dye. The length (nucleotide length) of the amplified fragment corresponding to each SSR marker (denoted by SSR ID number) is shown in a box below the analytical chart. FIG. 2 shows the R.I. Burbank, R.M. Norkoth, Ranger R.M. , And Alturas, are shown in the analysis chart by capillary electrophoresis of the amplified fragments corresponding to the eight SSR markers as subjects. The horizontal axis shows the nucleotide length (nt) of the amplified fragment, and the vertical axis shows the signal intensity of the amplified fragment peak. The eight SSR markers were amplified by multiplex PCR using primer pairs labeled with two fluorescent dyes of four colors each, and the reactants were analyzed by capillary electrophoresis. Each analytical chart shows a superposition of amplification fragments corresponding to all types of SSR markers (denoted by SSR ID number). The length of the amplified fragment (nucleotide length) corresponding to each SSR marker is shown in a box below the analytical chart. FIG. 3 is a diagram showing the genomic sequence (SEQ ID NO: 1) of the potato in the portion where the SSR marker of SSR ID 4026/4027 is located. The underline shows the SSR sequence, and the double underline shows the primer sequence used in the examples. FIG. 4 is a diagram showing the genomic sequence (SEQ ID NO: 2) of the potato in the portion where the SSR marker of SSR ID 8242 is located. The underline shows the SSR sequence, and the double underline shows the primer sequence used in the examples. FIG. 5 is a diagram showing the genomic sequence (SEQ ID NO: 3) of the potato in the portion where the SSR marker of SSR ID 12002 is located. The underline shows the SSR sequence, and the double underline shows the primer sequence used in the examples. FIG. 6 is a diagram showing the genomic sequence (SEQ ID NO: 4) of the potato in the portion where the SSR marker of SSR ID 16410 is located. The underline shows the SSR sequence, and the double underline shows the primer sequence used in the examples. FIG. 7 is a diagram showing the genomic sequence (SEQ ID NO: 5) of the potato in the portion where the SSR marker of SSR ID 31924 is located. The underline shows the SSR sequence, and the double underline shows the primer sequence used in the examples. FIG. 8 is a diagram showing the genomic sequence (SEQ ID NO: 6) of the potato in the portion where the SSR marker of SSR ID 35584 is located. The underline shows the SSR sequence, and the double underline shows the primer sequence used in the examples. FIG. 9 is a diagram showing the genomic sequence (SEQ ID NO: 7) of the potato in the portion where the SSR marker of SSR ID 43016 is located. The underline shows the SSR sequence, and the double underline shows the primer sequence used in the examples. FIG. 10 is a diagram showing the genomic sequence (SEQ ID NO: 8) of the potato in the portion where the SSR marker of SSR ID 46514 is located. The underline shows the SSR sequence, and the double underline shows the primer sequence used in the examples.

The present invention provides a method for identifying varieties of potatoes, tissues derived from them, or processed products thereof (hereinafter, abbreviated as "potassium and the like", if necessary).

As used herein, the term "valley" means a plant of potato and seeds and seedlings (eg, seed potatoes, seedlings, seeds) that grow into the plant of potato.

As used herein, the term "tissue derived from potato" shall mean a part of potato as defined above, and includes, for example, tubers, buds, roots, stems, leaves, flowers, fruits, and parts thereof. Be done.

As used herein, a "processed product" of a potato or a tissue derived from it is a processed product containing the potato or a tissue derived from it (eg, tuber), and the genomic DNA of the potato that can be used by the identification method of the present invention. Is not particularly limited as long as it can be extracted. Such processed products include, for example, foods (eg, potato flakes, potato chips, fried potatoes, potato salad, croquettes), feeds, starches (eg, potato starch, water and livestock paste products, instant noodles), processed starches (eg, instant noodles). , Iisomerized sugar), starched liquor (eg vodka, gin, aquabit, shochu).

Examples of varieties such as potatoes include Japanese varieties and American varieties. Examples of Japanese varieties include Nishiyutaka, Baron Yabu, Toyoshiro, Make-in, Kitaakali, Hokkaikogane, Inca Awakening, Musamaru, and Red Moon. Examples of US varieties include Russet Burbank (R. Burbank), Russet Norkotah (R. Norkotah), Ranger Russet (Ranger R.), Alturas, Umatilla Russet (Umatilla R.), Russet (Umatilla R.), Russet. Examples include Shepody, Clearwater, Cal white, and Western Russet (Western R.).

The identification method of the present invention includes identifying the cultivar based on the genetic polymorphism of one or more 4-base repeat SSRs in the test genomic DNA.
Here, one or more 4-base repeat SSRs are selected from the group consisting of:
(1) SSR ID 4026/4027;
(2) SSR ID 8242;
(3) SSR ID12002;
(4) SSR ID 16410;
(5) SSR ID 31924;
(6) SSR ID35584;
(7) SSR ID43016;
(8) SSR ID 46514.

The number of 4-base repeat SSRs used in the identification method of the present invention is not particularly limited as long as it is 1 or more, and is, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8. You may.

A 4-base repeat SSR (Simple Sequence Repeat) is a portion in which a 4-base motif sequence is repeated. The motif sequence in one SSR is often one type, but may include repetitions of a plurality of types (eg, two types) of motif sequences.

The 4-base repeat SSR specified by the SSR ID number can be accessed by the Spud DB Potato Genomics Resource At Michigan State University (http://solanaceae.plattobiology.musugoengo. The 4-base repeat SSR specified by the ID number registered in PGSC Data, "Putative SSRs on the PGSC Version 4.03 Pseudomethylules") is shown. The details of the 4-base repeating SSR used in the present invention are shown in Table 1. In addition, the genome sequences (SEQ ID NOs: 1 to 8; reference species: Solanum tuberosum group Pureja DM1-3) in the vicinity where these 4-base repeating SSRs are located are shown in FIGS. 3 to 10. These sequences can be found in the potato genome database (eg, Spud DB Genome Brown Potato (Solanum tuberosum group Potato DM1-3) PGSC v4.03 Pseudomolecules / URL http: // It can be viewed by accessing potato /).

In the identification method of the present invention, the detection of a genetic polymorphism of 4-base repeating SSR from a subject (potassium or the like) can be performed, for example, by a gene amplification method (eg, PCR) on the genomic DNA of the subject. The amplified fragment obtained by the gene amplification method can be analyzed by, for example, electrophoresis or sequencing. When a plurality of amplified fragments are detected, it may be difficult to analyze by sequencing, so it is preferable to analyze by electrophoresis (eg, capillary electrophoresis).

Primers for detecting genetic polymorphisms of 4-base repeat SSR by gene amplification method can amplify genomic regions containing genetic polymorphisms of 4-base repeat SSR, anneal to target sites, and analyze in 1-nucleotide length units. It can be designed without particular limitation as long as it produces an amplified fragment of a length (eg, 50 to 1000 nt) as long as possible. The nucleotide length of the primer may be, for example, 15 nt or more, 16 nt or more, 17 nt or more, 18 nt or more, or 20 nt or more, and may be 50 nt or less, 40 nt or less, or 30 nt or less. Primers may be labeled with a fluorescent material to detect amplified fragments. Examples of the fluorescent substance include NED, 6-FAM, VIC and PET. When analyzing genetic polymorphisms of multiple types of 4-base repeat SSRs at the same time, in order to distinguish each genetic polymorphism, each primer pair is a fluorescent substance having a different wavelength (eg, NED, 6-FAM). , VIC, PET) and / or may be designed as a primer pair with different lengths of gene amplification.

Primer pairs for detecting genetic polymorphisms in 4-base repeat SSRs with SSR ID 4026/4027 are preferably:
(1a) A first primer having at least a part of the base sequence of the 1st to 500th base sequences in the base sequence shown in SEQ ID NO: 1 or an equivalent base sequence thereof, and 561 in the base sequence shown in SEQ ID NO: 1. A combination of a second primer having a complementary sequence of at least a part of the base sequence of the 1060th base sequence or an equivalent base sequence thereof; or (1b) the 1st to 500th bases in the base sequence shown in SEQ ID NO: 1. The first primer having at least a part of the base sequence of the sequence or a complementary sequence of an equivalent base sequence thereof, and at least a part of the base sequence of the 561-1060th base sequence in the base sequence shown in SEQ ID NO: 1 or a base sequence thereof. A combination of second primers having an even base sequence.
More preferably, such a primer pair is a combination of a first primer having the base sequence shown in SEQ ID NO: 9 and a second primer having the base sequence shown in SEQ ID NO: 10.

Primer pairs for detecting genetic polymorphisms in 4-base repeat SSRs with SSR ID 8242 are preferably:
(2a) A first primer having at least a part of the base sequence of the 1st to 500th base sequences in the base sequence shown in SEQ ID NO: 2 or an equivalent base sequence thereof, and 933 in the base sequence shown in SEQ ID NO: 2. A combination of a second primer having at least a part of the base sequence of ~ 1432 or a complementary sequence of the equivalent base sequence; or (2b) bases 1 to 500 in the base sequence shown in SEQ ID NO: 2. The first primer having a complementary sequence of at least a part of the sequence or an equivalent base sequence thereof, and the base sequence of at least a part of the base sequences 933 to 1432 in the base sequence shown in SEQ ID NO: 2 or a base sequence thereof. A combination of second primers having an even base sequence.
More preferably, such a primer pair is a combination of a first primer having the base sequence shown in SEQ ID NO: 11 and a second primer having the base sequence shown in SEQ ID NO: 12.

Primer pairs for detecting genetic polymorphisms in the 4-base repeat SSR of SSR ID 12002 are preferably:
(3a) A first primer having at least a part of the base sequence of the 1st to 500th base sequences in the base sequence shown in SEQ ID NO: 3 or an equivalent base sequence thereof, and 533 in the base sequence shown in SEQ ID NO: 3. A combination of a second primer having a complementary sequence of at least a part of the base sequence of positions 1032 or an equivalent base sequence thereof; or (3b) the bases of positions 1 to 500 in the base sequence shown in SEQ ID NO: 3. The first primer having a complementary sequence of at least a part of the sequence or an equivalent base sequence thereof, and the base sequence of at least a part of the base sequences 533 to 1032 in the base sequence shown in SEQ ID NO: 3 or a base sequence thereof. A combination of second primers having an even base sequence.
More preferably, such a primer pair is a combination of a first primer having the base sequence shown in SEQ ID NO: 13 and a second primer having the base sequence shown in SEQ ID NO: 14.

Primer pairs for detecting genetic polymorphisms in the 4-base repeat SSR of SSR ID 16410 are preferably:
(4a) A first primer having at least a part of the base sequence of the 1st to 500th base sequences in the base sequence shown in SEQ ID NO: 4 or an equivalent base sequence thereof, and 585 in the base sequence shown in SEQ ID NO: 4. A combination of a second primer having a complementary sequence of at least a part of the base sequence of the 1084th base sequence or an equivalent base sequence thereof; or (4b) the 1st to 500th bases in the base sequence shown in SEQ ID NO: 4. A first primer having a complementary sequence of at least a part of the sequence or an equivalent base sequence thereof, and at least a part of the base sequence of the 585th to 1084th base sequence in the base sequence shown in SEQ ID NO: 4 or a base sequence thereof. A combination of second primers having an even base sequence.
More preferably, such a primer pair is a combination of a first primer having the nucleotide sequence shown in SEQ ID NO: 15 and a second primer having the nucleotide sequence shown in SEQ ID NO: 16.

Primer pairs for detecting genetic polymorphisms in the 4-base repeat SSR of SSR ID 31924 are preferably:
(5a) A first primer having at least a part of the 1st to 501st base sequences in the base sequence shown in SEQ ID NO: 5 or an equivalent base sequence thereof, and 729 in the base sequence shown in SEQ ID NO: 5. A combination of a second primer having at least a part of the base sequence of the 1228th base sequence or a complementary sequence of the equivalent base sequence thereof; or (5b) the 1st to 501st bases in the base sequence shown in SEQ ID NO: 5. A first primer having a complementary sequence of at least a part of the sequence or an equivalent base sequence thereof, and at least a part of the base sequence of positions 729 to 1228 in the base sequence shown in SEQ ID NO: 5 or a base sequence thereof. A combination of second primers having an even base sequence.
More preferably, such a primer pair is a combination of a first primer having the nucleotide sequence shown in SEQ ID NO: 17 and a second primer having the nucleotide sequence shown in SEQ ID NO: 18.

Primer pairs for detecting genetic polymorphisms in the 4-base repeat SSR of SSR ID 35584 are preferably:
(6a) The first primer having at least a part of the base sequence of the 1st to 500th base sequences in the base sequence shown in SEQ ID NO: 6 or an equivalent base sequence thereof, and 549 in the base sequence shown in SEQ ID NO: 6. A combination of a second primer having a complementary sequence of at least a part of the base sequence of the 1048th base sequence or an equivalent base sequence thereof; or (6b) the 1st to 500th bases in the base sequence shown in SEQ ID NO: 6. A first primer having a complementary sequence of at least a part of the sequence or an equivalent base sequence thereof, and at least a part of the base sequence of the 549th to 1048th base sequences in the base sequence shown in SEQ ID NO: 6 or a base sequence thereof. A combination of second primers having an even base sequence.
More preferably, such a primer pair is a combination of a first primer having the base sequence shown in SEQ ID NO: 19 and a second primer having the base sequence shown in SEQ ID NO: 20.

Primer pairs for detecting genetic polymorphisms in 4-base repeat SSRs with SSR ID 43016 are preferably:
(7a) A first primer having at least a part of the base sequence of the 1st to 500th base sequences in the base sequence shown in SEQ ID NO: 7 or an equivalent base sequence thereof, and 541 in the base sequence shown in SEQ ID NO: 7. A combination of a second primer having a complementary sequence of at least a part of the base sequence of the 1040th base sequence or an equivalent base sequence thereof; or (7b) the 1st to 500th bases in the base sequence shown in SEQ ID NO: 7. The first primer having a complementary sequence of at least a part of the sequence or an equivalent base sequence thereof, and at least a part of the base sequence of the 541st to 1040th base sequences in the base sequence shown in SEQ ID NO: 7 or a base sequence thereof. A combination of second primers having an even base sequence.
More preferably, such a primer pair is a combination of a first primer having the base sequence shown in SEQ ID NO: 21 and a second primer having the base sequence shown in SEQ ID NO: 22.

Primer pairs for detecting genetic polymorphisms in the 4-base repeat SSR of SSR ID 46514 are preferably:
(8a) The first primer having at least a part of the base sequence of the 1st to 500th base sequences in the base sequence shown in SEQ ID NO: 8 or an equivalent base sequence thereof, and 545 in the base sequence shown in SEQ ID NO: 8. A combination of a second primer having a complementary sequence of at least a part of the base sequence of the 1044th base sequence or an equivalent base sequence thereof; or (8b) the 1st to 500th bases in the base sequence shown in SEQ ID NO: 8. The first primer having a complementary sequence of at least a part of the sequence or an equivalent base sequence thereof, and at least a part of the base sequence of the 545th to 1044th base sequences in the base sequence shown in SEQ ID NO: 8 or a base sequence thereof. A combination of second primers having an even base sequence.
More preferably, such a primer pair is a combination of a first primer having the base sequence shown in SEQ ID NO: 23 and a second primer having the base sequence shown in SEQ ID NO: 24.

The above-mentioned "uniform base sequence" can be a base sequence corresponding to variations in the base sequence between varieties such as potatoes. Therefore, a base sequence that is uniform to a predetermined base sequence is a modification (substitution) of several bases (for example, 1 to 10, preferably 1 to 5, more preferably 1, 2 or 3) in the predetermined base sequence. , Deletion, insertion or addition). Such uniform base sequences are obvious to those skilled in the art and can be found by reference to databases that register genetic information for potato varieties, or on the genome of chromosome numbers and bases in potato varieties. It can be determined by decoding the base sequence of the vicinity region of the genetic polymorphism of the 4-base repeat SSR specified at the position of.

The gene amplification method may be carried out by either a single-plex gene amplification method in which a gene is amplified in a reaction system containing a single primer pair, or a multiplex gene amplification method in which a gene is amplified in a reaction system containing a plurality of primer pairs. In the case of the multiplex gene amplification method, primer pairs labeled with fluorescent substances having different wavelengths (eg, NED, 6-FAM, VIC, PET) may be mixed, and the gene amplification products labeled with the same fluorescent substance may be mixed. Primer pairs of different lengths may be mixed. In the case of the multiplex gene amplification method, a maximum of primer pairs corresponding to the genetic polymorphisms of all types of 4-base repeat SSRs used may be mixed, for example, the genetic polymorphisms of 8 types of 4-base repeat SSRs. When using a mold, 2 to 8 types of primer pairs may be mixed.

When the amplified fragment is analyzed by electrophoresis, the amplified fragment corresponding to the genetic polymorphism of a single 4-base repeating SSR may be electrophoresed, and corresponds to the genetic polymorphism of a plurality of 4-base repeating SSRs. A mixture of amplified fragments may be electrophoresed. Amplified fragments corresponding to the genetic polymorphisms of each 4-base repeat SSR were detected as peaks by electrophoresis, and the length (nucleotide length) of each peak was measured to correspond to the genetic polymorphisms of the 4-base repeat SSRs. Obtain peak pattern data.

The peak pattern corresponding to the genetic polymorphism of each 4-base repeat SSR reflects the genetic polymorphism of the genetic polymorphism of the 4-base repeat SSR. Varieties of subjects (such as potatoes) can be identified based on the genetic polymorphism represented by the peak pattern. The varieties may be identified by conducting an experiment using a potato or the like whose varieties are known in advance and collating the peak patterns, and the peak patterns known for each variety (eg, shown in Table 3). It may be performed by collating with the peak pattern).

In one embodiment, the identification method of the present invention can be performed utilizing a genetic polymorphism of a particular 4-base repeating SSR that produces a peak pattern peculiar to a particular variety of potatoes. According to this embodiment, a specific variety of potato can be easily identified based on the genetic polymorphism of one kind of 4-base repeating SSR. In such cases, the identification method of the present invention can be performed, for example, by:
(A) Identification of Toyoshiro, Makein, Kitaakali, Musamar, Alturas, Russet Bannock, Shepody, Clearwater, or Cal white based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 4026/4027;
(B) Identification of Toyoshiro, Makein, Kitaakali, Musamaru, Red Moon, Russet Burbank, Russet Norkotah, Alturas, Shepody, Clearwater, or Calwite based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 8242.
(C) Nishiyutaka, Toyoshiro, Make-in, Inca Awakening, Musamaru, Red Moon, Russet Burbank, Russet Norkotah, Alturas, Umatilla Russet, or Shep, based on the genetic polymorphism of the 4-base repeat SSR of SSR ID12002.
(D) Nishiyutaka, Baron Yabu, Toyoshiro, Make-in, Kitaakali, Hokkaikogane, Inca Awakening, Red Moon, Russet Burbank, Russet Norkoth, Shepody, based on the genetic polymorphism of 4-base repeat SSR of SSR ID 16410. identification;
(E) Identification of Nishiyutaka, Baron, Hokaikogane, Inca Awakening, Red Moon, Russet Burbank, Russet Norkotah, Ranger Russet, or Western Russet, based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 31924.
(F) Identification of Musamar, Red Moon, Russet Burbank, or Cal white based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 35584;
Identification of Nishiyutaka, Makein, Red Moon, Russet Burbank, or Alturas based on the genetic polymorphism of the 4-base repeat SSR of (G) SSR ID43016; or to the genetic polymorphism of the 4-base repeat SSR of (H) SSR ID46514 Identification of Make-in, Hokkaikogane, Red Moon, Russet Norkotah, Umatilla Russet, Shepody, or Western Russet based on.

In another embodiment, the identification method of the present invention can be performed utilizing a genetic polymorphism of a particular 4-base repeating SSR that produces a single peak peculiar to a particular variety of potatoes. According to this embodiment, not only electrophoresis but also analysis by sequencing can be easily performed. In such cases, the identification method of the present invention can be performed, for example, by:
(A') Identification of Alturas, or Clearwater, based on genetic polymorphisms of 4-base repeat SSR with SSR ID 4026/4027;
(B') Identification of Clearwater based on genetic polymorphism of 4-base repeat SSR with SSR ID 8242;
(C') Inca awakening, or Musamal identification based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 12002; or (E') Baron 薯 based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 31924 , Or identification of the Inca awakening.

In yet another embodiment, Japanese varieties may be identified based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 16410. The genetic polymorphism of the 4-base repeat SSR of SSR ID 16410 can distinguish Japanese varieties except Musamar from US varieties. If identification of Musamar is desired, one or more 4 bases selected from the group consisting of SSR ID4026 / 4027, SSR ID8242, SSR ID12002, and SSR ID35584, in addition to the genetic polymorphism of 4-base repeating SSR of SSR ID16410. Musamar can be identified by analyzing the genetic polymorphisms of repetitive SSRs.

In another embodiment, the identification method of the present invention can be used to evaluate the possibility of mixing other varieties in a potato of a designated variety or a tissue derived from the specified variety, or a processed product containing the designated variety. .. For example, when a potato of a specified variety or a tissue derived from it, or a processed product containing the specified variety shows a genetic polymorphism other than the genetic polymorphism peculiar to the 4-base repeated SSR of the specified variety (eg,). (If a genetic polymorphism specific to 4-base repeat SSR of other varieties is detected), the possibility of mixing other varieties, and / or the specified varieties may be substituted by other varieties. On the other hand, if no genetic polymorphism other than the genetic polymorphism peculiar to the 4-base repeat SSR of the specified variety is shown, the possibility of mixing other varieties and / or the specified variety Can be evaluated as having no or low possibility of being substituted by other varieties.

Specifically, for the evaluation of the possibility of mixing other varieties as described above, a single 4-base repeating SSR may be used depending on the specified varieties, or two or more (eg, 2, 2,). A combination of 3, 4, 5, 6, 7 or 8, preferably 2) 4-base repeating SSR may be used. For example, for such an evaluation, the single 4-base repeat SSR listed in Table 2 and the minimum combination of 4-base repeat SSRs can be used.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1: Protocol for cultivar identification from potato tubers (1) Subject R. Burbank, R.M. Norkotah, Ranger R.M. , And tubers of Alturas were used as subjects to identify varieties.
(2) DNA extraction DNA was extracted from each tuber using GM quicker 2 (Nippon Gene) according to the instructions of the kit. After measuring the concentration of the extracted DNA, it was diluted with sterilized water to a concentration of 2.5 ng / μL.
(3) Primers The primers shown in Table 3 were used as primers for detecting genetic polymorphisms of 8 kinds of 4-base repeated SSRs. As the F primer, a 6-FAM, VIC, NED or PET fluorescent dye labeled at the 5'end was used.

(4) PCR reaction The PCR reaction was carried out as 8-Plex PCR in which 8 to 16 primers were mixed and used at the concentrations used in Table 3. The reaction solution contains the above-mentioned concentration of primer, 5 ng of extracted DNA, 1xPCR Buffer for KOD-Multi & Epi-® (Toyobo), and 0.2 U of KOD-Multi & Epi-® (Toyobo). It was set to 10 μL. The temperature cycle conditions were 94 ° C. for 2 minutes and 40 cycles of 98 ° C. for 10 seconds; 63 ° C. for 30 seconds; 68 ° C. for 30 seconds.
(5) Preparation of Electrophoretic Sample After the PCR was completed, the reaction solution was diluted 100-fold with sterilized water. 1.0 μL of diluted PCR reaction solution, 0.3 μL of GeneScan ™ 500 LIZ ™ dye Size Standard (Thermo Fisher), 14.7 μL of Hi-Di ™ Formamide (Thermo Fisher) mixed. It was subjected to heat denaturation treatment and used as an electrophoresis sample.
(6) Capillary Electrophoresis and Amplified Fragment Analysis Electrophoresis samples were subjected to ABI PRISM® 310 Genetic Analyzer using a 36 cm capillary, POP-4 polymer, and a dedicated buffer (Genetic Analyzer Buffer with EDTA (Thermo Fisher)). , Capillary electrophoresis according to the protocol. Analysis of the results was performed using Gene Mapper® software (Thermo Fisher) (Fig. 1). Peaks of amplified fragments with nucleotide lengths specific to the varieties used were detected corresponding to the genetic polymorphisms of each 4-base repeat SSR.

Example 2: Genotype identification of potato varieties According to the protocol described in Example 1, fragments amplified by the primer pairs shown in Table 3 were analyzed for each cultivar. Table 4 shows the results of the peak size (nucleotide length) patterns of the amplified fragments detected corresponding to the genetic polymorphisms of each 4-base repeat SSR for each variety. In the table, square marks indicate that peaks have been detected.

Since the genome of the potatoes other than "Inca awakening" is tetraploid and can be heterozygous, a maximum of four peaks are detected depending on the length of the amplified fragment. Since the "Inca awakening" is diploid, a maximum of two peaks are detected depending on the length of the amplified fragment.

As a result, various varieties of potatoes (Japanese varieties and SSR ID46514) were produced by eight kinds of 4-base repeating SSRs (SSR ID4026 / 4027, SSR ID8242, SSR ID12002, SSR ID16410, SSR ID31924, SSR ID35584, SSR ID43016, and SSR ID46514). Foreign varieties) could be identified (Table 4). Among them, the specific 4-base repeat SSR produced a peak pattern (eg, multiple peaks, single peak) peculiar to a specific variety of potatoes. Therefore, it has been shown that the identification method of the present invention utilizing such a genetic polymorphism of 4-base repeat SSR is useful for identification of various varieties of potatoes.

Example 3: Identification of potatoes or tissues derived from them, or varieties of their processed products The potatoes or tissues derived from them, or their processed products were used as subjects and conformed to Examples 1 (2) to (6). Follow the procedure to detect the peak of the fragment. By collating the detected peaks with the information shown in Table 4, the potatoes or the tissues derived from them, or the varieties of their processed products are identified.

Reference Example 1: Identification of 4-base repeating SSR for identification of potatoes or tissues derived from them, or varieties of their processed products 4 bases for identification of potatoes or tissues derived from them, or varieties of their processed products Identification of the repetitive SSR was performed as follows.
(1) Extraction of 4-base repeating SSR from the potato genome A region (SSR) having 4 base repetitions and 8 or more repetitions was extracted from the potato genome. The reason for extracting those with 8 or more repetitions is that, in the experience so far, it is easier to obtain polymorphisms among varieties when the number of repetitions is large, and from the relationship with the number of extracted SSRs. The lower limit of iterations was set to 8. By searching the genome sequence, 56 4-base repeat SSRs were extracted.
(2) Selection (a) First, for the extracted 56 4-base repeating SSRs, an amplification primer pair was prepared, and PCR was performed on several varieties of potatoes using the amplification primer pair, and then agarose gel electrophoresis was performed. The polymorphism was analyzed by electrophoresis, and 4-base repeating SSRs in which the polymorphism was observed were selected.
(B) Next, for a 4-base repeat SSR in which a polymorphism was observed, a fluorescently labeled primer pair was prepared, and PCR was performed on potatoes of several varieties (excluding Nishiyutaka) using this fluorescently labeled primer pair. Polymorphisms were analyzed by capillary electrophoresis and 4-base repeat SSRs were selected. In this selection, (i) polymorphisms are observed as in agarose gel electrophoresis, (ii) stutter peaks do not appear, and (iii) "no amplification" results are not obtained. This was done using one as an index.
(C) To ensure marker independence, adjacent 4-base repeat SSRs on the genomic region were excluded. Specifically, in this case, one 4-base repeating SSR was selected for each chromosome.
(D) Finally, in order to enable rapid, simple and easy analysis by multiplex gene amplification method (eg, PCR), 4-base repeat SSRs that are easy to adjust to obtain amplification products of different sizes were selected. ..
As a result, eight 4-base repeating SSRs, SSR ID4026 / 4027, SSR ID8242, SSR ID12002, SSR ID16410, SSR ID31924, SSR ID35584, SSR ID43016, and SSR ID46514, were subjected to potatoes or tissues derived from them, or processed products thereof. It could be identified as a marker that is particularly useful for identifying varieties of.

As described above, the identification method of the present invention can identify potatoes or tissues derived from them, or varieties of their processed products (particularly Japanese varieties and US varieties), and potato fruits and vegetables and their varieties. It can be expected to be used for variety inspection of processed products and seedling management of potatoes.

SEQ ID NOs: 1 to 8 indicate the genomic sequence of the portion of SSR ID4026 / 4027, SSR ID8242, SSR ID12002, SSR ID16410, SSR ID31924, SSR ID35584, SSR ID43016, and SSR ID46514 where the SSR markers are located.
SEQ ID NOs: 9 and 10 show the sequences of primer pairs (Forward and Revase primers) for amplifying the SSR marker of SSR ID 4026/4027.
SEQ ID NOs: 11 and 12 show the sequences of primer pairs (Forward and Revase primers) for amplifying the SSR marker of SSR ID 8242.
SEQ ID NOs: 13 and 14 show the sequences of primer pairs (Forward and Revase primers) for amplifying the SSR marker of SSR ID 12002.
SEQ ID NOs: 15 and 16 show the sequence of primer pairs (Forward and Revase primers) for amplifying the SSR marker of SSR ID 16410.
SEQ ID NOs: 17 and 18 show the sequences of primer pairs (Forward and Revase primers) for amplifying the SSR marker of SSR ID 31924.
SEQ ID NOs: 19 and 20 show the sequences of primer pairs (Forward and Revase primers) for amplifying the SSR marker of SSR ID 35584.
SEQ ID NOs: 21 and 22 show the sequences of primer pairs (Forward and Revase primers) for amplifying the SSR marker of SSR ID 43016.
SEQ ID NOs: 23 and 24 show the sequences of primer pairs ((Forward and Revase primers)) for amplifying the SSR marker of SSR ID 46514.

Claims (3)

A method for identifying varieties of potatoes or tissues derived from them, or their processed products.
In the test genomic DNA, including identifying the varieties based on the genetic polymorphism of one or more 4-base repeat SSRs.
The one or more 4-base repeat SSRs are as follows:
(1) SSR ID 4026/4027;
(2) SSR ID 8242;
(3) SSR ID12002;
(4) SSR ID 16410;
(5) SSR ID 31924;
(6) SSR ID35584;
(7) SSR ID43016;
(8) SSR ID 46514;
It is selected from the group consisting of
Identification is done by:
(A) Identification of Toyoshiro, Makein, Kitaakali, Musamar, Alturas, Russet Bannock, Shepody, Clearwater, or Cal white based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 4026/4027;
(B) Identification of Toyoshiro, Makein, Kitaakali, Musamaru, Red Moon, Russet Burbank, Russet Norkotah, Alturas, Shepody, Clearwater, or Calwite based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 8242.
(C) Nishiyutaka, Toyoshiro, Make-in, Inca Awakening, Musamaru, Red Moon, Russet Burbank, Russet Norkotah, Alturas, Umatilla Russet, or Shep, based on the genetic polymorphism of the 4-base repeat SSR of SSR ID12002.
(D) Nishiyutaka, Baron Yabu, Toyoshiro, Make-in, Kitaakali, Hokkaikogane, Inca Awakening, Red Moon, Russet Burbank, Russet Norkoth, Shepody, based on the genetic polymorphism of 4-base repeat SSR of SSR ID 16410. identification;
(E) Identification of Nishiyutaka, Baron, Hokkaikogane, Inca Awakening, Red Moon, Russet Burbank, Russet Norkotah, Ranger Russet, or Western Russet, based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 31924.
(F) Identification of Musamar, Red Moon, Russet Burbank, or Cal white based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 35584;
Identification of Nishiyutaka, Makein, Red Moon, Russet Burbank, or Alturas based on the genetic polymorphism of the 4-base repeat SSR of (G) SSR ID43016; or to the genetic polymorphism of the 4-base repeat SSR of (H) SSR ID46514 Identification of Make-in, Hokkaikogane, Red Moon, Russet Norkotah, Umatilla Russet, Shepody, or Western Russet based on;
(I) Identification of Japanese varieties based on genetic polymorphisms of 4-base repeat SSR of SSR ID 16410. The identification method according to claim 1, wherein the identification is performed by the following:
(A') Identification of Alturas, or Clearwater, based on genetic polymorphisms of 4-base repeat SSR with SSR ID 4026/4027;
(B') Identification of Clearwater based on genetic polymorphism of 4-base repeat SSR with SSR ID 8242;
(C') Inca awakening, or Musamal identification based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 12002; or (E') Baron 薯 based on the genetic polymorphism of the 4-base repeat SSR of SSR ID 31924 , Or identification of the Inca awakening. The identification according to claim 1 or 2, wherein the following inspection is performed in order to evaluate the possibility of mixing of other varieties in the assessment of the designated varieties or the tissue derived from the specified varieties, or the processed products containing the designated varieties. Law:
(1) When the designated variety is Nishiyutaka, it is examined whether SSR ID31924 or SSR ID43016 shows a genetic polymorphism other than the genetic polymorphism peculiar to Nishiyutaka;
(2) When the designated variety is baron potato, whether or not a single 4-base repeating SSR having SSR ID 16410 or a combination containing the same shows a genetic polymorphism other than the genetic polymorphism peculiar to baron potato. To inspect;
(3) If the designated variety is Toyoshiro, inspect whether SSR ID 16410 shows a genetic polymorphism other than the genetic polymorphism peculiar to Toyoshiro;
(4) If the designated variety is make-in, check whether SSR ID 16410 shows a genetic polymorphism other than the genetic polymorphism peculiar to make-in;
(5) When the designated variety is Kitaakali, it is examined whether or not the combination of SSR ID 8242 and SSR ID 12002 shows a genetic polymorphism other than the genetic polymorphism peculiar to Kita Akari;
(6) When the designated variety is Hokkaikogane, it is examined whether or not SSR ID 16410 or SSR ID 46514 shows a genetic polymorphism other than the genetic polymorphism peculiar to Hokkaikogane;
(7) If the designated variety is an Inca awakening, inspect whether SSR ID12002 or SSR ID16410 shows a genetic polymorphism other than the Inca awakening-specific genetic polymorphism;
(8) If the designated variety is Musamaru, check whether SSR ID 4026/4027 or SSR ID 12002 shows a genetic polymorphism other than the genetic polymorphism peculiar to Musamaru;
(9) When the designated variety is Red Moon, the combination of SSR ID4026 / 4027 and SSR ID8242, the combination of SSR ID4026 / 4027 and SSR ID12002, the combination of SSR ID8242 and SSR ID12002, the combination of SSR ID8242 and SSR ID16410, SSR Whether the combination of ID 8242 and SSR ID 31924, the combination of SSR ID 8242 and SSR ID 46514, the combination of SSR ID 12002 and SSR ID 16410, or the combination of SSR ID 12002 and SSR ID 35584 show a genetic polymorphism other than the genetic polymorphism peculiar to Red Moon. To inspect
(10) When the designated variety is Russet Burbank, the combination of SSR ID4026 / 4027 and SSR ID8242, the combination of SSR ID4026 / 4027 and SSR ID16410, the combination of SSR ID8242 and SSR ID31924, the combination of SSR ID8242 and SSR ID46514, SSR Whether the combination of ID12002 and SSR ID31924, the combination of SSR ID16410 and SSR ID31924, the combination of SSR ID16410 and SSR ID46514, or the combination of SSR ID31924 and SSR ID46514 show genetic polymorphisms other than the genetic polymorphism peculiar to Russet Burbank. To inspect
(11) When the designated variety is Russet Norkotah, the combination of SSR ID4026 / 4027 and SSR ID16410, the combination of SSR ID12002 and SSR ID31924, or the combination of SSR ID16410 and SSR ID31924, other than the genetic polymorphism peculiar to Russet Norkotah. Testing for genetic polymorphisms;
(12) When the designated variety is Ranger Russet, the combination of SSR ID4026 / 4027 and SSR ID8242, the combination of SSR ID4026 / 4027 and SSR ID12002, the combination of SSR ID8242 and SSR ID31924, the combination of SSR ID8242 and SSR ID43016, SSR Combination of ID8242 and SSR ID46514, combination of SSR ID12002 and SSR ID31924, combination of SSR ID12002 and SSR ID43016, combination of SSR ID16410 and SSR ID31924, combination of SSR ID16410 and SSR ID43016, combination of SSR ID31610 and SSR ID43016, SSR ID31924 and SSR ID35 To test whether the combination of SSR ID46514, SSR ID35584 and SSR ID43016, or the combination of SSR ID43016 and SSR ID46514 shows genetic polymorphisms other than those specific to Ranger Russet;
(13) When the designated variety is Alturas, it is examined whether or not SSR ID 4026/4027 or SSR ID 12002 shows a genetic polymorphism other than the genetic polymorphism peculiar to Alturas;
(14) When the designated variety is Umatilla Russet, it is examined whether or not SSR ID 12002 shows a genetic polymorphism other than the genetic polymorphism peculiar to Umatilla Russet;
(15) When the designated variety is Russet Bannock, whether or not the combination of SSR ID 8242 and SSR ID 12002 or the combination of SSR ID 12002 and SSR ID 16410 shows a genetic polymorphism other than the genetic polymorphism peculiar to Russet Bannock. To inspect;
(16) When the designated variety is Shepody, it is examined whether or not SSR ID 16410 shows a genetic polymorphism other than the Shepody-specific genetic polymorphism;
(17) When the designated variety is Clearwater, it is examined whether or not SSR ID 4026/4027 or SSR ID 8242 shows a genetic polymorphism other than the genetic polymorphism peculiar to Clearwater;
(18) If the designated cultivar is Cal white, check whether SSR ID 4026/4027 shows a genetic polymorphism other than the genetic polymorphism peculiar to Cal white; or (19) The designated cultivar is In the case of Western Russet, the combination of SSR ID8242 and SSR ID31924, the combination of SSR ID8242 and SSR ID35584, the combination of SSR ID8242 and SSR ID43016, the combination of SSR ID12002 and SSR ID31924, the combination of SSR ID12002 and SSR ID3584 To test whether the combination of ID 31924, SSR ID 31924 and SSR ID 35584, or the combination of SSR ID 31924 and SSR ID 46514 shows a genetic polymorphism other than the genetic polymorphism peculiar to Western Russet.

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