JP7401911B2 - How to identify citrus varieties - Google Patents

Description

The present invention relates to a technology that can quickly and accurately identify the citrus variety "Mihaya" from other citrus varieties, and that can be widely introduced at inspection sites.

In recent years, cases have emerged in which excellent new varieties cultivated by the National Agriculture and Food Research Organization, such as the citrus Mihaya and the grape Shine Muscat, have been leaked overseas and grown without permission. In the future, there are concerns that rights infringement will occur due to these products, so applications for variety registration will be filed in countries where rights infringement is expected, and DNA variety identification technology will be developed to prove rights infringement. It is progressing. In order to monitor violations of Mihaya's breeder's rights, it is important to prevent imports at the border, such as customs. For this reason, in addition to existing product identification technology, there is a need for the development of technology that allows product product identification to be performed easily and quickly at inspection sites such as customs offices.

The Fruit Tea Division of the National Agriculture and Food Research Organization has developed a variety identification technology using CAPS markers for citrus seedlings distributed in Japan. Regarding this technology, our department has created a manual for DNA variety identification technology for citrus varieties that complies with ISO (International Organization for Standardization) standards (Non-Patent Document 1). Here, CAPS marker analysis technology cuts a PCR amplified fragment targeting a specific genomic region with a specific restriction enzyme, and then removes the restrictions caused by polymorphisms in genomic DNA such as base substitutions, deletions, and insertions that exist between varieties. This is a technology that takes advantage of differences in the size of enzyme fragments. Analysis using this technology does not require expensive equipment, making it highly pervasive in testing sites.
However, in variety identification for fruits and processed products, it is difficult to obtain high-quality DNA samples due to the contamination of contaminants such as sugars, acids, and polyphenols, as well as heat and chemical treatments during processing. Therefore, the CAPS analysis method, which uses relatively long PCR amplified fragments to detect polymorphisms using restriction enzymes, has problems with the analysis results due to contaminants inhibiting enzymes during PCR and restriction enzyme treatment and reducing PCR amplification efficiency. There is an issue of instability.

In addition to the CAPS method, the department has also developed a variety identification technology using SNP markers for fruits and processed products (Non-Patent Document 2). The SNP analysis method is a technology that uses fluorescently labeled oligonucleotide probes to detect single nucleotide polymorphisms in PCR amplified fragments targeting a specific genomic region of approximately 100 bp. It has the advantage that the time required is extremely short.
In this regard, the SNP analysis method is recognized as a technology that overcomes the problems of analysis accuracy with the CAPS method, but on the other hand, the running costs of special modified probe synthesis and reagents used to detect SNP markers are expensive. Issues are recognized. Furthermore, in order to easily perform the SNP method, it is necessary to introduce a real-time PCR device.

DNA variety identification technology for 22 citrus varieties using CAPS markers, National Agriculture and Food Research Organization, March 29, 2019 First edition Breeding Science, (2020) https://doi.org/10.1270/jsbbs.19142; T. Endo et al.; TaqMan-MGB SNP genotyping assay to identify 48 citrus cultivars distributed in the Japanese market Tree Genetics & Genomes, (2014) 10, 1001-1013; T. Shimada et al.; Construction of a citrus framework genetic map anchored by 708 gene-based markers Plant Molecular Biology Reporter, (2018) https://doi.org/10.1007/s11105-018-1111-1; Q. Fang et al.; Development of Species-Specific InDel Markers in Citrus

The present invention has been made in view of the above-mentioned circumstances of the prior art, and its object is to provide a technology that can quickly and accurately identify the citrus variety "Mihaya" from other citrus varieties, and, The aim is to provide technology that can be widely introduced at inspection sites.

In the situation of the above-mentioned prior art, the present inventors conducted a study focusing on 185 types of indel markers present in the genomic DNA of citrus plants, and found that allelic polymorphisms specific to the cultivar "Mihaya" were found. An indel marker IND131 was found.
Therefore, the present inventors investigated the allele type of the indel marker IND131, including 47 varieties that cover more than 98% of the distribution volume of citrus fruit in Japan, and found that the insertion type sequence was found in both homologous chromosomes regarding IND131. It was found that the only citrus cultivar that showed a homozygous type (insertion type sequence homozygous type) was the cultivar "Mihaya." Here, the survey result was recognized as one that satisfies the accuracy of variety identification specified in the ISO 13495 international standard from the perspective of distribution volume in Japan. That is, it was shown that the variety "Mihaya" could be identified by finding the above allele type from the gene locus of the indel marker IND131 of the citrus plant to be identified.
In addition, the present inventors conducted a study on the indel marker IND44 separately from the above-mentioned IND131, and found that among the major citrus varieties investigated above, 23% of the citrus varieties including the variety "Mihaya" had IND44. It was found that the allele type containing the deletion type sequence can be distinguished from the allele type of the remaining 77% of citrus varieties. That is, regarding the citrus identification technology using allele polymorphism analysis of IND131, we have found that by further performing allele polymorphism analysis regarding IND44, it is possible to further improve the identification accuracy of the variety "Mihaya".

The present inventors have completed the present invention based on the above findings. The present invention specifically relates to the inventions described below.
[Section 1]
A method for identifying citrus varieties, including the steps described in (1) and (2) below:
(1) Detecting homozygous allele polymorphism of the insertion type sequence of the indel marker IND131 from the genomic DNA of the citrus plant to be identified;
(2) If homozygous allele polymorphism of the insertion type sequence of IND131 is detected in the step described in (1) above, the variety of the citrus plant that is the target sample is the variety "Mihaya", or , a step of determining that IND131 is a progeny variety of the variety "Mihaya" that exhibits the same allele type as the variety "Mihaya";
;
Regarding the indel marker IND131 of the citrus plant genome described in (1) above,
(1a) When shown in the genome of sweet orange (Citrus sinensis), a standard variety of citrus, this is the locus of the indel marker present in the 3'UTR of the cell wall-related receptor kinase gene on chromosome 1; When shown by the base sequence described in SEQ ID NO: 1, which includes the sweet orange IND131 deletion type sequence, the 5527th base "A" indicates the most upstream base of the IND131 gene locus, and the 5528th base "G" indicates the most downstream base of the IND131 locus,
(1b) Regarding the IND131 locus of the citrus plant genome that is the target sample, if the nucleotide sequence has an inserted sequence between the most upstream base "A" and the most downstream base "G" of the IND131 gene locus, the IND131 insertion type In the case of a base sequence in which there is no inserted sequence between the most upstream base "A" and the most downstream base "G" of the IND131 gene locus, an IND131 deletion type sequence is shown.
[Section 2]
Furthermore, it includes the step described in (1') below, and
The step described in (2) above is performed when a homozygous allelic polymorphism of the insertion type sequence of IND131 is detected in the step described in (1) above, and in the step described in (1') below, a deletion of IND44 is detected. If an allelic polymorphism containing a lost sequence is detected, the variety of the citrus plant that is the target sample is the variety "Mihaya", or the allele type of the citrus plant that is the target sample is the same as the variety "Mihaya" regarding IND131 and IND44. This is the process of determining that it is a descendant variety of the designated variety "Mihaya".
Method for identifying citrus varieties as described in Section 1:
(1') Detecting an allelic polymorphism containing a deleted sequence of the indel marker IND44 from the genomic DNA of the citrus plant to be identified;
;
Regarding the indel marker IND44 of the citrus plant genome described in (1') above,
(1'a) When shown in the genome of sweet orange (Citrus sinensis), a standard variety of citrus, it is an indel marker locus present in the downstream region of the RALF-like peptide precursor gene on chromosome 1, When shown by the base sequence described in SEQ ID NO: 7, which includes the IND44 insertion type sequence of the sweet orange, the 4175th base "A" indicates the most upstream base of the IND44 gene locus, and the 4749th base "A" indicates the most downstream base of the IND44 gene locus, the base sequence from the 4176th base to the 4748th base indicates the inserted sequence,
(1'b) Regarding the IND44 locus of the citrus plant genome that is the target sample, if the nucleotide sequence has an inserted sequence between the most upstream base "A" and the most downstream base "A" of the IND44 gene locus, IND44 An insertion type sequence is shown, and in the case of a base sequence in which there is no insertion sequence between the most upstream base "A" and the most downstream base "A" of the IND44 gene locus, an IND44 deletion type sequence is shown.
[Section 3]
The step of detecting the allele polymorphism is a step including a PCR reaction using a citrus plant, a processed product of the plant, or DNA extracted from the plant or processed product as a target sample. The method for identifying citrus varieties according to 1 or 2.
[Section 4]
comprising using the method for identifying citrus varieties according to any one of items 1 to 3;
A method for cultivating a plant of the variety "Mihaya" or a progeny variety of the variety "Mihaya" that exhibits the same allele type as the variety "Mihaya" with respect to IND131,
A method for producing fruit of the variety "Mihaya" or a progeny variety of the variety "Mihaya" that exhibits the same allele type as the variety "Mihaya" with respect to IND131, or
A method for producing a processed fruit product of the variety "Mihaya" or a progeny of the variety "Mihaya" which exhibits the same allele type as the variety "Mihaya" with respect to IND131.
[Section 5]
comprising using the citrus variety identification method described in Section 2;
A method for cultivating a plant of the variety "Mihaya" or a progeny variety of the variety "Mihaya" that exhibits the same allele type as the variety "Mihaya" with respect to IND131 and IND44,
A method for producing fruit of the variety "Mihaya" or a progeny variety of the variety "Mihaya" that exhibits the same allele type as the variety "Mihaya" with respect to IND131 and IND44, or
A method for producing a processed fruit product of the variety "Mihaya" or a progeny of the variety "Mihaya" which exhibits the same allele type as the variety "Mihaya" with respect to IND131 and IND44.

The present invention is a technology that can quickly and accurately identify the citrus variety "Mihaya" from other citrus varieties, and also makes it possible to provide a technology that can be widely introduced at inspection sites and the like.

It is a photographic image of the appearance, longitudinal section, and cross section of the fruit of the citrus variety "Mihaya." This is a detailed photographic image of a cross section of a fruit of the citrus variety "Mihaya." FIG. 2 is a schematic diagram showing information regarding the gene locus of the indel marker IND131 and its surrounding region. FIG. 2 is a schematic diagram showing information regarding the gene locus of the indel marker IND44 and its surrounding region. FIG. 2 is an explanatory diagram showing that, in this experimental example, primers were designed at positions sandwiching the indel marker gene locus in order to detect indel marker allele polymorphisms. FIG. 2 is an explanatory diagram showing that primers including an indel marker gene locus were designed in order to detect an indel marker deletion type sequence in this experimental example. FIG. 3 is a diagram showing the detection results of allelic polymorphisms related to IND131 in relation to the variety identification test based on the detection of allelic polymorphisms in Experimental Example 2. “●” in the figure indicates a sample of the variety “Mihaya”. "ins." and "del." indicate an "insertion type sequence" and a "deletion type sequence," respectively. "+" indicates a sample in which an allele polymorphism band indicating a homozygous insertion type sequence was detected. FIG. 7 is a diagram showing the detection results of allelic polymorphism regarding IND44 in relation to the variety identification test based on the detection of allelic polymorphism in Experimental Example 3. “●” in the figure indicates a sample of the variety “Mihaya”. "ins." and "del." indicate an "insertion type sequence" and a "deletion type sequence," respectively. "+" indicates a sample in which an allelic polymorphism band containing the deleted sequence was detected. FIG. 4 is a diagram showing the detection results of allelic polymorphisms regarding IND131 and IND44 in relation to the variety identification test by detecting allelic polymorphisms in Experimental Example 4. “●” in the figure indicates a sample of the variety “Mihaya”. "ins." and "del." indicate an "insertion type sequence" and a "deletion type sequence," respectively. FIG. 9A shows the detection results of a homozygous type of the IND131 insertion type sequence, and "+" in the figure indicates a sample in which an allelic polymorphism band indicating a homozygous type of the IND131 insertion type sequence was detected. FIG. 9B shows the detection results of the IND44 deletion type sequence, and "+" in the figure indicates a sample in which the IND44 deletion type sequence was detected.

Embodiments of the present invention will be described in detail below. Note that the technical scope of the present invention is not limited to embodiments that include all of the configurations described below. Further, the technical scope of the present invention does not exclude aspects including configurations other than those described below.

[Explanation of terms]
As used herein, the term "mutation in a base sequence" is used to mean a mutation due to substitution, deletion, and/or insertion of bases constituting a base sequence.
As used herein, the term "breed" refers to a subclassification of "species", and is characterized by external and/or physiological characteristics (phenotype) that can be distinguished from other breeds (or lines) as a group. The term is used to refer to a population that has sufficiently similar characteristics of the same generation and is stable enough that the characteristics are maintained over generations.
In this specification, the expression **th base upstream or downstream from a certain base refers to the base that is one base upstream or downstream of that base, counting it as the first base. means the **th base.
In this specification, the **th base upstream (from) the base at the 3' end of a given base sequence refers to the base upstream one base upstream from the base at the 3' end, counting as the first base. means the **th base.

1. Method for identifying citrus varieties The present invention relates to a technology that can quickly and accurately identify the citrus variety "Mihaya" or its successor varieties from other citrus varieties, and that can be widely introduced at inspection sites.

[Identification target]
The citrus variety identification technology according to the present invention relates to a technology for identifying citrus variety "Mihaya" or its successor varieties from plants of other citrus varieties.

"Citrus plants" to which the technology of the present invention is applied refers to plants belonging to the genus Citrus. The fruits of plants belonging to the citrus genus are composed of a structure in which pouches filled with pulp are lined up in a radial manner inside the pericarp, and are often an important agricultural resource that is used as edible food. In addition, plants of the genus Citrus are evergreen shrubs (trees) that do not shed leaves, and have properties suitable for cultivation in warm regions, and are also suitable for cultivation in warm regions of Japan.
Here, in the present specification, "Citrus (also known as Citrus)" refers to a taxonomic group belonging to the family Rutaceae, Citrus family. Plants belonging to the citrus genus (genus Citrus) are classified as 'species' in some cases, but in many cases they have expanded their distribution as cultivated species through vegetative reproduction from a single tree that has undergone mutation, and has virtually no size. In most cases, genetic differences are only at the breed (strain) level. Recent studies comparing genotypes suggest that there are only about four original species of the citrus genus, and that most other species are hybrids. In this respect, the genus Citrus is a taxonomic group in which interspecific hybrids and grafting are relatively easy, and it is recognized as a taxonomic group consisting of species with similar genetic backgrounds (basic characteristics as plant species).
As for plants belonging to the genus Citrus, recent research comparing genotypes has shown that most of the species in the genus Citrus are genetically related, and the theory that they were created through hybridization is most likely. Species classified as subgenus Citrus are recognized to be extremely closely related.
From these viewpoints, each plant belonging to the genus Citrus (citrus plant) in this specification is treated as a distinguishable classification unit as a "variety." Furthermore, in the present invention, various groups such as varieties and lines that can be distinguished from other groups are treated as having the same meaning as long as they belong to the genus Citrus (i.e., they are treated as classified groups at the same level as cultivars). , it is possible to use it as an identification target.

In the citrus variety identification technology of the present invention, the variety "Mihaya" can be distinguished from other citrus varieties by detecting the homozygous allele polymorphism of the IND131 insertion type sequence. Here, the variety "Mihaya" is a new citrus breeding variety created by the present applicant, and is registered as a variety registration number: No. 23722.
The average variety characteristics of the variety "Mihaya" include the following characteristics.
The tree's appearance is between upright and spreading, and its vigor is moderate. Since it can be harvested within the year, cultivation of this variety is possible in most citrus cultivation areas. If canker and scab diseases are controlled in the same manner as for general citrus plants, there will be no problems in cultivating the variety.
It is an early variety that reaches maturity from late November and bears large fruits with an average fruit weight of around 190g. The fruit has an overall reddish-orange appearance and can be clearly distinguished from Unshu mandarin orange. The sugar content of the pulp of the fruit is about 12.2%, and the acidity is about 0.6%. It has high sugar content, low acidity, and excellent taste. Over two-thirds of the fruit is seedless and the capsule membrane is slightly soft and easy to eat, making it a highly marketable fruit. In addition, the fruit pulp contains the functional component β-cryptoxanthin in an amount comparable to that of Unshu mandarin orange.

In addition, in the citrus variety identification technology of the present invention, from the viewpoint of the identification principle, the present invention also applies to citrus varieties that exhibit the same allele type as the variety "Mihaya" with respect to IND131 among the progeny varieties of the variety "Mihaya". Using technology, it will be possible to distinguish it from other citrus varieties.
Here, examples of progeny varieties of the variety "Mihaya" include varieties derived from the variety "Mihaya" and varieties created based on the variety "Mihaya." Examples of such progeny varieties include progeny varieties produced by hybridization, mutation, gene introduction, genome editing, and the like. The progeny varieties also include varieties that were registered as varieties after this application was filed. The progeny varieties also include populations such as progeny lines of the variety "Mihaya," production lines derived from the variety "Mihaya," mutant lines derived from the variety "Mihaya," and the like.
In addition, the allele type that is the same as the variety "Mihaya" indicates the following allele polymorphism at the IND131 locus. Furthermore, if the allele type is derived from the variety "Mihaya", even if mutations occur in the base sequences constituting these loci and their surrounding regions, the variety identification technology of the present invention cannot be applied. It is possible.

In the identification technology of the present invention, examples of "other citrus varieties" that can be distinguished from the variety "Mihaya" include citrus varieties in which the IND131 gene locus does not exhibit the following allelic polymorphism. In citrus plants belonging to other citrus varieties, the allele type of IND131 is different from that of the variety "Mihaya".
Here, other citrus varieties that can be distinguished from the variety "Mihaya" using the identification technology of the present invention include, for example, unshu mandarin orange, grapefruit, sweet orange shown in the following experimental example, Lemon, Shiranui, Iyo (Iyokan), Natsumikan, Hassaku, Ponkan, Rinoka, Asumi, Asuki, Reiko, Tsunoki, Southwest Hikari, Tsunozomu, Haruhi, Kiyomi, Setoka, Harumi, Ha Rehime, Kanpei, Beni Madonna, Yuzu, Tosa Buntan, Kawachi Bankan, Hyuga Natsu, Sudachi, Shikuwasa, Kara Mandarin (Kara), Tankan, Seminole, Kabosu, Amakusa, Kogankan, Kishu Mandarin, Sweet Spring, Sanbokan, Examples include Tamami, Nishinoka, Saga Mandarin, Murcott, Nanka, Amaka, Citrus Intermediate Mother Book No. 6, and Cubs.
These 46 citrus varieties, together with the ``Mihaya'' variety (47 types in total), account for more than 98% of the citrus fruit market in Japan. In this regard, the variety identification technology specified in the ISO 13495 international standard is based on technology that can identify 90% or more of the products in circulation, which indicates that the technology satisfies the international standard. This point shows that the identification technology of the present invention is a product identification technology that enables the identification of the "Mihaya" product variety with very high accuracy.
Furthermore, considering the results of the following experimental examples and their phylogenetic relationships, other citrus varieties that can be distinguished from the variety "Mihaya" using the identification technology of the present invention are those whose IND131 locus has the following allele. It becomes possible to list citrus varieties that do not exhibit polymorphism as other citrus varieties.

In addition, with this identification technology, it is possible to identify the Mihaya variety with high accuracy as shown above by detecting the following allele type at the IND131 locus. By detecting the indication, it is also possible to further improve the identification accuracy. In this case, examples of "other citrus varieties" that can be distinguished from the variety "Mihaya" include citrus varieties in which neither the IND131 gene locus nor the IDN44 gene locus exhibits the following allelic polymorphisms.

[Detection process of allelic polymorphism regarding indel marker]
The citrus identification technology according to the present invention is a technology related to detecting allelic polymorphisms characteristic of the variety "Mihaya" regarding the following indel markers.
Specifically, the citrus identification technology according to the present invention includes (Step 1) detecting homozygous allelic polymorphism of the insertion type sequence of the indel marker IND131 from the genomic DNA of the citrus plant to be identified. Concerning methods for identifying varieties.
Furthermore, as the citrus identification technology according to the present invention, in addition to performing the above (step 1), (step 1') a deleted sequence of the indel marker IND44 is extracted from the genomic DNA of the citrus plant to be identified. It is also possible to provide an embodiment of a method for identifying citrus varieties, which includes a step of detecting allelic polymorphisms.

Here, the step of detecting allelic polymorphism of an indel marker from citrus plant genomic DNA can be performed by using existing nucleic acid detection technology. For example, a technique using PCR method, a technique using hybridization method, etc. can be used. It is also possible to use polymorphism detection techniques such as real-time PCR using a combination of probes and primers, and dot blotting using two types of probes, insertion type and deletion type.
The indel marker allele polymorphism detection step according to the present invention is carried out using ordinary PCR using a primer set that can specifically amplify the base sequence containing the indel marker gene locus, from the viewpoint of ease and rapidity. It is preferable to use a technique using the law. There are no particular restrictions on the reaction reagents, reaction conditions, etc. used in the PCR method, as long as the reaction reagents, reaction conditions, etc. can yield primer-specific PCR amplified fragments, and any known method can be used. be.
As means for detecting PCR amplified fragments, conventional methods or novel methods can be used. For example, it is possible to detect a PCR amplified fragment using ethidium bromide, a fluorescent substance, a coloring substance, a luminescent substance, a radioactive isotope, etc., but there are no particular limitations on these.

Sample The identification technique according to the present invention includes the step of detecting allelic polymorphism of an indel marker from the genomic DNA of a citrus plant to be identified.
Detection of allelic polymorphism in the detection step can be performed using a sample containing the citrus plant itself or its genomic DNA. Specifically, in the detection step, detection can be performed using a citrus plant, a processed product of the plant, or DNA extracted from the plant or processed product.
The citrus plant used for detecting allelic polymorphisms in the detection step includes all or any parts and tissues contained in the citrus plant, plants belonging to all or any growth stages, etc. It can be used as a sample. More specifically, growing buds, side buds, growing branches, branches, leaves, petioles, thorns, stems, flower buds, flowers, fruits, seeds, roots, etc. can be mentioned. Further examples include the exocarp, endocarp, pulp, seeds, etc. that constitute the fruit. As the sample, it is preferable to use tissues and organs such as leaves and buds, which contain few contaminants such as polyphenols and are easy to perform DNA extraction or PCR amplification. It is also of course possible to collect each component tissue from the fruit to be identified and use it as a sample for the detection step.
Moreover, in the detection step, it is also possible to use the above-mentioned processed product of the citrus plant as a sample. The processed product includes products at various processing stages and processing stages, but it is preferable to use processing processing and processing stages that allow DNA amplification by PCR. Examples include plant juice, plant extract, juice residue, extraction residue, processed products of the plant itself (cut plant products, crushed products, pastes, purees, dried products, Examples include powdered products, frozen processed products, various chemical treatments, etc.). In particular, as specific examples of these processed products, it is preferable to use fruit processed products that utilize fruits and exhibit these processing modes. Suitable examples of the processed fruit products include fruit processed products such as straight juice containing fruit pulp, canned fruit pulp soaked in syrup, and dried fruits.

IND131 gene locus In the indel marker allele detection step in this identification technology, (Step 1) detecting homozygous allele polymorphism of the insertion type sequence of IND131 from the genomic DNA of the citrus plant to be identified. include.
Here, the indel marker IND131 is an indel marker present in the 3'UTR of the cell wall-related receptor kinase gene, which is a type of serine/threonine protein kinase gene on chromosome 1 of the citrus genome (FIG. 3). When shown by the base sequence shown in SEQ ID NO: 1, which shows the IND131 deletion type sequence of sweet orange (Citrus sinensis), from the most upstream base of the 5'UTR representing the cell wall-related receptor kinase gene to the most downstream base of the 3'UTR The region shown by the base sequence up to is the region corresponding to the base sequence from the 3001st base to the 5756th base in the base sequence shown in SEQ ID NO: 1. Additionally, the ORF region (the region indicated by the base sequence from the first base of the first exon to the last base of the final exon) is the region from the 3083rd base to the 5478th base in the base sequence shown in SEQ ID NO: 1. This corresponds to the region shown in the base sequence up to.
Here, the 3'UTR of the gene corresponds to the base sequence from the 5479th base to the 5756th base in the base sequence shown in SEQ ID NO: 1, and the region downstream from the 5756th base ( The region shown by the base sequence from the 5757th base to the 8756th base of SEQ ID NO: 1) corresponds to the downstream region of the gene.
When shown by the base sequence shown in SEQ ID NO: 1, which shows the IND131 deletion type sequence of sweet orange, the 5527th base "A" is the most upstream base of the IND131 gene locus, and the most downstream base of the IND131 gene locus. There is no inserted sequence between the 5528th base "G".

On the other hand, in an insertion type sequence having an insertion sequence at the IND131 gene locus (IND131 insertion type sequence), the most upstream base "A" of the IND131 gene locus and the most downstream base "G" of the IND131 gene locus present in the 3'UTR. There is an inserted sequence of 233 bp or equivalent base length between them. The variety "Mihaya" indicates an allele type that has the type of sequence in which the insertion sequence exists (IND131 insertion type sequence) in a homologous chromosome. The nucleotide sequence containing the IND131 insertion type sequence isolated from the variety "Mihaya" can be shown as the nucleotide sequence set forth in SEQ ID NO: 2.
When shown by the base sequence shown in SEQ ID NO: 2, which shows the IND131 insertion type sequence of the variety "Mihaya", the most upstream base "A" of the IND131 locus corresponds to the 62nd base, and the most downstream base "A" corresponds to the 62nd base. G" corresponds to the 296th base, and there is a 233 bp insertion sequence (region shown by the base sequence from the 63rd base to the 295th base of SEQ ID NO: 2) between them.
Note that, unlike the variety "Mihaya", other citrus varieties exhibit allele types containing the IND131 deletion type sequence regarding the IND131 gene locus. Here, in the IND131 deletion type sequence, the region sandwiched between the most upstream base "A" and the most downstream base "G" of the IND131 locus with respect to the base sequence of the 3'UTR of the cell wall-related receptor kinase gene ( The nucleotide sequence (two bases of "AG") in which the above-mentioned inserted sequence) has been deleted is shown (Figure 3).

That is, in the present invention, the following definition can be made regarding the indel marker IND131 of the citrus plant genome:
When shown in the genome of sweet orange (Citrus sinensis), a standard variety of citrus, this is the locus of the indel marker present in the 3'UTR of the cell wall-related receptor kinase gene on chromosome 1; In the case of the base sequence shown in SEQ ID NO: 1, which includes the IND131 deletion type sequence, the 5527th base "A" indicates the most upstream base of the IND131 gene locus, and the 5528th base "G" indicates the IND131 gene locus. Indicates the most downstream base of the locus,
Regarding the IND131 gene locus of the citrus plant genome, which is the target sample, if the base sequence has an inserted sequence between the most upstream base "A" and the most downstream base "G" of the IND131 gene locus, it is indicated as an IND131 insertion type sequence. , a base sequence in which there is no inserted sequence between the most upstream base "A" and the most downstream base "G" of the IND131 gene locus indicates an IND131 deletion type sequence.

Here, the IND131 gene locus in the citrus plant genome, which is the target sample, is a locus that has an orthologous relationship with the above-mentioned IND131 in the sweet orange genome (a genetic locus that has the same origin in a common ancestor). refers to
For example, regarding the base sequence constituting the IND131 locus and its surrounding region in the citrus plant genome that is the target sample, for example, from the most upstream base of the 5'UTR of the cell wall-related receptor kinase gene in the sweet orange genome to the most upstream base of the IND131 gene locus. The sequence identity is 85% or more, 90% or more, or 95% or more with respect to the base sequence up to and the base sequence from the most downstream base of the IND131 locus to the 500th (preferably the 250th) base downstream thereof. If so, it can be determined that they correspond to mutually corresponding arrays.

Primer set for detecting allelic polymorphisms of IND131 Detecting allelic polymorphisms of IND131 in the detection step uses oligonucleotide primers designed to enable PCR amplification of a region containing the base sequence of the IND131 gene locus. This can be suitably carried out by a method that utilizes the PCR method.

(Primer set that spans the gene locus)
As an oligonucleotide primer capable of detecting allelic polymorphism of IND131 in the detection step, for example, a primer set consisting of a pair of oligonucleotide primers (a pair of common region primers for detecting IND131) located on both sides of the IND131 gene locus is used. An example of this is to design an oligonucleotide primer that makes it possible to detect a PCR amplified fragment of an IND131 insertion type sequence and/or an IND131 deletion type sequence (see Figures 3 and 5). .
When PCR amplification is performed using a primer set containing the pair of oligonucleotide primers, if an IND131 insertion type sequence is present in the genome, a long DNA fragment containing the insertion sequence is amplified. On the other hand, when the IND131 deletion type sequence is present in the genome, a short DNA fragment with a length corresponding to the inserted sequence is amplified.
Therefore, if the detection step is performed using the primer set and only a long DNA fragment containing the length corresponding to the inserted sequence is amplified as a single band, the allele type of the IND131 locus in the genomic DNA of the target sample is , it becomes possible to determine that the allele type has the IND131 insertion type sequence in a homozygous form.

Here, specifically, the primer set consisting of "a pair of common region primers for IND131 detection" (forward primer and reverse primer) is one that can amplify a DNA fragment including the following IND131 gene locus. It is possible to use a pair of oligonucleotide primers (p1) and (p2):
(p1) An oligonucleotide containing a base sequence of 16 or more bases included in the base sequence from the 1000th base upstream of the most upstream base "A" of the IND131 gene locus of the citrus plant genome to the most upstream base "A" Primer, and
(p2) A base sequence of 16 or more bases included in the complementary sequence of the base sequence from the most downstream base "G" to the 1000th base downstream of the IND131 locus in the citrus plant genome, and which is included in the above (p1). An oligonucleotide primer comprising the described primer and a base sequence capable of forming a primer pair in a direction and position sandwiching the IND131 gene locus.
Here, the oligonucleotide primers described in (p1) and (p2) above are used as a primer set of these oligonucleotide primers as base sequences to exhibit their primer functions, and are used as samples 1 to 2 in Tables 1 to 2. When PCR reaction is performed on the genomic DNA of the citrus varieties listed as No. 24 (total of 24 types), preferably the PCR reaction is performed on the genomic DNA of the citrus varieties listed in Tables 1 to 4 (total of 47 types). Preferred embodiments include oligonucleotide primers having base sequences that can amplify IND131 insertion type sequences and/or IND131 deletion type sequences for all citrus varieties when a reaction is performed. .

(Primer design matters)
The design conditions for the oligonucleotide primers mentioned above are that (p1) and (p2) are primers of the same primer set that form a primer pair, so if (p1) is a forward primer, (p2) is a reverse primer. , (p1) is a reverse primer, (p2) is a forward primer.

Furthermore, as a design condition for the primer, it is preferable that the primer contains at least 16 consecutive base sequences included in the base sequence constituting the IND131 gene locus and its surrounding region, or its complementary sequence. Suitable base lengths (mers) include 16 bases or more, 17 bases or more, 18 bases or more, 19 bases or more, 20 bases or more, or 21 bases or more for the base sequence contained in the base sequence or its complementary sequence. It is preferable that the primer contains the following. In addition, there is no particular restriction on the upper limit of the base length of the oligonucleotide primer as long as it does not deviate from the range that functions as a primer, but for example, the base length of the above base sequence is 50 bases or less or 40 bases or less. can be mentioned.
In addition, in order to increase sequence specificity and reduce non-specific PCR amplification, it is necessary to match the Tm value (annealing temperature), GC content, etc. of the primers forming the primer pair to approximate values, and then It is preferred to design suitable primers of long length.
Furthermore, the 5' end of the oligonucleotide primer may have a restriction enzyme site for use in vector insertion, etc., a modified base sequence for introducing various vectors, or a fluorescent substance, a labeling substance, etc. It may also be in the form of a combination of Furthermore, the base at the 3' end of the oligonucleotide primer is preferably a base sequence that completely matches the base sequence constituting the IND131 gene locus and its surrounding region or its complementary sequence.

The region in which the oligonucleotide primer described above can be designed is preferably a region in which the base length of the amplification product is suitable for PCR amplification. Specifically, from the viewpoint of ease of PCR amplification, a primer set (gene It is preferable to design a primer set that sandwiches the loci.
More preferably, the primer set is designed at a position where the base length of the PCR amplified fragment is 2 kbp or less, 1.5 kbp or less, 1.2 kbp or less, 1 kbp or less, 750 bp or less, or 500 bp or less. As a lower limit of the length of the PCR amplified fragment, for example, from the viewpoint of differentiation from non-specific amplification, it is preferable to design a primer set at a position where the base length of the PCR amplified fragment is 50 bp or more or 100 bp or more. Embodiments include, for example, a range of 50 to 2000 bp, 100 to 1000 bp, or 100 to 500 bp.

Considering the technical aspects of PCR, from the viewpoint of ease of PCR amplification, the regions (Fig. 3, Fig. 5) where common region primers for IND131 detection described in (p1) and (p2) can be designed include: Specifically, the region shown by the base sequence from the 1000th base upstream of the most upstream base "A" of the IND131 gene locus to the most upstream base "A", or the most downstream base "G" of the IND131 gene locus. '' to the 1000th base downstream thereof. Preferably, the region up to the 1000th, 900th, 800th, 750th, 500th, or 250th base can be preferably mentioned upstream or downstream.
Further, as an embodiment of the primer design, for example, it is possible to use these corresponding regions in the variety "Mihaya" or the standard variety Sweet Orange as the primer design region.

Regarding the primers described in (p1) and (p2) above, regarding the various primer design conditions described above, primers containing a base sequence that enables the amplification of the IND131 gene locus and its surrounding region on the target sample genomic DNA are used. It can be employed as the above-mentioned primer that can be used in the citrus identification technology of the invention. Furthermore, even if the primer contains a base sequence that has one or several mutations with respect to the base sequence constituting the IND131 gene locus and its surrounding region or its complementary sequence, the IND131 gene on the target sample genomic DNA Any primer containing a base sequence that allows amplification of the locus and its surrounding region can be used as the above primer.
Regarding the base sequence constituting the primer, the number of mutations to the base sequence constituting the IND131 locus and its surrounding region or its complementary sequence makes it possible to amplify the IND131 locus and its surrounding region on the target sample genomic DNA. There is no particular restriction as long as it is within the range, but examples include 3 bases or less, 2 bases or less, or 1 base or less. Particularly preferred are those in which the number of mutations is 2 or less bases.
In the embodiment of the primer containing the mutant sequence, preferably, the base sequence from the 10th base upstream of the 3' end base of the primer constituent bases to the 3' end base is the IND131 locus and its 3' end base. Examples include primers that match the base sequence in the surrounding region or the base sequence on its complementary sequence. In this embodiment, even if the base sequence has a mutation on the 5' side, sequence-specific binding ability on the 3' side is ensured, so it can be used as a primer with guaranteed sequence detection function. becomes possible. Preferably, the regions in the primer that match the base sequence of the IND131 gene locus and its surrounding region or the base sequence of its complementary sequence include the 12th, 15th, and Preferred examples include the region represented by the base sequence from the 16th, 17th, 18th, 19th, or 20th base to the 3' end base.
In addition, as for the primer including the mutated sequence, the entire primer constituent base sequence is 16 bases or more, 17 bases or more, excluding the mutation site for the base sequence constituting the above-mentioned IND131 gene locus and its surrounding region, or its complementary sequence. , 18 or more bases, 19 or more bases, or 20 or more bases preferably include a base sequence that matches the base sequence constituting the IND131 locus and its surrounding region or its complementary sequence.

Regarding the primers described in (p1) and/or (p2), the nucleotide sequence of the variety "Mihaya", the nucleotide sequence having a mutation in the nucleotide sequence of the variety "Mihaya", or the complementary sequence thereof The present invention provides a primer having a corresponding base sequence, which exhibits a PCR amplification mode similar to that of the IND131 insertion type sequence and IND131 deletion type sequence shown in the following experimental example. It can be suitably employed as a primer that can be used in citrus identification technology.
Note that the expression "same as the PCR amplification pattern shown in the following experimental example (omitted)" refers to whether the detection pattern is the same depending on the presence or absence of PCR amplification for the citrus cultivar in the experimental example below, and refers to the position of the primer design. Differences in the base length of amplified fragments due to differences in the like are described as acceptable content.
Similarly, the primers described in (p1) and/or (p2) correspond to a sweet orange base sequence, a base sequence having a mutation to the sweet orange base sequence, or a complementary sequence thereof. A primer having a base sequence that exhibits a PCR amplification mode similar to that of the IND131 insertion type sequence and IND131 deletion type sequence shown in the following experimental example is used with the citrus of the present invention. It can be suitably employed as a primer that can be used in identification technology.

IND44 gene locus In the indel marker allele detection step in this identification technology, (step 1') detecting an allelic polymorphism containing a deletion type sequence of IND44 from the genomic DNA of a citrus plant to be identified. It is possible to include an embodiment.
Here, the indel marker IND44 is an indel marker present in the downstream region of the RALF-like peptide precursor gene on chromosome 1 of the citrus genome (FIG. 4). When shown by the base sequence shown in SEQ ID NO: 7, which shows the IND44 insertion type sequence of sweet orange (Citrus sinensis), from the most upstream base of the 5'UTR representing the RALF-like peptide precursor gene to the most downstream base of the 3'UTR The region represented by the base sequence corresponds to the base sequence from the 2992nd base to the 4132nd base in the base sequence shown in SEQ ID NO: 7. In addition, the ORF region (because the ORF region of the gene consists of only one exon, the region indicated by the base sequence from the first base to the last base of the exon) has the base sequence shown in SEQ ID NO: 7. This corresponds to the region shown by the base sequence from the 3331st base to the 3825th base in .
Here, the 3'UTR of the gene corresponds to the base sequence from the 3826th base to the 4132nd base in the base sequence shown in SEQ ID NO: 7, and the region downstream from the 4132nd base ( The region shown by the base sequence from the 4133rd base to the 7132nd base of SEQ ID NO: 7) corresponds to the downstream region of the gene.

In the insertion type sequence having an insertion sequence in the IND44 gene locus (IND44 insertion type sequence), the most upstream base "A" of the IND44 gene locus, which is present in the downstream region of the RALF-like peptide precursor gene, and the most downstream base of the IND44 gene locus. There is an insertion sequence between "A". Here, there are three types of insertion sequences with different lengths as the insertion sequence of IND44: insertion sequence A of 573 bp or equivalent base length, insertion sequence B of 249 bp or equivalent base length, and insertion sequence B of 99 bp. There are three types of insertion sequences, as shown by C.
When shown by the base sequence set forth in SEQ ID NO: 7, which shows the IND44 insertion type sequence of sweet orange, the 4175th base "A" is the most upstream base of the IND44 gene locus, and the most downstream base of the IND44 gene locus. A 573 bp insertion sequence exists between the 4749th base "A", and this insertion sequence corresponds to insertion sequence A. As the sweet orange IND44 gene locus, an allele type having insertion sequence A and insertion sequence C in a heterologous manner on a homologous chromosome (hetero allele type of insertion sequence A/insertion sequence C of IND44) is shown.

On the other hand, the variety "Mihaya", unlike sweet orange, exhibits an allele type that includes a type of sequence in which no inserted sequence exists (IND44 deletion type sequence). Specifically, the variety "Mihaya" is an allele type that has a heterozygous deletion type sequence and an insertion type sequence of IND44 (more specifically, an allele type that has a heterozygous type of IND44 deletion type sequence and insertion type sequence C) shows.
Here, in the IND44 deletion type sequence, regarding the base sequence of the downstream region of the RALF-like peptide precursor gene, the region sandwiched between the most upstream base "A" and the most downstream base "A" of the IND44 locus (the above The nucleotide sequence (two bases of "AA") in which the inserted sequence) has been deleted is shown (Figure 4). The nucleotide sequence containing the IND44 deletion type sequence isolated from the variety "Mihaya" can be shown as the nucleotide sequence set forth in SEQ ID NO: 8. When shown by the base sequence shown in SEQ ID NO: 8, which shows the IND44 deletion type sequence of the variety "Mihaya", the most upstream base "A" of the IND44 gene locus corresponds to the 89th base, and the most downstream base "A" corresponds to the 90th base.

That is, in the present invention, the following definition can be made regarding the indel marker IND44 of the citrus plant genome:
When shown in the genome of sweet orange (Citrus sinensis), a standard variety of citrus, this is the locus of an indel marker located in the downstream region of the RALF-like peptide precursor gene on chromosome 1, and IND44 of the sweet orange. In the case of the base sequence shown in SEQ ID NO: 7, which includes an insertion type sequence, the 4175th base "A" indicates the most upstream base of the IND44 locus, and the 4749th base "A" indicates the most upstream base of the IND44 locus. The most downstream base is shown, and the base sequence from the 4176th base to the 4748th base represents the insertion sequence,
Regarding the IND44 gene locus of the citrus plant genome, which is the target sample, if the base sequence has an insertion sequence between the most upstream base "A" and the most downstream base "A" of the IND44 gene locus, it is indicated as an IND44 insertion type sequence. , a base sequence in which no inserted sequence exists between the most upstream base "A" and the most downstream base "A" of the IND44 gene locus indicates an IND44 deletion type sequence.

Here, the IND44 gene locus in the citrus plant genome, which is the target sample, is a locus that has an orthologous relationship with the above-mentioned IND44 in the sweet orange genome (a gene locus that has the same origin in a common ancestor) in the citrus plant genome, which is the target sample. refers to
For example, regarding the base sequence constituting the IND44 locus and its surrounding region in the citrus plant genome that is the target sample, the base sequence from the most upstream base of the 5'UTR of the RALF-like peptide precursor gene to the most upstream base of the IND44 gene locus. and the nucleotide sequence from the most downstream base of the IND44 gene locus to the 500th (preferably the 250th) base downstream thereof, if the sequence identity is 85% or more, 90% or more, or 95% or more, It can be determined that it corresponds to the array corresponding to .

Primer set for detecting allelic polymorphisms of IND44 The detection of allelic polymorphisms of IND44 in the detection step uses oligonucleotide primers designed to enable PCR amplification of a region containing the base sequence of the IND44 gene locus. This can be suitably carried out by a method that utilizes the PCR method.

(Primer set that spans the gene locus)
As an oligonucleotide primer capable of detecting an allele type containing an IND44 deletion type sequence in the detection step, for example, a primer set consisting of a pair of oligonucleotide primers (a pair of IND44 detection An example of this is to design an oligonucleotide primer that makes it possible to detect a PCR amplified fragment of an IND44 insertion type sequence and/or an IND44 deletion type sequence (Fig. 4, (See Figure 5).
When PCR amplification is performed using a primer set containing the pair of oligonucleotide primers, if any of the IND44 insertion type sequences A to C exists in the genome, each DNA fragment containing the insertion sequence is amplified. do. On the other hand, when the IND44 deletion type sequence is present in the genome, a DNA fragment with a length corresponding to the inserted sequence is amplified.
Therefore, if the detection step is performed using the primer set and a base-length DNA fragment that does not contain the inserted sequence is amplified as a band, the allele type of the IND44 locus in the genomic DNA of the target sample is the IND44 deletion type sequence. It becomes possible to determine that the allele type includes

Here, specifically, the primer set consisting of "a pair of common region primers for IND44 detection" (forward primer and reverse primer) is one that can amplify a DNA fragment including the following IND44 gene locus. It is possible to use a pair of oligonucleotide primers (p3) and (p4):
(p3) An oligonucleotide containing a base sequence of 16 or more bases included in the base sequence from the 1000th base upstream of the most upstream base "A" of the IND44 gene locus of the citrus plant genome to the most upstream base "A" Primer, and
(p4) A base sequence of 16 or more bases included in the complementary sequence of the base sequence from the most downstream base "A" to the 1000th base downstream of the IND44 gene locus in the citrus plant genome, and which is included in the above (p3). An oligonucleotide primer comprising the described primer and a base sequence capable of forming a primer pair in a direction and position sandwiching the IND44 gene locus.
Here, the oligonucleotide primers described in (p3) and (p4) above are used as a primer set of these oligonucleotide primers as base sequences to exhibit their primer functions, and are used as samples 1 to 2 in Tables 1 to 2. When PCR reaction is performed on the genomic DNA of the citrus varieties listed as No. 24 (total of 24 types), preferably the PCR reaction is performed on the genomic DNA of the citrus varieties listed in Tables 1 to 4 (total of 47 types). Preferred embodiments include oligonucleotide primers having base sequences that can amplify an IND44 insertion type sequence and/or an IND44 deletion type sequence when a reaction is performed.

(Primer set including deletion type sequence-specific primer)
In addition, in the detection step, oligonucleotide primers capable of detecting alleles containing the IND44 deletion type sequence may be used, for example, to ensure that PCR amplification occurs only when the IND44 deletion type sequence is present. One example is the design of an IND44 deletion type sequence-specific primer consisting of a base sequence connecting the most upstream base and the most downstream base of the disrupted IND44 gene locus (see FIG. 6).
When PCR amplification is performed using a primer set containing the IND44 deletion type sequence-specific primer and a common region primer for IND44 detection that forms a primer pair with this, IND44 deletion is detected for the indel marker insertion type sequence. No annealing of type sequence-specific primers occurs and no PCR amplified fragment is detected. On the other hand, annealing of the IND44 deletion type sequence-specific primer occurs to the indel marker deletion type sequence, and a PCR amplified fragment is detected.
Therefore, if the detection step is performed using the primer set and a DNA fragment with a base length that matches the IND44 deletion type sequence predicted from the primer design position is amplified, the IND44 locus in the genomic DNA of the target sample is amplified. , it becomes possible to determine that an IND44 deletion type sequence exists.

Here, the "IND44 deletion type sequence-specific primer" and the paired "IND44 detection common region primer" are specifically oligonucleotides that can specifically amplify only the IND44 deletion type sequence. It is possible to use a primer (p13) and a common region primer for IND44 detection (p14) that forms a primer pair with this primer:
(p13) A base sequence of 16 bases or more contained in the base sequence of a deletion type sequence in which the insertion sequence of IND44 in the citrus plant genome does not exist, and the base sequence of 16 bases or more is the IND44 gene locus.2 bases (2 bases consisting of the most upstream base “A” and the most downstream base “A” of the IND44 gene locus) An oligonucleotide primer containing a base sequence containing two bases that are the IND44 gene locus between the 10th base and the 3' end base, or a deletion type in which there is no inserted sequence of IND44 in the citrus plant genome. A base sequence of 16 or more bases included in the complementary sequence of the base sequence of the sequence, where the base sequence of 16 bases or more is the 2 bases of the IND44 gene locus (the most upstream base "A" and the most downstream base of the IND44 gene locus). A base sequence consisting of two complementary bases (corresponding two bases in a complementary strand) of the base "A" (two bases consisting of the base "A"), which is located upstream from the 3' end base of the base sequence of 16 or more bases. An oligonucleotide primer comprising a base sequence containing two complementary bases of the IND44 gene locus between the 10th base and the 3' end base, and
(p14) A base sequence of 16 or more bases included in the complementary sequence of the base sequence from the most downstream base "A" of the IND44 gene locus in the citrus plant genome to the 1000th base downstream thereof, or the IND44 gene in the citrus plant genome A base sequence of 16 or more bases included in the base sequence from the 1000th base upstream of the most upstream base "A" of the locus to the most upstream base "A", which is the primer described in (p. 13) above. An oligonucleotide primer containing a base sequence capable of forming a primer pair.
Here, the oligonucleotide primers described in (p. 13) and (p. 14) above are used as a primer set of these oligonucleotide primers as base sequences to exhibit their primer functions, and are used as samples 1 to 2 in Tables 1 to 2. When a PCR reaction was performed on the genomic DNA of the citrus varieties listed as samples 6, 7, 8, 10, and 11 (24 types in total), the IND44 deletion type was detected for the citrus varieties shown as samples 6, 7, 8, 10, and 11. Preferred embodiments include an oligonucleotide primer having a base sequence that allows sequence amplification and does not cause PCR amplification for citrus varieties indicated by other sample numbers. Preferably, when the PCR reaction is performed on the genomic DNA of the citrus varieties listed in Tables 1 to 4 (total of 47 types), samples 6, 7, 8, 10, 11, 26, 27, 28, It is possible to amplify the IND44 deletion type sequence for the citrus varieties indicated as 36, 38, and 39, and PCR amplification does not occur for the citrus varieties indicated by other sample numbers. A preferred embodiment is an oligonucleotide primer having a base sequence.

(Primer design matters)
The design conditions for the oligonucleotide primers mentioned above are that (p3) and (p4) are primers of the same primer set that form a primer pair, so if (p3) is a forward primer, (p4) is a reverse primer. , (p3) is a reverse primer, (p4) is a forward primer. Similarly, (p13) and (p14) are primers of the same primer set that form a primer pair, so if (p13) is a forward primer, (p14) is a reverse primer, and (p13) is a reverse primer. In this case, (p14) becomes the forward primer.

Further, as a design condition for the primer, it is preferable that the primer contains at least 16 consecutive base sequences included in the base sequence constituting the IND44 gene locus and its surrounding region, or its complementary sequence. Suitable base lengths (mers) include 16 bases or more, 17 bases or more, 18 bases or more, 19 bases or more, 20 bases or more, or 21 bases or more for the base sequence contained in the base sequence or its complementary sequence. It is preferable that the primer contains the following. In addition, there is no particular restriction on the upper limit of the base length of the oligonucleotide primer as long as it does not deviate from the range that functions as a primer. can be mentioned.
In addition, in order to increase sequence specificity and reduce non-specific PCR amplification, it is necessary to match the Tm value (annealing temperature), GC content, etc. of the primers forming the primer pair to approximate values, and then It is preferred to design suitable primers of long length.
Furthermore, the 5' end of the oligonucleotide primer may have a restriction enzyme site for use in vector insertion, etc., a modified base sequence for introducing various vectors, or a fluorescent substance, a labeling substance, etc. It may also be in the form of a combination of Furthermore, the base at the 3' end of the oligonucleotide primer is preferably a base sequence that completely matches the base sequence constituting the IND44 gene locus and its surrounding region, or its complementary sequence.

The region in which the oligonucleotide primer described above can be designed is preferably a region in which the base length of the amplification product is suitable for PCR amplification. Specifically, from the viewpoint of ease of PCR amplification, a primer set (gene It is preferable to design a primer set that includes the IND44 deletion type sequence-specific primer and a common region primer for IND44 detection (a primer set that includes deletion type sequence-specific primers).
More preferably, the primer set is designed at a position where the base length of the PCR amplified fragment is 2 kbp or less, 1.5 kbp or less, 1.2 kbp or less, 1 kbp or less, 750 bp or less, or 500 bp or less. As a lower limit of the length of the PCR amplified fragment, for example, from the viewpoint of differentiation from non-specific amplification, it is preferable to design a primer set at a position where the base length of the PCR amplified fragment is 50 bp or more or 100 bp or more. Embodiments include, for example, a range of 50 to 2000 bp, 100 to 1000 bp, or 100 to 500 bp.

Considering the technical aspects of PCR, the common region primers for IND44 detection described in (p3), (p4), and (p14) can be designed for regions (Figures 4 and 5) that are easy to amplify by PCR. From the viewpoint of sex, specifically, the region shown by the base sequence from the 1000th base upstream of the most upstream base "A" of the IND44 gene locus to the most upstream base "A", or the most downstream base "A". The region shown by the base sequence from "A" to the 1000th base downstream thereof can be preferably mentioned. Preferably, the region up to the 1000th, 900th, 800th, 750th, 500th, or 250th base can be preferably mentioned upstream or downstream.
Further, as an embodiment of the primer design, for example, it is possible to use these corresponding regions in the variety "Mihaya" or the standard variety Sweet Orange as the primer design region.

The IND44 deletion type sequence-specific primer described in (p13) includes two bases consisting of the most upstream base "A" and the most downstream base "A" of the IND44 gene locus, or two bases complementary to the two bases, with respect to the IND44 deletion type sequence. (corresponding two bases in the complementary strand) (Figure 6). It is preferable that the primer has a base sequence including the two bases or two complementary bases thereof between the base and the base at the 3' end. Preferably, the two bases or their complementary two bases are placed between the 8th, 6th, 4th, or 3rd base upstream of the 3'-end base of the primer constituent base sequence and the 3'-end base. It is preferable that the primer has a base sequence containing the following.
Further, as an embodiment of the primer design, for example, these corresponding regions in the variety "Mihaya" etc. can be used as the primer design region.

The primers described in (p3), (p4), (p13), and (p14) above are capable of amplifying the IND44 locus and its surrounding region on the target sample genomic DNA under the various primer design conditions described above. A primer containing the base sequence can be employed as the above-mentioned primer that can be used in the citrus identification technology of the present invention. Furthermore, even if the primer contains a nucleotide sequence that has one or more base mutations with respect to the nucleotide sequence constituting the IND44 locus and its surrounding region or its complementary sequence, the IND44 locus on the target sample genomic DNA may be Any primer can be used as the above primer as long as it includes a base sequence that enables amplification of the region and its surrounding region.
Regarding the base sequence constituting the primer, the number of mutations to the base sequence constituting the IND44 locus and its surrounding region or its complementary sequence makes it possible to amplify the IND44 locus and its surrounding region on the target sample genomic DNA. There is no particular restriction as long as it is within the range, but preferably 3 bases or less, 2 bases or less, or 1 base or less. Particularly preferred are those in which the number of mutations is 2 or less bases.
Here, as an embodiment of the primer including the mutant sequence, for example, the base sequence from the 10th base upstream of the 3' end base of the primer constituent bases to the 3' end base is the IND44 gene locus. Preferred examples include primers that match the nucleotide sequence of the nucleotide sequence and its surrounding region or the nucleotide sequence of its complementary sequence. In this embodiment, even if the base sequence has a mutation on the 5' side, sequence-specific binding ability on the 3' side is ensured, so it can be used as a primer with guaranteed sequence detection function. becomes possible. Preferably, the regions in the primer that match the base sequence of the IND44 gene locus and its surrounding region or the base sequence of its complementary sequence include the 12th, 15th, and Preferred examples include the region represented by the base sequence from the 16th, 17th, 18th, 19th, or 20th base to the 3' end base.
In addition, as for the primer including the mutated sequence, the entire primer constituent base sequence is 16 bases or more, 17 bases or more, excluding the mutation site for the base sequence constituting the IND44 gene locus and its surrounding region or its complementary sequence. , 18 or more bases, 19 or more bases, or 20 or more bases preferably include a base sequence that matches the base sequence constituting the IND44 locus and its surrounding region or its complementary sequence.

Regarding the primers described in (p3), (p4), (p13), and/or (p14), the base sequence of the variety "Mihaya" and the base having a mutation with respect to the base sequence of the variety "Mihaya" or a nucleotide sequence corresponding to these complementary sequences, which exhibits a PCR amplification pattern similar to that of the IND44 insertion type sequence and IND44 deletion type sequence shown in the experimental example below. A primer containing the base sequence can be suitably employed as a primer that can be used in the citrus identification technology of the present invention.
Note that the expression "same as the PCR amplification pattern shown in the following experimental example (omitted)" refers to whether the detection pattern is the same depending on the presence or absence of PCR amplification for the citrus cultivar in the experimental example below, and refers to the position of the primer design. Differences in the base length of amplified fragments due to differences in the like are described as acceptable content.
Similarly, the primers described in (p3), (p4), (p13), and/or (p14) include a sweet orange base sequence, a base sequence having a mutation with respect to the sweet orange base sequence, Or, a primer having a nucleotide sequence corresponding to these complementary sequences, which exhibits a PCR amplification pattern similar to that of the IND44 insertion type sequence and IND44 deletion type sequence shown in the following experimental example. A primer containing the following can be suitably employed as a primer that can be used in the citrus identification technology of the present invention.

Aspects of the step of detecting allelic polymorphisms of indel markers The variety "Mihaya" to be identified according to the present invention is a citrus variety whose IND131 allele type is "homotype of insertion type sequence." Therefore, in the identification technique according to the present invention, the step of detecting allelic polymorphism of an indel marker can be performed as (step 1) the step of detecting a homozygous allelic polymorphism of the insertion type sequence of IND131.

Furthermore, in the identification technology of the variety "Mihaya" according to the present invention, in addition to the above (step 1), (step 1') a step of detecting an allelic polymorphism containing a deletion type sequence from the IND44 gene locus. It is also possible to perform this as a technique that further includes.
Here, as (Step 1'), the step of detecting an allelic polymorphism including the IND44 deletion type sequence can be performed as the step of detecting the IND44 deletion type sequence. In addition, the step (Step 1') includes a step of detecting a heterozygous allelic polymorphism between the deletion type sequence and the insertion type sequence of IND44 (more specifically, the step of detecting the heterozygous allelic polymorphism between the deletion type sequence and the insertion type sequence C of IND44). It is also possible to perform this as a step of detecting heterozygous allele polymorphism.

When the allele polymorphism detection step is performed by PCR, it is possible to perform the step using a primer set that includes a primer set sandwiching the above-described indel marker gene locus.
In addition, when performing the IND44 allele polymorphism detection step by "detection of deletion type sequences", a primer set containing deletion type sequence-specific primers that can detect only deletion type sequences as amplified fragments is used. It is preferable to perform this step.

In the citrus identification technology according to the present invention, as the order of steps, (step 1) is performed and then (step 2) is performed.
Further, in an embodiment including (Step 1') as the citrus identification technology according to the present invention, (Step 2) is performed after (Step 1) and (Step 1') are performed. Here, there is no particular restriction on the order of (Step 1) and (Step 1'); 1) is possible. Moreover, in addition to the mode in which the above-mentioned (Step 1) and (Step 1') are carried out separately, a mode in which (Step 1) and (Step 1') are carried out simultaneously is also possible.
It is also possible to carry out the detection steps of (Step 1) and (Step 1') using the same sample solution. For example, when performing using the PCR method, it is possible to carry out (Step 1) and (Step 1') using the same PCR reaction solution. In this embodiment, a PCR reaction solution containing a primer set designed to sandwich the IND131 gene locus described above and a primer set for detecting only the deleted sequence of IND44 are prepared in the same solution, and An example is an embodiment (multiplex method) in which the amplification of the allelic polymorphism of IND131 and the amplification of the IND44 deletion type sequence are carried out simultaneously in a PCR reaction.

When the allele polymorphism detection step was performed by multiplex PCR, four types of primers were included: a primer set designed at positions sandwiching the IND131 gene locus, and a primer set containing an IND44 deletion type sequence-specific primer. It is preferable to prepare a PCR reaction solution in the following state and perform the step.
In addition, when performing a PCR reaction using the multiplex method, the base length of the PCR amplified fragment with a primer set designed on both sides of the IND131 gene locus, and the base length of a PCR amplified fragment with a primer set containing an IND44 deletion type sequence-specific primer. It is preferable to design the primer so that the base length of the amplified fragment is similar to the base length that can be distinguished from each other by electrophoresis. Further, it is preferable to design the primers so that the Tm value (annealing temperature), GC content, etc. of these four types of primers have similar values.
If this embodiment is adopted, by simply electrophoresing one PCR reaction solution using the multiplex method, allelic polymorphisms of IND131 (one or two bands depending on the presence or absence of an inserted sequence) and IND44 allele polymorphisms (deletion) can be detected. 0 or 1 band depending on the presence or absence of a lost sequence) can be determined at once.

[Judgment process]
The citrus identification technology according to the present invention performs the above-described allele polymorphism detection step of the indel marker, and then determines from the detection result whether the citrus plant to be identified is the variety "Mihaya" or its successor variety. including the step of
In the citrus identification technology of the present invention, after performing the IND131 allele polymorphism detection step described in (step 1) described above, the determination step (step 2) is performed based on the detection result of the allele type.
In this regard, the present inventors investigated 47 major citrus varieties distributed in Japan and found that there were no alleles with a homozygous IND131 insertion type sequence other than the Mihaya variety. . These methods account for more than 98% of the distribution volume of citrus fruits in Japan, and demonstrate that the technology is a variety identification technology that satisfies the distribution volume of 90% or more as defined by the ISO 13495 international standard.

That is, regarding the indel marker IND131 used in this identification technology, citrus varieties belonging to the genus Citrus other than the variety "Mihaya" have the IND131 deletion type sequence in either or both of the homologous chromosomes of the IND131 gene locus. Indicates the allele type containing. On the other hand, as shown in the results of the experimental examples described below, the variety "Mihaya" does not have an IND131 deletion type sequence at the IND131 gene locus, but exhibits an allele type having a homozygous IND131 insertion type sequence. Therefore, in this identification technology, if an allele having a homozygous IND131 insertion type sequence is detected from the DNA of a citrus plant to be identified, the citrus plant to be identified is of the variety "Mihaya". It becomes possible to judge.
That is, the citrus identification technology according to the present invention provides the following method: (Step 2): When a homozygous allele polymorphism of the insertion type sequence of IND131 is detected in the step described in (Step 1) above, the citrus identification technology of the present invention A citrus variety comprising the step of determining that the plant variety is the variety "Mihaya" or is a progeny variety of the variety "Mihaya" that exhibits the same allele type as the variety "Mihaya" regarding IND131. Regarding the identification method.

Furthermore, in this identification technology, in addition to the IND131 allele polymorphism detection step described in (step 1) above, after performing the IND44 allele polymorphism detection step described in (step 1'), those allele types are detected. It is also possible to perform the determination step (Step 2) based on the detection result.
In this regard, regarding the indel marker IND44, many citrus varieties belonging to the genus Citrus (approximately 77% of the 47 major varieties mentioned above) exhibit allele types that do not contain the IND44 deletion type sequence at the IND44 gene locus. On the other hand, as shown by the results of the experimental examples described later, some citrus varieties (approximately 23% of the 47 major varieties mentioned above), including the variety "Mihaya", have the IND44 deletion type sequence at the IND44 gene locus. Indicates the allele type. Therefore, in this identification technology, when an allele type having an IND44 deletion type sequence is detected from the DNA of a citrus plant to be identified, the citrus plant to be identified falls into the variety group that includes the variety "Mihaya". By combining this with the detection results of the allelic polymorphism of the IND131 insertion type sequence homozygous type described above (Step 1), the identification accuracy of the variety "Mihaya" can be further improved. It becomes possible to do so.
That is, in the citrus identification technology according to the present invention, (Step 2) in an embodiment including (Step 1') in addition to (Step 1), in the step described in (Step 1) above, the insertion type sequence of IND131 is If a homozygous allelic polymorphism is detected, and an allelic polymorphism containing a deletion type sequence of IND44 is detected in the step described above (step 1'), the variety of the citrus plant that is the target sample is It is possible to perform a process of determining that the variety is "Mihaya" or is a progeny of the variety "Mihaya" that exhibits the same allele type as the variety "Mihaya" regarding IND131 and IND44. .

In addition, in the citrus identification technology according to the present invention, the above (step 1') allele polymorphism detection step regarding IND44 is performed using the deletion type sequence/insertion type sequence of IND44 (more specifically, the deletion type sequence/insertion type sequence of IND44). It can also be carried out as a step of detecting a heterozygous type sequence C).
When the IND44 allele polymorphism detection step is carried out in this manner, the determination step (Step 2) is carried out when a homozygous allele polymorphism of the insertion type sequence of IND131 is detected in the step described in (Step 1) above. , and a heterozygous allelic polymorphism of the deletion type sequence and insertion type sequence of IND44 (or the deletion type sequence and insertion type sequence C of IND44) was detected in the step described in the above (step 1'). In this case, the variety of the citrus plant that is the target sample is the variety "Mihaya", or it is a progeny variety of the variety "Mihaya" that exhibits the same allele type as the variety "Mihaya" regarding IND131 and IND44. , it is possible to perform the process of determining .

[Other processes, etc.]
The citrus identification technology according to the present invention may include steps other than the above (step 1) and (step 2). Moreover, it is also possible to set it as an aspect including the process other than the said (process 1), (process 1'), and (process 2).
For example, it is possible to include another step before (Step 1) and/or (Step 1'), and it is possible to include another step after (Step 2). . Moreover, it is also possible to set it as the aspect which includes another process between (process 1), (process 1'), and/or (process 2).
Other steps include steps involving known and/or new technical matters, and are not particularly limited; Selection process of target sample plants based on growth characteristics, fruit morphology and quality, characteristics of metabolites and secreted substances, etc.) Variety identification process based on other DNA identification techniques (analysis of SNP, SSR, CAPS, RFLP, etc.) , etc. are also possible.

2. Inventions and Applied Inventions Related to Citrus Variety Identification Techniques The inventions related to this application include those described in paragraph 1 above. This invention includes inventions related to the citrus variety identification technology described in , inventions related to technical features for using the identification technology, inventions related to the use of the identification technology, etc.

[Inventions related to various methods]
The citrus identification technology related to the present invention includes an invention related to a method for identifying citrus varieties. Specifically, the present invention includes the use of the variety "Mihaya" or the progeny of the variety "Mihaya" that exhibits the same allele type as the variety "Mihaya" with respect to IND131 (more preferably IND131 and IND44) with other citrus varieties. This includes inventions related to methods for identifying varieties.
Further, inventions using the citrus identification technology according to the present invention include inventions relating to various methods, including the use of the above-mentioned method for identifying the citrus variety "Mihaya" or its successor varieties. For example, methods for cultivating plants of the variety "Mihaya" or its successors, methods for producing fruits of the variety "Mihaya" or its successors, or methods for cultivating plants of the variety "Mihaya" or its successors; Inventions related to methods of producing processed fruit products, etc. are included.
As the method for cultivating the plant, the method for producing fruit, and the method for producing processed fruit products, conventional methods or known methods can be used, except for using the citrus variety identification method according to the present invention. . Furthermore, regarding the inventions of these various methods, the descriptions in the above paragraphs can be referred to or quoted regarding various steps, conditions, methods, means, etc. Moreover, with respect to various steps, conditions, methods, means, etc. other than the characteristic parts of the present invention, conventional methods and known methods can be adopted.
In addition, in the method for producing processed citrus fruit products, it is possible to refer to or quote the description regarding processed products in the above paragraph as citrus fruit processed products that fall under the products, but for example, straight juice containing pulp, Examples include processed fruit products such as canned fruit pulp soaked in syrup, dried fruits, etc.

[Invention related to citrus variety identification kit]
The present invention includes an invention related to a citrus variety identification kit for identifying the variety "Mihaya" or its successor varieties, as an invention related to technical features for using the above-mentioned citrus identification technology.
That is, the present invention includes the invention related to the following citrus variety identification kit:
A citrus plant containing the primer set described in (A) below, for identifying the citrus variety "Mihaya" or a progeny variety of the variety "Mihaya" that exhibits the same allele type as the variety "Mihaya" regarding IND131. Variety identification kit:
(A) A primer set capable of detecting homozygous allele polymorphism of the insertion type sequence of IND131 described in (a-1) below:
(a-1) A primer set consisting of the oligonucleotide primer shown in (p1) above and the oligonucleotide primer shown in (p2) above.
The citrus variety identification kit related to this identification technology is a primer set for identifying the above-mentioned variety "Mihaya" or its successor varieties, and detects homozygous allele polymorphism of the insertion type sequence of IND131 described in (A). This invention relates to a citrus cultivar identification kit, including a primer set for the identification of citrus varieties. Here, as a primer set for detecting homozygous allele polymorphism of the insertion type sequence of IND131 described in (A), a primer set consisting of two types of primers (p1) and (p2) (IND131 gene locus It is possible to include a primer set designed between the two sides.

Furthermore, the present invention includes inventions related to the following citrus variety identification kits:
The citrus cultivar "Mihaya" or the cultivar "Mihaya" which exhibits the same allelic type as the cultivar "Mihaya" with respect to IND131 and IND44, which contains the primer set described in (A) below and the primer set described in (B) below. Citrus cultivar identification kit for identifying progeny cultivars:
(A) A primer set capable of detecting homozygous allele polymorphism of the insertion type sequence of IND131 described in (a-1) below:
(a-1) A primer set consisting of the oligonucleotide primer shown in (p1) above and the oligonucleotide primer shown in (p2) above;
(B) A primer set capable of detecting an allelic polymorphism containing the IND44 deletion type sequence described in (b-1) or (b-2) below:
(b-1) A primer set consisting of the oligonucleotide primer shown in (p3) above and the oligonucleotide primer shown in (p4) above,
(b-2) A primer set consisting of the oligonucleotide primer shown in (p13) above and the oligonucleotide primer shown in (p14) above.

The citrus variety identification kit related to the present identification technology of this embodiment uses a homozygous type of the insertion type sequence of IND131 described in (A) above as a primer set for identifying the above-mentioned variety "Mihaya" or its successor variety. In addition to the primer set for detecting allelic polymorphisms, the primer set for detecting allelic polymorphisms containing the IND44 deletion type sequence described in (B), both primer sets (a total of 4 types of primers) are used. ) concerning a citrus variety identification kit.
Here, as a primer set for detecting the allelic polymorphism containing the deletion type sequence of IND44 described in (B), a primer set consisting of two types of primers (p3) and (p4) (IND44 gene locus (a primer set designed to sandwich the IND44 deletion type sequence-specific primer), or a primer set consisting of two types of primers (p13) and (p14) (a primer set containing an IND44 deletion type sequence-specific primer). is possible. In addition, as a primer set for detecting the IND44 deletion type sequence described in (B), a primer set consisting of two types of primers (p13) and (p14) (including a primer specific for the IND44 deletion type sequence) is used. A preferred embodiment includes a primer set). In this aspect, since the kit includes a primer set that can amplify "only the deletion type sequence" regarding IND44, it becomes possible to more easily determine the PCR amplification of the deletion type sequence.

Furthermore, the citrus variety identification kit according to the present invention can be configured as a kit that includes various components in addition to the primer set described above. For example, it is also possible to form a kit containing various reagents, enzymes, a primer set serving as a positive control such as a primer set for amplifying the rbcL gene, and the like.

The present invention will be explained below with reference to Examples, but the scope of the present invention is not limited thereto.

[Experimental method]
Regarding the tests and analyzes conducted in the following experimental examples, unless otherwise specified, the tests and analyzes were carried out using the methods and conditions described below.

(1) “DNA extraction and PCR reaction”
The exocarp was cut from the fruit of a citrus plant, the sample was frozen using liquid nitrogen in a mortar, and the sample was crushed using a pestle. Total DNA was extracted from the obtained powdered sample using a DNA extraction kit, DNeasy Plant Mini Kit (Qiagen). The total DNA obtained by this method includes genomic DNA and chloroplast DNA.

Using the extracted total DNA as a template, a DNA fragment was amplified by PCR reaction using KOD DNA polymerase (Toyobo) and the primer set described below. The PCR reaction was performed using a PCR thermal cycler (ProFlex PCR System, Thermo Fisher Scientific) and a 96-well plate or 8-tube under the following conditions.
Furthermore, the primers for amplifying the indel marker IND131 or IND44 used in the following experimental examples related to the cultivar identification test were designed based on the nucleotide sequence of the cultivar "Mihaya"; It was confirmed in the following experimental examples that the primers were able to amplify the region containing the IND131 or IND44 locus of the citrus varieties used in Experimental Examples 1 to 4 (Tables 1 to 4, Fig. 7-9).

(PCR reaction solution)
2×KOD One PCR Master Mix Blue 5μL
Primer solution (4 pmol/μL each) 1μL
Sample DNA (5ng/μL) 1μL
Sterile ultrapure water 3μL
(Total 10μL)

(PCR conditions)
98℃ 1 second 32 cycles x (98℃ 10 seconds → 56℃ 5 seconds → 68℃ 2 seconds)
68℃ 30 seconds
4℃ until sample collection

Loading dye (blue dye) was added to the reaction solution after the above PCR reaction and applied to a 2% agarose gel, and electrophoresis was performed in 1×TAE buffer. A size marker, 100bp ladder or 200bp ladder, was also electrophoresed at the same time.
The gel after electrophoresis was stained in an EtBr solution for 20 minutes and irradiated with ultraviolet light to detect amplified DNA fragments separated according to size as bands.
In addition, regarding the photographic image of electrophoresis shown in the drawing, "each number" shown on the lane indicates the sample number indicating the citrus variety. Further, "M2" indicates a lane in which a 200 bp ladder DNA size marker was electrophoresed, and "M1" indicates a lane in which a 100 bp ladder DNA size marker was electrophoresed.

[Experiment Example 1] “Selection of indel markers that can identify the variety “Mihaya””
Regarding indel markers for plants of the genus Citrus, we searched for indel markers that exhibit allele types unique to the cultivar "Mihaya." The indel marker used in this study is a well-known indel marker in plants of the genus Citrus (non-patent documents 3, 4), and the allele type possessed by the genomic DNA of each variety of plants of the genus citrus. is not known.

(1) “Search for indel marker polymorphisms that can identify the variety “Mihaya””
Regarding 185 types of indel markers in plants of the genus Citrus, PCR primers were set at positions sandwiching the indel marker gene loci, and a PCR reaction was performed on total DNA extracted from the cultivar "Mihaya". The base length of the PCR amplified fragment amplified from genomic DNA using the set was detected by electrophoresis. The methods and conditions for DNA extraction, PCR reaction, DNA detection, etc. were performed according to the above description.

As a result, when performing a PCR reaction using a primer set related to IND131 among the 185 types of indel markers investigated above, all varieties except the variety "Mihaya" had an allele polymorphism with a deletion type sequence. It was shown that one type of PCR amplified fragment corresponding to the insertion type sequence was obtained for the variety "Mihaya". In addition, when performing a PCR reaction using a primer set related to IND44, many varieties show allelic polymorphism that does not include the deletion type sequence, but the variety ``Mihaya'' shows a PCR reaction corresponding to the deletion type sequence. It was shown that an amplified fragment could be obtained.
From these results, the cultivar "Mihaya" is a citrus cultivar that has a homozygous sequence (IND131 insertion type sequence) at the IND131 indel marker gene locus on the homologous chromosome. It has been shown. Furthermore, the cultivar "Mihaya" was shown to be a citrus cultivar having a deletion type sequence at the IND44 indel marker gene locus (IND44 deletion type sequence).

(2) “Base sequence information of IND131 and IND44”
Information regarding the nucleotide sequences of the citrus plant indel markers IND131 and IND44 is shown below.

(IND131)
The indel marker IND131 is located on the first chromosome of the sweet orange genome (http://citrus.hzau.edu.cn/orange/index.php), which is one of the standard varieties of citrus whose genome information has been decoded. , an indel marker present in the 3'UTR of the cell wall-associated receptor kinase gene (gene present within the base sequence shown in SEQ ID NO: 1, accession number: Cs1g13880.1), which is a type of serine/threonine protein kinase gene. Yes (Figure 3).
Here, the base sequence containing the IND131 deletion type sequence of sweet orange (Citrus sinensis) can be shown as the base sequence set forth in SEQ ID NO: 1. Regarding the sweet orange cell wall-related receptor kinase gene, the region shown in the base sequence from the most upstream base of the 5'UTR to the most downstream base of the 3'UTR is the 3001st base in the base sequence shown in SEQ ID NO: 1. This region corresponds to the base sequence from the base to the 5756th base. In addition, the ORF region (the region indicated by the base sequence from the first base of the first exon to the last base of the final exon) is the region from the 3083rd base to the 5478th base in the base sequence shown in SEQ ID NO: 1. This region corresponds to the base sequence up to.
Here, the 3'UTR of the gene corresponds to the base sequence from the 5479th base to the 5756th base in the base sequence shown in SEQ ID NO: 1, and the region downstream from the 5756th base ( The region shown by the base sequence from the 5757th base to the 8756th base of SEQ ID NO: 1) corresponds to the downstream region of the gene.
In the sweet orange IND131 deletion type sequence, the 5527th base "A" which is the most upstream base of the IND131 locus shown in SEQ ID NO: 1 and the 5528th base "A" which is the most downstream base of the IND131 locus shown in SEQ ID NO: 1. There is no inserted sequence between "G" and "G".

As a result of the selection test in this example, the cultivar "Mihaya", unlike Sweet Orange, showed an allele type that did not contain the IND131 deletion type sequence in the homologous chromosome and had a homologous D131 insertion type sequence in the homologous chromosome. . The nucleotide sequence containing the IND131 insertion type sequence isolated from the variety "Mihaya" can be shown as the nucleotide sequence set forth in SEQ ID NO: 2. When shown by the base sequence shown in SEQ ID NO: 2, which shows the IND131 insertion type sequence of the cultivar "Mihaya", the most upstream base "A" of the IND131 locus corresponds to the 62nd base, and the most downstream base "A" corresponds to the 62nd base. G" corresponds to the 296th base, and a 233 bp insertion sequence (region shown by the base sequence from the 63rd base to the 295th base of SEQ ID NO: 2) exists between them.
On the other hand, unlike the variety "Mihaya", other citrus varieties including sweet orange exhibit allele types containing the IND131 deletion type sequence regarding the IND131 gene locus. Here, in the IND131 deletion type sequence, the region sandwiched between the most upstream base "A" and the most downstream base "G" of the IND131 locus with respect to the 3'UTR of the cell wall-associated receptor kinase gene (the above inserted Figure 3 shows the deleted base sequence.

(IND44)
The indel marker IND44 is located on the first chromosome of the sweet orange genome, which is one of the standard varieties of citrus whose genome information has been decoded (http://citrus.hzau.edu.cn/orange/index.php). This is an indel marker present in the downstream region of the RALF-like peptide precursor gene (gene present within the base sequence shown in SEQ ID NO: 7, accession number: Cs1g22550.1) (FIG. 4).
Here, the base sequence containing the IND44 insertion type sequence of sweet orange (Citrus sinensis) can be shown as the base sequence set forth in SEQ ID NO: 7. Regarding the sweet orange RALF-like peptide precursor gene, the region shown in the base sequence from the most upstream base of the 5'UTR to the most downstream base of the 3'UTR is the 2992nd base in the base sequence shown in SEQ ID NO:7. This region corresponds to the base sequence from the base to the 4132nd base. In addition, the ORF region (because the ORF region of the gene consists of only one exon, the region indicated by the base sequence from the first base to the last base of the exon) has the base sequence shown in SEQ ID NO: 7. This region corresponds to the base sequence from the 3331st base to the 3825th base in .
Here, the 3'UTR of the gene corresponds to the base sequence from the 3826th base to the 4132nd base in the base sequence shown in SEQ ID NO: 7, and the region downstream from the 4132nd base ( The region shown by the base sequence from the 4133rd base to the 7132nd base of SEQ ID NO: 7) corresponds to the downstream region of the gene.

In the insertion type sequence having an insertion sequence in the IND44 gene locus (IND44 insertion type sequence), the most upstream base "A" of the IND44 gene locus, which is present in the downstream region of the RALF-like peptide precursor gene, and the most downstream base of the IND44 gene locus. There is an insertion sequence between "A". Here, there are three types of insertion sequences with different lengths as the insertion sequence of IND44: insertion sequence A of 573 bp or equivalent base length, insertion sequence B of 249 bp or equivalent base length, and insertion sequence B of 99 bp. There are three types of insertion sequences, as shown by C.
When shown by the base sequence set forth in SEQ ID NO: 7, which shows the IND44 insertion type sequence of sweet orange, the 4175th base "A" is the most upstream base of the IND44 gene locus, and the most downstream base of the IND44 gene locus. Between the 4749th base "A", there is an inserted sequence of 573 bp (the region shown by the base sequence from the 4176th base to the 4748th base of SEQ ID NO: 7), and the inserted sequence is This corresponds to array A.

As a result of the selection test in this example, the cultivar "Mihaya", unlike sweet orange, showed an allele type containing a type of sequence in which no inserted sequence was present (IND44 deletion type sequence). Here, in the IND44 deletion type sequence, regarding the base sequence of the RALF-like peptide precursor gene and its downstream region, the region sandwiched between the most upstream base "A" and the most downstream base "A" of the IND44 locus ( The nucleotide sequence in which the above inserted sequence) has been deleted is shown (Figure 4). The nucleotide sequence containing the IND44 deletion type sequence isolated from the variety "Mihaya" can be shown as the nucleotide sequence set forth in SEQ ID NO: 8. When shown by the base sequence shown in SEQ ID NO: 8, which shows the IND44 deletion type sequence of the variety "Mihaya", the most upstream base "A" of the IND44 gene locus corresponds to the 89th base, and the most downstream base "A" corresponds to the 90th base.

[Experiment Example 2] “Citrus variety identification test using IND131”
By conducting allele polymorphism analysis regarding the indel marker IND131 selected through the above test, we examined whether it was possible to distinguish the variety "Mihaya" from the major citrus varieties.

(1) “Allelic polymorphism analysis regarding IND131”
Total DNA was extracted from citrus varieties (samples 1 to 47) including the variety "Mihaya", and a primer set of oligonucleotide primers (SEQ ID NOs: 3 and 4) designed to detect allelic polymorphisms of IND131 was used. A PCR reaction was performed using the DNA fragment, and electrophoresis was performed to detect the size of the amplified DNA fragment. The methods and conditions for DNA extraction, PCR reaction, DNA detection, etc. were performed according to the above description. The primer design position for IND131 in this example was designed at a position sandwiching the indel marker gene locus, as shown in FIG. 5 (FIGS. 3 and 5).
Specifically, the upstream primer of the IND131 amplification primer set in this example is a 22-base oligonucleotide primer (IND131 detection common region primer 1) consisting of the base sequence shown in SEQ ID NO: 3. The primer is a forward primer and corresponds to the base sequence from the 1st base to the 22nd base of SEQ ID NO: 2, which indicates the genomic region including IND131 of the variety "Mihaya". Further, the downstream primer that forms a primer pair with the primer is a 20-base oligonucleotide primer (IND131 detection common region primer 2) consisting of the base sequence shown in SEQ ID NO: 4. The primer is a reverse primer and corresponds to the complementary sequence of the base sequence from the 501st base to the 520th base of SEQ ID NO: 2, which indicates the genomic region containing IND131 of the variety "Mihaya". .
Here, when performing PCR amplification using the primer set, if an indel marker insertion type sequence exists in the genome, a long DNA fragment containing the insertion sequence (520 bp corresponding to a long DNA fragment or something similar to this) It is expected that bands of similar base length) will be amplified. On the other hand, if an indel marker deletion type sequence exists in the genome, a short DNA fragment with a length corresponding to the inserted sequence is deleted (287 bp corresponding to the short DNA fragment or a band with a similar base length) ) is expected to increase.
Here, when PCR amplification was performed on the total DNA extracted from the standard variety sweet orange using the primer set, two bands (insertion type sequence/deletion type sequence) corresponding to long chain DNA fragment and short chain DNA fragment were detected. It was confirmed that a band (indicating a heterozygous type sequence) was amplified.

The results of PCR amplification regarding IND131 for the above-mentioned citrus varieties (samples 1 to 47) are shown in the table below and FIG. 7. As a result, PCR fragments corresponding to the IND131 deletion type sequence were amplified in all of the 47 citrus varieties investigated, except for the variety ``Mihaya'', and PCR fragments containing short DNA fragments were amplified. It showed an amplified band pattern. That is, all citrus varieties other than the variety "Mihaya" were shown to be alleles containing the IND131 deletion type sequence.
On the other hand, for the variety "Mihaya" (sample 11), only the PCR fragment of the IND131 insertion type sequence was amplified without the PCR fragment of the IND131 deletion type sequence being amplified, and the PCR amplification resulted in only the long DNA fragment being amplified. A band pattern was shown. That is, the variety "Mihaya" was shown to be an allele containing the IND131 insertion type sequence in a homozygous form.

(2) “Identification of the variety “Mihaya””
As shown in the results of the allelic polymorphism analysis for IND131 mentioned above, among the investigated citrus cultivars (samples 1 to 47), the citrus cultivars in which IND131 showed homozygous allele type of the insertion type sequence were the cultivars “Miha It was shown that only "Ya" (Sample 11) was present (Tables 1 to 4, FIG. 7). Furthermore, among the other citrus varieties investigated, no varieties were found that showed the same allelic polymorphism as the variety "Mihaya."
Here, a total of 47 types of citrus varieties, which are the citrus varieties (samples 1 to 47) investigated in this example, are citrus varieties that account for 98% of the citrus fruits distributed in Japan at the time of filing of this application. In this regard, the variety identification technology stipulated in the ISO 13495 international standard is based on technology that can identify 90% or more of the distribution volume, and the survey results of this example demonstrate that the technology in question satisfies the international standard. It was recognized that the results showed that
Based on the above results and findings, if polymorphism analysis of alleles related to IND131 shows an allele in which the IND131 locus has an insertion type sequence homologous on the homologous chromosome, with an accuracy that satisfies the ISO13495 international standard, It was shown that the citrus variety to be identified can be determined to fall under the variety "Mihaya."

[Experiment Example 3] “Citrus variety identification test using IND44”
By conducting allele polymorphism analysis regarding the indel marker IND44 selected through the above test, we investigated whether it is possible to distinguish the variety "Mihaya" from the major citrus varieties.

(1) “Allelic polymorphism analysis regarding IND44”
Using a primer set of oligonucleotide primers (SEQ ID NOs: 9 and 10) designed to be able to detect the allelic polymorphism of IND44, the total DNA of the citrus varieties (samples 1 to 47) prepared in Experimental Example 2 was used. A PCR reaction was performed, and electrophoresis was performed to detect the size of the amplified DNA fragment. The methods and conditions for DNA extraction, PCR reaction, DNA detection, etc. were performed according to the above description. The primer design position for IND44 in this example was designed at a position sandwiching the indel marker gene locus, as shown in FIG. 5 (FIGS. 4 and 5).
Specifically, the upstream primer of the IND44 amplification primer set in this example is a 19-base oligonucleotide primer (IND44 detection common region primer 1) consisting of the base sequence shown in SEQ ID NO: 9. The primer is a forward primer and corresponds to the base sequence from the 1st base to the 19th base of SEQ ID NO: 8, which indicates the genomic region including IND44 of the variety "Mihaya". Further, the downstream primer forming a primer pair with the primer is a 21-base oligonucleotide primer (IND44 detection common region primer 2) consisting of the base sequence shown in SEQ ID NO: 10. The primer is a reverse primer and corresponds to the complementary sequence of the base sequence from the 181st base to the 201st base of SEQ ID NO: 8, which indicates the genomic region containing IND44 of the variety "Mihaya". .
Here, when performing PCR amplification using the primer set, if an indel marker insertion type sequence exists in the genome, it is expected that DNA fragments containing insertion sequences of three different lengths will be amplified. Ru. Specifically, if insert sequence A exists, a band with a base length of 774 bp or the same base length, and if insert sequence B exists, a band with a base length of 450 bp or the same base length, and insert sequence C. In the presence of , bands of 300 bp or the same base length are expected to be amplified.
On the other hand, when an indel marker deletion type sequence is present in the genome, it is expected that a band of 201 bp or a base length equivalent to this, in which the length corresponding to the inserted sequence is deleted, will be amplified.
Here, when PCR amplification was performed on the total DNA extracted from the standard variety sweet orange using the primer set, two bands corresponding to the PCR amplified fragments of insert sequence A and insert sequence C (insert sequence A/ It was confirmed that a band indicating a heterozygous type of inserted sequence C) was amplified.

The results of PCR amplification regarding IND44 for the above-mentioned citrus varieties (samples 1 to 47) are shown in the table below and FIG. 8. As a result, 36 of the 47 citrus varieties investigated showed PCR amplified band patterns containing PCR fragments corresponding to either or two of the insertion sequences A to C of IND44. Ta. In other words, these citrus varieties were shown to be allelic types that do not contain the IND44 deletion type sequence.
On the other hand, 11 types of citrus varieties, including the variety "Mihaya" (sample 11), showed PCR amplification band patterns containing PCR fragments of the IND44 deletion type sequence. In other words, these 11 citrus varieties were shown to be allelic types containing the IND44 deletion type sequence. The more detailed allele type of IND44 exhibited by the variety "Mihaya" was an allele type having a deletion type sequence and an insertion type sequence C of IND44 in a heterozygous form.

(2) “Identification of the variety “Mihaya””
As shown in the above results of allelic polymorphism analysis regarding IND44, among the investigated citrus cultivars (samples 1 to 47), the citrus cultivars in which IND44 showed an allele type containing the deletion type sequence were the cultivars “Mihaya ” (Sample 11) (Tables 1 to 4, Figure 8). This result corresponded to 23.4% of the main varieties investigated. In this regard, although it is not possible to completely distinguish the Mihaya cultivar from other citrus cultivars only by performing allele polymorphism analysis of IND44, it is possible to exclude 76.6% of the main citrus cultivars surveyed. It has been shown.
This result was recognized as a result indicating that the identification accuracy of the variety "Mihaya" can be further improved when allele polymorphism analysis regarding IND44 is further performed in addition to allele polymorphism analysis regarding IND131.

[Organization of experimental results] “Results of experimental examples 2 and 3”
The results of the allele polymorphism analysis of Experimental Examples 2 and 3 described above are summarized and shown in Tables 1 to 4 below. In addition, a PCR reaction was performed on total DNA from the 47 citrus varieties investigated in this example using a primer set (SEQ ID NO: 13, 14) for PCR amplification of the rbcL gene as a positive control. However, PCR amplified fragments were detected in all the samples, and it was confirmed that there were no problems with the prepared DNA samples.

[Experiment Example 4] “Simplification of target PCR fragment amplification by improving primer design”
Regarding the above-mentioned homozygous allelic polymorphism of the IND131 insertion type sequence and allelic polymorphism including the IND44 deletion type sequence, we designed PCR primers that can be more easily identified by PCR amplified bands, and An identification test was conducted.

(1) “Detection of homozygous allele polymorphism of IND131 insertion type sequence”
In order to stably perform PCR amplification of the IND131 insertion type sequence and to more easily detect homozygous allele polymorphisms, even if the IND131 insertion type sequence is amplified at a position sandwiching the IND131 gene locus, A primer set was designed at a position where the amplified fragment length was 500 bp or less (Figures 3 and 5).
Specifically, the upstream primer of the IND131 amplification primer set in this example is a 25-base oligonucleotide primer (IND131 detection common region primer 3) consisting of the base sequence shown in SEQ ID NO: 5. The primer is a forward primer and corresponds to the base sequence from the 30th base to the 54th base of SEQ ID NO: 2, which indicates the genomic region including IND131 of the variety "Mihaya". Further, the downstream primer that forms a primer pair with the primer is a 26-base oligonucleotide primer (IND131 detection common region primer 4) consisting of the base sequence shown in SEQ ID NO: 6. The primer is a reverse primer and corresponds to the complementary sequence of the base sequence from the 481st base to the 506th base of SEQ ID NO: 2, which indicates the genomic region containing IND131 of the variety "Mihaya". .

A PCR reaction was performed using the above primer set using the total DNA of 24 types of citrus varieties shown in Samples 1 to 24 prepared in Experimental Example 2 as a template, and electrophoresis was performed to detect the amplified DNA fragments. The methods and conditions for DNA extraction, PCR reaction, DNA detection, etc. were performed according to the above description. The results are shown in Figure 9A.
The results showed that one 477 bp PCR amplified band was detected from the cultivar "Mihaya" (sample 11), which showed a homozygous type of the IND131 insertion type sequence in Experimental Example 2. On the other hand, PCR amplification patterns similar to those of the variety "Mihaya" were not detected from other citrus varieties other than the variety "Mihaya." This result was consistent with the polymorphism analysis results for Samples 1 to 24 of Experimental Example 2.
The above results showed that it is possible to design a primer set that sandwiches the IND131 gene locus at a position where the PCR amplification length of the IND131 insertion type sequence is 500 bp or less. This result was recognized as a result showing that when PCR amplification is performed using the primers, PCR amplification can be performed more easily even from a sample containing impurities.

(2) “Detection of allelic polymorphism including IND44 deletion type sequence”
In order to more easily detect allelic polymorphisms containing the IND44 deletion type sequence, bases that connect the fragmented common sequence to the insertion sequence are used so that PCR amplification occurs only when the IND44 deletion type sequence is present. An IND44 deletion type sequence-specific primer consisting of the sequence was designed (FIG. 4, FIG. 6).
The IND44 deletion type sequence-specific primer is a 22-base oligonucleotide primer consisting of the base sequence shown in SEQ ID NO: 11. The primer is a forward primer, and corresponds to the base sequence from the 71st base to the 92nd base of SEQ ID NO: 8, which indicates the genomic region containing the IND44 deletion type sequence of the variety "Mihaya". be. Among the bases constituting the primer, the second and third bases upstream from the 3' end base correspond to the two bases indicating the locus of the IND44 deletion type sequence.
Further, the downstream primer forming a primer pair with the primer is a 26-base oligonucleotide primer (IND44 detection common region primer 3) consisting of the base sequence shown in SEQ ID NO: 12. The primer is a reverse primer and corresponds to the complementary sequence of the base sequence from the 171st base to the 196th base of SEQ ID NO: 8, which indicates the genomic region containing IND44 of the variety "Mihaya". .

A PCR reaction was performed using the above primer set using the total DNA of 24 types of citrus varieties shown in Samples 1 to 24 prepared in Experimental Example 2 as a template, and electrophoresis was performed to detect the amplified DNA fragments. The methods and conditions for DNA extraction, PCR reaction, DNA detection, etc. were performed according to the above description. The results are shown in Figure 9B.
As a result, an amplified band of 126 bp or equivalent base length was found from five citrus cultivars, including the cultivar "Mihaya" (sample 11), which showed an allele type containing the IND44 deletion type sequence in Experimental Example 3. was detected in one band. On the other hand, no band representing a PCR amplified fragment was detected from the 19 types of citrus varieties that showed allele types that did not contain the deletion type sequence of IND44 in Experimental Example 3. This result was consistent with the polymorphism analysis results for Samples 1 to 24 of Experimental Example 3.
From the above results, by using a primer containing two bases constituting the most upstream base and the most downstream base of the IND44 gene locus in the primer sequence, IND44 deletion can be achieved without amplifying the base sequence containing the IND44 insertion type sequence. It has been shown that only the base sequence containing the type sequence can be specifically amplified and detected as a single band.

If agricultural products or processed products of the citrus variety "Mihaya" that have been leaked overseas are exported to Japan, it is necessary to submit evidence that the products are suspected of infringing in order to request a border detention at customs. . The identification technology for the citrus variety "Mihaya" based on the present invention is expected to be implemented by public institutions that conduct inspections, private inspection companies, etc.
It is also used by businesses engaged in citrus cultivation, seed distribution, fruit distribution, fruit processing, etc. to accurately identify the Mihaya variety for seeds, seedlings, fruits, processed products, etc. It is expected that

1. Indel marker insertion type sequence 11. IND131 insertion type sequence and its surrounding region 21. IND44 insertion type sequence and its surrounding region

2. Indel marker deletion type sequence 12. IND131 deletion type sequence and its surrounding region 22. IND44 deletion type sequence and its surrounding region

41. Cell wall-associated receptor kinase gene 51. Downstream region of cell wall-associated receptor kinase gene

42. RALF-like peptide precursor gene 52. Downstream region of RALF-like peptide precursor gene

3. Indel marker locus (region containing the insertion sequence in case of insertion type)
31. Most upstream base of indel marker locus 32. Most downstream base of indel marker locus 33. Insertion sequence 301. IND131 locus 302. IND44 locus

61. Upstream common sequence near indel marker 62. Downstream common sequence near indel marker

71. Common region primer for indel marker detection 72. Common region primer for indel marker detection 73. Indel marker deletion type sequence specific primer

101. Cross section of citrus fruit 102. Endocarp 103. exocarp

Claims (6)

A method for identifying citrus varieties using indel markers of citrus plants to be identified, characterized by comprising the steps described in (1), (1'), and (2) below: Citrus variety identification method to identify citrus variety “Mihaya”:
(1) detecting an IND131 insertion type sequence regarding the indel marker IND131 from the genomic DNA of the citrus plant to be identified;
(1′) detecting an IND44 deletion type sequence regarding the indel marker IND44 from the genomic DNA of the citrus plant to be identified;
(2) In the step described in (1) above, an IND131 deletion type sequence is not detected but an IND131 insertion type sequence is detected, and in the step described in (1') above, an IND44 deletion type sequence is detected. a step of determining that the variety of the citrus plant to be identified is "Mihaya"when
;
Regarding the indel marker IND131 of the citrus plant genome described in (1) above,
(1a) When shown in the genome of sweet orange (Citrus sinensis), a standard variety of citrus, this is the locus of the indel marker present in the 3'UTR of the cell wall-related receptor kinase gene on chromosome 1; When shown by the base sequence described in SEQ ID NO: 1, which includes the sweet orange IND131 deletion type sequence, the 5527th base "A" indicates the most upstream base of the IND131 gene locus, and the 5528th base "G" indicates the most downstream base of the IND131 locus,
(1b) Regarding the IND131 locus of the citrus plant genome to be identified, in the case of a base sequence in which there is an inserted sequence between the most upstream base "A" and the most downstream base "G" of the IND131 gene locus, IND131 is inserted. In the case of a base sequence in which there is no inserted sequence between the most upstream base "A" and the most downstream base "G" of the IND131 locus, it indicates an IND131 deletion type sequence.
;
Regarding the indel marker IND44 of the citrus plant genome described in (1') above,
(1'a) When shown in the genome of sweet orange (Citrus sinensis), a standard variety of citrus, it is an indel marker locus present in the downstream region of the RALF-like peptide precursor gene on chromosome 1, When shown by the base sequence described in SEQ ID NO: 7, which includes the IND44 insertion type sequence of the sweet orange, the 4175th base "A" indicates the most upstream base of the IND44 gene locus, and the 4749th base "A" indicates the most downstream base of the IND44 gene locus, the base sequence from the 4176th base to the 4748th base indicates the inserted sequence,
(1'b) Regarding the IND44 locus of the citrus plant genome to be identified, in the case of a base sequence in which an inserted sequence exists between the most upstream base "A" and the most downstream base "A" of the IND44 gene locus Indicates an IND44 insertion type sequence, and in the case of a base sequence in which there is no insertion sequence between the most upstream base "A" and the most downstream base "A" of the IND44 gene locus, indicates an IND44 deletion type sequence.
;
The step described in (1) above is a step comprising performing a PCR reaction on the genomic DNA of the citrus plant to be identified using a PCR primer set designed at positions sandwiching the IND131 gene locus,
The step described in (1') above includes performing a PCR reaction on the genomic DNA of the citrus plant to be identified using a PCR primer set containing an IND44 deletion type sequence-specific primer. be,
;
The PCR primer set designed at the position sandwiching the IND131 gene locus is
IND131 detection common region primer p1 (oligonucleotide primer consisting of the base sequence shown in SEQ ID NO: 5, or oligonucleotide whose base sequence on the 3' end side of the primer constituent bases matches the base sequence shown in SEQ ID NO: 5) nucleotide primer),
as well as,
IND131 detection common region primer p2 (oligonucleotide primer consisting of the base sequence shown in SEQ ID NO: 6, or oligonucleotide whose base sequence on the 3' end side of the primer constituent bases matches the base sequence shown in SEQ ID NO: 6) nucleotide primer),
A PCR primer set consisting of
;
A PCR primer set containing the IND44 deletion type sequence-specific primer,
IND44 deletion type sequence-specific primer p13 (an oligonucleotide primer consisting of the base sequence shown in SEQ ID NO: 11, or a base sequence in which the base sequence on the 3' end side of the primer constituent bases matches the base sequence shown in SEQ ID NO: 11) oligonucleotide primers),
as well as,
IND44 detection common region primer p14 (an oligonucleotide primer consisting of the base sequence shown in SEQ ID NO: 12, or an oligonucleotide whose base sequence on the 3' end side of the primer constituent bases matches the base sequence shown in SEQ ID NO: 12) nucleotide primer),
This is a PCR primer set consisting of. The PCR reaction in the step of detecting the IND131 insertion type sequence described in (1) above and the PCR reaction in the step of detecting the IND44 deletion type sequence described in (1') above are performed on the citrus plant to be identified. A PCR reaction using a plant, a processed product of the plant, or DNA extracted from the plant or processed product as a target sample,
;
The processed product of the plant is a frozen powdered product of the exocarp of a fruit of a citrus plant.
;
A citrus variety identification method for identifying the citrus variety "Mihaya" according to claim 1. Using the citrus variety identification method for identifying the citrus variety "Mihaya" according to claim 1 or 2,
A method for cultivating citrus cultivar "Mihaya" plants . A citrus variety identification kit for use in the citrus variety identification method for identifying the citrus variety "Mihaya" according to claim 1, comprising:
;
The kit is
A kit comprising as constituent articles a PCR primer set designed to sandwich the IND131 gene locus according to claim 1, and a PCR primer set comprising the IND44 deletion type sequence-specific primer according to claim 1.
;
Citrus variety identification kit for identifying the citrus variety "Mihaya". Using the citrus variety identification method for identifying the citrus variety "Mihaya" according to claim 1 or 2,
A method for producing fruit of the citrus variety "Mihaya". Using the citrus variety identification method for identifying the citrus variety "Mihaya" according to claim 1 or 2,
A method for producing processed products from the fruit of the citrus variety "Mihaya."

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