Citrus plants, methods for producing citrus plants, and uses thereof

Abstract

Provided are citrus plants, a method for producing citrus plants, and uses thereof. The present invention provides a method for producing citrus plants, which comprises the steps of: identifying genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 in a hybrid variety obtained by crossing citrus varieties with each other; or a progeny plant thereof; 2 is a method for producing a citrus plant, the method comprising a step of selecting plants having the following genotypes: Bf0158-3 is AB type, Bf0036-3 is AA type, Tf0300-3 is AB type, Tf0419-2 is AB type, Tf0420-2 is AA type, Al0302-2 is AB type, Mf0097-2 is AB type, Mf0090-2 is AA type, and Gn0073-2 is AA type.

Description

[Technical Field]

[0001] The present invention relates to citrus plants, methods for producing citrus plants, and uses thereof. [Background technology]

[0002] Seedlessness is essential for citrus fruits, as it contributes to their palatability, but stable seedless fruit production requires high levels of female sterility. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] DNA variety identification technology for 24 citrus varieties using CAPS markers, National Agriculture and Food Research Organization, published June 17, 2022 Summary of the Invention [Problem to be solved by the invention]

[0004] The National Agriculture and Food Research Organization (NARO) has focused on the female sterility derived from seedless Kishu and has promoted breeding, developing the citrus intermediate parental lines No. 5 and No. 6. However, these have not yet been widely cultivated commercially because they are small fruits weighing less than 100g. Therefore, there is a need to develop a mid-late maturing citrus variety that has the female sterility derived from seedless Kishu, is capable of stable seedless fruit production, and can produce relatively large, high-quality fruit. [Means for solving the problem]

[0005] A method for producing a citrus plant according to one aspect of the present invention includes: In hybrid varieties obtained by crossing citrus varieties or their progeny plants, genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 are shown below, respectively. Bf0158-3 is type AB, Bf0036-3 is the AA type, Tf0150-2 is type AB, Tf0271-2 is type AB, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type AB, Mf0090-2 is type AA, Gn0073-2 is AA type, and Gn0048-2 is type AA The method includes a step of selecting a plant having the genotype. Furthermore, a method for producing a citrus plant according to one aspect of the present invention is a method for producing a citrus plant, comprising: The hybridization process involves crossbreeding the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father. and a marker selection step of using at least one genetic marker selected from the citrus plants obtained by the hybridization step or their progeny to select candidate citrus plants that have different genotype patterns of the genetic markers from the parent variety "Harehime" and "Citrus intermediate parent No. 6."

[0006] Furthermore, in one aspect of the present invention, there is provided a citrus plant, wherein the genotype of the genetic marker in the citrus plant is: Bf0158-3 is type AB, Bf0036-3 is the AA type, Tf0150-2 is type AB, Tf0271-2 is type AB, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type AB, Mf0090-2 is type AA, Gn0073-2 is AA type, and Gn0048-2 is an AA type citrus plant. Furthermore, a citrus plant according to one aspect of the present invention is a citrus plant having the following traits (1), (2), (3), and (4): (1) It was obtained by crossbreeding the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father. (1) Seedless fruit production is possible, (2) Fruit weight is 160g or more, (3) The fruit has a high sugar content and good taste, and (4) It is distinguishable from other citrus cultivars, including "Harehime" and "Citrus Intermediate Parent No. 6," using at least one of the following genetic markers: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0007] A method for identifying a citrus plant according to one aspect of the present invention is a method for identifying a citrus plant, comprising: amplifying a region of the DNA of a citrus plant containing a genetic marker (CAPS marker) of the DNA using a primer set; and identifying the citrus plant based on base polymorphisms in the amplified DNA region, wherein the genetic markers (CAPS markers) are at least one of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0008] A citrus plant or a fruit thereof according to another aspect of the present invention is a citrus plant or a fruit thereof obtained by the method for producing a citrus plant.

[0009] A processed product according to one aspect of the present invention is a processed product produced using the citrus plant or its fruit. Furthermore, a fruit according to one aspect of the present invention is a fruit of the citrus plant. A processed product according to one aspect of the present invention is obtained from the citrus plant or the fruit of the citrus plant. [Effects of the Invention]

[0010] According to the present invention, a novel citrus plant, a method for producing a citrus plant, and uses thereof can be provided. [Brief explanation of the drawings]

[0011] [Figure 1] This is the pedigree diagram of "Citrus OK68". [Figure 2] This is a photo of the fruit of "Citrus OK68". [Figure 3] This is a photo of the tree "Citrus OK68". [Figure 4]FIG. 1 shows the genotype of CAPS markers in "Citrus OK68." [Figure 5] DNA extracted from "Citrus OK68" was PCR-amplified using the genetic markers (CAPS markers) Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, and Tf0419-2, digested with specific restriction enzymes, and then analyzed by agarose gel electrophoresis to determine the genotype of each genetic marker. In the figure, M indicates the electrophoretic pattern of a 100-bp ladder, and S indicates the electrophoretic pattern of DNA from "Citrus OK68" after restriction enzyme digestion. [Figure 6] DNA extracted from "Citrus OK68" was PCR-amplified using the genetic markers Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, or Mf0090-2, digested with specific restriction enzymes, and then analyzed by agarose gel electrophoresis to determine the genotype of each marker. In the figure, M indicates the electrophoretic pattern of a 100-bp ladder, and S indicates the electrophoretic pattern of DNA from "Citrus OK68" after restriction enzyme digestion. [Figure 7] DNA extracted from "Citrus OK68" was PCR-amplified using the genetic markers Gn0073-2 or Gn0048-2, digested with specific restriction enzymes, and then analyzed by agarose gel electrophoresis to determine the genotype of each genetic marker. In the figure, M indicates the electrophoretic pattern of a 100-bp ladder, and S indicates the electrophoretic pattern of DNA from "Citrus OK68" after restriction enzyme digestion. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below. All of the documents mentioned in this specification are incorporated herein by reference.

[0013] In this specification, the term "citrus plants" refers to plants of the genus Citrus L. in the family Rutaceae. "Citrus" is a general term for plants belonging to the genera Citrus (Citrus), Fortunella, and Poncirus in the subfamily Rutaceae of the family Rutaceae.

[0014] As used herein, the term "plant" may refer to a part or the whole of a plant body, and may refer to any stage of plant development.

[0015] Examples of plant parts include growing shoots, lateral buds, growing branches, branches, leaves, petioles, thorns, stems, roots, stalks, leaves, flowers, flower buds, petals, pollen, anthers, fruits, seeds, etc. Also included are the exocarp, endocarp, and flesh that make up fruits. Plant parts include cells or tissues of the plant, such as embryos, hypocotyls, meristematic cells, callus, and protoplasts obtained from cells of the plant.

[0016] As used herein, the term "traits" refers to the morphological and physiological characteristics of a plant. As used herein, CAPS means cleaved amplified polymorphic sequence (CAPS).

[0017] [Citrus plant production method 1] A method for producing a citrus plant according to one aspect of the present invention is a method for producing a citrus plant, comprising: In hybrid varieties obtained by crossing citrus varieties or their progeny plants, genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 are shown below, respectively. Bf0158-3 is type AB, Bf0036-3 is the AA type, Tf0150-2 is type AB, Tf0271-2 is type AB, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type AB, Mf0090-2 is type AA, Gn0073-2 is AA type, and Gn0048-2 is type AA The method for producing a citrus plant includes a step of selecting a plant having the genotype.

[0018] In one embodiment, the production method may further include a step of crossing the citrus cultivar "Harehime" as the mother and the citrus cultivar "Civil Fruit Intermediate Parent No. 6" as the father to obtain the hybrid cultivar. In one embodiment, the plant used in the selection process may be a hybrid variety obtained by crossing citrus varieties with each other, or a progeny plant thereof, but is preferably a hybrid variety obtained by crossing citrus varieties with each other. In one embodiment, the production method may further comprise a propagation step in which the citrus plant is vegetatively propagated.

[0019] (Marker-based selection process (identification process)) In one embodiment, the production method involves selecting hybrid varieties or their progeny plants obtained by crossing citrus varieties with each other using genetic markers (CAPS markers): Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0020] Variety identification using genetic markers can be performed by the method described in Non-Patent Document 1. The outline of the method is as follows. First, DNA is extracted using existing methods from a hybrid variety obtained by crossing citrus varieties with another citrus variety, or from a progeny plant. Next, PCR amplification of the extracted DNA is performed using pairs of forward and reverse primers corresponding to each genetic marker listed in the table below. The amplified DNA is cleaved with each restriction enzyme listed in the table below. The DNA after restriction enzyme treatment is subjected to agarose gel electrophoresis, and the electrophoresis pattern is confirmed. The genotype of each genetic marker can be determined based on differences in the electrophoresis pattern. This makes it possible to identify citrus varieties. Therefore, the production method may further include a step of extracting DNA from a hybrid variety obtained by crossing citrus varieties with each other, or from a progeny plant thereof. The production method may further include a step of amplifying the extracted DNA using a pair of forward and reverse primers corresponding to each genetic marker. The production method may further include a step of treating the amplified DNA with a restriction enzyme. The production method may further include a step of electrophoresing the DNA after the restriction enzyme treatment. The production method may further include a step of determining the genotype for each genetic marker from the electrophoresis pattern of DNA obtained by electrophoresis. The production method may further include a step of selecting a plant having the above-described genotype for each genetic marker based on the results of determining the genotype for each genetic marker.

[0021] [Table 1] TIFF0007756987000003.tif167155

[0022] The genotype of Bf0158-3 can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected near 352(±10)bp and no bands are detected near 149(±10)bp and 203(±10)bp. Type BB is when bands are detected near 149(±10)bp and 203(±10)bp and no bands are detected near 352(±10)bp. Type AB is when bands are detected near 149(±10)bp, 203(±10)bp, and 352(±10)bp.

[0023] The genotype of Bf0036-3 can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. If a band can be confirmed around 399 (±10) bp but no bands can be confirmed around 162 (±10) bp and 237 (±10) bp, it is called type AA. If bands can be confirmed around 162 (±10) bp and 237 (±10) bp but no bands can be confirmed around 399 (±10) bp, it is called type BB. If bands can be confirmed around 162 (±10) bp, 237 (±10) bp, and 399 (±10) bp, it is called type AB.

[0024] The Tf0150-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected near 385 (±10) bp and no bands are detected near 152 (±10) bp and 233 (±10) bp. Type BB is when bands are detected near 152 (±10) bp and 233 (±10) bp and no bands are detected near 385 (±10) bp. Type AB is when bands are detected near 152 (±10) bp, 233 (±10) bp, and 385 (±10) bp.

[0025] The Tf0271-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. AA type is when a band is detected around 400(±10)bp and no bands are detected around 157(±10)bp and 243(±10)bp. BB type is when bands are detected around 157(±10)bp and 243(±10)bp and no bands are detected around 400(±10)bp. AB type is when bands are detected around 157(±10)bp, 243(±10)bp, and 400(±10)bp.

[0026] The Tf0300-3 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected near 393 (±10) bp and no bands are detected near 166 (±10) bp and 227 (±10) bp. Type BB is when bands are detected near 166 (±10) bp and 227 (±10) bp and no bands are detected near 393 (±10) bp. Type AB is when bands are detected near 166 (±10) bp, 227 (±10) bp, and 393 (±10) bp.

[0027] The Tf0419-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected near 389 (±10) bp and no bands are detected near 167 (±10) bp and 222 (±10) bp. Type BB is when bands are detected near 167 (±10) bp and 222 (±10) bp and no bands are detected near 389 (±10) bp. Type AB is when bands are detected near 167 (±10) bp, 222 (±10) bp, and 389 (±10) bp.

[0028] The Tf0420-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected near 388(±10)bp and no bands are detected near 162(±10)bp and 226(±10)bp. Type BB is when bands are detected near 162(±10)bp and 226(±10)bp and no bands are detected near 388(±10)bp. Type AB is when bands are detected near 162(±10)bp, 226(±10)bp, and 388(±10)bp.

[0029] The Tf0318-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected around 400 (±10) bp and no bands are detected around 135 (±10) bp and 265 (±10) bp. Type BB is when bands are detected around 135 (±10) bp and 265 (±10) bp and no bands are detected around 400 (±10) bp. Type AB is when bands are detected around 135 (±10) bp, 265 (±10) bp, and 400 (±10) bp.

[0030] The Tf0293-4 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected near 394(±10)bp and no bands are detected near 157(±10)bp and 237(±10)bp. Type BB is when bands are detected near 157(±10)bp and 237(±10)bp and no bands are detected near 394(±10)bp. Type AB is when bands are detected near 157(±10)bp, 237(±10)bp, and 394(±10)bp.

[0031] The genotype of Al0302-2 can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. If a band can be confirmed around 383 (±10) bp but no bands can be confirmed around 167 (±10) bp and 216 (±10) bp, it is called AA type. If bands can be confirmed around 167 (±10) bp and 216 (±10) bp but no bands can be confirmed around 383 (±10) bp, it is called BB type. If bands can be confirmed around 167 (±10) bp, 216 (±10) bp, and 383 (±10) bp, it is called AB type.

[0032] The genotype of Mf0097-2 can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. If a band can be confirmed around 369 (±10) bp but no bands can be confirmed around 166 (±10) bp and 203 (±10) bp, it is called type AA. If bands can be confirmed around 166 (±10) bp and 203 (±10) bp but no bands can be confirmed around 369 (±10) bp, it is called type BB. If bands can be confirmed around 166 (±10) bp, 203 (±10) bp, and 369 (±10) bp, it is called type AB.

[0033] The Mf0090-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected around 400(±10)bp and no bands are detected around 139(±10)bp and 261(±10)bp. Type BB is when bands are detected around 139(±10)bp and 261(±10)bp and no bands are detected around 400(±10)bp. Type AB is when bands are detected around 139(±10)bp, 261(±10)bp, and 400(±10)bp.

[0034] The genotype of Gn0073-2 can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band can be confirmed around 394(±10)bp and no bands can be confirmed around 149(±10)bp and 245(±10)bp. Type BB is when bands can be confirmed around 149(±10)bp and 245(±10)bp and no bands can be confirmed around 394(±10)bp. Type AB is when bands can be confirmed around 149(±10)bp, 245(±10)bp, and 394(±10)bp.

[0035] The genotype of Gn0048-2 can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is observed around 380(±10)bp and no bands are observed around 134(±10)bp and 246(±10)bp. Type BB is when bands are observed around 134(±10)bp and 246(±10)bp and no bands are observed around 380(±10)bp. Type AB is when bands are observed around 134(±10)bp, 246(±10)bp, and 380(±10)bp.

[0036] In one embodiment, the method of making comprises: Bf0158-3 is type AB, Bf0036-3 is the AA type, Tf0150-2 is type AB, Tf0271-2 is type AB, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type AB, Mf0090-2 is type AA, Gn0073-2 is AA type, and Gn0048-2 is type AA The method includes a step of selecting a citrus plant having the genotype.

[0037] In one embodiment, the production method may further include a step of crossing the citrus cultivar "Harehime" as the mother and the citrus cultivar "Civil Fruit Intermediate Parent No. 6" as the father to obtain the hybrid cultivar. The parent varieties used in the crossbreeding, "Harehime" and "Citrus Intermediate Parent No. 6", are both varieties distributed on the market. "Harehime" is a variety with good taste and easy peeling, and is registered as Japanese Variety Registration Number: 12069. "Citrus Intermediate Parent No. 6" has female sterility derived from seedless Kishu, and is a variety capable of stable seedless fruit production with excellent taste, and was developed by the National Agriculture and Food Research Organization. "Citrus Intermediate Parent No. 6" is registered as Japanese Variety Registration Number: 12070. The citrus plants selected in the selection process have the genotype pattern of the above genetic markers. In contrast, in the other citrus cultivar "Harehime," Bf0036-3 is AB type, Tf0300-3 is BB type, Tf0419-2 is BB type, Mf0090-2 is AB type, and Gn0073-2 is AB type. Therefore, "Harehime" can be distinguished from other citrus cultivars using Bf0036-3, Tf0300-3, Tf0419-2, Mf0090-2, and / or Gn0073-2. In addition, in another citrus cultivar, "Citrus Intermediate Parent No. 6," Bf0158-3 is BB type, Bf0036-3 is AB type, Tf0420-2 is AB type, Al0302-2 is AA type, Mf0097-2 is BB type, and Gn0073-2 is AB type. Therefore, Bf0158-3, Bf0036-3, Tf0420-2, Al0302-2, Mf0097-2, and / or Gn0073-2 can be used to distinguish from other citrus cultivars, "Citrus Intermediate Parent No. 6." Furthermore, Bf0036-3 and / or Gn0073-2 can be used to distinguish it from both another citrus cultivar, "Harehime," and another citrus cultivar, "Citrus Intermediate Parent No. 6."

[0038] (Estimation of parent lineage) The genotypes listed in Tables 2 to 5 below represent the combination of alleles (allelic genes) detected for each variety at each genetic marker. Because citrus is typically a diploid plant, the two alleles found in the offspring must be inherited, one from the seed parent and one from the pollen parent. For example, if a variety has the AB genotype, it could be a breeding parent if either parent has an A or B allele, but if both parents are BB, this combination is unlikely to be a parent. Based on this principle, potential breeding parents can be estimated from information on 14 types of genetic markers. Citrus plants having the above genotypes selected through the selection process using the above genetic markers have different genotypes of genetic markers from other citrus varieties, as is clear from the following Tables 2 to 5. Therefore, citrus plants selected through the above selection process can be produced in a manner that distinguishes them from other citrus varieties.

[0039] [Table 2]

[0040] [Table 3]

[0041] [Table 4]

[0042] [Table 5]

[0043] In one embodiment, the method comprises the steps of selecting citrus plants having the trait of producing seedless fruit and / or selecting citrus plants having the trait of fruit weighing an average of 130g or more (preferably an average of 140g or more, more preferably an average of 150g or more, even more preferably an average of 160g or more, and even more preferably an average of 170g or more). Preferably, the method comprises the steps of selecting citrus plants having the trait of producing seedless fruit and selecting citrus plants having the trait of fruit weighing an average of 130g or more (preferably an average of 140g or more, more preferably an average of 150g or more, even more preferably an average of 160g or more, and even more preferably an average of 170g or more). "Harehime" is a seedless variety that utilizes male sterility, but seeds may be formed when pollinated with pollen from other varieties, limiting the stable production of seedless fruits. In contrast, the citrus plants of the present invention have female sterility, and will not form seeds even when pollinated with pollen from other varieties, making it possible to stably produce seedless fruits. Furthermore, "Nori No. 6" has small fruit weighing approximately 120g, while "Sakimeki" has slightly larger fruit weighing approximately 180g, making it a distinctly differentiable trait in terms of fruit size. Therefore, it can be further distinguished from "Harehime" and / or "Nori No. 6" by its traits of producing seedless fruit and large fruit weight.

[0044] In one embodiment, the production method may further include a preliminary selection step prior to the selection step, in which candidate citrus plants having different genotype patterns of the genetic markers from the breeding parents "Harehime" and "Citrus Intermediate Parent Nori No. 6" are selected by using at least one genetic marker (CAPS marker) selected from the genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0045] In the preliminary selection step, the genotype patterns of all 14 CAPS markers may be determined, but depending on the purpose of selection, only some of the 14 CAPS markers may be used. In one example, in the marker selection step, Bf0036-3 and / or Gn0073-2 are used to select candidate citrus plant lines that have genotype patterns of the genetic markers that differ from those of other citrus cultivars, "Harehime" and "Civil Intermediate Parent No. 6." In one embodiment, in the marker selection step, a combination of at least one genetic marker selected from Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2 with at least one genetic marker selected from Tf0300-3, Tf0419-2, and Mf0090-2, and at least one genetic marker selected from Bf0036-3 and Gn0073-2 is used to select candidate citrus plant lines that have different genotype patterns of the genetic markers from the parent varieties "Harehime" and "Citrus Intermediate Mother No. 6." In one embodiment, in the preliminary selection step, at least one genetic marker selected from Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2 is used to select a candidate citrus plant line that has a different genotype pattern of the genetic marker from that of the parent variety "Civil Fruit Intermediate Parent No. 6", and at least one genetic marker selected from Tf0300-3, Tf0419-2, and Mf0090-2 is used to select a candidate citrus plant line that has a different genotype pattern of the genetic marker from that of "Harehime", or At least one selected from at least one of the genetic markers Bf0036-3 and Gn0073-2 may be used to select a candidate citrus plant lineage that has a different genotype pattern of the genetic markers from the parent varieties "Harehime" and "Citrus Intermediate Parent No. 6." In one embodiment, the production method may further include a step of crossbreeding the citrus cultivar "Harehime" as the mother (seed parent) and the citrus cultivar "Citrus Intermediate Mother No. 6" as the father (pollen parent) to obtain the hybrid cultivar, and a preliminary selection step of selecting a candidate citrus plant line having the genotypes of the genetic markers Bf0036-3 as AA and Gn0073-2 as AA. In one embodiment, the production method may further include a step of crossing the citrus cultivar "Harehime" as the mother (seed parent) with the citrus cultivar "Citrus Intermediate Mother No. 6" as the father (pollen parent) to obtain the hybrid cultivar, and a preliminary selection step of selecting a candidate citrus plant line in which the genotypes of the genetic markers are as follows: Bf0158-3 is AB, Tf0420-2 is AA, Al0302-2 is AB, Mf0097-2 is AB, Tf0300-3 is AB, Tf0419-2 is AB, and Mf0090-2 is AA.

[0046] (Trait-based selection process) In one embodiment, the production method may further include a trait-based selection step. The trait-based selection step may be performed before, after, or simultaneously with the genetic marker-based selection step, but is preferably performed after the genetic marker-based selection step. By performing trait-based selection after genetic marker-based selection, citrus plants that are different from other varieties and have good traits can be more reliably selected.

[0047] The main traits used for selection include, for example, the ability to produce seedless fruit, fruit weight of 160g or more, high sugar content and good taste of fruit, soft pericarp, high alternate bearing ability, and fruit ripening period from late February to early March. The main traits used for selection include the trait of producing seedless fruit and / or the trait of fruit weight being 130 g or more on average (preferably 140 g or more on average, more preferably 150 g or more on average, even more preferably 160 g or more on average, and even more preferably 170 g or more on average). Preferably, the traits are the trait of producing seedless fruit and the trait of fruit weight being 130 g or more on average (preferably 140 g or more on average, more preferably 150 g or more on average, even more preferably 160 g or more on average, and even more preferably 170 g or more on average). Details of the above traits are described in the section below, "Citrus Plants." It is also preferable to use the traits described in the section on citrus plants for selection.

[0048] The method for producing a citrus plant according to one embodiment of the present invention may further include, for example, the preliminary selection step, primary selection step, and secondary selection step described in Method 2 for producing a citrus plant.

[0049] (Proliferation process) The method for producing a citrus plant according to one aspect of the present invention may further include a propagation step of vegetatively propagating the citrus plant cultivated through the selection step.

[0050] Asexual propagation methods include grafting and cuttings. , take They can be plantlets or cell cultures. Asexual propagation is also called unsexual propagation, asexual reproduction, or vegetative propagation. Asexual propagation does not require males or females, and is a method in which a single individual independently forms a new individual, with the reproductive cells produced by that single individual independently becoming a new individual. Furthermore, techniques for creating cell cultures of plant tissues and methods for regenerating plants from tissue cultures are carried out using known techniques.

[0051] In the present invention, the grafting On the treeThe rootstocks that can be used include various tree species that can be used as citrus trees or citrus rootstocks, such as trifoliate orange, satsuma mandarin, and shikuwasa. The type and size of the rootstock can be changed depending on the soil, climate, etc. For example, trifoliate orange can be used as the rootstock.

[0052] The cultivation type of the citrus plant according to the present invention is not particularly limited, but may be in the form of a high-grafted plant, a seedling, etc. dew Examples include ground cultivation, unheated greenhouse cultivation, and roofed greenhouse cultivation.

[0053] [Citrus plant production method 2] A method for producing a citrus plant according to one aspect of the present invention is a method for producing a citrus plant, comprising: The hybridization process involves crossbreeding the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father. and a marker selection step of using at least one genetic marker selected from the citrus plants obtained by the hybridization step or their progeny to select candidate citrus plants that have different genotype patterns of the genetic markers from the parent variety "Harehime" and "Citrus intermediate parent No. 6."

[0054] The creation method can also be referred to as a manufacturing method, a growing method, or a production method.

[0055] (Hybridization process) In the crossbreeding process, "Harehime" is used as the mother (seed parent) and "Citrus intermediate parent No. 6" is used as the father (pollen parent).

[0056] The parent varieties used in the crossbreeding, "Harehime" and "Citrus Intermediate Parent No. 6", are both varieties distributed on the market. "Harehime" is a variety with good taste and easy peeling, and is registered as Japanese Variety Registration Number: 12069. "Citrus Intermediate Parent No. 6" has female sterility derived from seedless Kishu, and is a variety capable of stable seedless fruit production with excellent taste, and was developed by the National Agriculture and Food Research Organization. "Citrus Intermediate Parent No. 6" is registered as Japanese Variety Registration Number: 12070.

[0057] (Marker-based selection process (identification process)) In one embodiment, the marker-assisted selection step uses at least one genetic marker (CAPS marker) selected from the citrus plants obtained by the hybridization step or their progeny plants, Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2, to identify and select candidate citrus plants that have a different genotype pattern of the genetic marker from the parent cultivar "Harehime" and "Citric intermediate parent Nori No. 6."

[0058] The identification of varieties from other varieties, including parent varieties, in marker selection will be described in detail in the section below entitled "Method for identifying citrus plants."

[0059] The genotype patterns of all 14 CAPS markers may be determined, but depending on the purpose of selection, only a portion of the 14 CAPS markers may be used. In one example, in the marker selection step, a combination of at least one genetic marker selected from Bf0158-3, Bf0036-3, Tf0300-3, Tf0419-2, Tf0420-2, Al0302-2, Mf0097-2, Mf0090-2, and Gn0073-2 is used to select candidate citrus plant lines that have different genotype patterns of the genetic markers from the parent cultivar "Harehime" and "Citrus Intermediate Parent No. 6." In one embodiment, in the marker selection step, a combination of at least one genetic marker selected from Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2 with at least one genetic marker selected from Tf0300-3, Tf0419-2, and Mf0090-2, and at least one genetic marker selected from Bf0036-3 and Gn0073-2 is used to select candidate citrus plant lines that have different genotype patterns of the genetic markers from the parent varieties "Harehime" and "Citrus Intermediate Mother No. 6."

[0060] In one embodiment, in the marker-based selection step, at least one genetic marker selected from Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2 is used to select a candidate citrus plant line that has a different genotype pattern of the genetic marker from that of the parent variety "Civil Fruit Intermediate Parent No. 6", and at least one genetic marker selected from Tf0300-3, Tf0419-2, and Mf0090-2 is used to select a candidate citrus plant line that has a different genotype pattern of the genetic marker from that of "Harehime", or Using at least one selected from at least one of the genetic markers Bf0036-3 and Gn0073-2, candidate citrus plant lines are selected that have different genotype patterns of the genetic markers from the parent varieties "Harehime" and "Citrus Intermediate Parent No. 6." In one embodiment, it is preferable to select a candidate citrus plant line in which the genotypes of the genetic markers Bf0036-3 are AA and Gn0073-2 are AA. In one embodiment, it is preferable to select a candidate citrus plant line having the following genotypes for genetic markers: Bf0158-3 is AB, Tf0420-2 is AA, Al0302-2 is AB, Mf0097-2 is AB, Tf0300-3 is AB, Tf0419-2 is AB, and Mf0090-2 is AA.

[0061] In a typical example of a citrus plant according to this embodiment, the genotype patterns of the 14 CAPS markers are as follows: Bf0158-3 is AB type, Bf0036-3 is AA type, Tf0150-2 is AB type, Tf0271-2 is AB type, Tf0300-3 is AB type, Tf0419-2 is AB type, Tf0420-2 is AA type, Tf0318-2 is BB type, Tf0293-4 is AA type, Al0302-2 is AB type, Mf0097-2 is AB type, Mf0090-2 is AA type, Gn0073-2 is AA type, and Gn0048-2 is AA type.

[0062] (Trait-based selection process) To obtain the citrus plant of this embodiment, a selection process based on specific traits among those listed below can be carried out on candidate citrus plants obtained by crossbreeding the citrus variety "Harehime" as the mother and the citrus variety "Civil Fruit Intermediate Mother No. 6" as the father. The selection step based on a specific trait can be carried out together with the selection step using a genetic marker as an indicator, or can be carried out before the selection step using a genetic marker as an indicator. In a preferred example, after the selection step using a genetic marker as an indicator, the selection step based on a specific trait is carried out on the sufficiently narrowed down candidate citrus plant lines.

[0063] The main traits used for selection include, for example, the ability to produce seedless fruit, fruit weight of 160g or more, high sugar content and good taste of fruit, soft pericarp, high alternate bearing ability, and fruit ripening period from late February to early March. The main traits used for selection include the trait of producing seedless fruit and / or the trait of fruit weight being 130 g or more on average (preferably 140 g or more on average, more preferably 150 g or more on average, even more preferably 160 g or more on average, and even more preferably 170 g or more on average). Preferably, the traits are the trait of producing seedless fruit and the trait of fruit weight being 130 g or more on average (preferably 140 g or more on average, more preferably 150 g or more on average, even more preferably 160 g or more on average, and even more preferably 170 g or more on average). Details of the above traits are described below in the section [Citrus plants].

[0064] The selection step in the method for producing a citrus plant according to one embodiment of the present invention is a step of selecting plants obtained in the hybridization step based on at least one of the criteria described in the Examples, for example. In the selection step, selection may be carried out multiple times over multiple years.

[0065] That is, the selection step may include, for example, a preliminary selection step, a first selection step, and a second selection step.

[0066] The preliminary selection step may be a step of selecting based on at least one of the following criteria (a) to (d): (a) Female sterility and seedless, (b) fruit weight is 150g or more on average, (c) sugar content is 12Brix% or more, and (d) peelability is "easy" or "slightly easy."

[0067] The primary selection process may be a process of selecting based on at least one of the following criteria (e) to (g): (e) annual bearing ability, yield ability, (f) evaluation of physiological disorders on the tree (peel disorders such as peeling and cracking), and (g) observation of the extent of disease occurrence.

[0068] Subsequently, the secondary selection step may be a step of selecting based on the following criterion (h): (h) Line adaptability test (regional adaptability evaluation).

[0069] (Proliferation process) The method for producing a citrus plant according to one embodiment of the present invention further comprises a propagation step of vegetatively propagating the citrus plant cultivated through the crossing step and the selection step.

[0070] Asexual propagation can be achieved by grafting, layering, or cell culture. Asexual propagation is also called asexual propagation, asexual reproduction, or vegetative propagation. Asexual propagation does not require males or females, and a single individual independently generates a new individual, with the reproductive cells produced by that single individual independently becoming a new individual. Furthermore, techniques for creating cell cultures of plant tissues and methods for regenerating plants from tissue cultures are carried out using known techniques.

[0071] In the present invention, the rootstock used for the grafting can be any of various tree species that can be used as citrus trees or citrus rootstocks, such as trifoliate orange, satsuma mandarin, and shikuwasa. The type and size of the rootstock used can be changed depending on the soil, climate, etc. For example, trifoliate orange can be used as the rootstock.

[0072] The cultivation type of the citrus plants according to the present invention is not particularly limited, but examples include open-field cultivation, unheated greenhouse cultivation, and roofed greenhouse cultivation in the form of high-grafted plants or seedlings.

[0073] [How to identify citrus plants] As mentioned above, the citrus plants of the present invention can be identified by specific cultivar identification markers.

[0074] To date, DNA variety identification technology has been developed. This technology is effective in monitoring infringement of breeder's rights or proving infringement of rights. In order to prevent imports at the border, such as at customs, development is underway to enable simple and rapid variety identification at inspection sites such as customs.

[0075] The Fruit and Tea Division of the National Agriculture and Food Research Organization (NARO) has developed a variety identification technology using CAPS (cleaved amplified polymorphic sequence) markers for citrus seeds and seedlings distributed in Japan. Regarding this technology, the division has compiled and published a DNA variety identification technology manual for citrus varieties that complies with ISO (International Organization for Standardization) standards (Non-Patent Document 1).

[0076] CAPS marker analysis technology is a technique that uses specific restriction enzymes to cleave PCR-amplified fragments targeting specific genomic regions, and utilizes the differences in size of restriction enzyme fragments that arise from genomic DNA polymorphisms such as base substitutions, deletions, and insertions that exist between varieties. Analysis using this technology does not require expensive equipment, making it highly applicable to testing sites.

[0077] CAPS marker analysis involves amplifying the target region by PCR, then treating the resulting product with a restriction enzyme that recognizes a specific base sequence and cleaves DNA to detect polymorphisms. The specific sequence of the target PCR amplification product differs depending on the variety, and the difference in product length (genotype) after cleavage with the restriction enzyme is confirmed by electrophoresis using agarose gel or other instruments. To identify varieties, it is necessary to combine the results of multiple markers ("Points to Note When Validating DNA Variety Identification Technology (1st Edition, Established March 6, 2023, National Agriculture and Food Research Organization, Seed and Seedling Management Center)").

[0078] The method for identifying citrus plants according to the present invention is a method in which the CAPS marker is used as an identification marker for the citrus "OK68" or its progeny plants to distinguish them from plants of other citrus varieties.

[0079] A method for identifying citrus plants according to one embodiment of the present invention includes the steps of (I) amplifying a region of the DNA of a citrus plant containing a CAPS marker using a primer set, and (II) identifying the citrus plant based on base polymorphisms in the amplified DNA region, wherein the CAPS marker is at least one of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. The genetic markers (CAPS markers) may be one, more than one, or all of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2, but it is preferable to use all of them. In one embodiment, the method for identifying citrus plants can distinguish and identify the citrus plants of the present invention from other citrus cultivars based on the polymorphism of bases in the region (DNA region) containing the amplified genetic markers. Examples of other citrus cultivars include citrus cultivars other than OK68 listed in Tables 2 to 5.

[0080] Since polymorphisms in CAPS are determined by the presence or absence of a restriction enzyme site, the allele without a restriction enzyme site is designated as A, and the allele with a restriction enzyme site is designated as B, and the genotype of the variety can be determined as AA, AB, or BB.

[0081] (Identification target) The method for identifying citrus plants according to the present invention distinguishes the citrus plants of the present invention from plants of other citrus varieties.

[0082] Furthermore, in the citrus plant identification technology of the present invention, from the perspective of its identification principle, citrus plants that exhibit the same allele type as "OK68" for the 14 identification markers designated as marker names Bf0158-3 to Gn0048-2 among the progeny plants of "OK68" can also be distinguished from other citrus varieties using this technology.

[0083] Furthermore, the same allele type as "OK68" refers to the allele type shown in Figure 4 for the 14 loci Bf0158-3 to Gn0048-2. As long as the allele type is derived from "OK68," the variety identification technology of the present invention can be applied even if mutations occur in the base sequences that make up these loci and their surrounding regions.

[0084] In the identification technique of the present invention, "other citrus varieties" that can be distinguished from "OK68" include citrus varieties that do not have the same genotype as "OK68" for at least one of the 14 identification markers. In citrus plants belonging to other citrus varieties, the 14 allele types of Bf0158-3 to Gn0048-2 are different from those of "OK68."

[0085] Examples of other citrus varieties that can be distinguished from "OK68" using the above-mentioned identification technology include "Unshu mandarin (Miyagawa Wase)," "Grapefruit (Duncan)," "Sweet orange (Trovita)," "Lemon (Lisbon)," "Shiranui," "Iyo (Miyauchi Iyokan)," "Natsumikan (Kawano Natsudaidai)," "Hassaku," "Ponkan (Ota Ponkan)," "Rinoka," "Mihaya," "Asumi," "Asuki," "Reiko," "Tsunohikari," "Seinan no Hikari," "Tsunozomi," "Haruhi," "Kiyomi," "Setoka," "Harumi," "Harehime," "Kanpei," and "Ehime Fruit Experiment No. 28 (Beni Madonna)," as shown in the manual of Non-Patent Document 1.

[0086] These 24 citrus varieties, together with "OK68" (25 varieties in total), account for more than 94% of the citrus fruit distribution volume in Japan.

[0087] Furthermore, other citrus varieties that can be distinguished from "OK68" using the identification technology of the present invention include, for example, yuzu, Kawachi bankan, Hyuganatsu, sudachi, shikuwasa, kara mandarin (kara), tankan, Seminole, kabosu, amakusa, koganekan, Kishu mikan, sweet spring, encore, Tamami, Nishinoka, Marcott, Nanko, Amaka, citrus intermediate parent No. 6, and bitter orange.

[0088] The identification method of the present invention enables identification of "OK68" with extremely high accuracy.

[0089] Furthermore, examples of other citrus varieties that can be distinguished from "OK68" using the identification technology of the present invention include citrus varieties that do not have the same genotype as "OK68" with respect to the 14 identification markers Bf0158-3 to Gn0048-2, taking into account the results of the Examples below. For example, pomelo and the like can also be recognized as other citrus varieties that can be distinguished from "OK68" from the fundamental standpoint of the identification technology of the present invention.

[0090] For example, to distinguish "OK68" from the parent variety "Harehime," at least one of markers Bf0036-3, Tf0300-3, Tf0419-2, Mf0090-2, and Gn0073-2 may be used.

[0091] Furthermore, to distinguish "OK68" from the parent variety "Citrus Intermediate Parent No. 6", at least one of markers Bf0158-3, Bf0036-3, Tf0420-2, Al0302-2, Mf0097-2, and Gn0073-2 may be used.

[0092] Furthermore, when distinguishing "OK68" from the parent varieties "Harehime" and "Citrus Intermediate Parent No. 6", a combination of at least one genetic marker from among Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2 with at least one genetic marker from among Tf0300-3, Tf0419-2, and Mf0090-2, as well as at least one selected from at least one genetic marker from among Bf0036-3 and Gn0073-2, can be used. When only "OK68" needs to be distinguished from its parents, at least one of Bf0036-3 and Gn0073-2 can be used.

[0093] In an identification method according to one embodiment of the present invention, the subject citrus plant is a candidate plant for breeding material, a plant obtained through a breeding process, etc. Candidate plants for breeding material include, for example, parent plants used in crossbreeding and plants used in molecular breeding using recombinant DNA technology. Furthermore, the subject citrus plant includes citrus plants used in molecular breeding using recombinant DNA technology and citrus plants obtained by molecular breeding.

[0094] An identification method according to one aspect of the present invention is a method for identifying a citrus plant of the present invention, comprising the steps of: (I) amplifying a partial region of DNA in the DNA of a citrus plant using a primer set capable of amplifying the CAPS marker region described above; and (II) A method comprising a step of identifying citrus plants based on the base polymorphism of the amplified DNA region.

[0095] The region in the DNA of a citrus plant specimen can be amplified by polymerase chain reaction (PCR) using DNA extracted from the citrus plant specimen as a template and a primer set that amplifies a region containing the CAPS marker.

[0096] When DNA obtained from a citrus plant is amplified by PCR using the primer set, and the length of the DNA fragment obtained by restriction enzyme treatment matches the length of the DNA fragment obtained by similar treatment in a DNA sample obtained from a known citrus variety, the plant can be determined to be the known citrus variety.

[0097] That is, in one embodiment of the identification method of the present invention, the step (II) of identifying a citrus plant based on the base polymorphism of the amplified DNA region includes the steps of (II-1) treating the amplified DNA fragment with a restriction enzyme, and (II-2) identifying the genotype based on the length of the DNA fragment obtained by the restriction enzyme treatment.

[0098] The genotype of the amplified fragment can be determined by a conventionally known method, and a preferred example is a method using agarose gel electrophoresis.

[0099] The length of a DNA fragment can be measured, for example, by its molecular weight, which can be measured, for example, by its migration distance in agarose gel electrophoresis or by DNA sequencing.

[0100] The reaction reagents and reaction conditions used in the PCR method are not particularly limited as long as they are capable of producing primer-specific PCR amplified fragments, and known methods can be used.

[0101] The means for detecting the PCR-amplified fragments is not particularly limited, but as an example, the PCR-amplified fragments can be detected using ethidium bromide, fluorescent substances, chromogenic substances, luminescent substances, etc.

[0102] The primers in the primer set are composed of a base sequence having 90% or more sequence identity to the base sequences of the nucleotides constituting each primer in the primer set. Modified primers composed of modified base sequences obtained by modifying some bases in the base sequence are also included in the scope of primers used in the identification method of the present invention. Furthermore, the modified primer may have, for example, a sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% with the base sequence of each primer. In principle, a modified primer is considered equivalent to each primer if it can hybridize with the complementary DNA strand of each primer. Furthermore, this modified primer considered to be equivalent may be a primer that functions as a primer that hybridizes with the complementary DNA strand of the original primer and is composed of nucleotides having a modified base sequence in which, for example, 0 to 3, 0 to 2, 1 to 3, or 1 or 2 bases have been added, deleted, inserted, or substituted in the base sequence of the oligonucleotides constituting each original primer.

[0103] The materials and methods described in the manual of Non-Patent Document 1 can be suitably used for the primers, reagents, reaction conditions, detection methods, and the like related to the PCR described above.

[0104] The method for identifying citrus plants of the present invention can accurately and quickly identify the citrus plant "OK68" or its progeny from other citrus varieties, and is a method that can be widely introduced in inspection sites, etc.

[0105] A method for identifying citrus plants according to one aspect of the present invention comprises: amplifying a region of the DNA of a citrus plant containing a genetic marker (CAPS marker) of the DNA using a primer set; and identifying the citrus plant based on the base polymorphism of the amplified DNA region; Preferably, the genetic marker (CAPS marker) is at least one of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. Furthermore, a method for identifying a citrus plant according to one aspect of the present invention comprises: amplifying a region of the DNA of a citrus plant containing a genetic marker (CAPS marker) of the DNA using a primer set; and identifying the citrus plant based on the base polymorphism of the amplified DNA region; More preferably, the genetic markers (CAPS markers) are Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. Furthermore, it is preferable to identify a citrus plant according to one aspect of the present invention when each genetic marker (CAPS marker) has the above-mentioned genotype.

[0106] [Citrus plants 1] In one aspect of the present invention, a citrus plant is a citrus plant having a genetic marker whose genotype is: Bf0158-3 is type AB, Bf0036-3 is the AA type, Tf0150-2 is type AB, Tf0271-2 is type AB, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type AB, Mf0090-2 is type AA, Gn0073-2 is AA type, and Gn0048-2 is type AA It is a citrus plant. As is clear from the above Tables 2 to 5, the citrus plant according to one embodiment of the present invention has a different genotype of genetic markers from other citrus varieties. Therefore, the citrus plant according to one embodiment of the present invention is a novel citrus plant. In one embodiment, the citrus plant may be a hybrid variety or a descendant plant of a citrus plant that has been crossbred using the citrus variety "Harehime" as the seed parent (mother) and the citrus variety "Citrus Intermediate Mother No. 6" as the pollen parent (father). In one embodiment, the plant used in the selection process may be a hybrid variety obtained by crossing citrus varieties with each other, or a progeny plant thereof, but is preferably a hybrid variety obtained by crossing citrus varieties with each other. In one embodiment, it is preferred that the citrus plant has the trait to produce seedless fruit and / or fruit weighing an average of 130g or more (preferably an average of 140g or more, more preferably an average of 150g or more, even more preferably an average of 160g or more, even more preferably an average of 170g or more), and it is more preferred that the citrus plant has the trait to produce seedless fruit and fruit weighing an average of 130g or more (preferably an average of 140g or more, more preferably an average of 150g or more, even more preferably an average of 160g or more, even more preferably an average of 170g or more).

[0107] [Citrus plants 2] A citrus plant according to one embodiment of the present invention is a citrus plant having the following traits (1), (2), (3), and (4): (1) It was obtained by crossbreeding the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father. (1) Seedless fruit production is possible, (2) Fruit weight is 160g or more, (3) The fruit has a high sugar content and good taste, and (4) It is distinguishable from other citrus cultivars, including "Harehime" and "Citrus Intermediate Parent No. 6," using at least one of the following genetic markers: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0108] Preferred embodiments of citrus plant 1 and / or citrus plant 2 will be described below. Furthermore, a citrus plant according to a further embodiment of the present invention is a citrus plant further having at least one of the following traits (5) to (7): (5) The scrotum is soft. (6) High alternate-bearing tendency (7) The fruit ripens from late February to early March.

[0109] The characteristics of the citrus plants according to the present invention may vary depending on the conditions under which they are grown or cultivated, such as the climate, soil, environment, cultivation method, and tree age. In addition, in this specification, limitations on the maturity period and other periods are based on cultivation in Japan, and for example, Shizuoka City, Shizuoka Prefecture, where the plants are grown in the examples, may be used as the standard.

[0110] Furthermore, a citrus plant according to one embodiment of the present invention is a plant that has at least one of the following characteristics as a main difference from the parent cultivar "Harehime" or "Citrus Intermediate Parent No. 6." The values ​​may be the average values ​​of measurements taken in each year for two or more years.

[0111] (Characteristics of the tree body) The branching angle is somewhat narrow, and the tree shape is "slightly upright." The tree vigor is "slightly strong." The branches are "dense," "long," and "thick." The branches (spring branches) have a high rate of thorns, but the length of the thorns is short.

[0112] The leaf blade is "spindle-shaped", the leaf size is "large", the length is "slightly long", and the width of the leaf blade is "medium". The leaf shape index is "small", and the leaf thickness is "thin". The alar leaves are "vestigial" and "narrow" in width. The petiole is "short" in length and "slightly thick" in thickness.

[0113] The flowers do not form an inflorescence but grow solitary. There are five white petals. The number of filaments is "few" and "partly fused". The amount of pollen is "slightly small". The style is "arch-shaped".

[0114] The fruit begins to color in late October and is fully colored in early to mid-December, although this varies from year to year. The flowering rate is medium, the alternate bearing rate is high, and late fruit drop is low and not a problem. The yield is medium.

[0115] (Fruit characteristics) The fruit is oblate, with a fruit shape index of around 127. The apex is flat, the stalk is truncated, and a short neck may develop depending on the year. There are no radial grooves at the apex, and the stalk is of medium size. The peel is deep orange. The surface is somewhat smooth. The peel is thin, about 2.5 mm thick, and peels somewhat easily. The peel has an orange-like fragrance.

[0116] The flesh is deep orange in color and has a high flesh content. The sepal membrane is soft and the flesh is somewhat soft. There are no complete seeds per fruit, and the percentage of seedless fruits is 100% under normal outdoor cultivation conditions. The fruit has female sterility derived from seedless Kishu, making stable seedless fruit production possible.

[0117] Although there was a small occurrence of sunburned fruit, there was no occurrence of peeling or cracking of fruit. In addition, as of the time of the survey in late January, there was no occurrence of physiological rosing.

[0118] An example of a citrus plant of the present invention is the citrus plant "Citrus OK68" or its progeny, as described in the Examples. Hereinafter, "Citrus OK68" will be referred to as "OK68." The citrus plant "OK68" of the Examples has the characteristics described in the Examples. For the morphological and physiological characteristics of "OK68," see Figures 2 and 3.

[0119] The characteristics of each of the above traits may be characteristic values ​​evaluated based on the Evaluation Standards for Other Citrus Fruits (Citrus L.) and Breeding Line Adaptability Test and Characteristic Test Survey Methods issued by the Ministry of Agriculture, Forestry and Fisheries of Japan (Fruit Tree Research Institute, National Agriculture and Food Research Organization, 2007).

[0120] Regarding the traits possessed by the citrus plant of the present invention, for example, trait (1) the ability to produce seedless fruit means that there are no complete seeds, and in an investigation of five or more fruits, the number of seeds with embryos that are more than half the size of normal seeds (complete seeds) is evaluated as follows: large: 5 or more seeds, medium: 3 to 5 seeds, small: 1 to 2 seeds or less, or none: 0 seeds. In one example, under natural pollination conditions in outdoor cultivation, there are almost no complete seeds, making it possible to produce seedless fruit. In one example, the proportion of seedless fruit is 100% under normal outdoor cultivation conditions, and the plant has female sterility derived from seedless Kishu, allowing for stable seedless fruit production.

[0121] The trait (2) of a fruit weight of 160 g or more possessed by the citrus plant of the present invention means the weight of the fruit measured at maturity using an electronic balance or the like, and the lower limit of the fruit weight is preferably 170 g or more, 180 g or more, 190 g or more, 200 g or more, 210 g or more, 220 g or more, 230 g or more, 240 g or more, 250 g or more, 260 g or more, 270 g or more, 280 g or more, 290 g or more, 300 g or more, 310 g or more, 320 g or more, 330 g or more, or 340 g or more. The upper limit of the fruit weight is not particularly limited, but is, provided that it is equal to or greater than any of the lower limits, for example, 360 g or less, 350 g or less, 340 g or less, 330 g or less, 320 g or less, 310 g or less, or 300 g or less.

[0122] Regarding the trait (3) that the citrus plant of the present invention has a high sugar content and good taste of the fruit, the "taste" can also be evaluated using the value of the sugar-acid ratio as an index.

[0123] In this specification, "sugar content" is synonymous with sweetness and refers to the sugar content reading of fruit juice as measured by a sugar content meter. For example, the sugar content of fruit juice in early January. The sugar content is measured using a refractometer. The unit of measurement is, for example, Brix%.

[0124] In this specification, "sourness" refers to the citric acid content in fruit juice. The sourness is measured by converting the titration value of the fruit juice with an aqueous solution of sodium hydroxide. The lower limit of the sugar content of the juice of the citrus plant of the present invention is preferably 12.0 Brix% or more, more preferably 13.0 Brix% or more, more preferably 14.0 Brix% or more, more preferably 15.0 Brix% or more, and even more preferably 16.0 Brix% or more. The upper limit of the sugar content is not particularly limited, but is, provided that it is equal to or greater than any of the lower limits, for example, 18 Brix% or less, 17 Brix% or less, or 16 Brix% or less.

[0125] The upper limit of the acid content of the fruit juice is, for example, 1.5 g / 100 ml or less, more preferably 1.40 g / 100 ml or less, more preferably 1.30 g / 100 ml or less, more preferably 1.20 g / 100 ml or less, more preferably 1.10 g / 100 ml or less, more preferably 1.00 g / 100 ml or less, more preferably 0.90 g / 100 ml or less, more preferably 0.850 g / 100 ml or less, more preferably 0.8 g / 100 ml or less, and even more preferably 0.7 g / 100 ml or less. The lower limit of the acid content is not particularly limited, but is, provided that it is equal to or less than any of the upper limits, for example, 0.5 g / 100 ml or more, 0.6 g / 100 ml or more, 0.7 g / 100 ml or more, or 0.8 g / 100 ml or more.

[0126] The "brix-acid ratio" is calculated as the saccharometer reading / citric acid content. The brix-acid ratio is, for example, 12.0 or more, 13.0 or more, 14.0 or more, or 15.0 or more.

[0127] For example, "good taste" includes a good balance between sugar content and acidity. Therefore, a product can be said to have good taste when its sugar-acid ratio is in the range of 12 to 14, and more preferably 14 or higher.

[0128] In the example, the sugar content of the "OK68" juice collected in early January was high at 15.0% on average. The acid content of the juice was approximately 1.14g / 100ml on average. The sugar-acid ratio was 13.2.

[0129] The trait (5) that the citrus plant of the present invention has, "the pericarp membrane is soft," refers to the degree of hardness of the pericarp membrane when eaten, and can be evaluated as follows, for example: hard: too hard to eat along with the pericarp; slightly hard: there is resistance when eating along with the pericarp; medium: the pericarp can be eaten along with the pericarp; slightly soft: the pericarp can be easily eaten along with the pericarp; soft: the pericarp breaks thinly and there is no resistance when eating.

[0130] (6) Regarding the high alternate bearing tendency, for example, when an observation survey is conducted over several years and the fruit is thinned appropriately from the third year onwards after the first fruit set, if the good and bad years are clear, it is evaluated as high (strong); if the good and bad years are moderate, it is evaluated as medium; and if the good and bad years are unknown, it is evaluated as low (low).

[0131] Regarding the trait (7) possessed by the citrus plant of the present invention, that the fruit ripens from late February to early March, preferably, when the cultivation type is, for example, top-grafted and grown outdoors, the ripening period is late February to early March, and depending on the growing area and cultivation conditions, the fruit ripens from early December to late March. That is, the ripening period is later than that of "Harehime," and in one example, about 4 to 12 weeks or 6 to 12 weeks later than that of "Harehime." Furthermore, the ripening period is slightly later than that of "Citrus Intermediate Parent Line Nou 6," and in one example, about 1 to 6 weeks or 2 to 4 weeks later than that of "Citrus Intermediate Parent Line Nou 6."

[0132] The citrus plant of the present invention may be a plant having characteristics equivalent to those of "OK68." A plant having equivalent characteristics means a plant that has the main characteristics of "OK68" when grown under the same environmental conditions. The main characteristics are (1) to (4) above. Preferably, the plant further has characteristics (5) to (7) above. Furthermore, the citrus plant of the present invention has, for example, three, two, or one of the characteristics (5) to (7) above. Furthermore, the citrus plant of the present invention also includes a plant that further has at least one or more characteristics from each of the characteristics listed under (Tree characteristics) and (Fruit characteristics).

[0133] Furthermore, the trait (4) possessed by the citrus plant of the present invention is distinguishable from other citrus cultivars, including "Harehime" or "Citrus Intermediate Parent No. 6," using at least one genetic marker selected from Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. The citrus plant of the present invention may be a plant in which the genotypes of the genetic markers are Bf0158-3 AB, Bf0036-3 AA, Tf0150-2 AB, Tf0271-2 AB, Tf0300-3 AB, Tf0419-2 AB, Tf0420-2 AA, Tf0318-2 BB, Tf0293-4 AA, Al0302-2 AB, Mf0097-2 AB, Mf0090-2 AA, Gn0073-2 AA, and Gn0048-2 AA.

[0134] Citrus plants of the present invention include "OK68" described in the Examples and its progeny plants. The progeny plants include the individual plants of the progeny plants or parts thereof.

[0135] In the present invention, the progeny plants also include plants obtained by crossing the "OK68" citrus plant with another citrus variety or other citrus plant, or by crossing the citrus plant with a wild citrus plant.

[0136] Progeny plants include individuals obtained using "OK68" as a parent line and their progeny, somatic hybrid plants and their progeny obtained by cell fusion between cells of the citrus plant and cells of other plant varieties, and individuals and their progeny obtained by grafting citrus plants as rootstocks or scions.

[0137] A citrus plant according to one aspect of the present invention has the above-described traits, and the citrus plant or its fruit obtained by the above-described production method is also within the scope of the present invention. Therefore, the citrus plant may be the fruit of a citrus plant. Furthermore, the citrus plant according to one aspect of the present invention also falls within the scope of the present invention in the form of vegetative propagation material such as seedlings or scions.

[0138] [Citrus plant processed products] The processed citrus plant product of one embodiment of the present invention may be a processed citrus plant product obtained from the citrus plant described above. Alternatively, the processed citrus plant product of one embodiment of the present invention may be a processed citrus fruit product obtained from the fruit of the citrus plant described above. The processed product is preferably an edible processed product. Examples of the processed citrus plant product of one embodiment of the present invention include squeezed juice, extract, squeezed residue, and extraction residue of the plant body. Other examples include processed products of the plant body itself, such as cutting, crushing, drying, powdering, freezing, various chemical treatments, paste formation, and puree formation. In particular, processed fruit products that utilize the fruit and exhibit the above-described processing modes are preferred. Suitable examples of such processed fruit products include fruit juice, fruit pulp, or juice containing fruit juice and fruit pulp, fruit pulp preserved in syrup, dried fruit, jelly, and powdered processed confectionery ingredients. Among these, the processed product is preferably at least one selected from the group consisting of juice, syrup-preserved products, jelly, and powdered confectionery ingredients, and more preferably at least one selected from the group consisting of juice and syrup-preserved products.

[0139] Fruits as part of the plant body of citrus plants will now be further described. Fruits may be seeded fruits (with seeds) or seedless fruits (without seeds). Citrus fruits may be in the form of a package wrapped in an appropriate packaging material. The form of the packaging material is not particularly limited, but examples thereof include films, sheets, bags, and sacks. The material of the packaging material is not particularly limited, but examples thereof include paper and resin (plastic). Packages of isolated citrus fruit pieces contain one or more fruits. Packages of citrus fruit may contain only one type of citrus fruit or a combination of multiple types of citrus fruit. Packages of citrus fruit may also contain agricultural products other than citrus fruit.

[0140] Citrus fruits (including citrus fruit packages) may be affixed with a product label. The product label records at least one piece of information selected from the following: the name of the citrus variety, intellectual property rights information related to the citrus, information about the citrus producer, information about the citrus production history (e.g., information about pesticide spraying), information about the citrus distribution history, product price, harvest date, and information about the ripeness period. This information may be recorded directly on the product label (by printing or, for example, electromagnetic recording), or may be recorded readably using a code format such as a one-dimensional code or a two-dimensional code.

[0141] The citrus plant may be in the form of vegetative propagation material such as a seedling or a scion. The seedling or scion may have a product label attached thereto. The product label contains at least one piece of information (product information) selected from the following: the name of the citrus variety, intellectual property rights information related to the citrus, individual identification information (ID number) of the seedling or scion, genetic marker information of the seedling or scion (e.g., the genotype pattern of the CAPS markers described above), producer information of the seedling or scion, production history information of the seedling or scion (e.g., information on pesticide spraying), distribution history information of the seedling or scion, and product price. In a typical example, at least the individual identification information (ID number) of the seedling or scion is recorded. In another typical example, at least the individual identification information (ID number) of the seedling or scion and genetic marker information of the seedling or scion (e.g., the genotype pattern of the CAPS markers described above) are recorded. This information may be directly recorded on the product label (either printed or readable electromagnetically), or readably recorded using a code format such as a one-dimensional code or a two-dimensional code. The product label may also be comprised of a small electromagnetic recording medium such as an IC chip, embedded inside the seedling or scion. The location where the small electromagnetic recording medium such as an IC chip is embedded is not particularly limited. However, in the case of a grafted seedling, a preferred example is within the rootstock, and another preferred example is within a predetermined distance from the grafting position. The predetermined distance from the grafting position may be, for example, within 10 cm, 5 cm, 4 cm, 3 cm, 2 cm, or 1 cm from the grafting position. The predetermined distance from the grafting position toward the rootstock may be maintained, or the predetermined distance from the grafting position toward the scion may be maintained.

[0142] [Product information management system for citrus plant seedlings or scions (vegetative propagation materials)] In the above section "Processed Products of Citrus Plants," we have explained the seedlings or scions of citrus plants according to this embodiment with a product label attached. The scope of the present invention also includes a product information management system for the seedlings or scions of citrus plants according to this embodiment with a product label attached.

[0143] An example of a product information management system includes at least 1) a seedling or scion of a citrus plant according to this embodiment with a product label attached thereto, and 2) a reader (an example of a client terminal) that reads the product information recorded on the product label. The product information management system may further include 3) a server. The product information management system may include a client terminal other than the reader, or may include a client terminal instead of the reader. These client terminals may not have the function of reading product information.

[0144] In one example, the client terminal (which may be a reader) transmits a set of information recorded on the product label (especially the individual identification information (ID number) of the seedling or scion) and location information of the client terminal to the server. The client terminal (which may be a reader) may also transmit to the server information about the owner of the seedling or scion (which may be the owner information of the client terminal if it matches the owner of the client terminal), location information of the seedling or scion (if different from the location information of the client terminal), etc. This allows information about the location, etc. of each individual seedling or scion to be managed. Furthermore, if the owner of the seedling or scion changes, if the location of the seedling or scion is relocated, or if the seedling or scion is discarded, or if the management environment of the seedling or scion changes, this information is transmitted from the client terminal to the server.

[0145] 〔summary〕 The present invention includes any of the following aspects. <1> 1. A method for producing a citrus plant, comprising: In hybrid varieties obtained by crossing citrus varieties or their progeny plants, genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 are shown below, respectively. Bf0158-3 is type AB, Bf0036-3 is the AA type, Tf0150-2 is type AB, Tf0271-2 is type AB, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type AB, Mf0090-2 is type AA, Gn0073-2 is AA type, and Gn0048-2 is type AA A method for producing a citrus plant, comprising the step of selecting a plant having a genotype. <2> The method further includes a step of crossbreeding the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father to obtain the hybrid variety. <1> 2. A method for producing a citrus plant according to claim 1. <3> The method further comprises a selection step of selecting citrus plants having the trait of producing seedless fruit and / or the trait of fruit having an average weight of 130 g or more. <1> or <2> The method for producing the strain described above. <4> The method further includes a selection step of selecting a citrus plant having the following traits (1), (2), and (3): <1> from <3> 1. The method for producing a gene according to any one of claims 1 to 9. (1) Seedless fruit production is possible, (2) Fruit weight is 160g or more, and (3) The fruit has a high sugar content and is delicious. <5> The method further comprises a propagation step of vegetatively propagating the citrus plant. <1> from <4> 1. A method for producing a citrus plant according to any one of claims 1 to 9. <6> A citrus plant, wherein the genotype of a genetic marker in the citrus plant is: Bf0158-3 is type AB, Bf0036-3 is the AA type, Tf0150-2 is type AB, Tf0271-2 is type AB, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type AB, Mf0090-2 is type AA, Gn0073-2 is AA type, and Gn0048-2 is type AA The citrus plant. <7> It is a hybrid variety or its descendants that was crossed between the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father. <6> The citrus plant described in <8> having the trait of producing seedless fruit and / or fruit that weighs an average of 130 g or more; <6> or <7> The citrus plant described in <9> The strain has the following characteristics (1), (2), (3), and (4): <6> from <8> A citrus plant according to any one of claims 1 to 5. (1) It was obtained by crossbreeding the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father. (1) Seedless fruit production is possible, (2) Fruit weight is 160g or more, (3) The fruit has a high sugar content and good taste, and (4) It is distinguishable from other citrus cultivars, including "Harehime" and "Citrus Intermediate Parent No. 6," using at least one of the following genetic markers: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. <10> Further having at least one of the following traits (5) and (6): <6> from <9> A citrus plant according to any one of claims 1 to 5. (5) The sac is soft (6) High alternate-bearing tendency. <11> The aforementioned <6> from <10> A method for identifying a citrus plant according to any one of the preceding claims, amplifying a region of the DNA of a citrus plant, the region including a genetic marker of the DNA, using a primer set; and identifying the citrus plant based on base polymorphisms in the amplified DNA region; The method, wherein the genetic markers are Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. <12> <1> from <5> A citrus plant or its fruit obtained by the method for producing a citrus plant according to any one of claims 1 to 4. <13> in the form of seedlings or scions, <6> from <10> 1. A citrus plant according to any one of claims 1 to 10. <14> <6> from <10> A fruit of the citrus plant according to any one of claims 1 to 4. <15> <6> From <10> A citrus plant according to any one of the preceding claims or <14> A processed product of a citrus plant or the fruit of a citrus plant, obtained from the fruit of the citrus plant described in 1. <16> At least one selected from the group consisting of fruit juice, fruit pulp, or juice containing fruit juice and fruit pulp, fruit pulp preserved in syrup, dried fruit, jelly, powdered confectionery raw materials, etc. <15> A processed product of the citrus plant or the fruit of the citrus plant described in 1.

[0146] <2-1> A method for producing a citrus plant, The hybridization process involves crossbreeding the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father. and a marker selection step of selecting, from the citrus plant obtained by the hybridization step or a progeny thereof, candidate citrus plants having a different genotype pattern of the genetic markers from those of the parent variety "Harehime" and "Citrus intermediate parent No. 6", using at least one genetic marker selected from Bf0158-3, Bf0036-3, Tf0300-3, Tf0419-2, Tf0420-2, Al0302-2, Mf0097-2, Mf0090-2, and Gn0073-2. <2-2> The production method according to <2-1>, wherein in the marker selection step, a candidate citrus plant line is selected that has a different genotype pattern of the genetic markers from the parent varieties “Harehime” and “Citrus Intermediate Mother Nori No. 6” using at least one selected from a combination of at least one genetic marker selected from Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2 with at least one genetic marker selected from Tf0300-3, Tf0419-2, and Mf0090-2, and at least one genetic marker selected from Bf0036-3 and Gn0073-2. <2-3> The method according to <2-1> or <2-2>, wherein a candidate citrus plant line is selected, in which the genotypes of the genetic markers are as follows: Bf0158-3 is AB, Bf0036-3 is AA, Tf0300-3 is AB, Tf0419-2 is AB, Tf0420-2 is AA, Al0302-2 is AB, Mf0097-2 is AB, Mf0090-2 is AA, and Gn0073-2 is AA. <2-4> The method according to any one of <2-1> to <2-3>, comprising a selection step of selecting a citrus plant having the following traits (1), (2) and (3):

[0147] (1) Seedless fruit production is possible, (2) Fruit weight is 160g or more, and (3) The fruit has a high sugar content and is delicious. <2-5> The method for producing a citrus plant according to any one of <2-1> to <2-4>, further comprising a propagation step of asexually propagating the citrus plant. <2-6> A citrus plant having the following traits (1), (2), (3), and (4): (1) It was obtained by crossbreeding the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father. (1) Seedless fruit production is possible, (2) Fruit weight is 160g or more, (3) The fruit has a high sugar content and good taste, and (4) It is distinguishable from other citrus cultivars, including "Harehime" and "Citrus Intermediate Parent No. 6," using at least one of the following genetic markers: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. <2-7> The citrus plant according to <2-6>, further having at least one of the following traits (5) and (6): (5) The sac is soft (6) High alternate-bearing tendency <2-8> The citrus plant according to <2-6> or <2-7>, wherein the genotypes of the genetic markers are as follows: Bf0158-3 is AB, Bf0036-3 is AA, Tf0150-2 is AB, Tf0271-2 is AB, Tf0300-3 is AB, Tf0419-2 is AB, Tf0420-2 is AA, Tf0318-2 is BB, Tf0293-4 is AA, Al0302-2 is AB, Mf0097-2 is AB, Mf0090-2 is AA, Gn0073-2 is AA, and Gn0048-2 are AA. <2-9> A method for identifying a citrus plant according to any one of <2-6> to <2-8>, amplifying a region of DNA of a citrus plant containing a CAPS marker using a primer set; and identifying the citrus plant based on base polymorphisms in the amplified DNA region; The method, wherein the CAPS marker is at least one of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. <2-10> A citrus plant or its fruit obtained by the method for producing a citrus plant according to any one of <2-1> to <2-5>. <2-11> The citrus plant according to any one of <2-6> to <2-8> or <2-10>, or a fruit thereof, in the form of a seedling or a scion. <2-12> A processed product produced using the citrus plant according to any one of <2-6> to <2-8> or the citrus plant according to <2-10> or its fruit.

[0148] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0149] [1. Development of citrus strains] (history) 1) Breeding method: Crossbreeding 2) Parents: "Harehime" x "Citrus Intermediate Parent No. 6" The cross was made using "Harehime," an early-maturing variety with good taste and peeling properties, as the seed parent and "Nou 6," a citrus intermediate parent that has female sterility derived from seedless Kishu and is capable of stable seedless fruit production with excellent taste, as the pollen parent. The pedigree diagram is shown in Figure 1.

[0150] (Development process) This line was developed through hybridization at the Fruit Tree Research Institute (now the Citrus Research Center, Fruit and Tea Research Division, National Agriculture and Food Research Organization) in May 2004 with the goal of developing a citrus tree with high sugar content and excellent flavor that would ripen after February and produce stable seedless fruit. In April 2005, it was grafted high onto a Satsuma mandarin interstock to promote fruit set. Its first fruit set occurred in 2011. Subsequent fruit quality studies revealed excellent fruit quality, leading to a primary selection in 2012 as a superior individual. Line OK68 began participating in the 12th Citrus Line Adaptability and Characteristics Certification Test in April 2017, with regional adaptability assessed at 25 test sites from Chiba Prefecture in the east to Kagoshima Prefecture in the south. As a result, the test results review committee (evergreen fruit trees) in August 2023 concluded that it was suitable as a candidate for a new variety.

[0151] (Selection process) The selection criteria for the selection process were as follows: Post-fruit evaluation (2010-2013) 1. Female sterility and seedlessness 2. Fruit weight is 150g or more on average 3. Sugar content of 12Brix% or more 4. Peelability is "easy" or "slightly easy" Based on the selection criteria 1. to 4. above, preliminary selection was conducted in 2012, and the selected plants were then grafted. 5. Consecutive year results and yield 6. Evaluation of physiological disorders on the tree (peel disorders such as peeling and cracking) 7. Observation of the extent of disease occurrence Based on the selection criteria 5 to 7 above, the first selection was carried out in 2017, and the selected plants were further grown. 8. Strain adaptability test (regional adaptability evaluation) Based on the selection criteria in 8 above, a second selection was conducted in 2023, and the selected plant was established as "OK68."

[0152] A photograph of the fruit of "OK68" is shown in Figure 2. A photograph of the tree of "OK68" is shown in Figure 3.

[0153] [2. Evaluation of characteristics of each trait in the growing area] Subsequently, the traits of the resulting varieties were evaluated.

[0154] The trait evaluation method was in accordance with the Evaluation Standards for Agricultural, Forestry and Fisheries Plant Types (Category: Other Citrus) (Ministry of Agriculture, Forestry and Fisheries) and the Breeding Line Adaptability Test and Characteristic Test Survey Method (Fruit Tree Research Institute, National Agriculture and Food Research Organization, 2007). The following traits are based on prototypes grown in Japan. The traits for each trait described above and the data described in the examples were evaluated in accordance with the Evaluation Standards for Category: Other Citrus (Citrus L.) published by the Ministry of Agriculture, Forestry and Fisheries of Japan (standards published by the Ministry of Agriculture, Forestry and Fisheries at the time of prototype development) and the Breeding Line Adaptability Test and Characteristic Test Survey Method (Fruit Tree Research Institute, National Agriculture and Food Research Organization, 2007).

[0155] (Characteristics overview) 1) The fruit averages about 180g and is oblate spheroidal. The skin is dark orange, the surface is somewhat smooth, and is thin at about 2.5mm. Peeling is somewhat easy. No peeling occurs. The flesh is dark orange, the flesh is somewhat soft, the sac is soft, and the flesh ratio is 80.7%. The sugar content of the juice is very high at 15% around February 20th, and the acid content is 1.14g / 100ml, making it tasty. The ripening period is estimated to be from early February to March. 2) The tree vigor is "slightly strong", and the shoots are "slightly upright" to "upright". The shoots are "dense". The alternate bearing rate is "high". There is a high incidence of thorns. There is almost no incidence of scab, and the tree is susceptible to canker, with "moderate" to "severe" incidence observed. 3) It has female sterility derived from seedless Kishu, and the average number of perfect seeds is 0.0. Even if varieties that produce a lot of pollen are planted nearby, almost no seeds are produced under outdoor cultivation conditions, making it possible to produce stable seedless fruit.

[0156] [Table 6] An overview of the features of the "OK68" is shown in Table 7 below.

[0157] [Table 7] (Suitable location and cultivation precautions) In previous strain adaptability tests, fruits with a sugar content of over 13% were produced from the ripening period in February onwards, suggesting that the variety has a wide range of adaptability to soil and weather conditions. However, since the ripening period is in the harsh winter months of February to March, it is best cultivated in areas with warm winters. Also, since it is susceptible to scab, it is best cultivated in an area that is not exposed to strong winds, with a windbreak, or under a roof to prevent the disease from spreading.

[0158] [2. Citrus plant identification using CAPS markers] We verified whether "OK68" can be identified using CAPS markers.

[0159] Using the CAPS markers published in "DNA variety identification technology for fruits of 24 citrus varieties using CAPS markers (Non-patent document 1)," we verified whether "OK68" could be distinguished from other citrus plants using previously reported CAPS markers.

[0160] The experiment was carried out according to the manual in Non-Patent Document 1. Specifically, the flavedo (outer pericarp) was excised from fresh fruit, and a DNA solution was obtained according to the manual in Non-Patent Document 1. For each CAPS marker, a PCR reaction was performed on the obtained DNA solution using the forward and reverse primers listed in Table 1, and an amplified product was obtained. The obtained amplified product was treated with each restriction enzyme listed in Table 1 to prepare a restriction enzyme reaction solution. The restriction enzyme reaction solution was applied to a 2% agarose gel, and polymorphism was confirmed by electrophoresis.

[0161] (result) The results of the polymorphism confirmed by electrophoresis are shown in Figures 5 to 7. Figure 4 shows the genotype of the CAPS marker of "OK68".

[0162] The genotype of the CAPS marker of "OK68" was found to be distinguishable from all 24 varieties targeted by this marker, and also from its parent varieties (Tables 2 to 5).

[0163] As described above, "OK68" could be distinguished from other varieties using the CAPS marker. [Industrial Applicability]

[0164] The present invention can be used in the fields of agriculture, plant breeding, and the like.

Claims

1. 1. A method for producing a citrus plant, comprising: A process of crossbreeding the citrus variety "Harehime" as a mother and the citrus variety "Citrus Intermediate Mother No. 6" as a father to obtain a hybrid variety; For the hybrid variety or its progeny plant, genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 were determined to be the following, respectively: Bf0158-3 is type AB, Bf0036-3 is type AA, Tf0150-2 is type AB, Tf0271-2 is type AB, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is type AA, Al0302-2 is AB type, Mf0097-2 is type AB, Mf0090-2 is type AA, Gn0073-2 is type AA, and Gn0048-2 is type AA A method for producing a citrus plant, comprising the step of selecting a plant having a genotype.

2. 2. The method for producing a citrus plant according to claim 1, further comprising a selection step of selecting a citrus plant having the trait of producing seedless fruit and / or the trait of fruit having an average weight of 130 g or more.

3. A production method as described in claim 1 or 2, further comprising a selection step of selecting citrus plants whose juice has a sugar content of 12.0 Brix% or more and / or a sugar-acid ratio of 12.0 or more.

4. The method for producing a citrus plant according to claim 1 or 2, further comprising a propagation step of vegetatively propagating the citrus plant.

5. 1. A method for producing a citrus plant, comprising: The hybridization process involves crossbreeding the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father. and a marker selection step of selecting, from the citrus plant obtained by the hybridization step or a progeny thereof, candidate citrus plants having a different genotype pattern of the genetic markers from those of the parent variety "Harehime" and "Citrus Intermediate Mother No. 6," using at least one genetic marker selected from Bf0158-3, Bf0036-3, Tf0300-3, Tf0419-2, Tf0420-2, Al0302-2, Mf0097-2, Mf0090-2, and Gn0073-2.

6. The method of claim 5, wherein in the marker selection step, a candidate citrus plant line is selected that has a different genotype pattern of the genetic markers from the parent varieties "Harehime" and "Citrus Intermediate Mother No. 6" using a combination of at least one genetic marker selected from Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2 with at least one genetic marker selected from Tf0300-3, Tf0419-2, and Mf0090-2, and at least one genetic marker selected from Bf0036-3 and Gn0073-2.

7. The method of claim 6, wherein candidate citrus plant lines are selected in which the genotypes of the genetic markers are Bf0158-3 AB type, Bf0036-3 AA type, Tf0300-3 AB type, Tf0419-2 AB type, Tf0420-2 AA type, Al0302-2 AB type, Mf0097-2 AB type, Mf0090-2 AA type, and Gn0073-2 AA type.

8. A production method described in any one of claims 5 to 7, including a selection step of selecting citrus plants whose juice has a sugar content of 12.0 Brix% or more and / or a sugar-acid ratio of 12.0 or more.

9. 6. The method for producing a citrus plant according to claim 5, further comprising a propagation step of vegetatively propagating the citrus plant.

10. A citrus plant that is a hybrid variety or a progeny thereof that has been crossed using the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Parent No. 6" as the father, wherein the genotype of a genetic marker in said citrus plant is: Bf0158-3 is type AB, Bf0036-3 is type AA, Tf0150-2 is type AB, Tf0271-2 is type AB, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is type AA, Al0302-2 is AB type, Mf0097-2 is type AB, Mf0090-2 is type AA, Gn0073-2 is type AA, and Gn0048-2 is type AA The citrus plant.

11. 11. The citrus plant of claim 10, having the trait of producing seedless fruit and / or fruit weighing an average of 130 g or more.

12. A citrus plant as described in claim 10, wherein the juice has a sugar content of 12.0 Brix% or more and / or a sugar-acid ratio of 12.0 or more.

13. 11. A method for identifying citrus plants according to claim 10, comprising: amplifying a region of the DNA of a citrus plant, the region including a genetic marker of the DNA, using a primer set; and and identifying the citrus plant based on base polymorphisms in the amplified DNA region; In the identifying step, the genotype of the genetic marker is Bf0158-3 is type AB, Bf0036-3 is type AA, Tf0150-2 is type AB, Tf0271-2 is type AB, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is type AA, Al0302-2 is AB type, Mf0097-2 is type AB, Mf0090-2 is type AA, Gn0073-2 is type AA, and The citrus plant is identified based on the fact that Gn0048-2 is type AA. The method.

14. A fruit of the citrus plant according to claim 10.

15. A processed product of a citrus plant or a fruit of a citrus plant, obtained from the fruit of the citrus plant according to claim 10 or the fruit of the citrus plant according to claim 14, The processed product is at least one selected from the group consisting of fruit juice, fruit pulp, or juice containing fruit juice and fruit pulp, fruit pulp preserved in syrup, dried fruit, jelly, and raw materials for powdered processed confectionery.

16. 11. The citrus plant of claim 10 in the form of a seedling or scion.

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