Citrus fruit plant, method for producing citrus fruit plant, and use thereof

Abstract

Provided are a citrus plant, a method for producing a citrus plant, and use thereof. The method for producing a citrus plant comprises a step for selecting, with respect to a hybrid variety obtained by crossing citrus varieties or a progeny plant thereof, plants having 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 which Bf0158-3 is type AB, Bf0036-3 is type AA, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Al0302-2 is type AB, Mf0097-2 is type AB, Mf0090-2 is type AA, and Gn0073-2 is type AA.

Description

Citrus plants, method for producing citrus plants, and use thereof

[0001] The present invention relates to citrus plants, a method for producing citrus plants, and use thereof.

[0002] In citrus, seedlessness related to ease of eating is essential, but in order to achieve stable production of seedless fruits, it is necessary to have a high degree of female sterility.

[0003] DNA variety identification technology for fruits of 24 citrus varieties using CAPS markers, National Agriculture and Food Research Organization, published on June 17, 2022

[0004] The National Agriculture and Food Research Organization focused on the female sterility derived from seedless Kishu and advanced breeding to develop citrus intermediate female parent cultivars No. 5 and No. 6. However, since their fruits are small fruits of 100 g or less, they have not been widely cultivated economically. Therefore, there is a demand for the development of mid-late-maturing citrus varieties that have female sterility derived from seedless Kishu, can produce stable seedless fruits, and can produce relatively large and high-quality fruits.

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

[0006] Furthermore, a citrus plant according to one aspect of the present invention is a citrus plant in which the genotypes of the genetic markers in the citrus plant are as follows: Bf0158-3 is of type AB, Bf0036-3 is of type AA, Tf0150-2 is of type AB, Tf0271-2 is of type AB, Tf0300-3 is of type AB, Tf0419-2 is of type AB, Tf0420-2 is of type AA, Tf0318-2 is of type BB, Tf0293-4 is of type AA, Al0302-2 is of type AB, Mf0097-2 is of type AB, Mf0090-2 is of type AA, Gn0073-2 is of type AA, and Gn0048-2 is of type AA. Furthermore, a citrus plant according to one aspect of the present invention is a citrus plant having the following characteristics (1), (2), (3), and (4): (1) obtained by crossing the citrus variety "Harehime" as the mother plant and the citrus variety "Citrus Intermediate Mother No. 6" as the father plant; (1) capable of producing seedless fruit; (2) having a fruit weight of 160 g or more; (3) having a fruit with high sugar content and good taste; (4) It is distinguishable from other citrus varieties, including "Harehime" and "Citrus Intermediate Parent No. 6", using a genetic marker which is at least one of the following: 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] Furthermore, a method for identifying citrus plants according to one aspect of the present invention is a method for identifying citrus plants, comprising the steps of: amplifying a region in the DNA of a citrus plant that includes a genetic marker (CAPS marker) using a primer set; and identifying the citrus plant based on the polymorphism of the bases in the amplified DNA region, wherein 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.

[0008] Furthermore, a citrus plant or its fruit according to one aspect of the present invention is a citrus plant or its fruit obtained by the method for producing the citrus plant described above.

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

[0010] According to the present invention, it is possible to provide new citrus plants, methods for creating citrus plants, and their uses.

[0011] This is a phylogenetic tree of "Citrus OK68". This is a photograph of the fruit of "Citrus OK68". This is a photograph of the tree of "Citrus OK68". This is a diagram showing the genotype of the CAPS marker of "Citrus OK68". This is a diagram showing the results of PCR amplification of DNA extracted from "Citrus OK68" using the genetic markers (CAPS markers) Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, or Tf0419-2, followed by digestion with a specific restriction enzyme, and then examination of the genotype of each genetic marker by agarose gel electrophoresis. In the diagram, M shows the electrophoresis pattern when a 100 bp ladder is electrophoresed, and S shows the electrophoresis pattern when the restriction enzyme-treated DNA of "Citrus OK68" is electrophoresed. This figure shows the results of examining the genotype of each genetic marker by agarose gel electrophoresis after PCR amplification of DNA extracted from "Citrus OK68" using the genetic markers Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, or Mf0090-2, followed by digestion with a specific restriction enzyme. In the figure, M shows the electrophoresis pattern when a 100 bp ladder is electrophoresed, and S shows the electrophoresis pattern when DNA from "Citrus OK68" after restriction enzyme treatment is electrophoresed. This figure shows the results of examining the genotype of each genetic marker by agarose gel electrophoresis after PCR amplification of DNA extracted from "Citrus OK68" using the genetic markers Gn0073-2 or Gn0048-2, followed by digestion with a specific restriction enzyme. In the figure, M shows the electrophoresis pattern when a 100 bp ladder is subjected to electrophoresis, and S shows the electrophoresis pattern when DNA treated with restriction enzyme "Citrus OK68" is subjected to electrophoresis.

[0012] The present invention will be described in detail below. All references cited herein are incorporated herein by reference.

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

[0014] In this specification, "plant" may refer to a part or all of a plant body, or to any stage in the development of a plant.

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

[0016] In this specification, “trait” means the morphological and physiological characteristics of a plant. In this specification, CAPS means Cleaved Amplified Polymorphic Sequence (CAPS).

[0017] [Method for Producing Citrus Plants 1] A method for producing citrus plants according to one aspect of the present invention is a method for producing citrus plants, wherein, in a hybrid variety obtained by crossing citrus varieties with each other, or in a descendant plant, 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 are as follows: Bf0158-3 is of type AB, Bf0036-3 is of type AA, Tf0150-2 is of type AB, The present invention relates to a method for producing citrus plants, comprising the step of selecting plants having a genotype in which Tf0271-2 is of type AB, Tf0300-3 is of type AB, Tf0419-2 is of type AB, Tf0420-2 is of type AA, Tf0318-2 is of type BB, Tf0293-4 is of type AA, Al0302-2 is of type AB, Mf0097-2 is of type AB, Mf0090-2 is of type AA, Gn0073-2 is of type AA, and Gn0048-2 is of type AA.

[0018] In one embodiment, the production method may further include a step of crossing the citrus variety "Harehime" as the mother plant and the citrus variety "Citrus Intermediate Mother Plant No. 6" as the father plant to obtain the hybrid variety. In one embodiment, the plants used in the selection step may be hybrid varieties obtained by crossing citrus varieties with each other, or they may be their progeny, but it is preferable that they be hybrid varieties obtained by crossing citrus varieties with each other. In one embodiment, the production method may further include a propagation step of asexually propagating citrus plants.

[0019] (Selection process using markers (identification process)) In one embodiment, the production method involves selecting hybrid varieties obtained by crossing citrus varieties with each other, or their offspring, 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 is as follows: First, DNA is extracted from a hybrid variety or its progeny obtained by crossing citrus varieties using an existing method. Next, PCR amplification of the extracted DNA is performed using forward primer and reverse primer pairs corresponding to each genetic marker listed in the table below. The amplified DNA is cut with each restriction enzyme listed in the table below. The DNA after restriction enzyme treatment is run on an agarose gel electrophoresis, and the electrophoresis pattern is confirmed. The genotype of each genetic marker can be determined by the difference in the electrophoresis pattern. This makes it possible to identify citrus varieties. Therefore, the production method may further include the step of extracting DNA from a hybrid variety or its progeny obtained by crossing citrus varieties. The production method may further include the step of amplifying the extracted DNA using forward primer and reverse primer pairs corresponding to each genetic marker. The production method may further include the step of treating the amplified DNA with restriction enzymes. Furthermore, the production method may further include a step of performing electrophoresis on the DNA after restriction enzyme treatment. Furthermore, the production method may further include a step of determining the genotype for each genetic marker from the electrophoretic pattern of the DNA obtained by electrophoresis. Furthermore, the production method may further include a step of selecting plants in which each genetic marker has the above genotype based on the genotype determination results for each genetic marker.

[0021]

[0022] The genotype of Bf0158-3 can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. Type AA is defined as when a band is observed around 352 (±10) bp and no bands are observed around 149 (±10) bp and 203 (±10) bp. Type BB is defined as when bands are observed around 149 (±10) bp and 203 (±10) bp and no band is observed around 352 (±10) bp. Type AB is defined as when bands are observed around 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 electrophoretic patterns. Type AA is defined as when a band is observed around 399 (±10) bp and no bands are observed around 162 (±10) bp and 237 (±10) bp. Type BB is defined as when bands are observed around 162 (±10) bp and 237 (±10) bp and no band is observed around 399 (±10) bp. Type AB is defined as when bands are observed around 162 (±10) bp, 237 (±10) bp, and 399 (±10) bp.

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

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

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

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

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

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

[0030] The genotype of Tf0293-4 can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. Type AA is defined as when a band is observed around 394 (±10) bp and no bands are observed around 157 (±10) bp and 237 (±10) bp. Type BB is defined as when bands are observed around 157 (±10) bp and 237 (±10) bp and no band is observed around 394 (±10) bp. Type AB is defined as when bands are observed around 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 electrophoretic patterns. Type AA is defined as when a band is observed around 383 (±10) bp and no bands are observed around 167 (±10) bp and 216 (±10) bp. Type BB is defined as when bands are observed around 167 (±10) bp and 216 (±10) bp and no band is observed around 383 (±10) bp. Type AB is defined as when bands are observed around 167 (±10) bp, 216 (±10) bp, and 383 (±10) bp.

[0032] The genotype of Mf0097-2 can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. Type AA is defined as when a band is observed around 369 (±10) bp and no bands are observed around 166 (±10) bp and 203 (±10) bp. Type BB is defined as when bands are observed around 166 (±10) bp and 203 (±10) bp and no band is observed around 369 (±10) bp. Type AB is defined as when bands are observed around 166 (±10) bp, 203 (±10) bp, and 369 (±10) bp.

[0033] The genotype of Mf0090-2 can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. Type AA is defined as when a band is observed around 400 (±10) bp and no bands are observed around 139 (±10) bp and 261 (±10) bp. Type BB is defined as when bands are observed around 139 (±10) bp and 261 (±10) bp and no band is observed around 400 (±10) bp. Type AB is defined as when bands are observed 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 electrophoretic patterns. Type AA is defined as when a band is observed around 394 (±10) bp and no bands are observed around 149 (±10) bp and 245 (±10) bp. Type BB is defined as when bands are observed around 149 (±10) bp and 245 (±10) bp and no band is observed around 394 (±10) bp. Type AB is defined as when bands are observed 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 electrophoretic patterns. Type AA is defined as 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 defined as when bands are observed around 134 (±10) bp and 246 (±10) bp and no band is observed around 380 (±10) bp. Type AB is defined as when bands are observed around 134 (±10) bp, 246 (±10) bp, and 380 (±10) bp.

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

[0037] In one embodiment, the production method may further include the step of obtaining the hybrid variety by crossing the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father. Both "Harehime" and "Citrus Intermediate Mother No. 6," which are parent varieties used in the cross, are varieties that are available on the market. "Harehime" is a variety with good taste and easy peeling, and is registered as Japanese variety registration number: No. 12069. "Citrus Intermediate Mother No. 6" has female sterility derived from seedless Kishu, is a variety that can produce seedless fruit stably and has excellent taste, and is a variety developed by the National Agriculture and Food Research Organization. "Citrus Intermediate Mother No. 6" is registered as Japanese variety registration number: No. 12070. The citrus plants selected in the selection process have the genotype pattern of the above-mentioned genetic markers. In contrast, other citrus varieties of "Harehime" have the following characteristics: Bf0036-3 is type AB, Tf0300-3 is type BB, Tf0419-2 is type BB, Mf0090-2 is type AB, and Gn0073-2 is type AB. Therefore, Bf0036-3, Tf0300-3, Tf0419-2, Mf0090-2, and / or Gn0073-2 can be used to distinguish it from other citrus varieties of "Harehime". Furthermore, in the other citrus variety "Citrus Intermediate Parent No. 6", Bf0158-3 is of type BB, Bf0036-3 is of type AB, Tf0420-2 is of type AB, Al0302-2 is of type AA, Mf0097-2 is of type BB, and Gn0073-2 is of type AB. Therefore, Bf0158-3, Bf0036-3, Tf0420-2, Al0302-2, Mf0097-2, and / or Gn0073-2 can be used to distinguish it from the other citrus variety "Citrus Intermediate Parent No. 6". Also, Bf0036-3, and / or Gn0073-2 can be used to distinguish it from both the other citrus variety "Harehime" and the other citrus variety "Citrus Intermediate Parent No. 6".

[0038] (Estimation of the Lineages of the Mating Parents) The genotypes described in Tables 2 to 5 below represent the combinations of alleles (alleles) for each variety detected at each genetic marker. Since citrus is usually a diploid plant, the two alleles found in the offspring must have been inherited one each from the seed parent and the pollen parent. For example, if a variety has a genotype of AB, either parent having the A or B allele could be a mating parent, but if both parents are of the BB type, the possibility that this combination is the parent is negated. Based on this principle, mating parent candidates can be estimated from the information of 14 genetic markers. The citrus plants having the genotype selected through the selection process using the above genetic markers have different genotypes of genetic markers from other citrus varieties, as is clear from Tables 2 to 5 below. Therefore, the citrus plants selected by the above selection process can be created separately from other citrus varieties.

[0039]

[0040]

[0041]

[0042]

[0043] In one embodiment, the production method includes the steps of selecting a citrus plant having the trait of producing seedless fruit and / or selecting a citrus plant having the trait of having a fruit weight of 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 method includes the steps of selecting a citrus plant having the trait of producing seedless fruit and selecting a citrus plant having the trait of having a fruit weight of 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). "Harehime" is a seedless variety that utilizes male sterility, but it may form seeds when pollinated with pollen from other varieties, and there are limitations to the stable production of seedless fruit. In contrast, the citrus plant of the present invention is female sterility and does not form seeds even when pollinated with pollen from other varieties, making it possible to stably produce seedless fruit. Furthermore, while "Intermediate Parent No. 6" has small fruits weighing about 120g, "Sakimeki" has slightly larger fruits weighing about 180g, and thus has characteristics that clearly distinguish it in terms of fruit size. Therefore, it can be further distinguished from "Harehime" and / or "Intermediate Parent No. 6" by its trait of producing seedless fruit and having large fruit weight.

[0044] In one embodiment, the production method may further include a preliminary selection step before the selection step, in which candidate citrus plants are selected by identifying those that have a different genotype pattern of the genetic marker from the parent plants "Harehime" and "Citrus Intermediate Parent No. 6" using a genetic marker (CAPS marker) which is at least one of 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 process, the genotype patterns of all 14 types of CAPS markers may be determined, but depending on the purpose of selection, only some of the 14 types of CAPS markers may be used. For example, in the marker selection process, Bf0036-3 and / or Gn0073-2 are used to select candidate citrus plant lines whose genotype patterns of the genetic markers differ from those of other citrus varieties, such as "Harehime" and "Citrus Intermediate Parent No. 6". In one embodiment, in the marker selection step, candidate citrus plant lines are selected that have a different genotype pattern of the genetic markers from the parent varieties "Harehime" and "Citrus Intermediate Parent No. 6" by using a combination of at least one genetic marker from Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2, and at least one genetic marker selected from Tf0300-3, Tf0419-2, and Mf0090-2, as well as at least one genetic marker selected from at least one genetic marker from Bf0036-3 and Gn0073-2. In one embodiment, in the preliminary selection process, at least one genetic marker from Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2 is used to select candidate citrus plant lines whose genotype patterns differ from those of the parent variety "Citrus Intermediate Mother No. 6", and at least one genetic marker from Tf0300-3, Tf0419-2, and Mf0090-2 is used to select candidate citrus plant lines whose genotype patterns differ from those of "Harehime", or at least one selected from at least one genetic marker from Bf0036-3 and Gn0073-2 is used to select candidate citrus plant lines whose genotype patterns differ from those of the parent varieties "Harehime" and "Citrus Intermediate Mother No. 6".In one embodiment, the production method further includes a step of crossing the citrus variety "Harehime" as the mother (seed parent) with the citrus variety "Citrus Intermediate Female Parent No. 6" as the father (pollen parent) to obtain the hybrid variety, and a preliminary selection step of selecting a line of candidate citrus plants in which the genotype of the genetic marker Bf0036-3 is AA and the genotype of Gn0073-2 is AA. In one embodiment, the production method further includes a step of crossing the citrus variety "Harehime" as the mother (seed parent) with the citrus variety "Citrus Intermediate Female Parent No. 6" as the father (pollen parent) to obtain the hybrid variety, and a preliminary selection step of selecting a line of candidate citrus plants in which the genotype of the genetic marker Bf0158-3 is AB, the genotype of Tf0420-2 is AA, the genotype of Al0302-2 is AB, the genotype of Mf0097-2 is AB, the genotype of Tf0300-3 is AB, the genotype of Tf0419-2 is AB, and the genotype of Mf0090-2 is AA.

[0046] (Selection step based on traits) In one embodiment, the production method may further include a selection step based on traits. The selection step based on traits may be performed before, after, or simultaneously with the selection step using genetic markers, but it is preferably performed after the selection step using genetic markers. By performing further selection based on traits after performing selection using genetic markers, it is possible to more reliably select citrus plants that are different from other varieties and have good traits.

[0047] The main traits used for selection include, for example, the ability to produce seedless fruit, a fruit weight of 160g or more, high sugar content and good taste, soft segment membranes, high biennial bearing, and a fruit maturation period from late February to early March. Furthermore, the main traits used for selection include the ability to produce seedless fruit and / or a fruit weight of 130g or more on average (preferably 140g or more on average, more preferably 150g or more on average, even more preferably 160g or more on average, and even more preferably 170g or more on average). Preferably, the traits are the ability to produce seedless fruit and a fruit weight of 130g or more on average (preferably 140g or more on average, more preferably 150g or more on average, even more preferably 160g or more on average, and even more preferably 170g or more on average). Details of these traits are described in the following section [Citrus Plants]. It is also preferable to use the traits described in the section on citrus plants for selection.

[0048] In a method for producing citrus plants according to one aspect of the present invention, the preliminary selection step, primary selection step, and secondary selection step described in, for example, Method for Producing Citrus Plants 2 may be further included.

[0049] (Propagation step) A method for producing citrus plants according to one aspect of the present invention may further include a propagation step of asexually propagating the citrus plants cultivated through the selection step.

[0050] Methods of asexual propagation include grafting, cuttings, layering, or cell culture. Asexual propagation is also called unsexual propagation, asexual reproduction, or vegetative propagation. In asexual propagation, male and female are not required; a single individual independently forms a new individual, with germ cells produced by a single individual independently becoming a new individual. Furthermore, techniques for creating cell cultures of plant tissue and methods for regenerating plants from tissue cultures are carried out using known techniques.

[0051] In this invention, the rootstock used for grafting can be various tree species that can be used as citrus trees or citrus rootstock, such as trifoliate orange, Satsuma mandarin, and shikwasa. Furthermore, the type and size of the rootstock used may be changed depending on the soil, climate, etc. For example, trifoliate orange can be used as the rootstock.

[0052] The cultivation methods for citrus plants according to the present invention are not particularly limited, but include grafted or seedling forms, and include open-field cultivation, unheated greenhouse cultivation, or covered greenhouse cultivation.

[0053] [Method for Producing Citrus Plants 2] A method for producing citrus plants according to one aspect of the present invention is a method for producing citrus plants, comprising: a crossing step of crossing the citrus variety "Harehime" as the mother plant and the citrus variety "Citrus Intermediate Mother No. 6" as the father plant; and a marker selection step of selecting candidate citrus plants from the citrus plants obtained by the crossing step or from the citrus plants of their progeny that have a different genotype pattern of the genetic marker from the parent varieties "Harehime" and "Citrus Intermediate Mother No. 6", using at least one of the genetic markers Bf0158-3, Bf0036-3, Tf0300-3, Tf0419-2, Tf0420-2, Al0302-2, Mf0097-2, Mf0090-2 and Gn0073-2.

[0054] Furthermore, the method of creation can also be referred to as the manufacturing method, cultivation method, or production method.

[0055] (Crossing process) In the crossing process, "Harehime" is used as the mother (seed parent) and "Citrus Intermediate Mother No. 6" is used as the father (pollen parent) for crossbreeding.

[0056] The parent varieties used in the crossbreeding, "Harehime" and "Citrus Intermediate Parent No. 6," are both varieties that are commercially available. "Harehime" is a variety with good taste and easy peeling, and is registered as Japanese variety registration number: No. 12069. "Citrus Intermediate Parent No. 6" is a variety that possesses female sterility derived from seedless Kishu, enabling stable seedless fruit production and having 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: No. 12070.

[0057] (Selection process using markers (identification process)) In the selection process using markers in one embodiment, candidate citrus plants are selected from the citrus plants obtained in the cross-pollination process or their progeny using a genetic marker (CAPS marker) which 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, by identifying candidate citrus plants that have a different genotype pattern of the genetic marker from the parent varieties "Harehime" and "Citrus Intermediate Parent No. 6".

[0058] The identification of parent varieties and other varieties in marker selection will be described in detail in the following section, [Methods for Identifying Citrus Plants].

[0059] While the genotype patterns of all 14 types of CAPS markers mentioned above may be determined, depending on the purpose of selection, only some of the 14 types of CAPS markers may be used. In one example, in the marker selection process, a combination of at least one genetic marker from among 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 whose genotype patterns differ from those of the parent varieties "Harehime" and "Citrus Intermediate Mother No. 6". In one embodiment, in the marker selection step, candidate citrus plant lines are selected that have a different genotype pattern of the genetic markers from the parent varieties "Harehime" and "Citrus Intermediate Parent No. 6" by using a combination of at least one genetic marker from Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2, and at least one genetic marker selected from Tf0300-3, Tf0419-2, and Mf0090-2, as well as at least one genetic marker selected from at least one genetic marker from Bf0036-3 and Gn0073-2.

[0060] In one embodiment, in the marker selection process, at least one genetic marker from Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2 is used to select candidate citrus plant lines whose genotype pattern differs from that of the parent variety "Citrus Intermediate Mother No. 6", and at least one genetic marker from Tf0300-3, Tf0419-2, and Mf0090-2 is used to select candidate citrus plant lines whose genotype pattern differs from that of "Harehime", or at least one selected from at least one genetic marker from Bf0036-3 and Gn0073-2 is used to select candidate citrus plant lines whose genotype pattern differs from that of the parent varieties "Harehime" and "Citrus Intermediate Mother No. 6". In one embodiment, it is preferable to select candidate citrus plant lines in which the genotypes of the genetic markers Bf0036-3 are AA and Gn0073-2 is AA. In another embodiment, it is preferable to select candidate citrus plant lines in which the genotypes of the genetic markers Bf0158-3 are 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 types of CAPS markers are as follows: 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 type AB, Mf0097-2 is type AB, Mf0090-2 is type AA, Gn0073-2 is type AA, and Gn0048-2 is type AA.

[0062] (Selection process based on traits) In order to obtain the citrus plants according to this embodiment, a selection process based on specific traits can also be performed on candidate citrus plants obtained by crossing the citrus variety "Harehime" as the mother plant and the citrus variety "Citrus Intermediate Mother Plant No. 6" as the father plant. The selection process based on specific traits can also be performed together with the selection process using genetic markers as indicators. It can also be performed before the selection process using genetic markers as indicators. In a preferred example, after performing the selection process using genetic markers as indicators, the selection process based on specific traits is performed on the lines of candidate citrus plants that have been sufficiently narrowed down.

[0063] The main traits used for selection include, for example, the ability to produce seedless fruit, a fruit weight of 160g or more, high sugar content and good taste, soft segment membranes, high biennial bearing, and a fruit maturation period from late February to early March. Furthermore, the main traits used for selection include the ability to produce seedless fruit and / or a fruit weight of 130g or more on average (preferably 140g or more on average, more preferably 150g or more on average, even more preferably 160g or more on average, and even more preferably 170g or more on average). Preferably, the traits are the ability to produce seedless fruit and a fruit weight of 130g or more on average (preferably 140g or more on average, more preferably 150g or more on average, even more preferably 160g or more on average, and even more preferably 170g or more on average). Details of these traits are described in the following section [Citrus Plants].

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

[0065] In other words, the selection process may include, for example, a preliminary selection process, a primary selection process, and a secondary selection process.

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

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

[0068] Next, the second selection process may be a process of selection based on the following criterion (h): (h) System adaptability test (regional adaptability assessment).

[0069] (Propagation step) A method for producing citrus plants according to one aspect of the present invention further includes a propagation step of asexually propagating the citrus plants that have been cultivated through the above-mentioned hybridization step and selection step.

[0070] Methods of asexual propagation include grafting, air layering, or cell culture. Asexual propagation is also called asexual propagation, asexual reproduction, or vegetative propagation. In asexual propagation, male and female are not required, and a single individual independently forms a new individual; germ cells produced by a single individual independently become a new individual. Furthermore, techniques for creating cell cultures of plant tissue and methods for regenerating plants from tissue cultures are carried out using known techniques.

[0071] In this invention, the rootstock used for grafting can be various tree species that can be used as citrus trees or citrus rootstock, such as trifoliate orange, Satsuma mandarin, and shikwasa. Furthermore, the type and size of the rootstock used may be changed depending on the soil, climate, etc. For example, trifoliate orange can be used as the rootstock.

[0072] The cultivation methods for citrus plants according to the present invention are not particularly limited, but include grafted or seedling forms, and include open-field cultivation, unheated greenhouse cultivation, or covered greenhouse cultivation.

[0073] [Method for Identifying Citrus Plants] As described above, the citrus plants of the present invention can be identified by specific variety identification markers.

[0074] Development of DNA variety identification technology has been underway. This technology is effective for monitoring or proving infringement of plant breeders' rights. In order to prevent imports at borders such as customs, development is underway to enable simple and rapid variety identification at customs inspection sites.

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

[0076] CAPS marker analysis technology is a technique that utilizes differences in the size of restriction enzyme fragments resulting from polymorphisms in genomic DNA, such as base substitutions, deletions, and insertions, that exist between varieties, by cutting PCR-amplified fragments targeting specific genomic regions with specific restriction enzymes. Analysis using this technology does not require expensive equipment, making it highly feasible for widespread adoption in testing facilities.

[0077] The CAPS marker analysis method detects polymorphisms by amplifying the target region using PCR and then treating the product with a restriction enzyme that recognizes a specific base sequence and cleaves the DNA. 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 an agarose gel, etc. In order to identify the variety, 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 Management Center").

[0078] The method for identifying citrus plants according to the present invention is a method for distinguishing the citrus variety "OK68" or its progeny from other citrus varieties by using a CAPS marker as an identification marker.

[0079] A method for identifying citrus plants according to one embodiment of the present invention comprises 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 the base polymorphism of 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 marker (CAPS marker) may be 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, or a combination of all of them, but it is preferable to use all of them. In one embodiment, a method for identifying citrus plants can distinguish the citrus plants of the present invention from other citrus varieties based on the polymorphism of bases in the region (DNA region) containing the amplified genetic marker. Examples of other citrus varieties include citrus varieties other than OK68 listed in Tables 2 to 5.

[0080] Since polymorphisms in CAPS are determined by the presence or absence of restriction enzyme sites, alleles without restriction enzyme sites are designated as A, and alleles with restriction enzyme sites are designated as B, allowing for the determination of the genotypes AA, AB, and BB of the varieties.

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

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

[0083] Furthermore, the allele type being the same as "OK68" refers to the allele type shown in Figure 4 for the 14 loci from Bf0158-3 to Gn0048-2. Even if mutations occur in the base sequences constituting these gene loci and their surrounding regions, the variety identification technology of the present invention can still be applied if the allele type originates from "OK68".

[0084] In the identification technology 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" in at least one of the 14 identification markers. In citrus plants belonging to other citrus varieties, the 14 allele types Bf0158-3 to Gn0048-2 show different allele types from "OK68".

[0085] Here, other citrus varieties that can be distinguished from "OK68" using the above-mentioned identification technology include, for example, "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", "Reikou", "Tsunohikari", "Seinan no Hikari", "Tsunono Nozomi", "Haruhi", "Kiyomi", "Setoka", "Harumi", "Harehime", "Kanpei", and "Ehime Fruit Experiment Station No. 28 (Beni Madonna)", as shown in the manual of Non-Patent Literature 1.

[0086] These 24 citrus varieties, when combined with "OK68" (a total of 25 varieties), account for more than 94% of the total 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, Hyuga Natsu, sudachi, shikwasa, calamondin (kara), tankan, seminole, kabosu, Amakusa, Ougonkan, Kishu Mikan, Sweet Spring, Encore, Tamami, Nishinoka, Murcott, Nanka, Amaka, Citrus Intermediate Parent No. 6, bitter orange, etc.

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

[0089] Furthermore, considering the results of the following examples, 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. For example, pomelos can also be recognized as other citrus varieties that can be distinguished from "OK68" from the principle of the identification technology of the present invention.

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

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

[0092] Furthermore, to distinguish "OK68" from the parent varieties "Harehime" and "Citrus Intermediate Mother No. 6", it is sufficient to use a combination of at least one genetic marker from among Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2 and 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. If it is sufficient to distinguish only "OK68" from its parents, it is sufficient to use at least one of Bf0036-3 and Gn0073-2.

[0093] In an identification method according to one aspect of the present invention, the citrus plant being tested is a candidate plant for breeding material, a plant obtained in the breeding process, etc. Candidate plants for breeding material include, for example, parent plants used in crossbreeding and plants used in molecular breeding using genetic engineering technology. Furthermore, the citrus plant being tested includes citrus plants used in molecular breeding using genetic engineering 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 citrus plants according to the present invention, comprising: (I) a step of amplifying a portion of the DNA of a citrus plant using a primer set capable of amplifying the CAPS marker region described above; and (II) a step of identifying the citrus plant based on the polymorphism of the bases in the amplified DNA region.

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

[0096] If the length of the DNA amplified by PCR using the primer set obtained from DNA obtained from a citrus plant matches the length of the DNA fragment obtained when the DNA amplified by the PCR method using the primer set is treated with restriction enzymes, then it can be determined that the DNA is from a known citrus variety.

[0097] In other words, in an identification method according to one aspect of the present invention, the step of (II) determining a citrus plant based on the polymorphism of bases in an amplified DNA region includes (II-1) a step of restriction enzyme treatment of the amplified DNA fragment, and (II-2) a step of identifying the genotype based on the length of the DNA fragment obtained by restriction enzyme treatment.

[0098] Conventional methods can be used to determine the genotype of the amplified fragment, and as an example, a method using agarose gel electrophoresis is preferred.

[0099] The length of a DNA fragment can be measured using, for example, its molecular weight. The molecular weight can be measured using, for instance, the migration distance in agarose gel electrophoresis. It can also be measured by DNA sequencing.

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

[0101] While there are no particular limitations on the means for detecting PCR amplification fragments, one example is the use of ethidium bromide, fluorescent substances, chromogenic substances, luminescent substances, etc.

[0102] The primers in the primer set described above consist of a base sequence having 90% or more sequence identity with the base sequence of the nucleotides constituting each primer in the primer set described above. Modified primers consisting of a modified base sequence obtained by modifying some of the bases in the base sequence are also included in the scope of primers used in the identification method of the present invention. Furthermore, the sequence identity of the modified primer with the base sequence of each primer may be 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%. In principle, a modified primer is considered equivalent to each primer if it can hybridize with the complementary DNA strand of each primer. Furthermore, these equivalent modified primers may function as primers that hybridize with the complementary DNA strand of the original primer, and may consist 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 are added, deleted, inserted, or substituted in the base sequence of the oligonucleotide constituting each of the original primers.

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

[0104] The present invention provides a method for identifying citrus plants that can accurately and quickly identify the citrus variety "OK68" or its offspring from other citrus varieties, and is also a method that can be widely introduced in inspection sites and other settings.

[0105] A method for identifying citrus plants according to one aspect of the present invention comprises the steps of: amplifying a region of the DNA of a citrus plant containing a genetic marker (CAPS marker) using a primer set; and identifying the citrus plant based on the base polymorphism of the amplified DNA region, wherein the genetic marker (CAPS marker) is preferably 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 citrus plants according to one aspect of the present invention comprises the steps of amplifying a region of the DNA containing a genetic marker (CAPS marker) in the DNA of a citrus plant using a primer set, and identifying the citrus plant based on the polymorphism of the bases in the amplified DNA region, wherein the genetic marker (CAPS marker) is preferably 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. It is also preferable to identify citrus plants according to one aspect of the present invention if each genetic marker (CAPS marker) has the above-mentioned genotype.

[0106] [Citrus Plant 1] A citrus plant according to one aspect of the present invention is a citrus plant in which the genotypes of the genetic markers in the citrus plant are as follows: Bf0158-3 is of type AB, Bf0036-3 is of type AA, Tf0150-2 is of type AB, Tf0271-2 is of type AB, Tf0300-3 is of type AB, Tf0419-2 is of type AB, Tf0420-2 is of type AA, Tf0318-2 is of type BB, Tf0293-4 is of type AA, Al0302-2 is of type AB, Mf0097-2 is of type AB, Mf0090-2 is of type AA, Gn0073-2 is of type AA, and Gn0048-2 is of type AA. As is clear from Tables 2 to 5 above, the citrus plant according to one aspect of the present invention has a different genetic marker genotype from other citrus varieties. Therefore, the citrus plant according to one aspect of the present invention is a novel citrus plant. In one embodiment, the citrus plant may be a hybrid variety or a progeny plant obtained by crossing 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, but it is preferable that it be a hybrid variety obtained by crossing citrus varieties with each other. In one embodiment, the citrus plant preferably has the trait of producing seedless fruit and / or the 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), and more preferably has the trait of producing seedless fruit and the 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).

[0107] [Citrus Plant 2] A citrus plant according to one aspect of the present invention is a citrus plant having the following characteristics (1), (2), (3), and (4): (1) obtained by crossing the citrus variety "Harehime" as the mother plant and the citrus variety "Citrus Intermediate Mother Plant No. 6" as the father plant; (1) capable of producing seedless fruit; (2) having a fruit weight of 160 g or more; (3) having a fruit with high sugar content and good taste; (4) It is distinguishable from other citrus varieties, including "Harehime" and "Citrus Intermediate Parent No. 6", using a genetic marker which is at least one of the following: 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 are described below. Furthermore, a citrus plant according to a further embodiment of the present invention is a citrus plant that further has at least one of the following traits (5) to (7): (5) soft segment membranes, (6) high biennial bearing, and (7) fruit maturation period is from late February to early March.

[0109] It should be noted that the characteristics of the citrus plants according to the present invention may vary depending on conditions such as climate, soil, environment, cultivation method, and tree age during cultivation or growing. Furthermore, in this specification, the time limitations such as the maturation period are based on cultivation in Japan, and for example, Shizuoka City, Shizuoka Prefecture, where the plants were grown in the examples, may serve as the standard.

[0110] Furthermore, a citrus plant according to one aspect of the present invention is a plant that has at least one of the following traits as its main characteristic that differs from the parent variety "Harehime" or "Citrus Intermediate Parent No. 6". Note that the numerical values ​​may be the average of measurements taken over two or more years.

[0111] (Characteristics of the tree) The branching angle is somewhat narrow, and the tree shape is "somewhat upright". The tree's vigor is "somewhat strong". The density of the branches and canopies is "dense", their length is "long", and their thickness is "thick". The rate of thorn development on the branches and canopies (spring branches) is high, but the length of the thorns is short.

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

[0113] The flowers are solitary and do not form inflorescences. They have five white petals. The number of filaments is sparse, and some are fused together. Pollen production is relatively low. The style is arch-shaped.

[0114] Furthermore, while the onset of fruit coloring varies from year to year, it is generally in late October, with full coloration occurring in early to mid-December. Flowering is moderate, biennial bearing is high, and late-season fruit drop is minimal and not problematic. Yield is moderate.

[0115] (Fruit characteristics) The fruit shape is "oblate," with a fruit shape index of approximately 127. The fruit apex is "flat," and the shape of the pedicel is "cut flat," although a short neck may occur depending on the year. There are no radial grooves at the fruit apex, and the pedicel is "medium" in size. The peel is "dark orange." The surface is "somewhat smooth." The peel is thin, about 2.5 mm thick, and is "somewhat easy" to peel. The peel has an orange-like aroma.

[0116] The flesh is a deep orange color, and the flesh content is high. The segment membranes are soft, and the flesh is somewhat soft. Each fruit contains no complete seeds, and the percentage of seedless fruits is 100% under normal open-field cultivation conditions. Because it possesses female sterility derived from seedless Kishu grapes, stable seedless fruit production is possible.

[0117] While there are a small number of sunburnt fruits, no instances of peeling or cracking have been observed. Furthermore, no physiological pithiness has been observed as of the late January survey.

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

[0119] The characteristics of each trait mentioned above may be characteristic values ​​evaluated based on the "Criteria for Examination of Other Citrus Fruits (Citrus L.) and Methods for Testing Adaptability and Trait Testing of Breeding Lines" (National Agriculture and Food Research Organization, Fruit Tree Research Institute, 2007), published by the Ministry of Agriculture, Forestry and Fisheries of Japan.

[0120] Regarding the traits of the citrus plant according to the present invention, for example, trait (1) the ability to produce seedless fruit means that there are no complete seeds. In a survey of five or more fruits, the number of seeds in which the embryo size has developed to more than half that of a normal seed (complete seeds) is evaluated as follows: many: 5 or more seeds, medium: 3 to 5 seeds, few: 1 to 2 seeds or less, or none: 0 seeds. In one example, under natural pollination conditions in open field cultivation, there were almost no complete seeds, making seedless fruit production possible. In another example, the proportion of seedless fruit was 100% under normal open field cultivation conditions, and it possesses female sterility derived from seedless Kishu, enabling stable seedless fruit production.

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

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

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

[0124] In this specification, "sourness" refers to the citric acid content in the fruit juice. The sourness is measured by converting it from the titration value of the fruit juice with a sodium compound aqueous solution. The lower limit of the sugar content of the citrus fruit juice of the present invention is preferably 12.0 Brix% or higher, more preferably 13.0 Brix% or higher, more preferably 14.0 Brix% or higher, more preferably 15.0% Brix% or higher, and even more preferably 16.0% Brix% or higher. The upper limit of the sugar content is not particularly limited, but assuming that it is above any of the lower limits, for example, it may be 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 assuming that it is less than or equal to any of the upper limits, it is, 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 "sugar-acid ratio" is calculated as the ratio of the sugar content meter reading to the citric acid content. Examples of sugar-acid ratios include 12.0 or higher, 13.0 or higher, 14.0 or higher, or 15.0 or higher.

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

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

[0129] Regarding the characteristic (5) soft segment membrane of the citrus plant according to the present invention, this refers to the degree of hardness of the segment membrane when eaten. For example, it can be evaluated as follows: hard: too hard to eat with the segment, slightly hard: there is resistance when eating with the segment, medium: can be eaten with the segment, slightly soft: can be easily eaten with the segment, soft: the segment breaks thinly and there is no resistance when eating.

[0130] (6) High biennial bearing is evaluated as follows: for example, if the on-year and off-year are clearly defined in observational studies over several years, based on the condition that fruit thinning is performed appropriately from the third year after the first fruit set, it is evaluated as high (strong); if it is moderate, it is evaluated as medium; and if the on-year and off-year are unclear, it is evaluated as low (few).

[0131] Regarding the characteristic (7) of the citrus plant according to the present invention, that the fruit matures from late February to early March, it is preferable that the cultivation method during growth is, for example, grafting and open-field cultivation, in which case the maturation period is from late February to early March, and the fruit maturation period may be from early December to late March depending on the growing area and cultivation conditions. In other words, the maturation period is later than 'Harehime', and in one example it is about 4 to 12 weeks or 6 to 12 weeks later than 'Harehime'. It is also slightly later than 'Citrus Intermediate Parent Line No. 6', and in one example it is about 1 to 6 weeks or 2 to 4 weeks later than 'Citrus Intermediate Parent Line No. 6'.

[0132] The citrus plant according to the present invention may be a plant having characteristics equivalent to "OK68". A plant having equivalent characteristics means a plant that, when grown under the same environmental conditions, possesses the main characteristics of "OK68". The main characteristics are those described in (1) to (4) above. It is also preferable to further possess the characteristics described in (5) to (7) above. Furthermore, the citrus plant according to the present invention may, for example, have three, two, or one of the characteristics described in (5) to (7) above. In addition, the citrus plant according to the present invention may further possess at least one of the characteristics described in (characteristics of the tree) and (characteristics of the fruit).

[0133] Furthermore, regarding the ability to distinguish the citrus plant according to the present invention from other citrus varieties including "Harehime" or "Citrus Intermediate Parent No. 6" using a genetic marker which is at least one of the traits (4) 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 is a plant in which the genotypes of the genetic markers are as follows: 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 type AB, Mf0097-2 is type AB, Mf0090-2 is type AA, Gn0073-2 is type AA, and Gn0048-2 is type AA.

[0134] The citrus plants of the present invention include "OK68" or its progeny described in the examples. The progeny include the individual plant or a part thereof.

[0135] In the present invention, the progeny include plants obtained from the cross between the "OK68" citrus plant and another citrus variety or other citrus plant, or from the cross between the citrus plant and a wild citrus plant.

[0136] Progeny include individuals obtained using "OK68" as the parent line and their offspring, somatic hybrid plants and their offspring resulting from cell fusion between cells of the citrus plant and cells of other plant varieties, and individuals and their offspring obtained by grafting with citrus plants as rootstock or scion.

[0137] A citrus plant according to one aspect of the present invention has the above-mentioned characteristics, and a citrus plant or its fruit obtained by the aforementioned production method is also within the scope of the present invention. Therefore, a citrus plant may also be the fruit of a citrus plant. Furthermore, a 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 a seedling or scion.

[0138] [Processed Citrus Products] A processed citrus product according to one aspect of the present invention may be a processed citrus product obtained from the citrus plants described above. Alternatively, a processed citrus product according to one aspect of the present invention may be a processed citrus fruit product obtained from the fruits of the citrus plants described above. The processed product is preferably an edible processed product. Examples of processed citrus products according to one aspect of the present invention include juice, extracts, juice residue, extraction residue, etc., of the plant body. Furthermore, processed products of the plant body itself, such as cutting, crushing, drying, powdering, freezing, various chemical treatments, pasteuring, and pureeing, can be cited. In particular, fruit processed products that utilize the fruits and exhibit the above-mentioned processing methods are preferred processed products. Suitable fruit processed products include, for example, fruit juice, fruit pulp, or juice containing both fruit juice and fruit pulp, fruit pulp preserved in syrup, dried fruit, jelly, and powdered confectionery raw materials. Among these, the processed product is preferably at least one selected from the group consisting of juice, syrup-preserved products, jellies, and powdered confectionery ingredients, and more preferably at least one selected from the group consisting of juice and syrup-preserved products.

[0139] Further explanation will be given regarding the fruit as part of the plant body of citrus trees. The fruit may be seeded or seedless. Citrus fruits may be in the form of a package, wrapped in appropriate packaging material. The form of the packaging material is not particularly limited, but examples include film, sheet, bag, or the like. The material of the packaging material is not particularly limited, but examples include paper or resin (plastic). A package of isolated citrus berries may contain one or more fruits. A package of citrus fruits may contain only one type of citrus fruit, or a combination of multiple types of citrus fruits. Furthermore, a package of citrus fruits may also contain agricultural products other than citrus fruits.

[0140] Citrus fruits (including packaging for citrus fruits) may have product labels attached. The product labels may record at least one piece of information selected from, for example, the citrus variety name, intellectual property rights information related to the citrus, information about the citrus producer, information about the citrus production history (e.g., information about pesticide application), information about the citrus distribution history, product price, harvest date, and information about the period during which the fruit is ripe. This information may be recorded directly on the product label (printed or, for example, electronically), or it may be recorded in a readable format using a code such as a one-dimensional code or a two-dimensional code.

[0141] Citrus plants may be in the form of vegetative propagation material such as seedlings or scions. Seedlings or scions may be labeled with a product label. The product label may contain at least one piece of information (product information) selected from, for example, the citrus variety name, intellectual property rights information relating 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 marker mentioned above), producer information of the seedling or scion, production history information of the seedling or scion (e.g., pesticide application information), 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 the genetic marker information of the seedling or scion (e.g., the genotype pattern of the CAPS marker mentioned above) are recorded. This information may be recorded directly on the product label (either printed or, for example, as a readable electromagnetic record), or it may be recorded in a readable format using a code such as a one-dimensional code or a two-dimensional code. The product label may also consist of a small electromagnetic recording medium such as an IC chip and be 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, but in the case of a grafted seedling, one preferred example is within the rootstock, and another preferred example is within a predetermined distance from the grafting site. A predetermined distance from the grafting site is, for example, within 10 cm, 5 cm, 4 cm, 3 cm, 2 cm, or 1 cm from the grafting site. The predetermined distance may be set either toward the rootstock from the grafting site or toward the scion from the grafting site.

[0142] [Product Information Management System for Citrus Seedlings or Scions (Vegetative Propagation Materials)] In the above section on [Processed Citrus Products], a product information management system for citrus seedlings or scions according to this embodiment, which are labeled with a product label, was described. The scope of the present invention also includes a product information management system for citrus seedlings or scions according to this embodiment, which are labeled with a product label.

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

[0144] In one example, a client terminal (which may be a reader) sends a set of information recorded on the product label (particularly the individual identification information (ID number) of the seedling or scion) and the location information of the client terminal to the server. The client terminal (which may be a reader) may also send to the server information such as the owner information of the seedling or scion (which may be the owner information of the client terminal if it matches the owner of the client terminal), and the location information of the seedling or scion (if it differs from the location information of the client terminal). This ensures that information regarding the location of each individual seedling or scion is managed. Furthermore, if the management environment of the seedling or scion changes, such as when the owner of the seedling or scion changes, the location of the seedling or scion moves, or the seedling or scion is discarded, this information is sent from the client terminal to the server.

[0145] [Summary] The present invention encompasses any of the following embodiments. <1> A method for producing citrus plants, wherein the hybrid varieties obtained by crossing citrus varieties with each other, or their offspring, have 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, respectively: Bf0158-3 is of type AB, Bf0036-3 is of type AA, Tf0150-2 is of type AB, Tf0271-2 is of type AB, Tf0300-3 is of type AB, A method for producing a citrus plant, comprising the step of selecting a plant having a genotype in which Tf0419-2 is of type AB, Tf0420-2 is of type AA, Tf0318-2 is of type BB, Tf0293-4 is of type AA, Al0302-2 is of type AB, Mf0097-2 is of type AB, Mf0090-2 is of type AA, Gn0073-2 is of type AA, and Gn0048-2 is of type AA. <2> The method for producing a citrus plant according to <1>, further comprising the step of crossing the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father to obtain the aforementioned hybrid variety. <3> A method for producing a citrus plant according to <1> or <2>, further comprising a selection step of selecting a citrus plant having the trait of producing seedless fruit and / or having a fruit weight of 130 g or more on average. <4> A method for producing a citrus plant according to any one of <1> to <3>, further comprising a selection step of selecting a citrus plant having the following traits: (1) capable of producing seedless fruit, (2) having a fruit weight of 160 g or more, and (3) having a fruit with high sugar content and good taste. <5> A method for producing a citrus plant according to any one of <1> to <4>, further comprising a propagation step of asexually propagating the citrus plant.<6> A citrus plant wherein the genotypes of the genetic markers in the citrus plant are as follows: Bf0158-3 is of type AB, Bf0036-3 is of type AA, Tf0150-2 is of type AB, Tf0271-2 is of type AB, Tf0300-3 is of type AB, Tf0419-2 is of type AB, Tf0420-2 is of type AA, Tf0318-2 is of type BB, Tf0293-4 is of type AA, Al0302-2 is of type AB, Mf0097-2 is of type AB, Mf0090-2 is of type AA, Gn0073-2 is of type AA, and Gn0048-2 is of type AA. <7> A citrus plant as described in <6>, which is a hybrid variety or a descendant plant obtained by crossing the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father. <8> A citrus plant as described in <6> or <7>, which has the trait of producing seedless fruit and / or having a fruit weight of 130 g or more on average. <9> A citrus plant as described in any one of <6> to <8>, which has the following traits (1), (2), (3) and (4): (1) Obtained by crossing the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father, (1) Capable of producing seedless fruit, (2) Fruit weight of 160 g or more, (3) Fruit has high sugar content and good taste, and (4) Distinguishable from other citrus varieties, including 'Harehime' and 'Citrus Intermediate Parent No. 6', using a genetic marker which 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. <10> A citrus plant according to any one of items <6> to <9>, further having at least one of the traits (5) and (6) below: (5) Soft segment membranes (6) High biennial bearing.<11> A method for identifying a citrus plant as described in any of <6> to <10> above, comprising the steps of: amplifying a region of the DNA of a citrus plant containing a genetic marker using a primer set; and identifying the citrus plant based on the polymorphism of the bases of the amplified DNA region, wherein the genetic marker is 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> A citrus plant or its fruit obtained by a method for producing a citrus plant as described in any one of <1> to <5>. <13> A citrus plant as described in any one of <6> to <10>, in the form of a seedling or scion. <14> The fruit of a citrus plant as described in any one of <6> to <10>. <15> A processed product of a citrus plant or the fruit of a citrus plant obtained from a citrus plant as described in any one of <6> to <10> or the fruit of a citrus plant as described in <14>. <16> A processed product of a citrus plant or the fruit of a citrus plant as described in <15>, which is at least one selected from the group consisting of fruit juice, fruit pulp, or juice containing fruit juice and fruit pulp, fruit pulp in syrup, dried fruit, jelly, powdered confectionery raw materials, etc.

[0146] <2-1> A method for producing citrus plants, comprising: a crossing step of crossing the citrus variety "Harehime" as the mother plant and the citrus variety "Citrus Intermediate Mother No. 6" as the father plant; and a marker selection step of selecting candidate citrus plants from the citrus plants obtained by the crossing step or from the citrus plants of their progeny that have a different genotype pattern of the genetic marker from the parent varieties "Harehime" and "Citrus Intermediate Mother No. 6", using at least one of the genetic markers Bf0158-3, Bf0036-3, Tf0300-3, Tf0419-2, Tf0420-2, Al0302-2, Mf0097-2, Mf0090-2 and Gn0073-2. <2-2> The method for producing the new variety described in <2-1>, wherein in the marker selection step, a combination of at least one genetic marker from Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2 with at least one genetic marker from Tf0300-3, Tf0419-2, and Mf0090-2, and at least one selected from at least one genetic marker from Bf0036-3 and Gn0073-2, is used to select a line of candidate citrus plants that have a different genotype pattern of the genetic marker from the parent varieties "Harehime" and "Citrus Intermediate Mother No. 6". <2-3> A method for producing a citrus plant according to <2-1> or <2-2>, wherein 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> A method for producing a citrus plant according to any one of <2-1> to <2-3>, further comprising a selection step for selecting a citrus plant having the traits (1), (2) and (3) below.

[0147] (1) Seedless fruit production is possible, (2) Fruit weight is 160g or more, and (3) Fruit has high sugar content and good taste. <2-5> A method for producing a citrus plant as described in any 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) Obtained by crossing the citrus variety "Harehime" as the mother plant and the citrus variety "Citrus Intermediate Mother No. 6" as the father plant, (1) Seedless fruit production is possible, (2) Fruit weight is 160g or more, (3) Fruit has high sugar content and good taste, (4) It is distinguishable from other citrus varieties, including 'Harehime' and 'Citrus Intermediate Parent No. 6', using a genetic marker which 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. <2-7> A citrus plant described in <2-6> that further has at least one of the traits (5) and (6) below: (5) Soft segment membranes (6) High biennial bearing <2-8> The citrus plant as described in <2-6> or <2-7>, wherein the genotypes of the aforementioned genetic markers are: 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.A method for identifying a citrus plant as described in any of <2-6> to <2-8>, comprising the steps of: amplifying a region of the DNA containing a CAPS marker in the DNA of a citrus plant using a primer set; and identifying the citrus plant based on the base polymorphism of 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. <2-10> Citrus plants or their fruits obtained by the method for producing citrus plants described in any of <2-1> to <2-5>. <2-11> Citrus plants described in any of <2-6> to <2-8> or citrus plants described in <2-10> or their fruits in the form of seedlings or scions. <2-12> Processed products manufactured using citrus plants described in any of <2-6> to <2-8> or citrus plants described in <2-10> or their fruits.

[0148] The present invention is not limited to the embodiments described above, 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.

[0149] [1. Creation of Citrus Lines] (History) 1) Breeding method: Hybrid breeding method 2) Parents: "Harehime" × "Citrus Intermediate Mother No. 6" "Harehime," which is early-maturing, has good taste and is easy to peel, was used as the seed parent, and "Citrus Intermediate Mother No. 6," which has female sterility derived from seedless Kishu, enables stable seedless fruit production and has excellent taste, was used as the pollen parent for the cross. The lineage diagram is shown in Figure 1.

[0150] (Development History) In May 2004, the Fruit Tree Research Institute (now the Citrus Research Center of the Fruit Tree and Tea Research Division, National Agriculture and Food Research Organization) developed this strain through crossbreeding with the goal of creating a citrus variety that matures after February, produces stable seedless fruit, has high sugar content, and excellent taste. In April 2005, it was grafted onto an intermediate rootstock of Satsuma mandarin to promote fruit setting. The first fruit set occurred in 2011. Subsequently, investigations into fruit quality revealed its superior quality, and it was selected as a superior individual in the first selection in 2012. Strain OK68 was subjected to the 12th Citrus Strain Adaptability and Characteristic Test from April 2017, and its regional adaptability was examined at 25 test sites from Chiba Prefecture in the east to Kagoshima Prefecture in the south. As a result, at the test results review meeting (evergreen fruit trees) in August 2023, it was concluded that it was suitable as a candidate for a new variety.

[0151] (Selection Process) The selection criteria in the selection process were carried out according to the following procedure. Post-fruiting evaluation (2010-2013) 1. Female sterility and seedless 2. Average fruit weight of 150g or more 3. Sugar content of 12 Brix% or more 4. Peelability (ease of peeling) is "easy" or "somewhat easy" Based on the above selection criteria 1 to 4, preliminary selection was carried out in 2012, and grafting was performed on the selected plants. 5. Consecutive fruiting ability and yield 6. Evaluation of physiological disorders on the tree (peeling, cracking, and other peel disorders) 7. Observation of the degree of disease occurrence Based on the above selection criteria 5 to 7, primary selection was carried out in 2017, and the selected plants were further grown. 8. System adaptability test (regional adaptability evaluation) Above 8. Based on the selection criteria, a second selection was conducted in 2023, and the selected plants were established as "OK68".

[0152] Figure 2 shows a photograph of the fruit of "OK68". Figure 3 shows a photograph of the "OK68" tree.

[0153] [2. Evaluation of the characteristics of each trait in the breeding area] Next, the traits of the obtained varieties were evaluated.

[0154] The evaluation method for traits followed the "Criteria for Examination of Agricultural and Forestry Plant Species (Category: Other Citrus)" (Ministry of Agriculture, Forestry and Fisheries) and the "Methods for Testing Adaptability of Breeding Lines and Testing Characteristics" (National Agriculture and Food Research Organization, Fruit Tree Research Institute, 2007). The traits described below are based on trial cultivation in Japan. The traits for each trait mentioned above and the traits described in the data for the examples were evaluated based on the "Criteria for Examination of Other Citrus (Citrus L.)" (standards published by the Ministry of Agriculture, Forestry and Fisheries at the time of trial cultivation) and the "Methods for Testing Adaptability of Breeding Lines and Testing Characteristics" (National Agriculture and Food Research Organization, Fruit Tree Research Institute, 2007), issued by the Ministry of Agriculture, Forestry and Fisheries of Japan.

[0155] (Summary of characteristics) 1) The fruit weighs approximately 180g on average and is oblate in shape. The peel is dark orange, the surface is somewhat smooth, and it is thin, about 2.5mm thick. Peeling is relatively easy. There is no occurrence of loose skin. The flesh is dark orange, the texture is somewhat soft, the segment hardness 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 1.14 g / 100ml, resulting in good taste. The ripening period is estimated to be from early February to March. 2) The tree vigor is moderately strong, and the branching characteristics are somewhat upright to upright. The branching density is dense. The tendency for biennial bearing is high. There are many thorns. Scab is rarely observed, but it is susceptible to canker, with moderate to severe cases observed. 3) It possesses female sterility derived from seedless Kishu, with an average of 0.0 complete seeds. Even if varieties that produce a lot of pollen are planted nearby, seeds are almost never produced under open-field cultivation conditions, making stable seedless fruit production possible.

[0156] Table 7 below provides an overview of the features of "OK68".

[0157] (Suitable locations and cultivation precautions) In previous variety adaptability tests, high-sugar content fruits with a sugar content of 13% or more have been produced from February onwards, the maturation period, suggesting a wide range of adaptability to soil and weather conditions. However, since the maturation period is from February to March, which falls during the harsh winter, cultivation in areas with mild winters is preferable if possible. Also, because it is susceptible to canker, it is desirable to cultivate it in orchards that are not exposed to strong winds, orchards with windbreaks, or in facilities such as covered areas to prevent the disease from occurring.

[0158] [2. Identification of citrus plants using CAPS markers] We verified whether "OK68" could 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" can be distinguished from other citrus plants using previously reported CAPS markers.

[0160] The experimental method was carried out according to the manual in Non-Patent Document 1. Specifically, the flavedo (exocarp) was cut from fresh fruit, and a DNA solution was obtained according to the manual in Non-Patent Document 1. For each CAPS marker, the obtained DNA solution was subjected to a PCR reaction using the forward primers and reverse primers listed in Table 1 to obtain amplified products. The obtained amplified products were treated with the restriction enzymes listed in Table 1 to prepare restriction enzyme reaction solutions. The restriction enzyme reaction solutions were applied to a 2% agarose gel, and polymorphisms were confirmed by electrophoresis.

[0161] (Results) Figures 5 to 7 show the polymorphisms confirmed by electrophoresis. Figure 4 also shows the genotype of the "OK68" CAPS marker.

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

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

[0164] This invention can be used in fields such as agriculture and plant breeding.

Claims

1. A method for producing citrus plants, wherein the hybrid varieties obtained by crossing citrus varieties or their offspring are such that 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 are as follows: Bf0158-3 is of type AB, Bf0036-3 is of type AA, Tf0150-2 is of type AB, Tf0271-2 is of type AB, Tf0300-3 is of type AB, A method for producing citrus plants, comprising the step of selecting plants having a genotype in which Tf0419-2 is of type AB, Tf0420-2 is of type AA, Tf0318-2 is of type BB, Tf0293-4 is of type AA, Al0302-2 is of type AB, Mf0097-2 is of type AB, Mf0090-2 is of type AA, Gn0073-2 is of type AA, and Gn0048-2 is of type AA.

2. A method for producing a citrus plant according to claim 1, further comprising the step of crossing the citrus variety "Harehime" as the mother plant with the citrus variety "Citrus Intermediate Mother No. 6" as the father plant to obtain the aforementioned hybrid variety.

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

4. A method for producing citrus fruits according to claim 1 or 2, further comprising a selection step of selecting citrus plants having the following characteristics: (1) capable of producing seedless fruit, (2) having a fruit weight of 160 g or more, and (3) having a fruit with high sugar content and good taste.

5. A method for producing citrus plants according to claim 1 or 2, further comprising a propagation step of asexually propagating the citrus plants.

6. A method for producing citrus plants, comprising: a crossing step of crossing the citrus variety "Harehime" as the mother plant and the citrus variety "Citrus Intermediate Mother No. 6" as the father plant; and a marker selection step of selecting candidate citrus plants from the citrus plants obtained by the crossing step or from the citrus plants of their progeny that have a different genotype pattern of the genetic marker from the parent varieties "Harehime" and "Citrus Intermediate Mother No. 6," using at least one of the genetic markers Bf0158-3, Bf0036-3, Tf0300-3, Tf0419-2, Tf0420-2, Al0302-2, Mf0097-2, Mf0090-2 and Gn0073-2.

7. The method for producing a citrus plant according to claim 6, wherein in the marker selection step, a combination of at least one genetic marker from Bf0158-3, Tf0420-2, Al0302-2, and Mf0097-2 and at least one genetic marker from Tf0300-3, Tf0419-2, and Mf0090-2, and at least one selected from at least one genetic marker from Bf0036-3 and Gn0073-2, is used to select candidate citrus plant lines that have a different genotype pattern of the genetic marker from the parent varieties "Harehime" and "Citrus Intermediate Mother No. 6".

8. The method for producing a citrus plant according to claim 7, wherein the genotypes of the genetic markers are as follows: Bf0158-3 is type AB, Bf0036-3 is type AA, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Al0302-2 is type AB, Mf0097-2 is type AB, Mf0090-2 is type AA, and Gn0073-2 is type AA.

9. A method for producing citrus fruits according to any one of claims 6 to 8, comprising a selection step of selecting citrus plants having the following traits: (1) capable of producing seedless fruit, (2) having a fruit weight of 160 g or more, and (3) having a fruit with high sugar content and good taste.

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

11. A citrus plant wherein the genotypes of the genetic markers in the citrus plant 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.

12. The citrus plant according to claim 11, which is a hybrid variety or a descendant plant obtained by crossing the citrus variety "Harehime" as the mother and the citrus variety "Citrus Intermediate Mother No. 6" as the father.

13. The citrus plant according to claim 11, having the trait of producing seedless fruit and / or having a fruit weight of 130 g or more on average.

14. The citrus plant according to claim 11, having the following traits (1), (2), (3), and (4): (1) Obtained by crossing the citrus variety "Harehime" as the mother plant and the citrus variety "Citrus Intermediate Mother Plant No. 6" as the father plant, (1) Capable of producing seedless fruit, (2) Fruit weight of 160 g or more, (3) Fruit has high sugar content and good taste, and (4) It is distinguishable from other citrus varieties, including "Harehime" and "Citrus Intermediate Parent No. 6", using a genetic marker which is at least one of the following: 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.

15. The citrus plant according to claim 14, further having at least one of the following traits (5) and (6): (5) having soft segment membranes; (6) having high biennial bearing.

16. A method for identifying a citrus plant according to claim 11, comprising the steps of: amplifying a region of the DNA of a citrus plant containing a genetic marker using a primer set; and identifying the citrus plant based on the base polymorphism of the amplified DNA region, wherein the genetic marker is 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.

17. The fruit of the citrus plant according to claim 11.

18. A processed product of a citrus plant or the fruit of a citrus plant, obtained from the citrus plant described in claim 11 or the fruit of the citrus plant described in claim 17.

19. The citrus plant or processed product of the fruit of a citrus plant according to claim 18, which 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, powdered confectionery ingredients, etc.

20. The citrus plant according to claim 11, in the form of a seedling or scion.

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