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

Abstract

Provided are a citrus plant, a method for producing a citrus plant, and use thereof. This method for producing a citrus plant includes a step of selecting, with respect to hybrid varieties obtained by crossing citrus varieties with each other or progeny plants thereof, a plant having a genotype in which 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 respectively as follows: Bf0158-3 is of BB type, Bf0036-3 is of AB type, Tf0150-2 is of AB type, Tf0271-2 is of AA type, Tf0300-3 is of AB type, Tf0419-2 is of BB type, Tf0420-2 is of AA type, Tf0318-2 is of BB type, Tf0293-4 is of AA type, Al0302-2 is of AB type, Mf0097-2 is of BB type, Mf0090-2 is of BB type, Gn0073-2 is of AA type, and Gn0048-2 is of AA type.

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] Conventionally, mid- to late-season citrus varieties that mature from late January to February have been cultivated. However, the varieties shipped from late December to the first and middle ten days of January are mainly iyo and ponkan, and the cultivation and distribution of high-quality mid-season varieties are not sufficient. In addition, the increase in imported citrus fruits is increasing the risk of invading the domestic citrus market.

[0003] In addition, β-cryptoxanthin, a type of carotenoid contained in citrus plants such as unshu mikan, is known for its effects as a health functional ingredient, and the demand for citrus varieties in various applications including raw consumption is increasing.

[0004] DNA variety identification technology for fruits of 24 citrus varieties using CAPS markers, National Agriculture and Food Research Organization, published on June 17, 2022

[0005] As described above, it is desired to cultivate high-quality mid-season varieties that mature from late December to the first and middle ten days of January.

[0006] In addition, the daily intake of β-cryptoxanthin as a functional ingredient is set at 3 to 6 mg, and it is considered that if three unshu mikan are eaten, the amount of the functional ingredient per day can be sufficiently ingested. However, in daily life, it is difficult to consume three unshu mikan raw fruits per day in terms of quantity and cost.

[0007] Therefore, an attempt was made to cultivate a mid-season variety that matures from late December to the first and middle ten days of January, has a good taste, can be differentiated from imported citrus fruits, and is rich in health functional ingredients that meet the health orientation of consumers.

[0008] 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 BB, Bf0036-3 is of type AB, Tf0150-2 is of type AB, Tf0271-2 is of type AA, Tf0300-3 is of type AB, The process includes selecting plants having a genotype in which Tf0419-2 is of type BB, 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 BB, Mf0090-2 is of type BB, 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 includes a crossing step of crossing the citrus variety "Tsunobo" as the mother plant and the citrus variety "Setoka" 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 offspring that have a different genotype pattern of the genetic marker from the parent varieties "Tsunobo" and "Setoka" using at least one of the genetic markers Bf0036-3, Tf0150-2, Al0302-2 and Gn0048-2.

[0009] 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 BB, Bf0036-3 is of type AB, Tf0150-2 is of type AB, Tf0271-2 is of type AA, Tf0300-3 is of type AB, Tf0419-2 is of type BB, 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 BB, Mf0090-2 is of type BB, 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), and (3): (1) obtained by crossing the citrus variety "Tsunobo" as the mother plant and the citrus variety "Setoka" as the father plant; (2) having a fruit in which the pulp contains β-cryptoxanthin with a content of 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less; (3) It is distinguishable from other citrus varieties, including "Tsunobo" and "Setoka", 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.

[0010] Furthermore, an identification method according to one aspect of the present invention is a method for identifying a citrus plant, comprising the steps of: amplifying a region in the DNA of a citrus plant that includes a genetic marker (CAPS marker) of the DNA using a primer set; and identifying the citrus plant based on the base polymorphism of the amplified DNA region, 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.

[0011] 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.

[0012] 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.

[0013] According to the present invention, it is possible to provide new citrus plants, methods for creating citrus plants, and their uses.

[0014] This is a phylogenetic tree of "Citrus KN53". This is a comparative chart of the β-cryptoxanthin content in the pulp of "Citrus KN53" and Satsuma mandarin. This is a diagram of the fruit of "Citrus KN53". This is a diagram of the tree of "Citrus KN53". This is a diagram of the genotype of the CAPS marker of "Citrus KN53". This is a diagram showing the results of PCR amplification of DNA extracted from "Citrus KN53" using the genetic markers (CAPS markers) Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, or Tf0419-2, followed by cleavage with specific restriction enzymes, and then examination of the genotype of each genetic marker by agarose gel electrophoresis. 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 KN53" after restriction enzyme treatment is electrophoresed. The figure shows the results of PCR amplification of DNA extracted from "Citrus KN53" using genetic markers Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, and Mf0090-2, followed by cleavage with specific restriction enzymes, and then agarose gel electrophoresis to determine the genotype for each genetic marker. 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 KN53" after restriction enzyme treatment is electrophoresed. This figure shows the results of PCR amplification of DNA extracted from "Citrus KN53" using genetic markers Gn0073-2 and Gn0048-2, followed by cleavage with specific restriction enzymes, and then agarose gel electrophoresis to determine the genotype for each genetic marker. In the figure, M shows the electrophoresis pattern when a 100 bp ladder is electrophoresed, and S shows the electrophoresis pattern when the DNA of "Citrus KN53" after restriction enzyme treatment is electrophoresed.

[0015] The present invention will be described in detail below. All references cited herein are incorporated herein by reference.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] In this specification, “trait” means the morphological and physiological characteristics of a plant. In this specification, CAPS means Cleaved Amplified Polymorphic Sequence (CAPS). In this specification, FW is an abbreviation for Fresh Weight. gFW indicates fresh weight in grams.

[0020] [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 plant that is a descendant of such a hybrid, 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 BB, Bf0036-3 is of type AB, 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 AA, Tf0300-3 is of type AB, Tf0419-2 is of type BB, 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 BB, Mf0090-2 is of type BB, Gn0073-2 is of type AA, and Gn0048-2 is of type AA. In one embodiment, the production method may further include the step of obtaining the hybrid variety by crossing "Tsunobo" as the mother and "Setoka" as the father. In one embodiment, the plants used in the selection process 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, a second selection process may be included before, simultaneously with, or after the selection process for selecting citrus plants that have fruits containing β-cryptoxanthin in the pulp at a content of 2.00 mg / 100 g FW or more (preferably 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less). In one embodiment, the production method may further include a preliminary selection process before the selection process for selecting candidate citrus plant lines whose genotypes of the genetic markers are Bf0036-3 is type AB, Tf0150-2 is type AB, Al0302-2 is type AB, and Gn0048-2 is type AA.The preliminary selection step may be performed before, after, or simultaneously with the second selection step. In one embodiment, the production method may further include a propagation step of asexually propagating citrus plants.

[0021] (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.

[0022] 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.

[0023]

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] In one embodiment, the production method includes the step of selecting a citrus plant having a genotype in which Bf0158-3 is of type BB, Bf0036-3 is of type AB, Tf0150-2 is of type AB, Tf0271-2 is of type AA, Tf0300-3 is of type AB, Tf0419-2 is of type BB, 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 BB, Mf0090-2 is of type BB, Gn0073-2 is of type AA, and Gn0048-2 is of type AA.

[0039] In one embodiment, the production method may further include the step of crossing "Tsunobo" as the mother and "Setoka" as the father to obtain the hybrid variety. "Tsunobo" is an early-maturing variety with good taste and excellent fruit-setting ability, and is registered as Japanese variety registration number: No. 20788. "Setoka" is a mid-season variety with good taste, aroma and excellent appearance, and is registered as Japanese variety registration number: No. 9398. The citrus plants selected in the selection process have the genotype pattern of the above-mentioned genetic markers. In contrast, other citrus varieties of "Tsunobo" have Bf0036-3 of type AA and Al0302-2 of type AA. Therefore, Bf0036-3 and / or Al0302-2 can be used to distinguish it from other citrus varieties of "Tsunobo". Furthermore, in the other citrus variety "Setoka," Tf0150-2 is of type BB, Al0302-2 is of type BB, and Gn0048-2 is of type AB. Therefore, it is possible to distinguish it from other citrus varieties "Setoka" using Tf0150-2, Al0302-2, and / or Gn0048-2. Furthermore, it is possible to distinguish it from both other citrus varieties "Tsunobo" and other citrus varieties "Setoka" using Bf0036-3, Tf0150-2, Al0302-2, and / or Gn0048-2. Furthermore, it is possible to distinguish it from both other citrus varieties "Tsunobo" and other citrus varieties "Setoka" using Bf0036-3, Tf0150-2, Al0302-2, and Gn0048-2. Furthermore, Al0302-2 can be used to distinguish it from both the other citrus variety "Tsunobo" and the other citrus variety "Setoka".

[0040] (Estimation of the lineages of the mating parents) The genotypes described in Tables 2 to 5 below represent the combinations of alleles (alleles) detected for each variety 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, for a variety with the genotype AB, if either parent has the A or B allele, there is a possibility that it is a mating parent, but if both parents are of the BB type, the possibility that this combination is the parent is excluded. Based on this principle, mating parent candidates can be estimated from the information of 14 genetic markers.

[0041] The citrus plants having the genotype selected through the selection process using the above genetic markers have a different genotype of the genetic marker 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.

[0042]

[0043]

[0044]

[0045]

[0046] In one embodiment, the production method may include a second selection step, performed before, simultaneously with, or after the selection step using a genetic marker, in which citrus plants having fruits with a β-cryptoxanthin content of 2.00 mg / 100 g FW or more (preferably 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less) in the pulp are selected. The second selection step may be performed before, simultaneously with, or after the selection step using a genetic marker, but it is preferable that it be performed after the selection step using a genetic marker. The preferred range of β-cryptoxanthin content is as described later. Citrus fruits selected using a genetic marker may be rich in β-cryptoxanthin. β-cryptoxanthin is known to have health-promoting effects, and producing β-cryptoxanthin-rich citrus fruits is extremely useful in industry.

[0047] In one embodiment, the production method may further include a preliminary selection step before the selection step, in which candidate citrus plant lines are selected in which the genotypes of the genetic markers are Bf0036-3 type AB, Tf0150-2 type AB, Al0302-2 type AB, and Gn0048-2 type AA. Alternatively, the production method may further include a preliminary selection step in which candidate citrus plant lines are selected in which Al0302-2 type AB. The preliminary selection step may be performed before, after, or simultaneously with the second selection step. In one embodiment, the production method may further include the steps of obtaining the hybrid variety by crossing "Tsunomochi" as the mother and "Setoka" as the father, and a preliminary selection step of selecting candidate citrus plant lines in which the genotypes of the genetic markers are Bf0036-3 is type AB, Tf0150-2 is type AB, Al0302-2 is type AB, and Gn0048-2 is type AA. In one embodiment, the production method may further include the steps of obtaining the hybrid variety by crossing "Tsunomochi" as the mother and "Setoka" as the father, and a preliminary selection step of selecting candidate citrus plant lines in which the genotype of the genetic marker is Al0302-2 is type AB.

[0048] (Selection process based on traits) In order to obtain citrus plants according to this embodiment, a selection process based on specific traits from among the following may also be performed. The selection process based on specific traits may be performed together with the selection process using genetic markers as indicators. It may also be performed before the selection process using genetic markers as indicators. It may also be performed after 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 sufficiently narrowed-down candidate citrus plant lines.

[0049] Furthermore, the main traits used for selection include, for example, having fruits with a β-cryptoxanthin content of 2.00 mg / 100 g FW or more (preferably 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less) in the pulp, the fruit maturing in early January, the fruit having a high sugar content and good taste, few thorns, low biennial bearing, and thin peel. Details of the above traits are described in the following section [Citrus Plants].

[0050] A method for producing citrus plants according to one aspect of the present invention may further include an additional selection step in which the hybrid varieties are selected based on, for example, at least one of the criteria described in the examples. The additional selection step may involve multiple selections over several years. The additional selection step is preferably performed after the selection step using genetic markers as indicators.

[0051] The additional selection process may include, for example, a preliminary selection process, a primary selection process, and a secondary selection process.

[0052] The preliminary selection process may be a process of selection based on at least one of the following criteria (a) to (e): (a) multi-year evaluation = sufficient fruit set, (b) average sugar content of 12 Brix% or higher, (c) sugar-acid ratio of 13 or higher and good taste, (d) average seed number of "few" or less, and (e) focus on individuals whose estimated value logBCR > 0 when estimated by a multiple regression equation of BCR content using a simple colorimeter (Nippon Denshoku Kogyo NF333) Lab (empirical estimation formula logBCR = -0.7775 - 0.0260 * L + 0.0881 * a + 0.029 * b).

[0053] The primary selection process may be a process of selection based on at least one of the following criteria (f) to (h): (f) recurring fruiting and yield, (g) evaluation of physiological disorders on the tree (e.g., peel disorders such as peeling and cracking), and (h) observation of the degree of disease occurrence (e.g., canker and scab).

[0054] Next, the secondary selection process may be a process of selection based on at least one of the following criteria (i) to (j): (i) higher content than Unshu mandarin in the evaluation of regional differences in carotenoid (β-cryptoxanthin) testing and (j) higher content than Unshu mandarin in the evaluation of regional differences.

[0055] (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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] [Method for Producing Citrus Plants 2] A method for producing citrus plants according to one aspect of the present invention includes a crossing step of crossing the citrus variety "Tsunobo" as the mother plant and the citrus variety "Setoka" 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 offspring that have a different genotype pattern of the genetic marker from the parent varieties "Tsunobo" and "Setoka" using at least one of the genetic markers Bf0036-3, Tf0150-2, Al0302-2 and Gn0048-2.

[0060] Furthermore, the method of creation can also be referred to as the manufacturing method, cultivation method, or production method.

[0061] (Crossing process) In the crossing process, "Tsunomochi" is used as the mother (seed parent) and "Setoka" is used as the father (pollen parent) for crossbreeding.

[0062] The parent varieties used in the crossbreeding, "Tsunobo" and "Setoka," are both varieties that are commercially available. "Tsunobo" is an early-maturing variety with good taste and excellent fruit-setting ability, and is registered as Japanese variety registration number: No. 20788. "Setoka" is a mid-season variety with good taste, fragrance, and excellent appearance, and is registered as Japanese variety registration number: No. 9398.

[0063] (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 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 "Tsunobo" and "Setoka".

[0064] 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].

[0065] While the genotype patterns of all 14 types of CAPS markers described 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, two, three, or four genetic markers from Bf0036-3, Tf0150-2, Al0302-2, and Gn0048-2 is used to select candidate citrus plant lines that have different genotype patterns of the genetic markers from the parent varieties "Tsunobo" and "Setoka". In one embodiment, in the marker selection process, a combination of at least one genetic marker from Tf0150-2 and Gn0048-2 with the genetic marker Bf0036-3, and at least one selected from the genetic marker Al0302-2, is used to select candidate citrus plant lines that have different genotype patterns of the genetic markers from the parent varieties "Tsunobo" and "Setoka". In one embodiment, in the marker selection step, candidate citrus plant lines with different genotype patterns of the genetic markers from the parent varieties "Tsunobo" and "Setoka" are selected using the genetic markers Tf0150-2, Gn0048-2, Bf0036-3, and Al0302-2.

[0066] In one embodiment, it is preferable to select a candidate citrus plant line in which the genotypes of the genetic markers are Bf0036-3 of type AB, Tf0150-2 of type AB, and Gn0048-2 of type AA. In another embodiment, it is preferable to select a candidate citrus plant line in which the genotype of the genetic marker Al0302-2 of type AB. 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 BB type, Bf0036-3 is AB type, Tf0150-2 is AB type, Tf0271-2 is AA type, Tf0300-3 is AB type, Tf0419-2 is BB type, Tf0420-2 is AA type, Tf0318-2 is BB type, Tf0293-4 is AA type, Al0302-2 is AB type, Mf0097-2 is BB type, Mf0090-2 is BB type, Gn0073-2 is AA type, and Gn0048-2 is AA type.

[0067] (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 "Tsunobo" as the mother plant and the citrus variety "Setoka" as the father plant. The selection process based on specific traits can 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.

[0068] The main traits used for selection include, for example, having fruits with a β-cryptoxanthin content of 2.00 mg / 100g FW or more and 2.50 mg / 100g FW or less in the fruit pulp, having a fruit maturation period in early January, having fruits with high sugar content and good taste, having few thorns, having low biennial bearing, and having thin peel. Among these, it is preferable to select citrus plants that have fruits with a β-cryptoxanthin content of 2.00 mg / 100g FW or more and 2.50 mg / 100g FW or less in the fruit pulp. Details of the above traits are described in the following section [Citrus Plants].

[0069] 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.

[0070] In other words, the selection process may include, for example, a preliminary selection process, a primary selection process, and a secondary selection process.

[0071] The preliminary selection process may be a process of selection based on at least one of the following criteria (a) to (e): (a) multi-year evaluation = sufficient fruit set, (b) average sugar content of 12 Brix% or higher, (c) sugar-acid ratio of 13 or higher and good taste, (d) average seed number of "few" or less, and (e) focus on individuals whose estimated value logBCR > 0 when estimated by a multiple regression equation of BCR content using a simple colorimeter (Nippon Denshoku Kogyo NF333) Lab (empirical estimation formula logBCR = -0.7775 - 0.0260 * L + 0.0881 * a + 0.029 * b).

[0072] The primary selection process may be a process of selection based on at least one of the following criteria (f) to (h): (f) recurring fruiting and yield, (g) evaluation of physiological disorders on the tree (e.g., peel disorders such as peeling and cracking), and (h) observation of the degree of disease occurrence (e.g., canker and scab).

[0073] Next, the secondary selection process may be a process of selection based on at least one of the following criteria (i) to (j): (i) higher content than Unshu mandarin in the evaluation of regional differences in carotenoid (β-cryptoxanthin) testing and (j) higher content than Unshu mandarin in the evaluation of regional differences.

[0074] (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.

[0075] Methods of asexual propagation include grafting, cuttings, 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.

[0076] 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.

[0077] 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.

[0078] [Method for Identifying Citrus Plants] As described above, the citrus plants of the present invention can be identified by specific variety identification markers.

[0079] 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.

[0080] 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).

[0081] 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.

[0082] 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").

[0083] The method for identifying citrus plants according to the present invention is a method for identifying the citrus variety "KN53" or its offspring plants from other citrus varieties by using a CAPS marker as an identification marker.

[0084] 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 KN53 listed in Tables 2 to 5.

[0085] 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.

[0086] (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.

[0087] 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 "KN53" can also be used to distinguish citrus plants that exhibit the same allelic type as "KN53" from other citrus varieties.

[0088] Furthermore, the allele type being the same as "KN53" refers to the allele type shown in Figure 5 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 "KN53".

[0089] In the identification technology of the present invention, "other citrus varieties" that can be distinguished from "KN53" include citrus varieties that do not have the same genotype as "KN53" 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 allele types different from those of "KN53".

[0090] Here, other citrus varieties that can be distinguished from "KN53" 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.

[0091] These 24 citrus varieties, when combined with "KN53" (a total of 25 varieties), account for more than 94% of the total citrus fruit distribution volume in Japan.

[0092] Furthermore, other citrus varieties that can be distinguished from "KN53" 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.

[0093] The identification method of the present invention enables the identification of "KN53" with very high accuracy.

[0094] Furthermore, considering the results of the following examples, other citrus varieties that can be distinguished from "KN53" using the identification technology of the present invention include citrus varieties that do not have the same genotype as "KN53" 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 "KN53" from the principle of the identification technology of the present invention.

[0095] For example, to distinguish between "KN53" and its parent variety "Tsunobo," at least one of the markers Bf0036-3 and Al0302-2 can be used.

[0096] Furthermore, to distinguish "KN53" from its parent variety "Setoka," at least one of the markers Tf0150-2, Al0302-2, and Gn0048-2 can be used.

[0097] Furthermore, to distinguish "KN53" from the parent varieties "Tsunobo" and "Setoka," it is sufficient to use a combination of at least one genetic marker from Tf0150-2 and Gn0048-2 with the genetic marker Bf0036-3, and at least one selected from the genetic marker Al0302-2. If it is sufficient to distinguish only "KN53" from its parents, at least Al0302-2 should be used.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] The present invention provides a method for identifying citrus plants that can accurately and quickly identify the citrus variety "KN53" or its offspring from other citrus varieties, and is also a method that can be widely introduced in inspection sites and other settings. 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.

[0110] [Citrus plant 1] A citrus plant according to one aspect of the present invention is characterized in that the genotypes of the genetic markers in the citrus plant are as follows: Bf0158-3 is of type BB, Bf0036-3 is of type AB, Tf0150-2 is of type AB, Tf0271-2 is of type AA, Tf0300-3 is of type AB, Tf0419-2 is of type BB, 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 BB, Mf0090-2 is of type BB, Gn0073-2 is of type AA, and Gn0048-2 is of type AA. It is a citrus plant. 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.

[0111] In one embodiment, the citrus plant may be a hybrid variety or a progeny plant obtained by crossing the citrus variety "Tsunobo" as the mother and the citrus variety "Setoka" as the 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, it is preferable that the citrus plant has fruit having a β-cryptoxanthin content of 2.00 mg / 100 g FW or more (preferably 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less) in the pulp. In one embodiment, the citrus plant is a hybrid variety or a descendant plant resulting from the cross between the citrus variety "Tsunobo" as the mother and the citrus variety "Setoka" as the father, and has fruit containing β-cryptoxanthin with a content of 2.00 mg / 100 g FW or more (preferably 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less) in the pulp, and the citrus plant may also be one in which the β-cryptoxanthin content in the pulp of the parent varieties "Tsunobo" and "Setoka" is less than 2.00 mg / 100 g FW.

[0112] [Citrus Plant 2] Furthermore, a citrus plant according to one aspect of the present invention is a citrus plant having the following characteristics (1), (2), and (3): (1) obtained by crossing the citrus variety "Tsunobo" as the mother and the citrus variety "Setoka" as the father, (2) having fruit in which the pulp contains β-cryptoxanthin with a content of 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less, and (3) It is distinguishable from other citrus varieties, including "Tsunobo" and "Setoka", 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.

[0113] The following describes preferred embodiments of citrus plant 1 and / or citrus plant 2.

[0114] A citrus plant according to a further aspect of the present invention is a citrus plant that further has at least one of the following traits (4) to (7): (4) The fruit has a high sugar content and good taste, (5) There are few thorns, (6) There is low biennial bearing, (7) The peel is thin. Furthermore, a citrus plant according to a further aspect of the present invention is a citrus plant that further has at least one of the following traits (4) to (8): (4) The fruit ripens in early January, (5) The fruit has a high sugar content and good taste, (6) There are few thorns, (7) There is low biennial bearing, (8) The peel is thin.

[0115] 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.

[0116] 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 distinguishes it from the parent varieties "Tsunobo" or "Setoka". Note that the numerical values ​​may be the average of measurements taken over two or more years.

[0117] (Characteristics of the tree) The tree's vigor is "slightly weak" to "moderate," about the same as or slightly stronger than "Tsunobochi" and "Setoka." The rate of thorn development on the branches (spring branches) is low, at about 5%, and the length of the thorns on the branches is also short. The size of the petals is smaller than that of "Tsunobochi" and "Setoka." The amount of pollen is "none," which is less than that of "Tsunobochi" and "Setoka." The style is "straight," and the degree of curvature is less compared to "Tsunobochi" and "Setoka."

[0118] The germination, flowering, and full coloring periods may vary depending on climatic conditions, but in the same growing area, the germination and peak flowering periods are the same as those of 'Tsunobo' and 'Setoka'. Also, although this may vary depending on climatic conditions, the fruit begins to color in early to mid-October, and full coloring occurs in mid-November. The number of flowers is "medium" to "slightly high," and the tendency for biennial bearing is about the same as or "lower" than that of 'Tsunobo' or 'Setoka'.

[0119] (Fruit characteristics) The peel is "deep orange" and slightly redder than "Setoka". The surface is "somewhat smooth". The peel is thin, about 2.0 mm thick.

[0120] The flesh is a deep orange color, and the average fruit weight is around 150g, smaller than both "Tsunobo" and "Setoka." The sugar content of the juice in early January is 13.6%, which is high, similar to "Tsunobo." The acid content is around 0.90g / 100ml, higher than "Tsunobo" but lower than "Setoka." The flavor is rich. The number of complete seeds per fruit is fewer than both "Tsunobo" and "Setoka." The proportion of seedless fruit is about the same as or higher than "Setoka" under normal open-field cultivation conditions, and higher than "Tsunobo." The seeds are polyembryonic. Based on the progress of coloring and the condition of the fruit, the maturation period is slightly later than "Tsunobo" and earlier than "Setoka," around early January.

[0121] An example of the citrus plant of the present invention is the citrus plant "Citrus KN53" or its progeny from the examples. Hereinafter, "Citrus KN53" will be referred to as "KN53". The citrus plant "KN53" from the examples has the characteristics described in the examples. The morphological and physiological characteristics of "KN53" can be found in Figures 2 to 4.

[0122] 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.

[0123] Regarding the characteristic (4) that the citrus plant possesses according to the present invention, the fruit matures in early January, preferably, when the cultivation method during growth is, for example, grafted and grown in open fields, the maturation period is in early January, and depending on the growing location and cultivation conditions, the fruit may mature from late December to mid-January. In other words, the maturation period is slightly later than 'Tsunobo', in one example being about 1 to 3 weeks or 1 to 2 weeks later than 'Tsunobo'. It is earlier than 'Setoka', in one example being about 1 to 6 weeks or 2 to 5 weeks earlier than 'Setoka'.

[0124] Furthermore, regarding the trait (5) of the citrus plant according to the present invention, which states that the fruit has a high sugar content and good taste, "taste" can also be evaluated using the sugar-acid ratio as an indicator.

[0125] 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%.

[0126] In this specification, "acidity" refers to the citric acid content in the fruit juice. The method for investigating acidity is to measure it by converting it from the titration value of the fruit juice with a sodium compound aqueous solution.

[0127] The lower limit of the sugar content of the citrus fruit juice according to the present invention is preferably 12.0 Brix% or higher, more preferably 13.0 Brix% or higher, more preferably 13.6 Brix% or higher, and even more preferably 14.0% 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 17 Brix% or less, 16 Brix% or less, or 15 Brix% or less.

[0128] The upper limit of the acid content of the fruit juice is, for example, 1.0 g / 100 ml or less, more preferably 0.90 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 below 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. The "sugar-acid ratio" is calculated as the sugar content meter reading / citric acid content value. The sugar-acid ratio is, for example, 12.0 or more, 13.0 or more, 14.0 or more, or 15.0 or more.

[0129] 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.

[0130] In the example, the sugar content of the juice of "KN53" in early January was high, averaging 13.6%. Furthermore, the acid content of the juice was low, averaging approximately 0.90 g / 100 ml. The sugar-acid ratio was 15.1.

[0131] (β-cryptoxanthin content) β-cryptoxanthin is a carotenoid found in citrus fruits and is known as a health-promoting functional ingredient that has effects such as preventing osteoporosis and lifestyle-related diseases such as diabetes and arteriosclerosis.

[0132] Regarding the trait (2) of the citrus plant according to the present invention, which is that the fruit pulp contains β-cryptoxanthin in a content of 2.00 mg / 100 g FW or more (preferably 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less), the lower limit of the β-cryptoxanthin content in the fruit pulp of the citrus plant according to the present invention at maturity is 2.00 mg / 100 g FW or more, preferably 2.10 mg / 100 g FW or more, more preferably 2.20 mg / 100 g FW or more, even more preferably 2.30 mg / 100 g FW or more, and most preferably 2.33 mg / 100 g FW or more. The upper limit of the β-cryptoxanthin content in the fruit pulp is not particularly limited, but it is assumed to be above one of the lower limits, for example, 2.45 mg / 100g FW or less, 2.40 mg / 100g FW or less, and 2.35 mg / 100g FW or less.

[0133] The β-cryptoxanthin content in the pulp of Satsuma mandarin is approximately 1.75 mg / 100g FW, the β-cryptoxanthin content in the pulp of the parent variety "Tsunobo" is approximately 1.82 mg / 100g FW, and the β-cryptoxanthin content in the pulp of the parent variety "Setoka" is approximately 1.51 mg / 100g FW.

[0134] Thus, the citrus plants according to the present invention have the characteristic of having a higher β-cryptoxanthin content in the pulp than Satsuma mandarins and their parent varieties "Tsunomochi" and "Setoka".

[0135] In one embodiment, the citrus plant may have fruit containing β-cryptoxanthin in the pulp at a content of 2.00 mg / 100 g FW or more (preferably 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less). In one embodiment, the citrus plant may be a hybrid variety or a descendant plant resulting from the cross between the citrus variety "Tsunobo" as the mother and the citrus variety "Setoka" as the father, and may have fruit containing β-cryptoxanthin in the pulp at a content of 2.00 mg / 100 g FW or more (preferably 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less). In one embodiment, the citrus plant is a hybrid variety or a descendant plant resulting from the cross between the citrus variety "Tsunobo" as the mother and the citrus variety "Setoka" as the father, and has fruit containing β-cryptoxanthin with a content of 2.00 mg / 100 g FW or more (preferably 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less) in the pulp, and the β-cryptoxanthin content in the pulp of the parent varieties "Tsunobo" and "Setoka" is less than 2.00 mg / 100 g FW. The preferred content of β-cryptoxanthin in the pulp of the citrus plant in one embodiment of the present invention is as described above. The β-cryptoxanthin content in the pulp of the parent varieties "Tsunobo" and "Setoka" is usually less than 2.00 mg / 100 g FW, typically 1.95 mg / 100 g FW or less, more typically 1.90 mg / 100 g FW or less, and even more typically 1.85 mg / 100 g FW or less. Therefore, according to further embodiments of the present invention, the β-cryptoxanthin content in the pulp of citrus plants can be used to distinguish them from the parent varieties "Tsunobo" and "Setoka".

[0136] The β-cryptoxanthin levels were measured using a known method (PLOS ONE | https: / / doi.org / 10.1371 / journal.pone.0246468 February 4, 2021). The values ​​mentioned above are the average values ​​from five or more fruits each year over a period of two to three years.

[0137] The citrus plant of the present invention yields citrus fruits containing 30% or more more β-cryptoxanthin than conventional varieties. In other words, by consuming two or fewer fruits of the present invention per day, it is possible to ingest the daily required amount (approximately 3 mg) of β-cryptoxanthin, which is expected to have beneficial effects such as preventing osteoporosis, thus providing health benefits.

[0138] Regarding the traits of citrus plants according to the present invention, for example, trait (6) low thorn development is evaluated by the number of thorns on the investigated branches ÷ total number of leaf-bearing nodes on the investigated branches × 100. Trait (7) low biennial bearing is evaluated by observing the plants for several years and determining whether the year is a good year or bad year based on moderate fruit thinning from the third year after the first fruit set, with a strong evaluation for good years and a moderate evaluation for bad years and a weak evaluation for bad years and a weak evaluation for bad years and bad years when it is unclear. Trait (8) thin peel is evaluated by measuring the thickness of the peel at the equator of the fruit using calipers and averaging the measurement of the thickness of the peel at the equator of the cross-section or longitudinal section of the fruit from five or more fruits.

[0139] According to one aspect of the present invention, the thickness of the fruit peel of a citrus plant is, for example, 1.3 mm or more and 2.5 mm or less.

[0140] The citrus plant according to the present invention may be a plant having characteristics equivalent to "KN53". A plant having equivalent characteristics means a plant that, when grown under the same environmental conditions, possesses the main characteristics of "KN53". The main characteristics are those described in (1) to (3) above. It is also preferable to further possess the characteristics described in (5) to (8) above. Furthermore, the citrus plant according to the present invention may, for example, have four, three, two, or one of the characteristics described in (5) to (8) 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).

[0141] Furthermore, the present invention makes it possible to distinguish the citrus plant from other citrus varieties, including "Tsunobo" and "Setoka," using a genetic marker which is at least one of the traits (3) 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 is one in which the genotypes of the aforementioned genetic markers are as follows: Bf0158-3 is BB type, Bf0036-3 is AB type, Tf0150-2 is AB type, Tf0271-2 is AA type, Tf0300-3 is AB type, Tf0419-2 is BB type, Tf0420-2 is AA type, Tf0318-2 is BB type, Tf0293-4 is AA type, Al0302-2 is AB type, Mf0097-2 is BB type, Mf0090-2 is BB type, Gn0073-2 is AA type, and Gn0048-2 is AA type.

[0142] Furthermore, the citrus plants of the present invention include "KN53" or its progeny described in the examples. The progeny include the individual plant or a part thereof.

[0143] In the present invention, the progeny include plants obtained from the cross between the "KN53" citrus plant and another citrus variety or other citrus plant, or from the cross between the citrus plant and a wild citrus plant.

[0144] Progeny include individuals obtained using "KN53" 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.

[0145] A citrus plant according to one aspect of the present invention has the above-described 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. 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.

[0146] [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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] [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.

[0151] 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.

[0152] 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.

[0153] [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 BB, Bf0036-3 is of type AB, Tf0150-2 is of type AB, Tf0271-2 is of type AA, 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 BB, 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 BB, Mf0090-2 is of type BB, 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 obtaining the hybrid variety by crossing "Tsunomochi" as the mother and "Setoka" as the father. <3> The method for producing citrus plants according to <1> or <2>, further comprising a second selection step of selecting citrus plants having fruits having a content of 2.00 mg / 100 g FW or more (preferably 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less) in the pulp before, simultaneously with, or after the selection step. <4> The method for producing citrus plants according to any one of <1> to <3>, further comprising a propagation step of asexually propagating the citrus plants.<5> A method for producing citrus plants, comprising: a hybridization step of crossing the citrus variety "Tsunobo" as the mother plant and the citrus variety "Setoka" as the father plant; and a marker selection step of selecting candidate citrus plants from the citrus plants obtained by the hybridization step or from the citrus plants of their offspring that have a different genotype pattern of the genetic marker from the parent varieties "Tsunobo" and "Setoka" using at least one of the genetic markers Bf0036-3, Tf0150-2, Al0302-2 and Gn0048-2. <6> The method for producing a new variety according to <5>, wherein in the marker selection step, a candidate citrus plant line is selected in which the genotype pattern of the genetic marker differs from that of the parent varieties "Tsunobo" and "Setoka" by using a combination of at least one genetic marker from Tf0150-2 and Gn0048-2 with the genetic marker Bf0036-3, and at least one selected from the genetic marker Al0302-2. <7> The method for producing a new variety according to <6>, wherein a candidate citrus plant line is selected in which the genotypes of the genetic markers are AB for Bf0036-3, AB for Tf0150-2, AB for Al0302-2, and AA for Gn0048-2. <8> A method for producing a citrus plant according to any one of <5> to <7>, comprising a selection step of selecting a citrus plant having a fruit having a β-cryptoxanthin content of 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less in the fruit pulp. <9> A method for producing a citrus plant according to any one of <5> to <8>, further comprising a propagation step of asexually propagating the citrus plant.<10> A citrus plant wherein the genotypes of the genetic markers in the citrus plant are as follows: Bf0158-3 is of type BB, Bf0036-3 is of type AB, Tf0150-2 is of type AB, Tf0271-2 is of type AA, Tf0300-3 is of type AB, Tf0419-2 is of type BB, 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 BB, Mf0090-2 is of type BB, Gn0073-2 is of type AA, and Gn0048-2 is of type AA. <11> The citrus plant described in <10>, which is a hybrid variety or a descendant plant obtained by crossing the citrus variety "Tsunobo" as the mother plant and the citrus variety "Setoka" as the father plant. <12> The citrus plant described in <10> or <11>, which has a fruit in which the pulp contains 2.00 mg / 100 g FW or more (preferably 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less) of β-cryptoxanthin. <13> A citrus plant that is a hybrid variety or a descendant plant resulting from the cross between the citrus variety "Tsunobo" as the mother and the citrus variety "Setoka" as the father, and has fruit having a pulp containing 2.00 mg / 100 g FW or more (preferably 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less) of β-cryptoxanthin, wherein the β-cryptoxanthin content in the pulp of the parent varieties "Tsunobo" and "Setoka" is less than 2.00 mg / 100 g FW, as described in any of <10> to <12>. <14> Citrus plants described in any of <10> to <13> that further possess at least one of the following traits (4) to (7): (4) Fruit has high sugar content and good taste (5) Few thorns (6) Low biennial bearing (7) Thin peel.<15> A method for identifying a citrus plant as described in any of <10> to <14>, comprising the steps of: amplifying a region containing a genetic marker in the DNA of a citrus plant using a primer set; and identifying the citrus plant based on the polymorphism of bases in 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. <16> A citrus plant as described in any of <10> to <14>, in the form of a seedling or scion. <17> The fruit of a citrus plant as described in any of <10> to <14>. <18> A processed product of a citrus plant or the fruit of a citrus plant obtained from a citrus plant as described in any of <10> to <14> or the fruit of a citrus plant as described in <17>. <19> A processed product of a citrus plant or the fruit of a citrus plant as described in <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 in syrup, dried fruit, jelly, powdered confectionery raw materials, etc.

[0154] <2-1> A method for producing citrus plants, comprising: a hybridization step of crossing the citrus variety "Tsunobo" as the mother plant and the citrus variety "Setoka" as the father plant; and a marker selection step of selecting candidate citrus plants from the citrus plants obtained by the hybridization step or from the citrus plants of their offspring that have a different genotype pattern of the genetic marker from the parent varieties "Tsunobo" and "Setoka" using at least one of the genetic markers Bf0036-3, Tf0150-2, Al0302-2 and Gn0048-2. <2-2> The method for producing a new variety according to <2-1>, wherein in the marker selection step, a candidate citrus plant line is selected in which the genotype pattern of the genetic marker differs from that of the parent varieties "Tsunobo" and "Setoka" by using a combination of at least one genetic marker from Tf0150-2 and Gn0048-2 with the genetic marker Bf0036-3, and at least one selected from the genetic marker Al0302-2. <2-3> The method for producing a new variety according to <2-2>, wherein a candidate citrus plant line is selected in which the genotypes of the genetic markers are AB for Bf0036-3, AB for Tf0150-2, AB for Al0302-2, and AA for Gn0048-2. <2-4> A method for producing a citrus plant according to any one of <2-1> to <2-3>, comprising a selection step of selecting a citrus plant having a fruit having a β-cryptoxanthin content of 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less in the fruit pulp. <2-5> A method for producing a citrus plant according to any one of <2-1> to <2-4>, further comprising a propagation step of asexually propagating the citrus plant.<2-6> Citrus plants having the following characteristics (1), (2), and (3): (1) Obtained by crossing the citrus variety "Tsunobo" as the mother and the citrus variety "Setoka" as the father, (2) Having fruits in which the pulp contains β-cryptoxanthin with a content of 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less, (3) It can be distinguished from other citrus varieties, including 'Tsunobo' and 'Setoka', 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 following traits (4) to (7): (4) The fruit has a high sugar content and good taste (5) There are few thorns (6) There is low biennial bearing (7) The peel is thin. <2-8> The citrus plant according to <2-6> or <2-7>, wherein the genotypes of the genetic markers are: Bf0158-3 is BB type, Bf0036-3 is AB type, Tf0150-2 is AB type, Tf0271-2 is AA type, Tf0300-3 is AB type, Tf0419-2 is BB type, Tf0420-2 is AA type, Tf0318-2 is BB type, Tf0293-4 is AA type, Al0302-2 is AB type, Mf0097-2 is BB type, Mf0090-2 is BB 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.

[0155] 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.

[0156] [1. Creation of Citrus Varieties] (History) 1) Breeding Method: Hybrid breeding method 2) Parents: "Tsunobo" × "Setoka" "Tsunobo," which is early-maturing, has good taste and excellent fruiting ability, was used as the mother plant, and "Setoka," which is mid-season-maturing, has good taste and aroma and excellent appearance, was used as the father plant for the hybridization. The lineage diagram is shown in Figure 1.

[0157] (Development History) This variety was developed in 2001 at the Fruit Tree Research Institute (now the Kuchinotsu Citrus Research Station of the Kyushu Okinawa Agricultural Research Center, National Agriculture and Food Research Organization) through crossbreeding with the goal of developing a citrus fruit that matures from December to January, has high sugar content and excellent taste, is seedless, has thin membranes that make it easy to eat, is aromatic, and has the ability to bear fruit year after year. In 2002, it was grafted onto Shikuwasa rootstock to promote fruiting. In 2005, it bore fruit for the first time, and fruit characteristics were investigated. In 2008, it was selected as a superior individual and grafted onto Satsuma mandarin as an intermediate rootstock. Subsequently, investigations into the fruit and tree characteristics of strain KN53 were continued, and due to its superior qualities, it was selected for the first round of selection in 2017. From April of the same year, it was subjected to the 12th Citrus Strain Adaptability and Characteristic Test, where its regional adaptability and dissemination potential were 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.

[0158] (Selection Process) The selection criteria in the selection process were carried out according to the following procedure. Primary Selection - Post-Fruiting Evaluation (2005-2008) 1. Multi-year evaluation = sufficient fruiting ability. 2. Average sugar content of 12 Brix% or higher. 3. Good taste with a sugar-acid ratio of 13 or higher. 4. Average number of seeds is "few" or less. 5. Individuals with an estimated value logBCR > 0, estimated by multiple regression equation of BCR content using a simple colorimeter (Nippon Denshoku Kogyo NF333) Lab (empirical estimation formula logBCR = -0.7775 - 0.0260 * L + 0.0881 * a + 0.0291 * b). Based on the selection criteria 1 to 5 above, preliminary selection was carried out in 2008, and grafting was performed on the selected plants. 6. Consecutive year bearing ability, yield ability 7. 8. Evaluation of physiological disorders on the tree (peel disorders such as peeling and cracking) Based on the selection criteria in 6. to 8. above, primary selection was carried out in 2017, and the selected plants were further grown. 9. System adaptability test (regional adaptability evaluation) 10. Evaluation of inter-regional differences in carotenoids (β-cryptoxanthin) with a selection criterion of higher content than Unshu mandarin Based on the selection criteria in 9. to 10. above, secondary selection was carried out in 2023, and the selected plants were established as "KN53".

[0159] A photograph of the fruit of "KN53" is shown in Figure 3. A photograph of the "KN53" tree is shown in Figure 4.

[0160] [2. Evaluation of the characteristics of each trait in the breeding area] Next, the traits of the obtained varieties were evaluated.

[0161] 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 from 2021 to 2023. The traits for each trait described 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 as of 2021-2023) and the "Methods for Testing Adaptability of Breeding Lines and Testing Characteristics" (National Agriculture and Food Research Organization, Fruit Tree Research Institute, 2007), which are issued by the Ministry of Agriculture, Forestry and Fisheries of Japan.

[0162] (Summary of characteristics) 1) The fruit weighs about 150g, the peel is "dark orange", the surface is "somewhat smooth", and the thickness is thin at about 2.0mm. Peeling is "somewhat easy" to "medium". There is no occurrence of puffy skin. The segment is "soft", and the flesh ratio is large at 83.9%. In a survey conducted in early January, the sugar content of the juice was high at over 13%, and the acid content was less than 1.0%, resulting in a rich flavor. The ripening period is early January. Under natural pollination conditions in open field cultivation, the average number of complete seeds is about 0.8, making seedless fruit production possible. 2) The tree vigor is "medium", and the branch characteristics are "intermediate" between upright and spreading. The density of branches and shoots is "dense". Biennial bearing is "low" to "medium", and fruit setting is relatively stable. There are few thorns. Scab disease was almost nonexistent, and citrus canker was observed only in mild cases on the branches and leaves. 3) The average content of β-cryptoxanthin, a health-promoting functional component, in the pulp was 2.33 mg / 100g FW, which is significantly higher than that of Satsuma mandarin (1.75 mg / 100g FW). The minimum value was 1.50 mg / 100g FW or higher, meaning that eating 'two' mandarins would provide the daily recommended intake of the functional component (3-6 mg) for which its effects have been reported.

[0163] Table 7 below provides an overview of the characteristics of "KN53".

[0164] (β-cryptoxanthin content) The β-cryptoxanthin content in the pulp of "KN53" was measured. As shown in Figure 2, the average β-cryptoxanthin content in the pulp was 2.33 mg / 100gFW, which was significantly higher than that of Satsuma mandarin (1.75 mg / 100gFW). Furthermore, the minimum value was 1.50 mg / 100gFW or higher, indicating that eating 'two' fruits would allow for the intake of the daily amount of the functional ingredient (3-6 mg) for which functional effects have been reported.

[0165] (Suitable locations and cultivation precautions) In previous variety adaptability tests, the variety produced high-quality fruit with an average sugar content of 13% in January during its maturation period in many test sites, indicating a wide range of adaptability to soil and weather conditions. "KN53" is male-sterile and produces very little pollen on its own, so care should be taken not to plant varieties with abundant pollen, such as Hassaku or Amanatsu, nearby. Also, to avoid promoting the occurrence of canker, it is desirable to cultivate it in orchards that are not exposed to strong winds, or in orchards with windbreaks.

[0166] [3. Identification of citrus plants using CAPS markers] We verified whether "KN53" could be identified using CAPS markers.

[0167] 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 "KN53" can be distinguished from other citrus plants using previously reported CAPS markers.

[0168] 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.

[0169] (Results) The polymorphisms confirmed by electrophoresis are shown in Figures 6 to 8. Figure 5 also shows the genotype of the CAPS marker "KN53".

[0170] The genotype of the CAPS marker for "KN53" was found to be distinguishable from all 24 varieties targeted by this marker, and also distinguishable from the parent varieties (Tables 2-5).

[0171] As described above, "KN53" could be distinguished from other varieties using the CAPS marker.

[0172] This invention can be used in fields such as agriculture and plant breeding.

Claims

1. A method for producing citrus plants, 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 BB, Bf0036-3 is of type AB, Tf0150-2 is of type AB, Tf0271-2 is of type AA, 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 BB, 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 BB, Mf0090-2 is of type BB, 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 crossbreeding a parent plant named "Tsunobo" with a father plant named "Setoka" to obtain the aforementioned hybrid variety.

3. The method for producing according to claim 1, further comprising a second selection step of selecting citrus plants having the characteristic of having fruits in which the pulp contains 2.00 mg / 100 g FW or more of β-cryptoxanthin.

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

5. A method for producing citrus plants, comprising: a hybridization step of crossing the citrus variety "Tsunobo" as the mother plant and the citrus variety "Setoka" as the father plant; and a marker selection step of selecting candidate citrus plants from the citrus plants obtained by the hybridization step or from the citrus plants of their offspring that have a different genotype pattern of the genetic marker from the parent varieties "Tsunobo" and "Setoka" using at least one of the genetic markers Bf0036-3, Tf0150-2, Al0302-2 and Gn0048-2.

6. The method for producing a new variety according to claim 5, wherein in the marker selection step, a combination of at least one genetic marker from Tf0150-2 and Gn0048-2 and the genetic marker Bf0036-3, and at least one selected from the genetic marker Al0302-2, is used to select a line of candidate citrus plants whose genotype pattern of the genetic marker differs from that of the parent varieties "Tsunobo" and "Setoka".

7. The method for producing according to claim 6, wherein the genotypes of the genetic markers are AB for Bf0036-3, AB for Tf0150-2, AB for Al0302-2, and AA for Gn0048-2.

8. A method for producing according to any one of claims 5 to 7, comprising a selection step of selecting citrus plants having fruits in which the pulp contains β-cryptoxanthin in a content of 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less.

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

10. A citrus plant wherein the genotypes of the genetic markers in the citrus plant are as follows: Bf0158-3 is of type BB, Bf0036-3 is of type AB, Tf0150-2 is of type AB, Tf0271-2 is of type AA, Tf0300-3 is of type AB, Tf0419-2 is of type BB, 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 BB, Mf0090-2 is of type BB, Gn0073-2 is of type AA, and Gn0048-2 is of type AA.

11. The citrus plant according to claim 10, which is a hybrid variety or a descendant plant obtained by crossing the citrus variety "Tsunobo" as the mother and the citrus variety "Setoka" as the father.

12. The citrus plant according to claim 10, wherein the fruit pulp contains β-cryptoxanthin in a content of 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less.

13. A citrus plant that is a hybrid variety or a descendant plant resulting from the cross between the citrus variety "Tsunobo" as the mother and the citrus variety "Setoka" as the father, wherein the fruit has pulp containing β-cryptoxanthin in a content of 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less, and the β-cryptoxanthin content in the pulp of the parent varieties "Tsunobo" and "Setoka" is less than 2.00 mg / 100 g FW, as described in claim 10.

14. The citrus plant according to claim 10, further having at least one of the following traits (4) to (7): (4) The fruit has a high sugar content and good taste (5) There are few thorns (6) There is low biennial bearing (7) The peel is thin.

15. A method for identifying a citrus plant according to claim 10, 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.

16. The citrus plant according to claim 10, in the form of a seedling or scion.

17. The fruit of the citrus plant according to claim 10.

18. A processed product of a citrus plant or the fruit of a citrus plant obtained from the citrus plant described in claim 10 or the fruit of a citrus plant described in claim 16.

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.

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