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

Abstract

Provided are citrus plants, a method for producing citrus plants, and uses thereof. [Solution] A method for producing citrus plants, comprising the step of selecting, from a hybrid variety obtained by crossing citrus varieties or a progeny plant thereof, plants having the following genotypes for genetic markers: 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.

Description

[Technical Field]

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

[0002] Until now, mid-late citrus varieties that ripen from late January to February have been developed, but the varieties shipped from late December to early-mid-January are mainly Iyo and Ponkan, and there has been insufficient development and distribution of high-quality mid-season varieties.In addition, there is a growing concern that the increase in imported citrus fruits will erode the domestic citrus market.

[0003] In addition, β-cryptoxanthin, a type of carotenoid found in citrus plants such as Satsuma mandarins, is known to have health-related functional properties, and there is growing demand for citrus varieties for various uses, including eating them raw. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] DNA variety identification technology for 24 citrus varieties using CAPS markers, National Agriculture and Food Research Organization, published June 17, 2022 Summary of the Invention [Problem to be solved by the invention]

[0005] As mentioned above, there is a desire to develop high-quality mid-season varieties that mature from late December to early-mid-January.

[0006] The daily intake of β-cryptoxanthin as a functional component is said to be 3-6 mg, and it is thought that eating three Satsuma mandarins would provide a sufficient daily intake of this functional component. However, in daily life, consuming three fresh Satsuma mandarins per day is difficult in terms of quantity and cost.

[0007] Therefore, we aimed to develop a variety that is mid-season, ripening from late December to early-mid January, has good taste, is differentiated from imported citrus fruits, and is high in health functional ingredients to meet consumers' health-conscious needs. [Means for solving the problem]

[0008] A method for producing a citrus plant according to one aspect of the present invention includes the steps of: In hybrid varieties obtained by crossing citrus varieties or their progeny plants, genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 are shown below, respectively. Bf0158-3 is the BB type, Bf0036-3 is type AB, Tf0150-2 is type AB, Tf0271-2 is type AA, Tf0300-3 is type AB, Tf0419-2 is type BB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type BB, Mf0090-2 is type BB, Gn0073-2 is AA type, and Gn0048-2 is type AA The method includes a step of selecting a plant having the genotype. Furthermore, a method for producing a citrus plant according to one aspect of the present invention includes the steps of: The hybridization process involves crossbreeding the citrus variety "Tsuno Nozomi" as the mother and the citrus variety "Setoka" as the father. and a marker selection process in which candidate citrus plants having different genotype patterns of the genetic markers from the parent varieties "Tsuno Nozomi" and "Setoka" are selected from the citrus plants obtained by the hybridization process or their progeny using at least one of genetic markers Bf0036-3, Tf0150-2, Al0302-2, and Gn0048-2.

[0009] Furthermore, in one aspect of the present invention, there is provided a citrus plant, wherein the genotype of the genetic marker in the citrus plant is: Bf0158-3 is the BB type, Bf0036-3 is type AB, Tf0150-2 is type AB, Tf0271-2 is type AA, Tf0300-3 is type AB, Tf0419-2 is type BB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type BB, Mf0090-2 is type BB, Gn0073-2 is AA type, and Gn0048-2 is an AA type citrus plant. Furthermore, a citrus plant according to one aspect of the present invention is a citrus plant having the following traits (1), (2), and (3): (1) It was obtained by crossbreeding the citrus cultivar "Tsuno Nozomi" as the mother and the citrus cultivar "Setoka" as the father. (2) having a fruit having a β-cryptoxanthin content of 2.00 mg / 100 gFW or more and 2.50 mg / 100 gFW or less in the flesh, (3) It can be distinguished from other citrus cultivars, including "Tsuno Nozomi" and "Setoka," using at least one of the following genetic markers: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0010] Furthermore, an identification method according to one aspect of the present invention is a method for identifying the citrus plant, comprising the steps of: amplifying a region of the DNA of a citrus plant containing a genetic marker (CAPS marker) of the DNA using a primer set; and identifying the citrus plant based on base polymorphisms in the amplified DNA region; The method is characterized in that 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] A citrus plant or a fruit thereof according to another aspect of the present invention is a citrus plant or a fruit thereof obtained by the method for producing a citrus plant.

[0012] A processed product according to one aspect of the present invention is a processed product produced using the citrus plant or its fruit. Furthermore, a fruit according to one aspect of the present invention is a fruit of the citrus plant. A processed product according to one aspect of the present invention is obtained from the citrus plant or the fruit of the citrus plant. [Effects of the Invention]

[0013] According to the present invention, a novel citrus plant, a method for producing a citrus plant, and uses thereof can be provided. [Brief explanation of the drawings]

[0014] [Figure 1] This is the pedigree diagram of "Citrus KN53". [Figure 2] FIG. 1 is a comparison diagram of the β-cryptoxanthin content in the flesh of "Citrus KN53" and Satsuma mandarin. [Figure 3] FIG. 1 is a diagram showing the fruit of "Citrus KN53." [Figure 4] FIG. 1 is a diagram showing the tree of "Citrus KN53." [Figure 5] FIG. 1 shows the genotype of CAPS markers in "Citrus KN53." [Figure 6] DNA extracted from "Citrus KN53" was PCR-amplified using the genetic markers (CAPS markers) Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, and Tf0419-2, digested with specific restriction enzymes, and then analyzed by agarose gel electrophoresis to determine the genotype of each genetic marker. In the figure, M indicates the electrophoretic pattern of a 100-bp ladder, and S indicates the electrophoretic pattern of "Citrus KN53" DNA after restriction enzyme digestion. [Figure 7] DNA extracted from "Citrus KN53" was PCR-amplified using the genetic markers Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, and Mf0090-2, digested with specific restriction enzymes, and then analyzed by agarose gel electrophoresis to determine the genotype of each marker. In the figure, M indicates the electrophoretic pattern of a 100-bp ladder, and S indicates the electrophoretic pattern of DNA from "Citrus KN53" after restriction enzyme digestion. [Figure 8]DNA extracted from "Citrus KN53" was PCR-amplified using the genetic markers Gn0073-2 and Gn0048-2, digested with specific restriction enzymes, and then subjected to agarose gel electrophoresis to examine the genotype of each genetic marker. In the figure, M indicates the electrophoretic pattern of a 100-bp ladder, and S indicates the electrophoretic pattern of the DNA of "Citrus KN53" after restriction enzyme treatment. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below. All of the documents mentioned in this specification are incorporated herein by reference.

[0016] In this specification, the term "citrus plants" refers to plants of the genus Citrus L. in the family Rutaceae. "Citrus" is a general term for plants belonging to the genera Citrus (Citrus), Fortunella, and Poncirus in the subfamily Rutaceae of the family Rutaceae.

[0017] As used herein, the term "plant" may refer to a part or the whole of a plant body, and may refer to any stage of plant development.

[0018] Examples of plant parts include growing shoots, lateral buds, growing branches, branches, leaves, petioles, thorns, stems, roots, stalks, leaves, flowers, flower buds, petals, pollen, anthers, fruits, seeds, etc. Also included are the exocarp, endocarp, and flesh that make up fruits. Plant parts include cells or tissues of the plant, such as embryos, hypocotyls, meristematic cells, callus, and protoplasts obtained from cells of the plant.

[0019] As used herein, the term "traits" refers to the morphological and physiological characteristics of a plant. As used herein, CAPS means cleaved amplified polymorphic sequence (CAPS). In this specification, FW is an abbreviation for Fresh Weight, and gFW indicates fresh weight in grams.

[0020] [Citrus plant production method 1] A method for producing a citrus plant according to one aspect of the present invention is a method for producing a citrus plant, comprising: In hybrid varieties obtained by crossing citrus varieties or their progeny plants, genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 are shown below, respectively. Bf0158-3 is the BB type, Bf0036-3 is type AB, Tf0150-2 is type AB, Tf0271-2 is type AA, Tf0300-3 is type AB, Tf0419-2 is type BB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type BB, Mf0090-2 is type BB, Gn0073-2 is AA type, and Gn0048-2 is type AA The method for producing a citrus plant includes a step of selecting a plant having the genotype. In one embodiment, the production method may further include a step of crossbreeding "Tsuno Nozomi" as the mother and "Setoka" as the father to obtain the hybrid variety. In one embodiment, the plant used in the selection process may be a hybrid variety obtained by crossing citrus varieties with each other, or a progeny plant thereof, but is preferably a hybrid variety obtained by crossing citrus varieties with each other. In one embodiment, a second selection step may be included before, simultaneously with, or after the selection step, in which citrus plants having the trait of having fruit with a β-cryptoxanthin content in the flesh 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) are selected. In one embodiment, the production method may further include a preliminary selection step, prior to the selection step, of selecting a candidate citrus plant line having the genotypes of the genetic markers Bf0036-3: AB, Tf0150-2: AB, Al0302-2: AB, and Gn0048-2: 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 comprise a propagation step in which the citrus plant is vegetatively propagated.

[0021] (Marker-based selection process (identification process)) In one embodiment, the production method involves selecting hybrid varieties or their progeny plants obtained by crossing citrus varieties with each other using genetic markers (CAPS markers): Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0022] Variety identification using genetic markers can be performed by the method described in Non-Patent Document 1. The outline of the method is as follows. First, DNA is extracted using existing methods from a hybrid variety obtained by crossing citrus varieties with another citrus variety, or from a progeny plant. Next, PCR amplification of the extracted DNA is performed using pairs of forward and reverse primers corresponding to each genetic marker listed in the table below. The amplified DNA is cleaved with each restriction enzyme listed in the table below. The DNA after restriction enzyme treatment is subjected to agarose gel electrophoresis, and the electrophoresis pattern is confirmed. The genotype of each genetic marker can be determined based on differences in the electrophoresis pattern. This makes it possible to identify citrus varieties. Therefore, the production method may further include a step of extracting DNA from a hybrid variety obtained by crossing citrus varieties with each other, or from a progeny plant thereof. The production method may further include a step of amplifying the extracted DNA using a pair of forward and reverse primers corresponding to each genetic marker. The production method may further include a step of treating the amplified DNA with a restriction enzyme. The production method may further include a step of electrophoresing the DNA after the restriction enzyme treatment. The production method may further include a step of determining the genotype for each genetic marker from the electrophoresis pattern of DNA obtained by electrophoresis. The production method may further include a step of selecting a plant having the above-described genotype for each genetic marker based on the results of determining the genotype for each genetic marker.

[0023] [Table 1] TIFF0007756986000003.tif146155

[0024] The genotype of Bf0158-3 can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected near 352(±10)bp and no bands are detected near 149(±10)bp and 203(±10)bp. Type BB is when bands are detected near 149(±10)bp and 203(±10)bp and no bands are detected near 352(±10)bp. Type AB is when bands are detected near 149(±10)bp, 203(±10)bp, and 352(±10)bp.

[0025] The genotype of Bf0036-3 can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. If a band can be confirmed around 399 (±10) bp but no bands can be confirmed around 162 (±10) bp and 237 (±10) bp, it is called type AA. If bands can be confirmed around 162 (±10) bp and 237 (±10) bp but no bands can be confirmed around 399 (±10) bp, it is called type BB. If bands can be confirmed around 162 (±10) bp, 237 (±10) bp, and 399 (±10) bp, it is called type AB.

[0026] The Tf0150-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected near 385 (±10) bp and no bands are detected near 152 (±10) bp and 233 (±10) bp. Type BB is when bands are detected near 152 (±10) bp and 233 (±10) bp and no bands are detected near 385 (±10) bp. Type AB is when bands are detected near 152 (±10) bp, 233 (±10) bp, and 385 (±10) bp.

[0027] The Tf0271-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. AA type is when a band is detected around 400(±10)bp and no bands are detected around 157(±10)bp and 243(±10)bp. BB type is when bands are detected around 157(±10)bp and 243(±10)bp and no bands are detected around 400(±10)bp. AB type is when bands are detected around 157(±10)bp, 243(±10)bp, and 400(±10)bp.

[0028] The Tf0300-3 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected near 393 (±10) bp and no bands are detected near 166 (±10) bp and 227 (±10) bp. Type BB is when bands are detected near 166 (±10) bp and 227 (±10) bp and no bands are detected near 393 (±10) bp. Type AB is when bands are detected near 166 (±10) bp, 227 (±10) bp, and 393 (±10) bp.

[0029] The Tf0419-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected near 389 (±10) bp and no bands are detected near 167 (±10) bp and 222 (±10) bp. Type BB is when bands are detected near 167 (±10) bp and 222 (±10) bp and no bands are detected near 389 (±10) bp. Type AB is when bands are detected near 167 (±10) bp, 222 (±10) bp, and 389 (±10) bp.

[0030] The Tf0420-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected near 388(±10)bp and no bands are detected near 162(±10)bp and 226(±10)bp. Type BB is when bands are detected near 162(±10)bp and 226(±10)bp and no bands are detected near 388(±10)bp. Type AB is when bands are detected near 162(±10)bp, 226(±10)bp, and 388(±10)bp.

[0031] The Tf0318-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected around 400 (±10) bp and no bands are detected around 135 (±10) bp and 265 (±10) bp. Type BB is when bands are detected around 135 (±10) bp and 265 (±10) bp and no bands are detected around 400 (±10) bp. Type AB is when bands are detected around 135 (±10) bp, 265 (±10) bp, and 400 (±10) bp.

[0032] The Tf0293-4 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected near 394(±10)bp and no bands are detected near 157(±10)bp and 237(±10)bp. Type BB is when bands are detected near 157(±10)bp and 237(±10)bp and no bands are detected near 394(±10)bp. Type AB is when bands are detected near 157(±10)bp, 237(±10)bp, and 394(±10)bp.

[0033] The genotype of Al0302-2 can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. If a band can be confirmed around 383 (±10) bp but no bands can be confirmed around 167 (±10) bp and 216 (±10) bp, it is called AA type. If bands can be confirmed around 167 (±10) bp and 216 (±10) bp but no bands can be confirmed around 383 (±10) bp, it is called BB type. If bands can be confirmed around 167 (±10) bp, 216 (±10) bp, and 383 (±10) bp, it is called AB type.

[0034] The genotype of Mf0097-2 can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. If a band can be confirmed around 369 (±10) bp but no bands can be confirmed around 166 (±10) bp and 203 (±10) bp, it is called type AA. If bands can be confirmed around 166 (±10) bp and 203 (±10) bp but no bands can be confirmed around 369 (±10) bp, it is called type BB. If bands can be confirmed around 166 (±10) bp, 203 (±10) bp, and 369 (±10) bp, it is called type AB.

[0035] The Mf0090-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is detected around 400(±10)bp and no bands are detected around 139(±10)bp and 261(±10)bp. Type BB is when bands are detected around 139(±10)bp and 261(±10)bp and no bands are detected around 400(±10)bp. Type AB is when bands are detected around 139(±10)bp, 261(±10)bp, and 400(±10)bp.

[0036] The genotype of Gn0073-2 can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band can be confirmed around 394(±10)bp and no bands can be confirmed around 149(±10)bp and 245(±10)bp. Type BB is when bands can be confirmed around 149(±10)bp and 245(±10)bp and no bands can be confirmed around 394(±10)bp. Type AB is when bands can be confirmed around 149(±10)bp, 245(±10)bp, and 394(±10)bp.

[0037] The genotype of Gn0048-2 can be divided into AA, AB, and BB types based on differences in electrophoresis patterns. Type AA is when a band is observed around 380(±10)bp and no bands are observed around 134(±10)bp and 246(±10)bp. Type BB is when bands are observed around 134(±10)bp and 246(±10)bp and no bands are observed around 380(±10)bp. Type AB is when bands are observed around 134(±10)bp, 246(±10)bp, and 380(±10)bp.

[0038] In one embodiment, the method of making comprises: Bf0158-3 is the BB type, Bf0036-3 is type AB, Tf0150-2 is type AB, Tf0271-2 is type AA, Tf0300-3 is type AB, Tf0419-2 is type BB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type BB, Mf0090-2 is type BB, Gn0073-2 is AA type, and Gn0048-2 is type AA The method includes a step of selecting a citrus plant having the genotype.

[0039] In one embodiment, the production method may further include a step of crossbreeding "Tsuno Nozomi" as the mother and "Setoka" as the father to obtain the hybrid variety. "Tsuno Nozomi" is an early-maturing variety with good taste and excellent fruiting ability, and is registered as Japan Variety Registration No. 20788. "Setoka" is a medium-maturing variety with good taste, fragrance, and excellent appearance, and is registered as Japan Variety Registration No. 9398. The citrus plants selected in the selection process have the genotype pattern of the above genetic markers. In contrast, in the other citrus cultivar "Tsu no Nozomi," Bf0036-3 is AA type and Al0302-2 is AA type. Therefore, "Tsu no Nozomi" can be distinguished from other citrus cultivars using Bf0036-3 and / or Al0302-2. In addition, in another citrus cultivar, "Setoka," Tf0150-2 is BB type, Al0302-2 is BB type, and Gn0048-2 is AB type. Therefore, Tf0150-2, Al0302-2, and / or Gn0048-2 can be used to distinguish "Setoka" from other citrus cultivars. Additionally, Bf0036-3, Tf0150-2, Al0302-2, and / or Gn0048-2 can be used to distinguish from both other citrus cultivars "Tsuno Nozomi" and other citrus cultivars "Setoka." Additionally, Bf0036-3, Tf0150-2, Al0302-2, and Gn0048-2 can be used to distinguish from both other citrus cultivars, "Tsuno Nozomi" and "Setoka." Furthermore, Al0302-2 can be used to distinguish it from both the other citrus cultivar "Tsuno Nozomi" and the other citrus cultivar "Setoka."

[0040] (Estimation of parent lineage) The genotypes listed in Tables 2 to 5 below represent the combination of alleles (allelic genes) detected for each variety at each genetic marker. Because citrus is typically a diploid plant, the two alleles found in the offspring must be inherited, one from the seed parent and one from the pollen parent. For example, if a variety has the AB genotype, it could be a breeding parent if either parent has an A or B allele, but if both parents are BB, this combination is unlikely to be a parent. Based on this principle, potential breeding parents can be estimated from information on 14 types of genetic markers.

[0041] Citrus plants having the above genotypes selected through the selection process using the above genetic markers have different genotypes of genetic markers from other citrus varieties, as is clear from the following Tables 2 to 5. Therefore, citrus plants selected through the above selection process can be produced in a manner that distinguishes them from other citrus varieties.

[0042] [Table 2]

[0043] [Table 3]

[0044] [Table 4]

[0045] [Table 5]

[0046] In one embodiment, the production method may include a second selection step, which is performed before, simultaneously with, or after the selection step using a genetic marker, to select citrus plants having a trait of producing fruit having a β-cryptoxanthin content in the flesh 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 second selection step may be performed before, simultaneously with, or after the selection step using a genetic marker, but is preferably performed after the selection step using a genetic marker. The preferred range of the β-cryptoxanthin content is described below. Citrus fruits selected using genetic markers can be enriched in β-cryptoxanthin, which is known to be a functional component with health benefits, and the creation of citrus fruits enriched in β-cryptoxanthin would be extremely useful in the industrial world.

[0047] In one embodiment, the production method may further include a preliminary selection step, prior to the selection step, of selecting a candidate citrus plant line 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. Alternatively, the production method may further include a preliminary selection step of selecting a candidate citrus plant line in which Al0302-2 is 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 a step of crossing "Tsuno Nozomi" as the mother and "Setoka" as the father to obtain the hybrid variety, and a preliminary selection step of selecting a candidate citrus plant line 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. In one embodiment, the production method may further include a step of crossing "Tsuno Nozomi" as the mother and "Setoka" as the father to obtain the hybrid variety, and a preliminary selection step of selecting a candidate citrus plant line in which the genotype of the genetic marker Al0302-2 is type AB.

[0048] (Trait-based selection process) In order to obtain a citrus plant according to this embodiment, a selection step based on specific traits among those listed below can also be carried out. The selection step based on a specific trait can be carried out together with the selection step using a genetic marker as an indicator, or can be carried out before the selection step using a genetic marker as an indicator, or can be carried out after the selection step using a genetic marker as an indicator. In a preferred example, after the selection step using a genetic marker as an indicator has been carried out, the selection step based on a specific trait is carried out on the candidate citrus plant lines that have been sufficiently narrowed down.

[0049] The main traits used for selection include, for example, fruit 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 flesh, fruit maturity in early January, fruit with high sugar content and good taste, low incidence of thorns, low alternate bearing, and thin skin. Details of the above traits are described below in the section [Citrus plants].

[0050] The method for producing a citrus plant according to one embodiment of the present invention may further include an additional selection step in which the hybrid variety is selected based on at least one of the criteria described in the Examples. The additional selection step may be carried out multiple times over multiple years. The additional selection step is preferably carried out after the selection step using genetic markers.

[0051] Additional selection steps may include, for example, a pre-selection step, a primary selection step, and a secondary selection step.

[0052] The preliminary selection step may be a step of selecting based on at least one of the following criteria (a) to (e): (a) Multi-year evaluation = sufficient fruiting, (b) average sugar content of 12 Brix% or more Focus on individuals with (c) a sugar-acid ratio of 13 or higher and good taste, (d) an average seed number of "few" or less, and (e) an estimated value of logBCR > 0 as estimated by multiple regression of BCR content using a simple color difference meter (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 selecting based on at least one of the following criteria (f) to (h): (f) annual bearing ability and yield, (g) evaluation of physiological disorders on the tree (e.g., peel disorders such as peeling and cracking), and (h) observation of the extent of disease occurrence (e.g., canker, scab).

[0054] Subsequently, the secondary selection process may be a process of selecting based on at least one of the following criteria (i) to (j): (i) a lineage adaptability test (regional adaptability evaluation), and (j) a higher carotenoid (β-cryptoxanthin) content than Satsuma mandarins in an evaluation of regional differences.

[0055] (Proliferation process) The method for producing a citrus plant according to one aspect of the present invention may further include a propagation step of vegetatively propagating the citrus plant cultivated through the selection step.

[0056] Asexual propagation can be achieved by grafting, cuttings, layering, or cell culture. Asexual propagation is also called unsexual propagation, asexual reproduction, or vegetative propagation. Asexual propagation does not require males or females, and a single individual independently generates a new individual, with the reproductive cells produced by that single individual independently becoming a new individual. Furthermore, techniques for creating cell cultures of plant tissues and methods for regenerating plants from tissue cultures are carried out using known techniques.

[0057] In the present invention, the rootstock used for the grafting can be any of various tree species that can be used as citrus trees or citrus rootstocks, such as trifoliate orange, satsuma mandarin, and shikuwasa. The type and size of the rootstock used can be changed depending on the soil, climate, etc. For example, trifoliate orange can be used as the rootstock.

[0058] The cultivation type of the citrus plant according to the present invention is not particularly limited, but may be in the form of a high-grafted plant, a seedling, etc. dew Examples include ground cultivation, unheated greenhouse cultivation, and roofed greenhouse cultivation.

[0059] [Citrus plant production method 2] A method for producing a citrus plant according to one embodiment of the present invention includes a hybridization step of crossing the citrus cultivar "Tsuno Nozomi" as a mother and the citrus cultivar "Setoka" as a father; and a marker selection process in which candidate citrus plants having different genotype patterns of the genetic markers from the parent varieties "Tsuno Nozomi" and "Setoka" are selected from the citrus plants obtained by the hybridization process or their progeny using at least one of genetic markers Bf0036-3, Tf0150-2, Al0302-2, and Gn0048-2.

[0060] The creation method can also be referred to as a manufacturing method, a growing method, or a production method.

[0061] (Hybridization process) In the crossbreeding process, "Tsuno Nozomi" is used as the mother (seed parent) and "Setoka" is used as the father (pollen parent).

[0062] The parent varieties used in the crossbreeding, "Tsuno Nozomi" and "Setoka," are both varieties distributed on the market. "Tsuno Nozomi" is an early-maturing variety with good taste and excellent fruiting ability, and is registered as Japan Variety Registration No. 20788. "Setoka" is a medium-maturing variety with good taste, fragrance, and excellent appearance, and is registered as Japan Variety Registration No. 9398.

[0063] (Marker-based selection process (identification process)) In one embodiment, in the marker-based selection process, a genetic marker (CAPS marker) selected from 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 is used to identify and select candidate citrus plants whose genotype patterns of the genetic markers differ from those of the parent varieties "Tsuno Nozomi" and "Setoka."

[0064] The identification of varieties from other varieties, including parent varieties, in marker selection will be described in detail in the section below entitled "Method for identifying citrus plants."

[0065] The genotype patterns of all 14 CAPS markers may be determined, but depending on the purpose of selection, only a portion of the 14 CAPS markers may be used. In one example, in the marker-based selection step, a combination of at least one, two, three, or four genetic markers from among 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 "Tsu no Nozomi" and "Setoka." In one embodiment, in the marker-based selection step, a combination of at least one genetic marker from among Tf0150-2 and Gn0048-2 with a genetic marker from Bf0036-3, and at least one genetic marker from Al0302-2 is used to select candidate citrus plant lines that have different genotype patterns of the genetic markers from the parent varieties "Tsu no Nozomi" and "Setoka." In one embodiment, in the marker selection step, genetic markers Tf0150-2, Gn0048-2, Bf0036-3, and Al0302-2 are used to select candidate citrus plant lines that have different genotype patterns of the genetic markers from the parent varieties "Tsuno Nozomi" and "Setoka."

[0066] In one embodiment, it is preferable to select a candidate citrus plant line in which the genotypes of the genetic markers are AB for Bf0036-3, AB for Tf0150-2, and AA for Gn0048-2. In one embodiment, it is preferable to select a candidate citrus plant line in which the genotype of the genetic marker Al0302-2 is AB type. 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] (Trait-based selection process) To obtain the citrus plant of this embodiment, a selection process based on specific traits among those listed below can be carried out on candidate citrus plants obtained by crossbreeding the citrus variety "Tsuno Nozomi" as the mother and the citrus variety "Setoka" as the father. The selection step based on a specific trait can be carried out together with the selection step using a genetic marker as an indicator, or can be carried out before the selection step using a genetic marker as an indicator. In a preferred example, after the selection step using a genetic marker as an indicator, the selection step based on a specific trait is carried out on the sufficiently narrowed down candidate citrus plant lines.

[0068] The main traits used for selection include, for example, fruit with a β-cryptoxanthin content of 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less in the flesh, fruit maturity in early January, fruit with high sugar content and good taste, low incidence of thorns, low alternate bearing, and thin skin. Among these, it is preferable to select citrus plants having the trait of producing fruit having a β-cryptoxanthin content in the flesh of not less than 2.00 mg / 100 g FW and not more than 2.50 mg / 100 g FW. Details of the above traits are described below in the section [Citrus plants].

[0069] The selection step in the method for producing a citrus plant according to one embodiment of the present invention is a step of selecting plants obtained in the hybridization step based on at least one of the criteria described in the Examples, for example. In the selection step, selection may be carried out multiple times over multiple years.

[0070] That is, the selection step may include, for example, a preliminary selection step, a first selection step, and a second selection step.

[0071] The preliminary selection step may be a step of selecting based on at least one of the following criteria (a) to (e): (a) Multi-year evaluation = sufficient fruiting, (b) average sugar content of 12 Brix% or more Focus on individuals with (c) a sugar-acid ratio of 13 or higher and good taste, (d) an average seed number of "few" or less, and (e) an estimated value of logBCR > 0 as estimated by multiple regression of BCR content using a simple color difference meter (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 selecting based on at least one of the following criteria (f) to (h): (f) annual bearing ability and yield, (g) evaluation of physiological disorders on the tree (e.g., peel disorders such as peeling and cracking), and (h) observation of the extent of disease occurrence (e.g., canker, scab).

[0073] Subsequently, the secondary selection process may be a process of selecting based on at least one of the following criteria (i) to (j): (i) a lineage adaptability test (regional adaptability evaluation), and (j) a higher carotenoid (β-cryptoxanthin) content than Satsuma mandarins in an evaluation of regional differences.

[0074] (Proliferation process) The method for producing a citrus plant according to one embodiment of the present invention further comprises a propagation step of vegetatively propagating the citrus plant cultivated through the crossing step and the selection step.

[0075] Asexual propagation can be achieved by grafting, cuttings, layering, or cell culture. Asexual propagation is also called asexual propagation, asexual reproduction, or vegetative propagation. Asexual propagation does not require males or females, and a single individual independently generates a new individual, with the reproductive cells produced by that single individual independently becoming a new individual. Furthermore, techniques for creating cell cultures of plant tissues and methods for regenerating plants from tissue cultures are carried out using known techniques.

[0076] In the present invention, the rootstock used for the grafting can be any of various tree species that can be used as citrus trees or citrus rootstocks, such as trifoliate orange, satsuma mandarin, and shikuwasa. The type and size of the rootstock used can be changed depending on the soil, climate, etc. For example, trifoliate orange can be used as the rootstock.

[0077] The cultivation type of the citrus plants according to the present invention is not particularly limited, but examples include open-field cultivation, unheated greenhouse cultivation, and roofed greenhouse cultivation in the form of high-grafted plants or seedlings.

[0078] [How to identify citrus plants] As mentioned above, the citrus plants of the present invention can be identified by specific cultivar identification markers.

[0079] To date, DNA variety identification technology has been developed. This technology is effective in monitoring infringement of breeder's rights or proving infringement of rights. In order to prevent imports at the border, such as at customs, development is underway to enable simple and rapid variety identification at inspection sites such as customs.

[0080] The Fruit and Tea Division of the National Agriculture and Food Research Organization (NARO) has developed a variety identification technology using CAPS (cleaved amplified polymorphic sequence) markers for citrus seeds and seedlings distributed in Japan. Regarding this technology, the division has compiled and published a DNA variety identification technology manual for citrus varieties that complies with ISO (International Organization for Standardization) standards (Non-Patent Document 1).

[0081] CAPS marker analysis technology is a technique that uses specific restriction enzymes to cleave PCR-amplified fragments targeting specific genomic regions, and utilizes the differences in size of restriction enzyme fragments that arise from genomic DNA polymorphisms such as base substitutions, deletions, and insertions that exist between varieties. Analysis using this technology does not require expensive equipment, making it highly applicable to testing sites.

[0082] CAPS marker analysis involves amplifying the target region by PCR, then treating the resulting product with a restriction enzyme that recognizes a specific base sequence and cleaves DNA to detect polymorphisms. The specific sequence of the target PCR amplification product differs depending on the variety, and the difference in product length (genotype) after cleavage with the restriction enzyme is confirmed by electrophoresis using agarose gel or other instruments. To identify varieties, it is necessary to combine the results of multiple markers ("Points to Note When Validating DNA Variety Identification Technology (1st Edition, Established March 6, 2023, National Agriculture and Food Research Organization, Seed and Seedling Management Center)").

[0083] The method for identifying citrus plants according to the present invention is a method in which the CAPS marker is used as an identification marker for the citrus "KN53" or its progeny plants to distinguish them from plants of other citrus varieties.

[0084] A method for identifying citrus plants according to one embodiment of the present invention includes the steps of (I) amplifying a region of the DNA of a citrus plant containing a CAPS marker using a primer set, and (II) identifying the citrus plant based on base polymorphisms in the amplified DNA region, wherein the CAPS marker is at least one of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. The genetic markers (CAPS markers) may be one, more than one, or all of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2, but it is preferable to use all of them. In one embodiment, the method for identifying citrus plants can distinguish and identify the citrus plants of the present invention from other citrus cultivars based on the polymorphism of bases in the region (DNA region) containing the amplified genetic markers. Examples of other citrus cultivars include citrus cultivars other than KN53 listed in Tables 2 to 5.

[0085] Since polymorphisms in CAPS are determined by the presence or absence of a restriction enzyme site, the allele without a restriction enzyme site is designated as A, and the allele with a restriction enzyme site is designated as B, and the genotype of the variety can be determined as AA, AB, or BB.

[0086] (Identification target) The method for identifying citrus plants according to the present invention distinguishes the citrus plants of the present invention from plants of other citrus varieties.

[0087] Furthermore, in the citrus plant identification technology of the present invention, from the perspective of its identification principle, citrus plants that exhibit the same allele type as "KN53" for the 14 identification markers designated as marker names Bf0158-3 to Gn0048-2 among progeny plants of "KN53" can also be distinguished from other citrus varieties using this technology.

[0088] Furthermore, the same allele type as "KN53" refers to the allele type shown in Figure 5 for the 14 loci Bf0158-3 to Gn0048-2. As long as the allele type is derived from "KN53," the variety identification technology of the present invention can be applied even if mutations occur in the base sequences that make up these loci and their surrounding regions.

[0089] In the identification technique 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" for at least one of the 14 identification markers. In citrus plants belonging to other citrus varieties, the 14 allele types of Bf0158-3 to Gn0048-2 are different from those of "KN53."

[0090] Examples of 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," "Reiko," "Tsunohikari," "Seinan no Hikari," "Tsunozomi," "Haruhi," "Kiyomi," "Setoka," "Harumi," "Harehime," "Kanpei," and "Ehime Fruit Experiment No. 28 (Beni Madonna)," as shown in the manual of Non-Patent Document 1.

[0091] These 24 citrus varieties, together with "KN53" (25 varieties in total), account for more than 94% of the 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, Hyuganatsu, sudachi, shikuwasa, kara mandarin (kara), tankan, Seminole, kabosu, amakusa, koganekan, Kishu mikan, sweet spring, encore, Tamami, Nishinoka, Marcott, Nanko, Amaka, citrus intermediate parent No. 6, and bitter orange.

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

[0094] Furthermore, examples of 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, taking into account the results of the Examples below. For example, pomelo and the like can also be recognized as other citrus varieties that can be distinguished from "KN53" from the fundamental standpoint of the identification technology of the present invention.

[0095] For example, to distinguish "KN53" from the parent variety "Tsuno Nozomi," at least one of markers Bf0036-3 and Al0302-2 may be used.

[0096] Furthermore, when distinguishing "KN53" from the parent cultivar "Setoka", at least one of markers Tf0150-2, Al0302-2, and Gn0048-2 may be used.

[0097] Furthermore, when distinguishing "KN53" from each of its parent varieties, "Tsuno Nozomi" 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; when it is only necessary to distinguish only "KN53" from its parents, it is sufficient to use at least Al0302-2.

[0098] In an identification method according to one embodiment of the present invention, the subject citrus plant is a candidate plant for breeding material, a plant obtained through a breeding process, etc. Candidate plants for breeding material include, for example, parent plants used in crossbreeding and plants used in molecular breeding using recombinant DNA technology. Furthermore, the subject citrus plant includes citrus plants used in molecular breeding using recombinant DNA technology and citrus plants obtained by molecular breeding.

[0099] An identification method according to one aspect of the present invention is a method for identifying a citrus plant of the present invention, comprising the steps of: (I) amplifying a partial region of DNA in the DNA of a citrus plant using a primer set capable of amplifying the CAPS marker region described above; and (II) A method comprising a step of identifying citrus plants based on the base polymorphism of the amplified DNA region.

[0100] The region in the DNA of a citrus plant specimen can be amplified by polymerase chain reaction (PCR) using DNA extracted from the citrus plant specimen as a template and a primer set that amplifies a region containing the CAPS marker.

[0101] When DNA obtained from a citrus plant is amplified by PCR using the primer set, and the length of the DNA fragment obtained by restriction enzyme treatment matches the length of the DNA fragment obtained by similar treatment in a DNA sample obtained from a known citrus variety, the plant can be determined to be the known citrus variety.

[0102] That is, in one embodiment of the identification method of the present invention, the step (II) of identifying a citrus plant based on the base polymorphism of the amplified DNA region includes the steps of (II-1) treating the amplified DNA fragment with a restriction enzyme, and (II-2) identifying the genotype based on the length of the DNA fragment obtained by the restriction enzyme treatment.

[0103] The genotype of the amplified fragment can be determined by a conventionally known method, and a preferred example is a method using agarose gel electrophoresis.

[0104] The length of a DNA fragment can be measured, for example, by its molecular weight, which can be measured, for example, by its migration distance in agarose gel electrophoresis or by DNA sequencing.

[0105] The reaction reagents and reaction conditions used in the PCR method are not particularly limited as long as they are capable of producing primer-specific PCR amplified fragments, and known methods can be used.

[0106] The means for detecting the PCR-amplified fragments is not particularly limited, but as an example, the PCR-amplified fragments can be detected using ethidium bromide, fluorescent substances, chromogenic substances, luminescent substances, etc.

[0107] The primers in the primer set are composed of a base sequence having 90% or more sequence identity to the base sequences of the nucleotides constituting each primer in the primer set. Modified primers composed of modified base sequences obtained by modifying some bases in the base sequence are also included in the scope of primers used in the identification method of the present invention. Furthermore, the modified primer may have, for example, a sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% with the base sequence of each primer. In principle, a modified primer is considered equivalent to each primer if it can hybridize with the complementary DNA strand of each primer. Furthermore, this modified primer considered to be equivalent may be a primer that functions as a primer that hybridizes with the complementary DNA strand of the original primer and is composed of nucleotides having a modified base sequence in which, for example, 0 to 3, 0 to 2, 1 to 3, or 1 or 2 bases have been added, deleted, inserted, or substituted in the base sequence of the oligonucleotides constituting each original primer.

[0108] The materials and methods described in the manual of Non-Patent Document 1 can be suitably used for the primers, reagents, reaction conditions, detection methods, and the like related to the PCR described above.

[0109] The method for identifying citrus plants of the present invention can accurately and quickly identify the citrus plant "KN53" or its progeny from other citrus varieties, and is a method that can be widely introduced in inspection sites, etc. A method for identifying citrus plants according to one aspect of the present invention comprises: amplifying a region of the DNA of a citrus plant containing a genetic marker (CAPS marker) of the DNA using a primer set; and identifying the citrus plant based on the base polymorphism of the amplified DNA region; Preferably, the genetic marker (CAPS marker) is at least one of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. Furthermore, a method for identifying a citrus plant according to one aspect of the present invention comprises: amplifying a region of the DNA of a citrus plant containing a genetic marker (CAPS marker) of the DNA using a primer set; and identifying the citrus plant based on the base polymorphism of the amplified DNA region; More preferably, the genetic markers (CAPS markers) are Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. Furthermore, it is preferable to identify a citrus plant according to one aspect of the present invention when each genetic marker (CAPS marker) has the above-mentioned genotype.

[0110] [Citrus plants 1] In one aspect of the present invention, a citrus plant is a citrus plant having a genetic marker whose genotype is: Bf0158-3 is the BB type, Bf0036-3 is type AB, Tf0150-2 is type AB, Tf0271-2 is type AA, Tf0300-3 is type AB, Tf0419-2 is type BB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type BB, Mf0090-2 is type BB, Gn0073-2 is AA type, and Gn0048-2 is type AA, It is a citrus plant. As is clear from the above Tables 2 to 5, the citrus plant according to one embodiment of the present invention has a different genotype of genetic markers from other citrus varieties. Therefore, the citrus plant according to one embodiment of the present invention is a novel citrus plant.

[0111] In one embodiment, the citrus plant may be a hybrid variety or a progeny thereof obtained by crossbreeding the citrus cultivar "Tsuno Nozomi" as the mother and the citrus cultivar "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 thereof, but is preferably a hybrid variety obtained by crossing citrus varieties with each other. In one embodiment, the citrus plant preferably has fruit having a β-cryptoxanthin content in the flesh 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 obtained by crossbreeding the citrus cultivar "Tsuno Nozomi" as a mother and the citrus cultivar "Setoka" as a father, or a progeny thereof, and the citrus plant has fruit having a β-cryptoxanthin content in the flesh 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 citrus plant may be one in which the β-cryptoxanthin content in the flesh of the parent cultivars "Tsuno Nozomi" and "Setoka" is less than 2.00 mg / 100 g FW.

[0112] [Citrus plants 2] Furthermore, a citrus plant according to one aspect of the present invention is a citrus plant having the following traits (1), (2), and (3): (1) It was obtained by crossbreeding the citrus cultivar "Tsuno Nozomi" as the mother and the citrus cultivar "Setoka" as the father. (2) having fruit having a β-cryptoxanthin content in the flesh of not less than 2.00 mg / 100 gFW and not more than 2.50 mg / 100 gFW; and (3) It can be distinguished from other citrus cultivars, including "Tsuno Nozomi" and "Setoka," using at least one of the following genetic markers: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0113] Preferred embodiments of citrus plant 1 and / or citrus plant 2 will be described below.

[0114] A citrus plant according to a further embodiment of the present invention is a citrus plant further having at least one of the following traits (4) to (7): (4) The fruit has a high sugar content and a good taste. (5) Less thorns (6) Low alternate-bearing ability (7) The skin is thin. Furthermore, a citrus plant according to a further embodiment of the present invention is a citrus plant further having at least one of the following traits (4) to (8): (4) The fruit ripens in early January. (5) The fruit has a high sugar content and a good taste. (6) Less thorns (7) Low alternate-bearing ability (8) The skin is thin.

[0115] The characteristics of the citrus plants according to the present invention may vary depending on the conditions under which they are grown or cultivated, such as the climate, soil, environment, cultivation method, and tree age. In addition, in this specification, limitations on the maturity period and other periods are based on cultivation in Japan, and for example, Shizuoka City, Shizuoka Prefecture, where the plants are grown in the examples, may be used as the standard.

[0116] Furthermore, a citrus plant according to one embodiment of the present invention is a plant that has at least one of the following characteristics as a main characteristic that differs from the parent cultivar "Tsuno Nozomi" or "Setoka." The values ​​may be the average values ​​of measurements taken in each year for two or more years.

[0117] (Characteristics of the tree body) The tree's vigor is "slightly weak" to "medium", and is about the same as or slightly stronger than "Tsu no Nozomi" and "Setoka". The rate at which thorns occur on the branch shoots (spring branches) is low at around 5%, and the length of the thorns on the branch shoots is also short. The size of the petals is smaller than "Tsu no Nozomi" and "Setoka". There is no pollen, and it is less than "Tsu no Nozomi" and "Setoka". The style is "straight", and is less curved than "Tsu no Nozomi" and "Setoka".

[0118] The germination period, flowering period, and full coloring period may vary depending on climatic conditions, but in the same growing area, the germination period and peak flowering period are the same as those of "Tsu no Nozomi" 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 alternate bearing rate is the same as or "lower" than "Tsu no Nozomi" or "Setoka."

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

[0120] The flesh is deep orange in color, and the average fruit weight is about 150g, smaller than "Tsu no Nozomi" and "Setoka." The sugar content of the juice in early January is 13.6%, which is high and similar to "Tsu no Nozomi." The acid content is also high, at about 0.90g / 100ml, higher than "Tsu no Nozomi" but lower than "Setoka." The flavor is rich. The number of intact seeds per fruit is lower than "Tsu no Nozomi" and "Setoka." The proportion of seedless fruits is the same as or higher than "Setoka" under normal outdoor cultivation conditions, and higher than "Tsu no Nozomi." The seeds are polyembryonic. Based on the progress of coloring and fruit quality, the ripening period is slightly later than "Tsu no Nozomi" and earlier than "Setoka," around early January.

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

[0122] The characteristics of each of the above traits may be characteristic values ​​evaluated based on the Evaluation Standards for Other Citrus Fruits (Citrus L.) and Breeding Line Adaptability Test and Characteristic Test Survey Methods issued by the Ministry of Agriculture, Forestry and Fisheries of Japan (Fruit Tree Research Institute, National Agriculture and Food Research Organization, 2007).

[0123] Regarding the trait (4) possessed by the citrus plant of the present invention, that the fruit ripens in early January, preferably, when the cultivation type is, for example, top-grafted and grown outdoors, the fruit ripens in early January, and depending on the growing area and cultivation conditions, the fruit ripens from late December to mid-January. That is, the citrus plant is slightly later in maturity than "Tsu no Nozomi," and in one example, about 1 to 3 weeks or 1 to 2 weeks later than "Tsu no Nozomi." It is earlier in maturity than "Setoka," and in one example, about 1 to 6 weeks or 2 to 5 weeks earlier than "Setoka."

[0124] Furthermore, the citrus plant according to the present invention has the trait (5) that the fruit has a high sugar content and a good taste. Regarding the "taste", the sugar-acid ratio can also be used as an index to evaluate the "taste".

[0125] In this specification, "sugar content" is synonymous with sweetness and refers to the sugar content reading of fruit juice as measured by a sugar content meter. For example, the sugar content of fruit juice in early January. The sugar content is measured using a refractometer. The unit of measurement is, for example, Brix%.

[0126] In this specification, "sourness" refers to the citric acid content in fruit juice. The sourness is measured by converting the titration value of the fruit juice with an aqueous solution of sodium hydroxide.

[0127] The lower limit of the sugar content of the juice of the citrus plant of the present invention is preferably 12.0 Brix% or more, more preferably 13.0 Brix% or more, more preferably 13.6 Brix% or more, and even more preferably 14.0 Brix% or more. The upper limit of the sugar content is not particularly limited, but is provided to be equal to or greater than any of the lower limits, and is, for example, 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 is, provided that it is equal to or less than any of the upper limits, for example, 0.5 g / 100 ml or more, 0.6 g / 100 ml or more, 0.7 g / 100 ml or more, or 0.8 g / 100 ml or more. The "brix-acid ratio" is calculated as the saccharometer reading / citric acid content. The brix-acid ratio is, for example, 12.0 or more, 13.0 or more, 14.0 or more, or 15.0 or more.

[0129] For example, "good taste" includes a good balance between sugar content and acidity. Therefore, a product can be said to have good taste when its sugar-acid ratio is in the range of 12 to 14, and more preferably 14 or higher.

[0130] In the example, the sugar content of the juice of "KN53" harvested in early January was high at 13.6% on average. The acid content of the juice was low at about 0.90g / 100ml on average. 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 component that is effective in preventing osteoporosis and lifestyle-related diseases such as diabetes and arteriosclerosis.

[0132] Regarding the trait (2) possessed by the citrus plant of the present invention, that is, having fruit with a β-cryptoxanthin content in the flesh 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 flesh of the citrus plant of 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 pulp is not particularly limited, but is, provided that it is equal to or greater than one of the lower limits, for example, 2.45 mg / 100 g FW or less, 2.40 mg / 100 g FW or less, or 2.35 mg / 100 g FW or less.

[0133] The content of β-cryptoxanthin in the flesh of Unshu mandarins is approximately 1.75 mg / 100 gFW, the content of β-cryptoxanthin in the flesh of the parent cultivar "Tsuno Nozomi" is approximately 1.82 mg / 100 gFW, and the content of β-cryptoxanthin in the flesh of the parent cultivar "Setoka" is approximately 1.51 mg / 100 gFW.

[0134] Thus, the citrus plant according to the present invention has the characteristic of having a higher β-cryptoxanthin content in the flesh than Satsuma mandarin and the parent cultivars "Tsuno Nozomi" and "Setoka."

[0135] In one embodiment, the citrus plant may have fruit having a β-cryptoxanthin content in the flesh 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 progeny thereof obtained by crossbreeding the citrus cultivar "Tsuno Nozomi" as the mother and the citrus cultivar "Setoka" as the father, and may be a citrus plant having fruit 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 flesh. In one embodiment, the citrus plant may be a hybrid variety or a progeny thereof obtained by crossbreeding the citrus cultivar "Tsuno Nozomi" as the mother and the citrus cultivar "Setoka" as the father, and the citrus plant has fruit having a β-cryptoxanthin content in the flesh 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), and the β-cryptoxanthin content in the flesh of the parent cultivar "Tsuno Nozomi" and the parent cultivar "Setoka" is less than 2.00 mg / 100 g FW. The preferred content of β-cryptoxanthin in the flesh of a citrus plant in one embodiment of the present invention is as described above. The β-cryptoxanthin content in the flesh of the parent cultivar "Tsuno Nozomi" and the parent cultivar "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, the β-cryptoxanthin content in the flesh of a citrus plant according to a further aspect of the present invention allows it to be distinguished from the parent cultivar "Tsuno Nozomi" and the parent cultivar "Setoka."

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

[0137] The citrus plant of the present invention produces citrus fruits containing β-cryptoxanthin that is 30% or more higher than conventional varieties. In other words, by consuming two or fewer fruits of the present invention per day, the required daily amount (approximately 3 mg) of β-cryptoxanthin can be consumed, which is expected to be effective in preventing osteoporosis and other conditions, and health functionality can be expected.

[0138] Regarding the traits possessed by the citrus plants of the present invention, for example, trait (6) low thorn occurrence is evaluated by dividing the number of thorns on the examined shoot by the total number of leaf-bearing nodes on the examined shoot × 100. Trait (7) low alternate bearing is evaluated as strong when the first and second years are clearly distinguished based on the state of moderate fruit thinning after the third year from the first fruit set, medium when the first and second years are clearly distinguished, and low when the first and second years are unclear based on the state of moderate fruit thinning after several years of observation. Trait (8) thin pericarp can be evaluated by measuring the thickness of the pericarp at the equator of the fruit with a vernier caliper and averaging the thickness of the pericarp at the equator of five or more fruits.

[0139] The thickness of the pericarp of a citrus plant according to one embodiment of the present invention is, for example, 1.3 mm or more and 2.5 mm or less.

[0140] The citrus plant of the present invention may be a plant having characteristics equivalent to those of "KN53." A plant having equivalent characteristics means a plant that has the main characteristics of "KN53" when grown under the same environmental conditions. The main characteristics are (1) to (3) above. Preferably, the plant further has characteristics (5) to (8) above. Furthermore, the citrus plant of the present invention has, for example, four, three, two, or one of the characteristics (5) to (8) above. Furthermore, the citrus plant of the present invention also includes a plant that further has at least one or more characteristics from each of the characteristics listed under (Tree characteristics) and (Fruit characteristics).

[0141] Furthermore, the present invention relates to the trait (3) possessed by the citrus plant of the present invention, that is, the plant can be distinguished from other citrus cultivars, including "Tsuno Nozomi" and "Setoka," using at least one genetic marker selected from the group consisting 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 citrus plant may be a plant in which the genotypes of the genetic markers are Bf0158-3 BB type, Bf0036-3 AB type, Tf0150-2 AB type, Tf0271-2 AA type, Tf0300-3 AB type, Tf0419-2 BB type, Tf0420-2 AA type, Tf0318-2 BB type, Tf0293-4 AA type, Al0302-2 AB type, Mf0097-2 BB type, Mf0090-2 BB type, Gn0073-2 AA type, and Gn0048-2 AA type.

[0142] The citrus plants of the present invention include "KN53" described in the Examples and its progeny plants. The progeny plants include individual plants of the progeny plants or parts thereof.

[0143] In the present invention, the progeny plants also include plants obtained by crossing the "KN53" citrus plant with another citrus variety or other citrus plant, or by crossing the citrus plant with a wild citrus plant.

[0144] Progeny plants include individuals obtained using "KN53" as a parent line and their progeny, somatic hybrid plants and their progeny obtained by cell fusion between cells of the citrus plant and cells of other plant varieties, and individuals and their progeny obtained by grafting using citrus plants as rootstocks or scions.

[0145] A citrus plant according to one aspect of the present invention has the above-described traits, and the citrus plant or its fruit obtained by the above-described production method is also within the scope of the present invention. Therefore, the citrus plant may be the fruit of a citrus plant. The citrus plant according to one aspect of the present invention also falls within the scope of the present invention in the form of vegetative propagation material such as seedlings or scions.

[0146] [Citrus plant processed products] The processed citrus plant product of one embodiment of the present invention may be a processed citrus plant product obtained from the above-mentioned citrus plant. Alternatively, the processed citrus plant product of one embodiment of the present invention may be a processed citrus plant fruit product obtained from the fruit of the above-mentioned citrus plant. The processed product is preferably an edible processed product. Examples of processed citrus plant products according to one embodiment of the present invention include squeezed juice, extracts, squeezed residue, and extracted residue from the plant. Other examples include processed products of the plant itself, such as cutting, crushing, drying, powdering, freezing, various chemical treatments, making into a paste, or puree. In particular, processed fruit products that utilize the fruit and exhibit the above-mentioned processing methods are preferred. Suitable examples of such processed fruit products include fruit juice, fruit pulp, or juice containing fruit juice and fruit pulp, fruit pulp in syrup, dried fruit, jelly, and powdered processed confectionery ingredients. Among these, the processed product is preferably at least one selected from the group consisting of juice, syrup-preserved products, jelly, and powdered confectionery ingredients, and more preferably at least one selected from the group consisting of juice and syrup-preserved products.

[0147] Fruits as part of the plant body of citrus plants will now be further described. Fruits may be seeded fruits (with seeds) or seedless fruits (without seeds). Citrus fruits may be in the form of a package wrapped in an appropriate packaging material. The form of the packaging material is not particularly limited, but examples thereof include films, sheets, bags, and sacks. The material of the packaging material is not particularly limited, but examples thereof include paper and resin (plastic). Packages of isolated citrus fruit pieces contain one or more fruits. Packages of citrus fruit may contain only one type of citrus fruit or a combination of multiple types of citrus fruit. Packages of citrus fruit may also contain agricultural products other than citrus fruit.

[0148] Citrus fruits (including citrus fruit packages) may be affixed with a product label. The product label records at least one piece of information selected from the following: the name of the citrus variety, intellectual property rights information related to the citrus, information about the citrus producer, information about the citrus production history (e.g., information about pesticide spraying), information about the citrus distribution history, product price, harvest date, and information about the ripeness period. This information may be recorded directly on the product label (by printing or, for example, electromagnetic recording), or may be recorded readably using a code format such as a one-dimensional code or a two-dimensional code.

[0149] The citrus plant may be in the form of vegetative propagation material such as a seedling or a scion. The seedling or scion may have a product label attached thereto. The product label contains at least one piece of information (product information) selected from the following: the name of the citrus variety, intellectual property rights information related to the citrus, individual identification information (ID number) of the seedling or scion, genetic marker information of the seedling or scion (e.g., the genotype pattern of the CAPS markers described above), producer information of the seedling or scion, production history information of the seedling or scion (e.g., information on pesticide spraying), distribution history information of the seedling or scion, and product price. In a typical example, at least the individual identification information (ID number) of the seedling or scion is recorded. In another typical example, at least the individual identification information (ID number) of the seedling or scion and genetic marker information of the seedling or scion (e.g., the genotype pattern of the CAPS markers described above) are recorded. This information may be directly recorded on the product label (either printed or readable electromagnetically), or readably recorded using a code format such as a one-dimensional code or a two-dimensional code. The product label may also be comprised of a small electromagnetic recording medium such as an IC chip, embedded inside the seedling or scion. The location where the small electromagnetic recording medium such as an IC chip is embedded is not particularly limited. However, in the case of a grafted seedling, a preferred example is within the rootstock, and another preferred example is within a predetermined distance from the grafting position. The predetermined distance from the grafting position may be, for example, within 10 cm, 5 cm, 4 cm, 3 cm, 2 cm, or 1 cm from the grafting position. The predetermined distance from the grafting position toward the rootstock may be maintained, or the predetermined distance from the grafting position toward the scion may be maintained.

[0150] [Product information management system for citrus plant seedlings or scions (vegetative propagation materials)] In the above section "Processed Products of Citrus Plants," we have explained the seedlings or scions of citrus plants according to this embodiment with a product label attached. The scope of the present invention also includes a product information management system for the seedlings or scions of citrus plants according to this embodiment with a product label attached.

[0151] An example of a product information management system includes at least 1) a seedling or scion of a citrus plant according to this embodiment with a product label attached thereto, and 2) a reader (an example of a client terminal) that reads the product information recorded on the product label. The product information management system may further include 3) a server. The product information management system may include a client terminal other than the reader, or may include a client terminal instead of the reader. These client terminals may not have the function of reading product information.

[0152] In one example, the client terminal (which may be a reader) transmits a set of information recorded on the product label (especially the individual identification information (ID number) of the seedling or scion) and location information of the client terminal to the server. The client terminal (which may be a reader) may also transmit to the server information about the owner of the seedling or scion (which may be the owner information of the client terminal if it matches the owner of the client terminal), location information of the seedling or scion (if different from the location information of the client terminal), etc. This allows information about the location, etc. of each individual seedling or scion to be managed. Furthermore, if the owner of the seedling or scion changes, if the location of the seedling or scion is relocated, or if the seedling or scion is discarded, or if the management environment of the seedling or scion changes, this information is transmitted from the client terminal to the server.

[0153] 〔summary〕 The present invention includes any of the following aspects. <1> 1. A method for producing a citrus plant, comprising: In hybrid varieties obtained by crossing citrus varieties or their progeny plants, genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 are shown below, respectively. Bf0158-3 is the BB type, Bf0036-3 is type AB, Tf0150-2 is type AB, Tf0271-2 is type AA, Tf0300-3 is type AB, Tf0419-2 is type BB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type BB, Mf0090-2 is type BB, Gn0073-2 is AA type, and Gn0048-2 is type AA A method for producing a citrus plant, comprising the step of selecting a plant having a genotype. <2> The step of crossbreeding "Tsuno Nozomi" as the mother and "Setoka" as the father to obtain the hybrid variety is further included. <1> 2. A method for producing a citrus plant according to claim 1. <3> The method further comprises a second selection step of selecting, before, simultaneously with, or after the selection step, citrus plants having a trait of having fruit having a β-cryptoxanthin content in the flesh 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); <1> or <2> The method for producing the strain described above. <4> The method further comprises a propagation step of vegetatively propagating the citrus plant. <1> from <3> 2. A method for producing a citrus plant according to any one of the above. <5> 1. A method for producing a citrus plant, comprising: The hybridization process involves crossbreeding the citrus variety "Tsuno Nozomi" as the mother and the citrus variety "Setoka" as the father. and a marker selection step of selecting candidate citrus plants that have different genotype patterns of the genetic markers from the citrus plants obtained by the hybridization step or their progeny using at least one of Bf0036-3, Tf0150-2, Al0302-2, and Gn0048-2, from the parent varieties "Tsuno Nozomi" and "Setoka." <6> In the marker selection step, a combination of at least one genetic marker selected from Tf0150-2 and Gn0048-2 with the Bf0036-3 genetic marker, and at least one selected from the Al0302-2 genetic marker, is used to select candidate citrus plant lines that have different genotype patterns of the genetic markers from the parent varieties "Tsuno Nozomi" and "Setoka." <5> The method for producing the strain described above. <7> Selecting a candidate citrus plant line 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; <6> The method for producing the strain described above. <8> The method includes a selection step of selecting a citrus plant having a trait of having fruit having a beta-cryptoxanthin content of 2.00 mg / 100 gFW or more and 2.50 mg / 100 gFW or less in the flesh; <5> from <7> 2. A method for producing a strain of a plant according to any one of the preceding claims. <9> The method further comprises a propagation step of vegetatively propagating the citrus plant. <5> from <8> 2. A method for producing a citrus plant according to any one of the above. <10> A citrus plant, wherein the genotype of a genetic marker in the citrus plant is: Bf0158-3 is the BB type, Bf0036-3 is type AB, Tf0150-2 is type AB, Tf0271-2 is type AA, Tf0300-3 is type AB, Tf0419-2 is type BB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is AA type, Al0302-2 is type AB, Mf0097-2 is type BB, Mf0090-2 is type BB, Gn0073-2 is AA type, and Gn0048-2 is type AA, The citrus plant. <11> It is a hybrid variety or its descendants that was crossed using the citrus variety "Tsuno Nozomi" as the mother and the citrus variety "Setoka" as the father. <10> The citrus plant described in <12> The fruit has a β-cryptoxanthin content of 2.00 mg / 100 gFW or more (preferably 2.00 mg / 100 gFW or more and 2.50 mg / 100 gFW or less) in the flesh; <10> or <11> The citrus plant described in <13> A citrus plant that is a hybrid variety or a progeny thereof obtained by crossbreeding the citrus variety "Tsuno Nozomi" as a mother and the citrus variety "Setoka" as a father, and that 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 flesh; The beta-cryptoxanthin content in the flesh of the parent varieties "Tsuno Nozomi" and "Setoka" is less than 2.00 mg / 100 gFW. <10> from <12> 1. A citrus plant according to any one of the preceding items. <14> Further having at least one of the following traits (4) to (7): <10> from <13> 1. A citrus plant according to any one of the preceding items: (4) The fruit has a high sugar content and a good taste. (5) Fewer thorns (6) Low alternate-bearing ability (7) The skin is thin. <15> <10> from <14> A method for identifying a citrus plant according to any one of the above, amplifying a region of the DNA of a citrus plant, the region including a genetic marker of the DNA, using a primer set; and identifying the citrus plant based on base polymorphisms in the amplified DNA region; The method, wherein the genetic markers are Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. <16> in the form of seedlings or scions, <10> from <14> 1. A citrus plant according to any one of the preceding items. <17> <10> from <14> The fruit of any of the citrus plants described above. <18> <10> from <14> A citrus plant according to any one of the above or <17> A processed product of a citrus plant or the fruit of a citrus plant, obtained from the fruit of the citrus plant described in 1. <19> At least one selected from the group consisting of fruit juice, fruit pulp, or juice containing fruit juice and fruit pulp, fruit pulp preserved in syrup, dried fruit, jelly, powdered confectionery raw materials, etc. <18> A processed product of the citrus plant or the fruit of the citrus plant described in 1.

[0154] <2-1> A method for producing a citrus plant, The hybridization process involves crossbreeding the citrus variety "Tsuno Nozomi" as the mother and the citrus variety "Setoka" as the father. and a marker selection step of selecting candidate citrus plants that have different genotype patterns of the genetic markers from the citrus plants obtained by the hybridization step or their progeny using at least one of Bf0036-3, Tf0150-2, Al0302-2, and Gn0048-2, from the parent varieties "Tsuno Nozomi" and "Setoka." <2-2> The production method according to <2-1>, wherein in the marker selection step, a combination of at least one genetic marker selected 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 those of the parent varieties "Tsuno Nozomi" and "Setoka." <2-3> The method 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> The method according to any one of <2-1> to <2-3>, comprising a selection step of selecting a citrus plant having a trait of having fruit having a β-cryptoxanthin content in the flesh of 2.00 mg / 100 gFW or more and 2.50 mg / 100 gFW or less. <2-5> The method for producing a citrus plant according to any one of <2-1> to <2-4>, further comprising a propagation step of vegetatively propagating the citrus plant. <2-6> A citrus plant having the following traits (1), (2), and (3): (1) It was obtained by crossbreeding the citrus cultivar "Tsuno Nozomi" as the mother and the citrus cultivar "Setoka" as the father. (2) having a fruit having a β-cryptoxanthin content of 2.00 mg / 100 gFW or more and 2.50 mg / 100 gFW or less in the flesh, (3) It can be distinguished from other citrus cultivars, including "Tsuno Nozomi" and "Setoka," using at least one of the following genetic markers: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. <2-7> The citrus plant according to <2-6>, further having at least one of the following traits (4) to (7): (4) The fruit has a high sugar content and a good taste. (5) Fewer thorns (6) Low alternate-bearing ability (7) The skin is thin. <2-8> The citrus plant according to <2-6> or <2-7>, wherein the genotypes of the genetic markers are as follows: Bf0158-3 is 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 are AA type. <2-9> A method for identifying a citrus plant according to any one of <2-6> to <2-8>, amplifying a region of DNA of a citrus plant containing a CAPS marker using a primer set; and identifying the citrus plant based on base polymorphisms in the amplified DNA region; The method, wherein the CAPS marker is at least one of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. <2-10> A citrus plant or its fruit obtained by the method for producing a citrus plant according to any one of <2-1> to <2-5>. <2-11> The citrus plant according to any one of <2-6> to <2-8> or <2-10>, or a fruit thereof, in the form of a seedling or a scion. <2-12> A processed product produced using the citrus plant according to any one of <2-6> to <2-8> or the citrus plant according to <2-10> or its fruit.

[0155] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0156] [1. Development of citrus strains] (history) 1) Breeding method: Crossbreeding 2) Parents: "Tsuno Nozomi" x "Setoka" The cross was made using the early-maturing, good-tasting, and excellent-fruiting variety "Tsuno Nozomi" as the mother and the mid-maturing, good-tasting, fragrant variety "Setoka" as the father. The pedigree diagram is shown in Figure 1.

[0157] (Development process) This line was developed through hybridization at the Fruit Tree Research Institute (now the Kuchinotsu Citrus Research and Development Site of the National Agriculture and Food Research Organization's Kyushu Okinawa Agricultural Research Center) in 2001 with the goal of developing a citrus tree that would mature between December and January, have high sugar content, excellent flavor, be seedless, have a thin sepal membrane for easy eating, have a fragrant aroma, and have the ability to bear fruit year after year. In 2002, it was grafted onto a Shikuwasa (Citrus depressa) rootstock to promote fruiting. It first bore fruit in 2005, and fruit characteristics were investigated. In 2008, a superior individual was pre-selected and grafted onto a Satsuma mandarin (Unshu mandarin) interstock. Subsequently, investigations of fruit and tree characteristics continued for line KN53. Based on its superiority, it underwent a first selection in 2017 and was submitted to the 12th Citrus Line Adaptability and Characteristics Certification Test in April of the same year. Regional adaptability and potential for widespread use were examined at 25 test sites from Chiba Prefecture in the east to Kagoshima Prefecture in the south. As a result, the test results review meeting (evergreen fruit trees) in August 2023 concluded that the variety was suitable as a candidate for a new variety.

[0158] (Selection process) The selection criteria for the selection process were as follows: First selection, post-fruit evaluation (2005-2008) 1. Multi-year evaluation = has sufficient fruiting potential. 2. Sugar content average 12Brix% or more 3. A sugar-acid ratio of 13 or more provides good flavor. 4. Seed count averages below "low" 5. Using a simple color difference meter (Nippon Denshoku Industries NF333) Lab, estimate the BCR content using a multiple regression equation and focus on individuals with an estimated value of logBCR > 0 (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 the selected plants were then subjected to high-grafting. 6. Consecutive year results and yield 7. Evaluation of physiological disorders on the tree (peel disorders such as peeling and cracking) 8. Observation of the extent of disease occurrence Based on the selection criteria 6 to 8 above, the first selection was carried out in 2017, and the selected plants were further grown. 9. Strain adaptability test (regional adaptability evaluation) 10. Evaluation of regional differences in carotenoid (β-cryptoxanthin) and selection criteria based on higher content than Satsuma mandarins. Based on the selection criteria 9 to 10 above, a second selection was conducted in 2023, and the selected plant was established as "KN53."

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

[0160] [2. Evaluation of characteristics of each trait in the growing area] Subsequently, the traits of the resulting varieties were evaluated.

[0161] The trait evaluation method followed the Evaluation Standards for Agricultural, Forestry, and Fisheries Plant Types (Category: Other Citrus) (Ministry of Agriculture, Forestry, and Fisheries) and the Breeding Line Adaptability Test and Characteristic Test Survey Method (Fruit Tree Research Institute, National Agriculture and Food Research Organization, 2007). The following traits are based on prototypes grown in Japan from 2021 to 2023. The traits for each trait described above and the data described in the examples were evaluated based on the Evaluation Standards for Category: Other Citrus (Citrus L.) published by the Ministry of Agriculture, Forestry, and Fisheries of Japan (standards published by the Ministry of Agriculture, Forestry, and Fisheries as of 2021-2023) and the Breeding Line Adaptability Test and Characteristic Test Survey Method (Fruit Tree Research Institute, National Agriculture and Food Research Organization, 2007).

[0162] (Characteristics overview) 1) The fruit weighs approximately 150g, has a deep orange skin, is somewhat smooth, and is thin at approximately 2.0mm thick. Peeling is moderately easy to moderate. No peeling occurs. The sac is soft, and the flesh content is large at 83.9%. A survey conducted in early January found that the sugar content of the juice was high at over 13%, and the acid content was below 1.0%, giving it a rich flavor. Ripening occurs in early January. Under natural pollination conditions in outdoor cultivation, the average number of complete seeds is approximately 0.8, and seedless fruit can be produced. 2) The tree has medium vigor and the branching is intermediate between upright and spreading. The density of the branches is dense. The alternate bearing is low to medium, and fruiting is relatively stable. There is little thorn development. There is almost no scab development, and mild canker development is seen on the branches and leaves. 3) The average content of beta-cryptoxanthin, a health-functional component, in the flesh of the fruit was 2.33 mg / 100 gFW, significantly higher than that of Unshu mandarins (1.75 mg / 100 gFW). Furthermore, the minimum value was over 1.50 mg / 100 gFW, and eating two of these mandarins would provide the daily intake of the reported functional component (3-6 mg).

[0163] [Table 6] An overview of the features of the "KN53" is shown in Table 7 below.

[0164] [Table 7] (β-cryptoxanthin content) The β-cryptoxanthin content in the flesh of "KN53" was measured. As shown in Figure 2, the average β-cryptoxanthin content in the flesh was 2.33 mg / 100gFW, which was significantly higher than that of Unshu mandarins (1.75 mg / 100gFW). Furthermore, the minimum value was over 1.50 mg / 100gFW, and it was found that eating two of these mandarins would provide the daily intake of the reported functional component (3-6 mg).

[0165] (Suitable location and cultivation precautions) In previous strain adaptability tests, KN53 has been shown to be a year-round bearer, producing high-quality fruit with an average sugar content of 13% in January, the ripening period, at many test sites, suggesting that it has a wide range of adaptability to soil and weather conditions. KN53 is male sterile and produces very little pollen itself, so care should be taken to avoid planting pollen-rich varieties such as Hassaku and Amanatsu in the vicinity. In addition, to avoid encouraging the occurrence of scab disease, it is best to cultivate it in an orchard that is not exposed to strong winds, or in an orchard with a windbreak.

[0166] [3. Citrus plant identification using CAPS markers] We verified whether "KN53" can 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" could be distinguished from other citrus plants using previously reported CAPS markers.

[0168] The experiment was carried out according to the manual in Non-Patent Document 1. Specifically, the flavedo (outer pericarp) was excised from fresh fruit, and a DNA solution was obtained according to the manual in Non-Patent Document 1. For each CAPS marker, a PCR reaction was performed on the obtained DNA solution using the forward and reverse primers listed in Table 1, and an amplified product was obtained. The obtained amplified product was treated with each restriction enzyme listed in Table 1 to prepare a restriction enzyme reaction solution. The restriction enzyme reaction solution was applied to a 2% agarose gel, and polymorphism was confirmed by electrophoresis.

[0169] (result) The results of the polymorphism confirmed by electrophoresis are shown in Figures 6 to 8. In addition, Figure 5 shows the genotype of the CAPS marker of "KN53".

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

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

[0172] The present invention can be used in the fields of agriculture, plant breeding, and the like.

Claims

1. 1. A method for producing a citrus plant, comprising: A process of crossbreeding the citrus variety "Tsuno Nozomi" as a mother and the citrus variety "Setoka" as a father to obtain a hybrid variety; For the hybrid variety or its progeny plant, genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 were determined to be the following, respectively: Bf0158-3 is type BB, Bf0036-3 is type AB, Tf0150-2 is type AB, Tf0271-2 is type AA, Tf0300-3 is type AB, Tf0419-2 is type BB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is type AA, Al0302-2 is AB type, Mf0097-2 is type BB, Mf0090-2 is type BB, Gn0073-2 is type AA, and Gn0048-2 is type AA A method for producing a citrus plant, comprising the step of selecting a plant having a genotype.

2. The method of claim 1, further comprising a second selection step of selecting citrus plants having a trait of producing fruit having a β-cryptoxanthin content of 2.00 mg / 100 g FW or more in the flesh, before, simultaneously with, or after the selection step.

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

4. 1. A method for producing a citrus plant, comprising: The hybridization process involves crossbreeding the citrus variety "Tsuno Nozomi" as the mother and the citrus variety "Setoka" as the father. and a marker selection step of selecting candidate citrus plants that have a different genotype pattern of the genetic markers from the parent varieties "Tsuno Nozomi" and "Setoka" using at least one of genetic markers Bf0036-3, Tf0150-2, Al0302-2, and Gn0048-2 from the citrus plants obtained by the hybridization step or from the progeny citrus plants.

5. The method of claim 4, wherein in the marker selection step, a combination of at least one genetic marker selected from Tf0150-2 and Gn0048-2 with the Bf0036-3 genetic marker, and at least one selected from the Al0302-2 genetic marker, is used to select candidate citrus plant lines that have different genotype patterns of the genetic markers from the parent varieties "Tsunomi" and "Setoka".

6. 6. The method according to claim 5, wherein candidate citrus plant lines are 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.

7. The method according to any one of claims 4 to 6, further comprising a selection step of selecting a citrus plant having a trait of having fruit having a β-cryptoxanthin content in the flesh of 2.00 mg / 100 g FW or more and 2.50 mg / 100 g FW or less.

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

9. A citrus plant that is a hybrid variety or a progeny thereof that has been crossed using the citrus variety "Tsuno Nozomi" as the mother and the citrus variety "Setoka" as the father, wherein the genotype of a genetic marker in said citrus plant is: Bf0158-3 is type BB, Bf0036-3 is type AB, Tf0150-2 is type AB, Tf0271-2 is type AA, Tf0300-3 is type AB, Tf0419-2 is type BB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is type AA, Al0302-2 is AB type, Mf0097-2 is type BB, Mf0090-2 is type BB, Gn0073-2 is type AA, and Gn0048-2 is type AA, The citrus plant.

10. 10. The citrus plant of claim 9, having fruit with a β-cryptoxanthin content in the flesh of greater than or equal to 2.00 mg / 100 g FW and less than or equal to 2.50 mg / 100 g FW.

11. A citrus plant that is a hybrid variety or a progeny thereof obtained by crossbreeding the citrus variety "Tsuno Nozomi" as a mother and the citrus variety "Setoka" as a father, and that has fruit having a β-cryptoxanthin content in the flesh of not less than 2.00 mg / 100 g FW and not more than 2.50 mg / 100 g FW; The β-cryptoxanthin content in the flesh of the parent varieties "Tsuno Nozomi" and "Setoka" is less than 2.00 mg / 100 g FW. The citrus plant of claim 9.

12. A citrus plant as described in claim 9, wherein the juice has an average sugar content of 12 Brix% or more and / or a sugar-acid ratio of 13 or more.

13. 10. A method for identifying citrus plants according to claim 9, comprising: amplifying a region of the DNA of a citrus plant, the region including a genetic marker of the DNA, using a primer set; and and identifying the citrus plant based on base polymorphisms in the amplified DNA region; The method, wherein the genetic markers are Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

14. 10. The citrus plant of claim 9 in the form of a seedling or scion.

15. A fruit of the citrus plant according to claim 9.

16. A processed product of a citrus plant or a fruit of a citrus plant obtained from the fruit of the citrus plant according to claim 9 or the fruit of the citrus plant according to claim 15, said processed product being at least one selected from the group consisting of fruit juice, fruit pulp, or juice containing fruit juice and fruit pulp, fruit pulp preserved in syrup, dried fruit, jelly, and raw materials for powdered confectionery.

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