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

Abstract

This invention provides citrus plants and a method for producing citrus plants. The method for producing citrus plants includes: selecting plants containing genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2, each possessing the following characteristics. Bf0158-3 is type AB, Bf0036-3 is type AA, Tf0150-2 is type BB, Tf0271-2 is type AA, Tf0300-3 is type AB, Tf0419-2 is type AB, Tf0420-2 is type AA, Tf0318-2 is type BB, Tf0293-4 is type AA, Al0302-2 is type AB, Mf0097-2 is type AB, Mf0090-2 is type AB, Gn0073-2 is type AA, and Gn0048-2 is type AA.

Description

Technical Field

[0001] This invention relates to citrus plants, methods for producing citrus plants, and their utilization. Background Technology

[0002] To date, the country has been cultivating mid-to-late ripening citrus varieties that mature from late January to February. The varieties available from late December to mid-January are mainly Iyokan and Ponkan, but the cultivation and distribution of high-quality, large-fruited mid-ripening varieties are insufficient. Furthermore, concerns are growing that the increase in imported citrus fruit is eroding the Japanese citrus market.

[0003] [Existing Technical Documents]

[0004] [Non-patent literature]

[0005] [Non-Patent Literature 1] DNA Variety Identification Technology for 24 Citrus Varieties Based on CAPS Markers, National Research and Development Agency for Agriculture, Food and Rural Development, published June 17, 2022. Summary of the invention

[0006] [The problem the invention aims to solve]

[0007] As mentioned above, the goal is to cultivate high-quality, large-fruited mid-ripening varieties that mature from late December to mid-January.

[0008] Therefore, the aim is to cultivate varieties with mid-ripening characteristics, high sugar content, good taste, and large fruit that mature in early to mid-January.

[0009] [Methods used to solve problems]

[0010] One embodiment of the present invention relates to a method for producing citrus plants, comprising:

[0011] For hybrid varieties or their offspring obtained by crossing citrus varieties with each other, the procedure for selecting plants with the following genotypes for each of the genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 is as follows:

[0012] Bf0158-3 is type AB.

[0013] Bf0036-3 is type AA.

[0014] Tf0150-2 is a BB type.

[0015] Tf0271-2 is type AA.

[0016] Tf0300-3 is type AB.

[0017] Tf0419-2 is type AB.

[0018] Tf0420-2 is type AA.

[0019] Tf0318-2 is of type BB.

[0020] Tf0293-4 is type AA.

[0021] Al0302-2 is type AB.

[0022] Mf0097-2 is type AB.

[0023] Mf0090-2 is type AB.

[0024] Gn0073-2 is type AA, and

[0025] Gn0048-2 is of type AA.

[0026] Furthermore, one embodiment of the present invention relates to a method for producing citrus plants, comprising:

[0027] The hybridization process of using the mating parent (A) as the female parent and the mating parent (B) as the male parent, as shown below, and...

[0028] Crossparent (A): Varieties obtained by crossing the citrus variety "Nanxiang" and the citrus variety "Amakusa".

[0029] mating parent (B): Citrus variety "Southwest Light"

[0030] A marker selection process is used to identify candidate citrus plants that are different from mating parents (A) and mating parents (B) on the genotype map of the genetic markers by using at least one of the following genetic markers: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0419-2, Tf0420-2, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2, from citrus plants or their progeny obtained by the hybridization process.

[0031] Furthermore, the genotype of the genetic marker in the citrus plant involved in one embodiment of the present invention is as follows:

[0032] Bf0158-3 is type AB.

[0033] Bf0036-3 is type AA.

[0034] Tf0150-2 is a BB type.

[0035] Tf0271-2 is type AA.

[0036] Tf0300-3 is type AB.

[0037] Tf0419-2 is type AB.

[0038] Tf0420-2 is type AA.

[0039] Tf0318-2 is of type BB.

[0040] Tf0293-4 is type AA.

[0041] Al0302-2 is type AB.

[0042] Mf0097-2 is type AB.

[0043] Mf0090-2 is type AB.

[0044] Gn0073-2 is type AA, and

[0045] Gn0048-2 is of type AA.

[0046] Furthermore, one embodiment of the present invention relates to a citrus plant having the following characteristics (1), (2), (3), and (4):

[0047] (1) The result obtained by hybridization using the parent (A) as the female parent and the parent (B) as the male parent, as shown below.

[0048] Crossparent (A): Varieties obtained by crossing the citrus variety "Nanxiang" and the citrus variety "Amakusa".

[0049] mating parent (B): Citrus variety "Southwest Light"

[0050] (2) The fruit has a sugar content of over 13 Brix and a good taste.

[0051] (3) The fruit weighs more than 200g, and,

[0052] (4) Can be identified with other citrus varieties containing the mating parent (A) and mating parent (B) by using at least one of the genetic markers of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2 and Gn0048-2.

[0053] Additionally, a method for identifying a citrus plant according to one embodiment of the present invention includes a step of amplifying a region containing a genetic marker (CAPS marker) in the DNA of the citrus plant using a primer set, and a step of identifying the citrus plant based on the polymorphism of the bases in the amplified DNA region, wherein the genetic marker (CAPS marker) is at least one of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0054] In another embodiment of the present invention, the citrus plant or its fruit is obtained by the method for producing the citrus plant.

[0055] In addition, one embodiment of the present invention relates to processed products made using the citrus plant or its fruit.

[0056] In addition, one embodiment of the present invention relates to the fruit of the citrus plant.

[0057] In addition, one embodiment of the present invention relates to a processed product obtained from the citrus plant or the fruit of the citrus plant.

[0058] [The effects of the invention]

[0059] This invention provides new citrus plants, methods for producing citrus plants, and their utilization. Brief description of the attached diagram

[0060]

【 Figure 1 This is the pedigree chart of "Citrus KN55".

[0061]

【 Figure 2 [Image of the fruit of "Citrus KN55"]

[0062]

【 Figure 3 [Image of a "Citrus KN55" tree]

[0063]

【 Figure 4 The image shows the genotypes of the CAPS marker for "Citrus KN55".

[0064]

【 Figure 5This graph shows the results of PCR amplification of DNA extracted from "Citrus KN55" using Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, or Tf0419-2 as genetic markers (CAPS markers), followed by genotyping of the DNA with specific restriction endonucleases and analysis of the genotypes in each genetic marker by agarose gel electrophoresis. In the graph, M represents the electrophoresis pattern when performing electrophoresis on a 100bp ladder, and S represents the electrophoresis pattern when performing electrophoresis on DNA treated with restriction endonucleases from "Citrus KN55".

[0065]

【 Figure 6 This graph shows the results of PCR amplification of DNA extracted from "Citrus KN55" using genetic markers Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, or Mf0090-2, followed by cleavage with specific restriction endonucleases, and then agarose gel electrophoresis to analyze the genotypes of each genetic marker. In the graph, M represents the electrophoresis pattern of a 100bp ladder, and S represents the electrophoresis pattern of DNA from "Citrus KN55" after restriction endonuclease treatment.

[0066]

【 Figure 7 This figure shows the results of PCR amplification of DNA extracted from "Citrus KN55" using the genetic markers Gn0073-2 or Gn0048-2, followed by cleavage with specific restriction endonucleases, and then agarose gel electrophoresis to analyze the genotypes of each genetic marker. In the figure, M represents the electrophoresis pattern of a 100bp ladder, and S represents the electrophoresis pattern of DNA treated with restriction endonucleases from "Citrus KN55". Detailed Implementation

[0067] The present invention will now be described in detail. Furthermore, all documents described in this specification are incorporated herein by reference.

[0068] In this application specification, the term "citrus plant" refers to plants of the genus *Citrus* L. within the family Rutaceae. Furthermore, "citrus" is a general term encompassing plants belonging to the genera *Citrus*, *Fortunella*, and *Poncirus* within the subfamily Rutaceae.

[0069] In this specification, "plant" may also refer to a part or all of a plant. Additionally, it may refer to any stage of plant development.

[0070] As part of a plant, examples include buds, lateral buds, shoots, branches, leaves, petioles, thorns, stems, roots, fruit stalks, leaves, flowers, buds, petals, pollen, anthers, fruits, and seeds. Additionally, examples include the exocarp, endocarp, and pulp that constitute a fruit. A part of a plant contains cells or tissues of the plant body, and also includes, for example, protoplasts derived from embryos, hypocotyls, dividing cells, callus tissue, or cells of the plant body.

[0071] In this specification, "traits" refers to the morphological and physiological characteristics of a plant.

[0072] In this manual, CAPS refers to Cleaved Amplified Polymorphic Sequence (CAPS).

[0073] Method 1 for processing citrus plants

[0074] One embodiment of the present invention relates to a method for producing citrus plants, which includes:

[0075] For hybrid varieties or their offspring obtained by crossing citrus varieties with each other, the procedure for selecting plants with the following genotypes for each of the genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 is as follows:

[0076] Bf0158-3 is type AB.

[0077] Bf0036-3 is type AA.

[0078] Tf0150-2 is a BB type.

[0079] Tf0271-2 is type AA.

[0080] Tf0300-3 is type AB.

[0081] Tf0419-2 is type AB.

[0082] Tf0420-2 is type AA.

[0083] Tf0318-2 is of type BB.

[0084] Tf0293-4 is type AA.

[0085] Al0302-2 is type AB.

[0086] Mf0097-2 is type AB.

[0087] Mf0090-2 is type AB.

[0088] Gn0073-2 is type AA, and

[0089] Gn0048-2 is of type AA.

[0090] In one embodiment, the method may also include the step of hybridizing the hybrid variety using mating parent (A) as the seed parent (maternal parent) and mating parent (B) as the pollen parent (paternal parent) as shown below:

[0091] Cross-parent (A): Varieties obtained by crossing the citrus variety "Nanxiang" as the seed parent (female parent) and the citrus variety "Tencho" as the pollen parent (male parent).

[0092] mating parent (B): Citrus variety "Southwest Light".

[0093] In one embodiment, the plant used in the selection process may be a hybrid obtained by crossing citrus varieties with each other, or its offspring plants, preferably a hybrid obtained by crossing citrus varieties with each other.

[0094] In one embodiment, the production method may also include a second selection step of selecting citrus plants with the trait of having fruit weighing more than 200g.

[0095] In one embodiment, the production method may also include a second selection step of selecting citrus plants having the following traits (1) and (2).

[0096] (1) The fruit has a sugar content of over 13 Brix%, a good taste, and...

[0097] (2) The fruit weighs more than 200g.

[0098] In one embodiment, the preparation method may also include a propagation step of causing citrus plants to reproduce asexually.

[0099] [From the selection process of the markers (identification process)]

[0100] In one embodiment, the method for producing hybrid varieties or their offspring obtained by crossing citrus varieties with each other uses Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 as genetic markers.

[0101] Variety identification based on genetic markers can be performed using the method described in Non-Patent Literature 1.

[0102] The process is outlined below. First, DNA is extracted from hybrids or their offspring obtained by crossing citrus varieties using existing methods. Next, the extracted DNA is amplified by PCR using pairs of forward and reverse primers corresponding to the genetic markers listed in the table below. The amplified DNA is then cut with the restriction endonucleases listed in the table below. The restriction endonuclease-treated DNA is then subjected to agarose gel electrophoresis to confirm the electrophoretic pattern. Differences in the electrophoretic patterns can be used to study the genotypes of each genetic marker. This allows for the identification of citrus varieties.

[0103] Therefore, the production method may also include the step of extracting DNA from hybrids or their offspring obtained by crossing citrus varieties with each other.

[0104] In addition, the preparation method may also include the step of amplifying the extracted DNA using forward and reverse primers corresponding to each genetic marker.

[0105] In addition, the preparation method may also include the step of treating the amplified DNA with a restriction endonuclease.

[0106] In addition, the preparation method may also include the step of electrophoresis on the DNA after treatment with restriction endonuclease.

[0107] In addition, the preparation method may also include the step of determining the genotype of each genetic marker by analyzing the electrophoretic pattern of the DNA obtained from electrophoresis.

[0108] In addition, the preparation method may also include the step of selecting plants that have the genotype of each genetic marker based on the determination results of the genotype of each genetic marker.

[0109] Table 1

[0110]

[0111]

[0112] The Bf0158-3 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by a band that can be identified near 352 (±10) bp but not near 149 (±10) bp and 203 (±10) bp. The BB type is characterized by a band that can be identified near 149 (±10) bp and 203 (±10) bp but not near 352 (±10) bp. The AB type is characterized by a band that can be identified near 149 (±10) bp, 203 (±10) bp, and 352 (±10) bp.

[0113] The Bf0036-3 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by a band that can be identified around 399 (±10) bp but not around 162 (±10) bp and 237 (±10) bp. The BB type is characterized by a band that can be identified around 162 (±10) bp and 237 (±10) bp but not around 399 (±10) bp. The AB type is characterized by a band that can be identified around 162 (±10) bp and around 237 (±10) bp and 399 (±10) bp.

[0114] The Tf0150-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by a band that can be identified around 385 (±10) bp but not around 152 (±10) bp and 233 (±10) bp. The BB type is characterized by a band that can be identified around 152 (±10) bp and 233 (±10) bp but not around 385 (±10) bp. The AB type is characterized by a band that can be identified around 152 (±10) bp, 233 (±10) bp, and 385 (±10) bp.

[0115] The Tf0271-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by bands that can be identified around 400 (±10) bp but not around 157 (±10) bp and 243 (±10) bp. The BB type is characterized by bands that can be identified around 157 (±10) bp, 243 (±10) bp, and 400 (±10) bp. The AB type is characterized by bands that can be identified around 157 (±10) bp, 243 (±10) bp, and 400 (±10) bp.

[0116] The Tf0300-3 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by a band that can be identified around 393 (±10) bp but not around 166 (±10) bp and 227 (±10) bp. The BB type is characterized by a band that can be identified around 166 (±10) bp and 227 (±10) bp but not around 393 (±10) bp. The AB type is characterized by a band that can be identified around 166 (±10) bp, 227 (±10) bp, and 393 (±10) bp.

[0117] The Tf0419-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by a band that can be identified around 389 (±10) bp but not around 167 (±10) bp and 222 (±10) bp. The BB type is characterized by a band that can be identified around 167 (±10) bp, 222 (±10) bp, and 389 (±10) bp. The AB type is characterized by a band that can be identified around 167 (±10) bp, 222 (±10) bp, and 389 (±10) bp.

[0118] The Tf0420-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by a band that can be identified around 388 (±10) bp but not around 162 (±10) bp and 226 (±10) bp. The BB type is characterized by a band that can be identified around 162 (±10) bp and 226 (±10) bp but not around 388 (±10) bp. The AB type is characterized by a band that can be identified around 162 (±10) bp, 226 (±10) bp, and 388 (±10) bp.

[0119] The Tf0318-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by bands that can be identified around 400 (±10) bp but not around 135 (±10) bp and 265 (±10) bp. The BB type is characterized by bands that can be identified around 135 (±10) bp, 265 (±10) bp, and 400 (±10) bp. The AB type is characterized by bands that can be identified around 135 (±10) bp, 265 (±10) bp, and 400 (±10) bp.

[0120] The Tf0293-4 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by a band that can be identified around 394 (±10) bp but not around 157 (±10) bp and 237 (±10) bp. The BB type is characterized by a band that can be identified around 157 (±10) bp and 237 (±10) bp but not around 394 (±10) bp. The AB type is characterized by a band that can be identified around 157 (±10) bp, 237 (±10) bp, and 394 (±10) bp.

[0121] The Al0302-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by a band that can be identified around 383 (±10) bp but not around 167 (±10) bp and 216 (±10) bp. The BB type is characterized by a band that can be identified around 167 (±10) bp, 216 (±10) bp, and 383 (±10) bp. The AB type is characterized by a band that can be identified around 167 (±10) bp, 216 (±10) bp, and 383 (±10) bp.

[0122] The Mf0097-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by a band that can be identified around 369 (±10) bp but not around 166 (±10) bp and 203 (±10) bp. The BB type is characterized by a band that can be identified around 166 (±10) bp and 203 (±10) bp but not around 369 (±10) bp. The AB type is characterized by a band that can be identified around 166 (±10) bp, 203 (±10) bp, and 369 (±10) bp.

[0123] The Mf0090-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by a band that can be identified around 400 (±10) bp but not around 139 (±10) bp and 261 (±10) bp. The BB type is characterized by a band that can be identified around 139 (±10) bp and 261 (±10) bp but not around 400 (±10) bp. The AB type is characterized by a band that can be identified around 139 (±10) bp, 261 (±10) bp, and 400 (±10) bp.

[0124] The Gn0073-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by a band that can be identified around 394 (±10) bp but not around 149 (±10) bp and 245 (±10) bp. The BB type is characterized by a band that can be identified around 149 (±10) bp and 245 (±10) bp but not around 394 (±10) bp. The AB type is characterized by a band that can be identified around 149 (±10) bp, 245 (±10) bp, and 394 (±10) bp.

[0125] The Gn0048-2 genotype can be divided into AA, AB, and BB types based on differences in electrophoretic patterns. The AA type is characterized by a band that can be identified around 380 (±10) bp but not around 134 (±10) bp and 246 (±10) bp. The BB type is characterized by a band that can be identified around 134 (±10) bp and 246 (±10) bp but not around 380 (±10) bp. The AB type is characterized by a band that can be identified around 134 (±10) bp, 246 (±10) bp, and 380 (±10) bp.

[0126] In one embodiment, the method includes a step of selecting citrus plants with the following genotypes:

[0127] Bf0158-3 is type AB.

[0128] Bf0036-3 is type AA.

[0129] Tf0150-2 is a BB type.

[0130] Tf0271-2 is type AA.

[0131] Tf0300-3 is type AB.

[0132] Tf0419-2 is type AB.

[0133] Tf0420-2 is type AA.

[0134] Tf0318-2 is of type BB.

[0135] Tf0293-4 is type AA.

[0136] Al0302-2 is type AB.

[0137] Mf0097-2 is type AB.

[0138] Mf0090-2 is type AB.

[0139] Gn0073-2 is type AA, and

[0140] Gn0048-2 is of type AA.

[0141] In one embodiment, the method may also include a step of hybridizing the hybrid variety by using the mating parent (A) as the seed parent (maternal parent) and the mating parent (B) as the pollen parent (paternal parent).

[0142] Cross-parent (A): Varieties obtained by crossing the citrus variety "Nanxiang" as the seed parent (female parent) and the citrus variety "Tencho" as the pollen parent (male parent).

[0143] mating parent (B): Citrus variety "Southwest Light".

[0144] In this specification, mating parent (A) is also referred to as "System A" below.

[0145] System A is an early-maturing, high-quality variety with relatively large and high fruit production. It was obtained by hybridization of Nanxiang as the seed parent and Tiancao as the pollen parent. The following are the types of hybrids: Bf0158-3 (AA), Bf0036-3 (AA), Tf0150-2 (BB), Tf0271-2 (AB), Tf0300-3 (AB), Tf0419-2 (AB), Tf0420-2 (AA), Tf0318-2 (BB), Tf0293-4 (AA), Al0302-2 (AA), Mf0097-2 (AB), Mf0090-2 (AB), Gn0073-2 (AA), and Gn0048-2 (AB).

[0146] "Nanxiang" is registered as a variety in Japan under variety registration number 1857.

[0147] "Amakusa" is registered as a variety in Japan under variety registration number 4596.

[0148] "Southwest Light" is a mid-ripening variety with a good flavor and aroma. It is early-ripening, easy to peel, has few seeds and is easy to eat, and has high sugar content and excellent taste. It is registered as a variety in Japan under variety registration number 17969.

[0149] The citrus plants selected through the selection process have genotype profiles of the aforementioned genetic markers. In contrast, System A consists of Bf0158-3 (AA), Tf0271-2 (AB), AI0302-2 (AA), and Gn0048-2 (AB). Therefore, Bf0158-3, Tf0271-2, AI0302-2, and / or Gn0048-2 can be used to distinguish System A.

[0150] Other citrus varieties of "Nanxiang" have the following type classifications: Bf0036-3 (AB), Tf0150-2 (AB), Tf0300-3 (AA), Tf0293-4 (AB), Al0302-2 (AA), Mf0097-2 (BB), and Gn0073-2 (AB). Therefore, Bf0036-3, Tf0150-2, Tf0300-3, Tf0293-4, Al0302-2, Mf0097-2, and / or Gn0073-2 can be used to distinguish them from other citrus varieties of "Nanxiang".

[0151] Other citrus varieties of "Amakusa" have the following blood types: Bf0036-3 (AB type), Tf0271-2 (AB type), and Gn0048-2 (AB type). Therefore, Bf0036-3, Tf0271-2, and / or Gn0048-2 can be used to distinguish them from other citrus varieties of "Amakusa".

[0152] Other citrus varieties of "Southwest Light" are: Bf0036-3 (AB), Tf0150-2 (AB), Tf0271-2 (AB), Tf0419-2 (BB), Tf0420-2 (AB), Al0302-2 (BB type), Mf0097-2 (BB type), Mf0090-2 (BB type), and Gn0073-2 (AB). Therefore, Bf0036-3, Tf0150-2, Tf0271-2, Tf0419-2, Tf0420-2, Al0302-2, Mf0097-2, Mf0090-2, and / or Gn0073-2 can be used to distinguish them from other citrus varieties of "Southwest Light".

[0153] [Presumption of mating kinship system]

[0154] Tables 2-5 below list genotypes representing combinations of alleles detected for each variety at each genetic marker. Since citrus is typically a diploid plant, it is necessary for offspring to inherit one of each of the two alleles found in their seed and pollen parents. For example, if a variety has the genotype AB, then any parent possessing either the A or B allele has the potential to be a mating parent. If both parents are BB, then their combination is unlikely to be a parent. Based on this principle, mating parent candidates can be deduced from information from the 14 genetic markers.

[0155] Citrus plants with the aforementioned genotype, selected through the genetic marker selection process, as shown in Tables 2-5 below, differ from other citrus varieties in their genotype of the genetic marker. Therefore, citrus plants selected through this process can be distinguished from other citrus varieties for production.

[0156] Table 2

[0157]

[0158] Table 3

[0159]

[0160] Table 4

[0161]

[0162] Table 5

[0163]

[0164] In one embodiment, the production method may also include a second selection step before, simultaneously with, or after the selection step using genetic markers, including selecting citrus plants with a fruit weight of 200g or more. The second selection step may be performed either before, simultaneously with, or after the selection step using genetic markers, but is preferably performed after the selection step using genetic markers. Preferred ranges for fruit weight are described below.

[0165] In one embodiment, the preparation method may also include, prior to the selection process, a preliminary selection step that uses at least one of the genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, Al0302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 as a genetic marker (CAPS marker) to identify candidate citrus plants that differ from "System A" and "Southwest Light" on the genotype map of the genetic markers.

[0166] In the preliminary selection process, the genotype profiles of all 14 CAPS markers can be determined, or only a subset of the 14 CAPS markers can be used for the selection purpose. In one example, during the marker selection process, at least one genetic marker from Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0419-2, Tf0420-2, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 is used to select candidate citrus plants that differ from "System A" and "Southwest Light" in the genotype profiles of the genetic markers. In one embodiment, in the marker selection process, a combination of at least one genetic marker selected from Bf0158-3 and Gn0048-2 and at least one genetic marker selected from Bf0036-3, Tf0150-2, Tf0419-2, Tf0420-2, Mf0097-2, Mf0090-2 and Gn0073-2, and at least one genetic marker selected from Tf0271-2 and Al0302-2 is used to select candidate citrus plants that are different from the genetic markers of "System A" and "Southwest Light" on the genotype map of the genetic markers.

[0167] In another embodiment, during the pre-selection process, at least one genetic marker among Bf0158-3 and Gn0048-2 is used to select candidate citrus plants that differ from "System A" in the genotype map of the genetic marker. Furthermore, at least one genetic marker among Bf0036-3, Tf0150-2, Tf0419-2, Tf0420-2, Mf0097-2, Mf0090-2, and Gn0073-2 is used to select candidate citrus plants whose genetic markers differ from those of the mating parent "Southwest Light" in the genotype map of the genetic marker.

[0168] Alternatively, a system of at least one genetic marker selected from Tf0271-2 and Al0302-2, which selects candidate citrus plants that are different from the genetic markers of "System A" and "Southwest Light" on the genotype map of the genetic markers, may be used.

[0169] [Characteristic-based selection process]

[0170] In one embodiment, the preparation method may also include a trait-based selection step. The trait-based selection step may be performed before, after, or simultaneously with the selection step of the genetic markers, but preferably after the selection step of the genetic markers.

[0171] The main traits used in selection include, for example, a sugar content of 13 Brix% or higher, a good taste, and a fruit weight of 200g or more. Among these, citrus plants with a fruit weight of 200g or more are preferred.

[0172] The detailed description of the aforementioned traits is provided in the following section [Citrus Plants].

[0173] In one embodiment of the present invention, the method for producing citrus plants may also include, for example, the pre-selection step, the first selection step, and the second selection step described in the method 2 for producing citrus plants.

[0174] [Proliferation Process]

[0175] In one embodiment of the present invention, the method for producing citrus plants may also include a propagation step of asexually propagating the citrus plants cultivated through the selection step.

[0176] Methods of asexual propagation include grafting, cuttings, wood sampling, and cell culture. Asexual propagation is also known as unsexual reproduction, asexual reproduction, or vegetative propagation. In asexual reproduction, male and female are not necessary; a new individual is formed from a single individual, and the reproductive cells produced from a single individual become the new individual. Furthermore, the techniques for creating plant tissue cell cultures and the methods for regenerating plant bodies from self-tissue cultures utilize well-known techniques.

[0177] In this invention, the rootstock suitable for grafting can be any tree species of citrus trees, such as trifoliate orange, Satsuma mandarin orange, and tangerine, or other citrus rootstocks. Furthermore, the type and size of the suitable rootstock can be changed due to factors such as soil and climate. For example, trifoliate orange can be used as rootstock.

[0178] The cultivation type of citrus plants involved in this invention is not particularly limited, and examples include top grafting, open-air cultivation in the form of seedlings, unheated facility cultivation, or roof-mounted facility cultivation.

[0179] [Method 2 for producing citrus products]

[0180] One embodiment of the present invention relates to a method for producing citrus plants, comprising:

[0181] The hybridization process of using the mating parent (A) as the female parent and the mating parent (B) as the male parent, as shown below, and...

[0182] Crossparent (A): Varieties obtained by crossing the citrus variety "Nanxiang" and the citrus variety "Amakusa".

[0183] mating parent (B): Citrus variety "Southwest Light"

[0184] A marker selection process is used to identify candidate citrus plants that are different from mating parents (A) and mating parents (B) on the genotype map of the genetic markers by using at least one of the following genetic markers: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0419-2, Tf0420-2, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2, from citrus plants or their progeny obtained by the hybridization process.

[0185] Hereinafter, in this specification, mating parent (A) will also be referred to as "system A".

[0186] Furthermore, the method of preparation can also be called the manufacturing method, cultivation method, or production method.

[0187] [Hybridization process]

[0188] In the hybridization process, "System A" is used as the female parent (seed parent) and "Southwest Light" is used as the male parent (pollen parent) for hybridization.

[0189] System A is an early-maturing, flavorful, relatively large-fruited, and high-yielding variety. It was obtained by hybridization of "Nanxiang" as the seed parent and "Amakusa" as the pollen parent. "Nanxiang" is registered as a variety in Japan under variety registration number 1857. "Amakusa" is registered as a variety in Japan under variety registration number 4596.

[0190] "Southwest Light" is a mid-ripening variety with a good flavor and aroma. It is early-ripening, easy to peel, has few seeds and is easy to eat, and has high sugar content and excellent taste. It is registered as a variety in Japan under variety registration number 17969.

[0191] The 'Southwest Light' used as the mating parent in the hybridization is a commercially available variety. Both 'Nanxiang' and 'Amakusa' are varieties developed by the National Research and Development Agency for Agriculture and Food Science and Technology, and are also commercially available varieties. Furthermore, it is also possible to identify and screen the mating parents using the well-known genetic markers (CAPS markers) mentioned later.

[0192] [From the selection process of the markers (identification process)]

[0193] In one embodiment, during the marker selection process, candidate citrus plants that differ from the mating parents “System A” and “Southwest Light” on the genotype map of the genetic markers are selected from citrus plants or their progeny plants obtained from the hybridization process using at least one of the following genetic markers (CAPS markers): Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0194] For the identification of other varieties with mating parents selected with markers, the following section on [Identification Methods for Citrus Varieties] is used for details.

[0195] The genotype maps of all 14 CAPS markers can be determined, or only a subset of the 14 CAPS markers can be used for the purpose of selection. In one example, during the marker selection process, at least one of the genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0419-2, Tf0420-2, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 is used to select candidate citrus plants that differ from their mating parents "System A" and "Southwest Light" on the genotype maps of the genetic markers. In one embodiment, in the marker selection process, a combination of at least one genetic marker selected from Bf0158-3 and Gn0048-2 and at least one genetic marker selected from Bf0036-3, Tf0150-2, Tf0419-2, Tf0420-2, Mf0097-2, Mf0090-2 and Gn0073-2, and at least one genetic marker selected from Tf0271-2 and A10302-2, is used to select candidate citrus plants that are different from the genetic markers of mating parents “System A” and “Southwest Light” on the genotype map of the genetic markers.

[0196] In another embodiment, during the marker selection process, at least one genetic marker among Bf0158-3 and Gn0048-2 is used to select candidate citrus plants that differ from "System A" in the genotype map of the genetic marker. Furthermore, at least one genetic marker among Bf0036-3, Tf0150-2, Tf0419-2, Tf0420-2, Mf0097-2, Mf0090-2, and Gn0073-2 is used to select candidate citrus plants whose genetic markers differ from those of the mating parent "Southwest Light" in the genotype map of the genetic marker. Alternatively,

[0197] A system for selecting candidate citrus plants that are different from the genetic markers of "System A" and "Southwest Light" on the genotype map of the genetic markers, using at least one genetic marker selected from at least one of Tf0271-2 and Al0302-2.

[0198] In a typical example of citrus plants involved in this embodiment, the genotype profiles of the 14 CAPS markers are as follows: Bf0158-3 is AB type, Bf0036-3 is AA type, Tf0150-2 is BB type, Tf0271-2 is AA type, Tf0300-3 is AB type, Tf0419-2 is AB type, Tf0420-2 is AA type, Tf0318-2 is BB type, Tf0293-4 is AA type, A10302-2 is AB type, Mf0097-2 is AB type, Mf0090-2 is AB type, Gn0073-2 is AA type, and Gn0048-2 is AA type.

[0199] [Characteristic-based selection process]

[0200] To obtain the citrus plant according to this embodiment, a selection process based on specific traits can be performed on candidate citrus plants obtained by crossing the citrus "System A" as the female parent and the citrus variety "Southwest Light" as the male parent. This selection process based on specific traits can also be performed together with a selection process using genetic markers as indicators. Alternatively, it can be performed before the selection process using genetic markers as indicators. In a preferred embodiment, after performing the selection process using genetic markers as indicators, a selection process based on specific traits is performed on the system of sufficiently selected candidate citrus plants.

[0201] The main traits used in the selection are, for example, a sugar content of 13 Brix% or higher, a good taste, and a fruit weight of 200g or more. Among these, citrus plants with a fruit weight of 200g or more are preferred.

[0202] The detailed description of the aforementioned traits is provided in the following section [Citrus Plants]. It is also preferable to use the traits described in the section on Citrus Plants during selection.

[0203] The method for producing citrus plants according to one embodiment of the present invention involves a selection step in which the plants obtained in the hybridization process are selected based on at least one of the criteria described in the examples. In this selection step, selection may be performed multiple times over several years.

[0204] That is, the selection process may also include, for example, a pre-selection process, a first selection process, and a second selection process.

[0205] The preliminary selection process may also be a selection process based on at least one of the criteria (a) to (d) below:

[0206] (a) Large fruits with an average weight exceeding 200g, (b) ripening in January (with an acid content of approximately 1.0–1.2% by December), (c) average sugar content of 12 Brix% or higher, and (d) few to no seeds.

[0207] The first selection process may also be a selection process based on at least one of the following criteria (e) to (g): (e) annual fruiting performance and yield, (f) evaluation of physiological obstacles on the tree (peel obstacles such as peeling and cracking), and (g) observation of the degree of disease occurrence.

[0208] Next, the second selection process can also be a selection process based on the following criteria (h): (h) System adaptability testing (regional adaptability evaluation).

[0209] [Proliferation Process]

[0210] In one embodiment of the present invention, the method for producing citrus plants further includes a propagation step of asexually propagating the citrus plants cultivated through the hybridization and selection steps.

[0211] Methods of asexual propagation include grafting, wood harvesting, and cell culture. Asexual propagation is also known as asexual reproduction, vegetative propagation, or vegetative propagation. In asexual reproduction, male and female are not necessary; it is a method where a single individual forms a new individual, and the reproductive cells produced from a single individual become the new individual. Furthermore, the techniques for creating plant tissue cell cultures and the methods for regenerating plant bodies from self-tissue cultures are carried out using well-known techniques.

[0212] In this invention, the rootstock suitable for grafting can be any tree species of citrus trees, such as trifoliate orange, Satsuma mandarin orange, and tangerine, or other citrus rootstocks. Furthermore, the type and size of the suitable rootstock can be changed due to factors such as soil and climate. For example, trifoliate orange can be used as rootstock.

[0213] The cultivation type of citrus plants involved in this invention is not particularly limited, and examples include top grafting, open-air cultivation in the form of seedlings, unheated facility cultivation, or roof-mounted facility cultivation.

[0214] Methods for identifying citrus plants

[0215] As previously stated, the citrus plants of the present invention can be identified by specific variety identification markers.

[0216] To this end, the development of DNA variety identification technology is being advanced. Existing technologies are effective in monitoring or proving infringements of breeder rights. To prevent imports at customs and other waterways, variety identification technology that can be easily and quickly identified at inspection sites such as customs offices is being developed.

[0217] The Fruit and Tea Division of the National Research and Development Agency for Agriculture, Food and Rural Affairs has developed a variety identification technology using CAPS (cleaved amplified polymorphic sequence) markers on domestically distributed citrus seedlings. Regarding prior art, a DNA variety identification technology manual for citrus varieties based on ISO (International Organization for Standardization) standards has been created and published by the division (Non-Patent Document 1).

[0218] CAPS biomarker analysis technology involves cutting PCR-amplified fragments targeting specific genomic regions with specific restriction endonucleases. The technology utilizes the size differences in restriction endonuclease fragments resulting from genomic DNA polymorphisms such as base substitutions, deletions, and insertions between varieties. This method requires minimal equipment and is highly accessible for on-site testing.

[0219] The CAPS marker analysis method involves amplifying the target region by PCR and then treating the DNA product with a restriction endonuclease that recognizes a specific base sequence to detect polymorphism. The specific sequence of the target PCR amplification product varies from variety to variety, and the differences in the length of the products after restriction endonuclease cutting (genotype) are confirmed by electrophoresis using agarose gels or similar methods. To identify varieties, it is necessary to combine the results of multiple markers ("Points to Note When Confirming the Appropriateness of DNA Variety Identification Technology" (1st Edition, March 6, 2023, Seedling Management Center, National Research and Development Agency of Agriculture and Food Science).

[0220] The present invention relates to a method for identifying citrus plants by using the CAPS marker as an identification marker for citrus "KN55" or its progeny plants, and a method for identifying plants from other citrus varieties.

[0221] One aspect of the present invention relates to a method for identifying citrus plants, comprising: (I) a step of amplifying a region containing a CAPS marker in the DNA of a citrus plant using a primer set; and (II) a step of identifying the citrus plant based on the polymorphism of the bases 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, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0222] Genetic markers (CAPS markers) can be one of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2, or multiple markers, or all of them, with the use of all of them being preferred.

[0223] In one embodiment, the method for identifying citrus plants can distinguish the citrus plants of the present invention from other citrus varieties based on the polymorphism of bases in the region (DNA region) containing the amplified genetic marker. Examples of other citrus varieties include citrus varieties other than KN55 listed in Tables 2-5.

[0224] Since polymorphism in CAPS is determined by the presence or absence of restriction endonuclease sites, alleles without restriction endonuclease sites can be used as A, and alleles with restriction endonuclease sites can be used as B to determine the genotypes of varieties AA, AB, and BB.

[0225] [Identify the object]

[0226] The method for identifying citrus plants according to the present invention relates to identifying the citrus plants of the present invention from other citrus varieties.

[0227] Furthermore, in the citrus plant identification technology of the present invention, from the viewpoint of its identification principle, the 14 identification markers shown as marker names Bf0158-3 to Gn0048-2 among the progeny plants of "KN55" can be used to identify citrus plants that show the same allele as "KN55" from other citrus varieties.

[0228] Additionally, the alleles identical to "KN55" refer to the 14 alleles from Bf0158-3 to Gn0048-2. Figure 4 The alleles. Furthermore, as long as the alleles are derived from "KN55", the variety identification technology of the present invention is applicable even if variations occur in the base sequences constituting these loci and their surrounding regions.

[0229] In the identification technology of this invention, "other citrus varieties" that can be distinguished from "KN55" can be exemplified by citrus varieties that do not share at least one of the 14 identification markers with "KN55". Among citrus plants belonging to other citrus varieties, the 14 alleles from Bf0158-3 to Gn0048-2 show alleles that are different from those of "KN55".

[0230] Among them, other citrus varieties that can be distinguished from "KN55" using the aforementioned identification technology can be exemplified by, for example, the following varieties shown in the manual of Non-Patent Document 1: "Wenzhou Mandarin (Miyagawa Wase)," "Grapefruit (Duncan)," "Sweet Orange (Trovita)," "Lemon (Lisbon)," "Shiranui," "Iyokan (Miyauchi Iyokan)," "Natsumikan (Kawano Natsumi)," "Hachisaku Mandarin," "Ponkan (Ota Ponkan)," "Rinoka," "Miwa," "Ashimi," "Ashiki," "Reikou," "Tsunoki," "Southwest Light," "Tsunoki," "Haruhi," "Kiyomi," "Setoka," "Harumi," "Haruhime," "Kanpei," and "Ehime Fruit Test No. 28 (Red Madonna)."

[0231] These 24 citrus varieties, together with "KN55" (a total of 25 varieties), account for more than 94% of the citrus fruit circulation in Japan.

[0232] In addition, other citrus varieties that can be distinguished from "KN55" using the identification technology of the present invention include, for example, pomelo, Hanoi late mandarin orange, Tenko Natsu, vinegar mandarin orange, Shiki mandarin orange, Kara mandarin orange, Bucket mandarin orange, Seminol mandarin orange, stinky orange, Amakusa mandarin orange, golden mandarin orange, Kishu mandarin orange, sweet spring mandarin orange, Anchor, Jumi, Nishi no Kaori, Murcott mandarin orange, Nan Kaori, Gan Kaori, the middle female parent of citrus Nong 6, and sour orange, etc.

[0233] The identification method of the present invention can identify "KN55" with very high accuracy.

[0234] Furthermore, considering the results of the following embodiments, other citrus varieties that can be distinguished from "KN55" using the identification technology of the present invention can be cited as examples of citrus varieties that do not belong to the same genotype as "KN55" based on the 14 identification markers from Bf0158-3 to Gn0048-2. For example, even regarding grapefruit, from the principle viewpoint of the identification technology of the present invention, other citrus varieties that can be distinguished from "KN55" can be identified.

[0235] For example, when distinguishing between "KN55" and mating parent "System A", it is sufficient to use at least one of the markers Bf0158-3, Tf0271-2, Al0302-2, and Gn0048-2.

[0236] In addition, when distinguishing between "KN55" and its mating parent "Southwest Light", it is sufficient to use at least one of the following markers: Bf0036-3, Tf0150-2, Tf0271-2, Tf0419-2, Tf0420-2, Al0302-2, Mf0097-2, Mf0090-2, and Gn0073-2.

[0237] In addition, when distinguishing between "KN55" and the two mating parents "System A" and "Southwest Light", at least one genetic marker selected from Bf0158-3 and Gn0048-2 and at least one genetic marker selected from Bf0036-3, Tf0150-2, Tf0419-2, Tf0420-2, Mf0097-2, Mf0090-2 and Gn0073-2, and at least one genetic marker selected from Tf0271-2 and Al0302-2 can be used, as long as it is only necessary to identify "KN55" between the two parents, at least one of Tf0271-2 and Al0302-2 can be used.

[0238] In one embodiment of the identification method of the present invention, the citrus plant used as the test subject is a candidate plant for breeding materials, a plant obtained during the breeding process, etc. Candidate plants for breeding materials include, for example, parent plants used in mating, and plants used in molecular breeding using gene recombination technology. Furthermore, the citrus plant used as the test subject includes citrus plants used in molecular breeding using gene recombination technology and citrus plants obtained through molecular breeding.

[0239] One embodiment of the present invention relates to an identification method for identifying citrus plants of the present invention, comprising:

[0240] (I) A procedure for amplifying a portion of the DNA of a citrus plant using a primer set capable of amplifying the CAPS marker region: and

[0241] (II) A process for identifying citrus plants based on the polymorphism of the bases in the amplified DNA region.

[0242] The amplification of the region described in the DNA of the citrus plant specimen can be performed by polymerase chain reaction (PCR) using DNA extracted from the citrus plant specimen as a template and a primer set for amplifying the region containing the CAPS marker.

[0243] For DNA obtained from citrus plants, the length of the DNA fragment when subjected to restriction endonuclease treatment of the DNA amplified by PCR using the primer set is consistent with the length of the DNA fragment when subjected to the same treatment in a DNA sample obtained from a known citrus variety, thus identifying it as the known citrus variety.

[0244] That is, in the identification method according to one embodiment of the present invention, the step of determining the citrus plant based on the polymorphism of the bases in the amplified DNA region in step (II) includes the step of treating the amplified DNA fragment with a restriction endonuclease and the step of identifying the genotype based on the length of the DNA fragment obtained by the restriction endonuclease treatment in step (II-2).

[0245] For determining the genotype in the amplified fragment, previously known methods can be used; for example, agarose gel electrophoresis is a suitable method.

[0246] The length of a DNA fragment can be indicated by, for example, its molecular weight. The molecular weight can be determined using, for example, the electrophoretic distance in agarose gel electrophoresis. Alternatively, it can be determined by DNA sequencing.

[0247] There are no particular restrictions on the reaction reagents or reaction conditions used in PCR, as long as they are methods that produce primer-specific PCR amplification fragments; any known methods can be used.

[0248] As a method for detecting PCR amplified fragments, there are no particular limitations. For example, ethidium bromide, fluorescent substances, chromogenic substances, luminescent substances, etc., can be used to detect PCR amplified fragments.

[0249] Primers in the primer set are composed of base sequences that have at least 90% sequence identity with the base sequences of the nucleotides constituting each primer in the primer set. Modified primers, which are composed of modified base sequences obtained by changing a portion of the base sequences, are also included in the scope of primers used in the identification method of the present invention. Furthermore, the sequence identity of the modified primer with the base sequences of each primer may be, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. In principle, any modified primer that can hybridize with the complementary DNA strand of each primer is considered equivalent to the primer. Furthermore, the equivalent altered primer can also be a primer that functions as a primer for hybridization with the complementary DNA strand of the original primer, and is composed of nucleotides with altered base sequences, such as 0-3, 0-2, 1-3, or 1 or 2 bases, added, deleted, inserted, or substituted in the base sequence of the oligonucleotides constituting the original primers.

[0250] For the primers, reagents, reaction conditions, detection methods, etc. of the PCR shown above, the materials and methods described in the manual of Non-Patent Document 1 may be used appropriately.

[0251] The citrus plant identification method of the present invention enables the high-precision and rapid identification of citrus "KN55" or its offspring plants from other citrus varieties, and can be widely adopted even in field inspections.

[0252] The method for identifying the citrus plant according to an embodiment of the present invention preferably includes:

[0253] The procedure of amplifying regions containing genetic markers (CAPS markers) of citrus plant DNA using primer sets, and

[0254] The process of identifying the citrus plant based on the base polymorphism of the amplified DNA region.

[0255] 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, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0256] Furthermore, the method for identifying citrus plants according to one embodiment of the present invention more preferably includes:

[0257] The procedure of amplifying regions containing genetic markers (CAPS markers) of citrus plant DNA using primer sets, and

[0258] The process of identifying the citrus plant based on the base polymorphism of the amplified DNA region.

[0259] The genetic markers (CAPS markers) are Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0260] In addition, it is preferred that each genetic marker (CAPS marker) has the genotype described above as a citrus plant according to an embodiment of the present invention.

[0261]

Citrus Plant 1

[0262] The genotype of the genetic marker in citrus plants involved in one embodiment of the present invention is as follows:

[0263] Bf0158-3 is type AB.

[0264] Bf0036-3 is type AA.

[0265] Tf0150-2 is a BB type.

[0266] Tf0271-2 is type AA.

[0267] Tf0300-3 is type AB.

[0268] Tf0419-2 is type AB.

[0269] Tf0420-2 is type AA.

[0270] Tf0318-2 is of type BB.

[0271] Tf0293-4 is type AA.

[0272] Al0302-2 is type AB.

[0273] Mf0097-2 is type AB.

[0274] Mf0090-2 is type AB.

[0275] Gn0073-2 is type AA, and

[0276] Gn0048-2 is of type AA.

[0277] As shown in Tables 2-5, the citrus plant according to one embodiment of the present invention differs from other citrus varieties in its genotype of genetic markers. Therefore, the citrus plant according to one embodiment of the present invention is a novel citrus plant.

[0278] In another embodiment, the citrus plant may be a hybrid variety obtained by crossing a mating parent (A) as the seed parent (maternal parent) and a mating parent (B) as the pollen parent (paternal parent), or it may be its offspring plant, preferably a hybrid variety obtained by crossing citrus varieties with each other:

[0279] Cross-parent (A): Varieties obtained by crossing the citrus variety "Nanxiang" as the seed parent (female parent) and the citrus variety "Tencho" as the pollen parent (male parent).

[0280] mating parent (B): Citrus variety "Southwest Light".

[0281] In one embodiment, the citrus plant preferably has the trait of having a fruit weight of 200g or more.

[0282]

Citrus Plants 2

[0283] One embodiment of the present invention relates to a citrus plant having the following characteristics (1) to (4):

[0284] (1) The citrus variety “Southwest Light” was obtained by crossing citrus “System A” as the female parent and citrus variety “Southwest Light” as the male parent.

[0285] (2) The fruit has a sugar content of over 13 Brix and a good taste.

[0286] (3) The fruit weighs more than 200g, and

[0287] (4) Can be identified with other citrus varieties containing “System A” and “Southwest Light” by using at least one of the genetic markers 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.

[0288] Hereinafter, preferred embodiments are described for citrus plant 1 and / or citrus plant 2.

[0289] Furthermore, in a further embodiment of the present invention, the citrus plant involved is a citrus plant that also has at least one of the following traits (5) to (7):

[0290] (5) Seedless fruit production is possible.

[0291] (6) No floating skin fruit occurs.

[0292] (7) The fruit ripens in early January.

[0293] Furthermore, the characteristics of the citrus plants involved in this invention can vary depending on the conditions of the climate, soil, environment, cultivation method, tree age, etc., during cultivation or planting. Additionally, the time-related limitations on maturity periods, etc., in this specification are based on cultivation within Japan; for example, Shizuoka City, Shizuoka Prefecture, the cultivation location in the embodiments, can be used as a reference.

[0294] Furthermore, in one embodiment of the present invention, the citrus plant is one that possesses at least one of the following traits as a major characteristic that differs from "System A" or "Southwest Light". Moreover, the value can be the average of measurements taken over two or more years.

[0295] Characteristics of a tree

[0296] The branches branch at wide angles, giving the tree an "open" shape. The tree vigor is "medium," stronger than "Southwest Light." Thorns on the branch tips (spring branches) have a high rate of occurrence and are short in length. It does not form inflorescences, but grows singly. The petals are shorter and wider than those of "Southwest Light." The number of filaments is "few," and they are "separate." The amount of pollen is "absent," less than that of "Southwest Light." The anthers are degenerate, exhibiting high male sterility. The style is "curved," with a greater degree of curvature compared to "Southwest Light."

[0297] The budding, flowering, and full coloring periods vary depending on annual climatic conditions, but within the same cultivation area, the budding and peak flowering periods are roughly concurrent with "Southwest Light." Furthermore, although variations occur year by year, fruit coloring begins in mid-to-late October, and full coloring occurs in mid-to-late November. Flowering rate is "slightly high," and the fruiting rate the following year is "higher" than "Southwest Light." Late-season fruit drop is "none." Yield is measured every two years, averaging "medium."

[0298] Characteristics of the fruit

[0299] The fruit shape is "oblate," with a shape index of approximately 119. The top of the fruit is "flat," with a "short neck" at the stem end. There are no radial grooves at the top, and the stem end appears to be of "medium" thickness. The peel is "orange" to "deep orange," slightly thinner than the "Southwest Light" variety. The smoothness of the fruit surface is "medium." The peel thickness is approximately 2.1 mm, and the peeling difficulty is "medium," more difficult than the "Southwest Light." The peel has an aroma similar to that of an orange.

[0300] The flesh is a deep orange color, with a flesh content of approximately 85.8%, higher than "Southwest Light". The juice vesicle membranes and flesh are "soft" to "slightly soft". The average fruit weight is around 250g, larger than "Southwest Light" and "System A". The sugar content of the juice in early January is approximately 14.0%, higher than the two-year average. Furthermore, although the acid content varies from year to year, it is around 1.00g / 100ml and has a rich flavor. There are "none" intact seeds per fruit, and the proportion of seedless fruit can be achieved under open-field cultivation conditions. The seeds are polyembryonic. Regarding the full coloring period and fruit quality, although the ripening period varies due to acid content and year, it is later than "Southwest Light" and "System A", estimated to be around early January. Sunburn and cracking are "few", and puffy skin is not observed. Additionally, no physiological increase is observed in early January.

[0301] An example of the citrus plant of the present invention can be given by the citrus plant "Citrus KN55" or its progeny. Hereinafter, "Citrus KN55" will be referred to as "KN55". Furthermore, the citrus plant "KN55" of the embodiment has the characteristics described in the embodiment. The morphological and physiological characteristics of "KN55" can be found in [reference needed]. Figure 2 and Figure 3 .

[0302] The characteristics mentioned can be attributed to the characteristic values ​​evaluated based on the "Distinction between Other Citrus (Citrus L.) Examination Criteria and Breeding System Adaptability Test and Characteristic Test Survey Method" issued by the Ministry of Agriculture, Forestry and Fisheries of Japan (Fruit Research Institute, National Agricultural and Food Science and Technology Agency, 2007).

[0303] The citrus plant involved in this invention has the following characteristics (2) The sugar content of the fruit is above 13Brix%, and the taste is good. Next, the "taste" can also be evaluated as an indicator by the sugar-acid ratio.

[0304] In this instruction manual, "sweetness" is synonymous with sweetness and refers to the sweetness indicated by a saccharimeter. For example, the sweetness of juice in early January. The method for determining sweetness is by measuring sweetness using a crisscross saccharimeter. The unit of measurement is, for example, Brix%.

[0305] In this specification, "sourness" refers to the citric acid content in the fruit juice. The method for investigating sourness is to measure it by converting the titration value of the fruit juice into a sodium bicarbonate solution.

[0306] The lower limit of the sugar content of the citrus juice of the present invention is 13.0 Brix% or more, more preferably 13.9 Brix% or more, and even more preferably 14.0% Brix% or more. The upper limit of the sugar content is not particularly limited, and any value above the lower limit is taken as a premise, for example, below 17 Brix%, below 16 Brix%, or below 15 Brix%.

[0307] The upper limit of the acid content in the fruit juice is, for example, below 1.0g / 100ml, more preferably below 0.90g / 100ml, even more preferably below 0.8g / 100ml, and even more preferably below 0.7g / 100ml. The lower limit of the acid content is not particularly limited, and is based on any value below the upper limit, for example, above 0.5g / 100ml, above 0.6g / 100ml, above 0.7g / 100ml, or above 0.8g / 100ml.

[0308] The "sugar-acid ratio" is calculated as the ratio of the saccharimeter reading to the citric acid content. A sugar-acid ratio of, for example, 12.0 or higher, 13.0 or higher, 14.0 or higher, or 15.0 or higher is acceptable.

[0309] In one example, "good taste" means a good balance between sugar and acidity. Therefore, a sugar-acid ratio in the range of 12 to 14, preferably above 14, can be described as having a good taste.

[0310] In the example, the sugar content of the juice from "KN55" in early January averaged as high as 13.9%. Additionally, the acid content of the juice averaged as low as approximately 0.99 g / 100 ml. The sugar-acid ratio was 14.0.

[0311] For the aforementioned trait (3), fruit weight (preferably average) of 200g or more, this refers to the weight of the fruit measured at maturity using an electronic balance. The lower limit of fruit weight is preferably 210g or more, 220g or more, 230g or more, 240g or more, 250g or more, 260g or more, 270g or more, 280g or more, 290g or more, 300g or more, 310g or more, 320g or more, 330g or more, or 340g or more. The upper limit of fruit weight is not particularly limited; any value above the lower limit is acceptable, for example, below 360g, below 350g, below 340g, below 330g, below 320g, below 310g, or below 300g. In one example, in cultivation without heating facilities, large fruits weighing 250g or more with reduced acidity can be quickly marketed within the year.

[0312] For example, the fruit weight of other citrus varieties, such as "Southwest Light," is around 180g. Therefore, fruit weight can distinguish it from its parent variety, "Southwest Light." The ripening period for citrus fruits is generally based on the point when the peel is fully colored and the pulp acidity reaches below 1.0%. An acidity of 1.0% refers to a state where 100g of juice contains approximately 1g of organic acid (equivalent to citric acid), indicating a decrease in fruit acidity and a improved flavor.

[0313] In addition, the fruit of Amakusa weighs around 200g, while the fruit of Nanxiang weighs around 120g.

[0314] For the citrus plants involved in this invention, for example, the possibility of seedless fruit production for trait (5) refers to the absence of intact seeds, evaluated by surveying 5 or more fruits, with the size of the embryo being more than half that of a normal seed, indicating a high number of developed seeds: more than 10, medium: 5 to 10 (approximately 6 to 8), low: less than 5, or none: no seeds. In one example, under natural pollination conditions in open cultivation, almost no intact seeds were found, making seedless fruit production possible.

[0315] Furthermore, regarding the trait (6) of no loose peel in the citrus plant involved in this invention, it refers to the degree of loose peel on the fruit, as determined by observation and investigation, for example...

[0316] When setting "no" to 0, "light" to 1, "medium" to 2, and "very" to 3, the numerical evaluation can be calculated as ((number of light results × 1) + (number of medium results × 2) + (number of very severe results × 3)) ÷ (number of survey results × 3) × 100.

[0317] Furthermore, regarding the citrus plant involved in this invention, the fruit's ripening period (7) is early January. Preferably, the ripening period is early January in the case of open-field cultivation through, for example, top grafting. Depending on the cultivation site or conditions, the fruit's ripening period can be from mid-December to late January. That is, the ripening period is slightly later than "System A," in one example, about 1 to 3 weeks or 1 to 2 weeks later than "System A." In addition, it is later than "Southwest Light," in one example, about 1 to 6 weeks or 2 to 5 weeks later than "Southwest Light."

[0318] The citrus plant involved in this invention can be a plant with characteristics equivalent to "KN55". A plant with equivalent characteristics refers to a plant that, when grown under the same environmental conditions, possesses the main characteristics of "KN55". These main characteristics are (1) to (4). Furthermore, it is preferable that it also possesses characteristics (5) to (7). Additionally, the citrus plant of this invention, for example, has, for example, three, two, or one of the characteristics (5) to (7). Furthermore, the citrus plant of this invention further possesses one or more of the traits shown in (Tree Characteristics) and (Fruit Characteristics).

[0319] Furthermore, the trait (3) of the citrus plant involved in this invention can be identified with other citrus varieties containing "System A" and "Southwest Light" by using at least one of the genetic markers among Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2. The plants can be those whose genotypes of the genetic markers are Bf0158-3 (AB type), Bf0036-3 (AA type), Tf0150-2 (BB type), Tf0271-2 (AA type), Tf0300-3 (AB type), Tf0419-2 (AB type), Tf0420-2 (AA type), Tf0318-2 (BB type), Tf0293-4 (AA type), A10302-2 (AB type), Mf0097-2 (AB type), Mf0090-2 (AB type), Gn0073-2 (AA type), and Gn0048-2 (AA type).

[0320] The citrus plant of the present invention includes "KN55" or its progeny plants as described in the embodiments. The progeny plants include individual plants or portions thereof.

[0321] In this invention, the offspring plants also include plants obtained from hybridization of the “KN55” citrus plant with other citrus varieties or other citrus plants, or hybridization of the citrus plant with wild citrus plants.

[0322] The offspring plants include individuals and their offspring obtained with "KN55" as the parent system, somatic cell hybrids and their offspring resulting from the fusion of cells from the citrus plant genus and cells from other plant varieties, and individuals and their offspring obtained by grafting with citrus plants as rootstock or scion.

[0323] The citrus plant according to one embodiment of the present invention is a plant having the aforementioned characteristics, obtained by the aforementioned production method. Citrus plants or their fruits obtained by the production method described later are also within the scope of the present invention. Thus, the citrus plant can also be the fruit of a citrus plant. Furthermore, the morphology of vegetative propagation materials such as seedlings or scions of the citrus plant according to one embodiment of the present invention is also within the scope of the present invention.

[0324] Processed products made from citrus fruits

[0325] The processed citrus plant product according to one embodiment of the present invention may be a processed citrus plant product obtained from the aforementioned citrus plant. Alternatively, the processed citrus plant product according to one embodiment of the present invention may be a processed citrus fruit product obtained from the fruit of the aforementioned citrus plant. As a processed product, an edible processed product is preferred.

[0326] Examples of processed citrus plant products according to one embodiment of the present invention include plant juice, extracts, juice residue, and extraction residue. Other examples of processed plant products include those obtained by cutting, crushing, drying, pulverizing, freezing, various chemical treatments, gelatinization, and mud-like processing. In particular, suitable processed products are fruit products using the processing embodiments described above. Suitable examples of such fruit products include, for instance, fruit juice, pulp, or juice containing both juice and pulp, pulp soaked in syrup, dried fruit, jelly, and powdered confectionery ingredients.

[0327] Among these, as processed products, it is preferred to select at least one of the raw materials for desserts made from juice or syrup, jelly, or powder, and more preferably to select at least one of the raw materials made from juice or syrup.

[0328] Further explanation is given regarding the fruit, which is part of the citrus plant. The fruit can be a drupe (containing seeds) or a seedless fruit (without seeds). Citrus fruits can also be packaged in suitable packaging materials. The form of the packaging material is not particularly limited, and examples include films, sheets, bags, or pouches. The material of the packaging material is not particularly limited, and examples include paper or resin (plastic). Furthermore, the packaging of isolated citrus berries may contain one or more fruits. Moreover, the packaging of citrus fruits may contain only one type of citrus fruit or a combination of fruits from multiple citrus varieties. Additionally, the packaging of citrus fruits may also include agricultural products other than citrus fruits.

[0329] Product labels may also be affixed to the citrus fruit (including the packaging containing the citrus fruit). The product label records at least one piece of information, such as the citrus variety name, intellectual property information about the citrus, producer information, production history information (e.g., pesticide distribution information), distribution history information, product price, harvest date, and consumption period information. This information may be recorded directly on the product label (in addition to printing, for example, electromagnetic recording), or it may be recorded in a readable encoding format, such as a barcode or QR code.

[0330] Citrus plants can also be propagated as seedlings or scions. Product labels can also be attached to seedlings or scions. The product label records, for example, at least the following information: the citrus variety name, intellectual property information about the citrus, individual identification information (ID number) of the seedling or scion, genetic marker information of the seedling or scion (e.g., a genotypic map of the CAPS marker), producer information of the seedling or scion, production history information of the seedling or scion (e.g., pesticide distribution information), distribution history information of the seedling or scion, and product price (product information). In one 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) and genetic marker information of the seedling or scion (e.g., a genotypic map of the CAPS marker) are recorded. This information can be recorded directly on the product label (in addition to printed text, for example, as a readable electromagnetic record), or it can be recorded in a readable encoding format, such as a barcode or QR code. Alternatively, the product label may be made of a small electromagnetic recording medium, such as an IC chip, embedded inside the seedling or scion. The location of the embedded small electromagnetic recording medium, such as an IC chip, is not particularly limited. In the case of seedling grafting, in a preferred example, it is within the rootstock; in other preferred examples, it is within a range specified from the grafting position. The range specified from the grafting position means, for example, within 10 cm, 5 cm, 4 cm, 3 cm, 2 cm, or 1 cm from the grafting position. Furthermore, the specified distance can be taken from the grafting position toward the rootstock or from the grafting position toward the scion.

[0331] [Commodity Information Management System for Citrus Plant Seedlings or Scions (Vegetative Propagation Material)]

[0332] In the section on "Processed Citrus Products," the seedlings or scions of the citrus plants involved in this embodiment, which are affixed to product labels, are described. Within the scope of this invention, a product information management system for the seedlings or scions of the citrus plants involved in this embodiment, which are affixed to product labels, is also included.

[0333] An example of a product information management system includes at least (1) a seedling or scion of the citrus plant involved in this embodiment with a product label attached, and (2) a reader (an example of a client terminal) for reading product information recorded on the product label. The product information management system may also include (3) a server. The product information management system may have a client terminal other than a reader, or it may have a client terminal in place of a reader. These client terminals may not have the function of reading product information.

[0334] In one example, the client terminal (which may be a reader) sends a combination of information recorded on the product label (including the individual identification information (ID number) of the seedling or scion) and the client terminal's location information to the server. The client terminal (which may be a reader) may also further send the server the seedling or scion's owner information (which may also be the owner information of the client terminal if it matches the owner), the seedling or scion's location information (which may differ from the client terminal's location information), etc. Thus, information regarding the location of the seedling or scion is managed individually. Furthermore, this information is sent from the client terminal to the server when the owner of the seedling or scion changes, when the location of the seedling or scion is moved, when the seedling or scion is discarded, or when the management environment of the seedling or scion changes.

[0335]

in conclusion

[0336] This invention includes any of the following embodiments.

[0337] <1> Methods for processing citrus plants, including:

[0338] For hybrid varieties or their offspring obtained by crossing citrus varieties with each other, the procedure for selecting plants with the following genotypes for each of the genetic markers Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2 is as follows:

[0339] Bf0158-3 is type AB.

[0340] Bf0036-3 is type AA.

[0341] Tf0150-2 is a BB type.

[0342] Tf0271-2 is type AA.

[0343] Tf0300-3 is type AB.

[0344] Tf0419-2 is type AB.

[0345] Tf0420-2 is type AA.

[0346] Tf0318-2 is of type BB.

[0347] Tf0293-4 is type AA.

[0348] Al0302-2 is type AB.

[0349] Mf0097-2 is type AB.

[0350] Mf0090-2 is type AB.

[0351] Gn0073-2 is type AA, and

[0352] Gn0048-2 is of type AA.

[0353] <2> also includes the process of hybridizing the citrus plant described in <1> using mating parent (A) as the seed parent (maternal parent) and mating parent (B) as the pollen parent (paternal parent) to obtain the hybrid variety:

[0354] Cross-parent (A): Varieties obtained by crossing the citrus variety "Nanxiang" as the seed parent (female parent) and the citrus variety "Tencho" as the pollen parent (male parent).

[0355] mating parent (B): Citrus variety "Southwest Light".

[0356] <3> also includes the production method described in <1> or <2>, which is a second selection step of selecting citrus plants with the trait that the fruit weighs more than 200g.

[0357] <4> also includes the production method described in any one of <1> to <3> of the second selection step of selecting citrus plants having the following traits (1) and (2).

[0358] (1) The fruit has a sugar content of over 13 Brix%, a good taste, and...

[0359] (2) The fruit weighs more than 200g.

[0360] <5> also includes a method for producing citrus plants according to any one of <1> to <4> of the propagation process for asexual propagation of the citrus plants.

[0361] <6> The mating parent (A) mentioned above is a variety obtained by hybridization of "Nanxiang" as seed parent and "Tiancao" as pollen parent, and the genetic markers Bf0158-3 is AA type, Bf0036-3 is AA type, Tf0150-2 is BB type, Tf0271-2 is AB type, Tf0300-3 is AB type, Tf0419-2 is AB type, Tf0420-2 is AA type, Tf0318-2 is BB type, Tf0293-4 is AA type, A10302-2 is AA type, Mf0097-2 is AB type, Mf0090-2 is AB type, Gn0073-2 is AA type, and Gn0048-2 is AB type. The method of producing citrus plants described in any one of <2> to <5>.

[0362] <7> Methods for processing citrus plants, including:

[0363] The hybridization process of using the mating parent (A) as the female parent and the mating parent (B) as the male parent, as shown below, and...

[0364] Cross-parent (A): Varieties obtained by crossing the citrus variety "Nanxiang" as the seed parent (female parent) and the citrus variety "Tencho" as the pollen parent (male parent).

[0365] mating parent (B): Citrus variety "Southwest Light"

[0366] A marker selection process is used to identify candidate citrus plants that are different from mating parents (A) and mating parents (B) on the genotype map of the genetic markers by using at least one of the following genetic markers: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0419-2, Tf0420-2, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2, from citrus plants or their progeny obtained by the hybridization process.

[0367] <8> In the marker selection process, a combination of at least one genetic marker selected from Bf0158-3 and Gn0048-2 and at least one genetic marker selected from Bf0036-3, Tf0150-2, Tf0419-2, Tf0420-2, Mf0097-2, Mf0090-2 and Gn0073-2, and at least one genetic marker selected from Tf0271-2 and A10302-2, is used to select candidate citrus plants that are different from the mating parents (A) and (B) on the genotype map of the genetic markers, as described in <7>.

[0368] <9> The method described in <8> is used to systematically select candidate citrus plants with the following genotypes for the genetic markers: Bf0158-3 (AB type), Bf0036-3 (AA type), Tf0150-2 (BB type), Tf0271-2 (AA type), Tf0419-2 (AB type), Tf0420-2 (AA type), Al0302-2 (AB type), Mf0097-2 (AB type), Mf0090-2 (AB type), Gn0073-2 (AA type), and Gn0048-2 (AA type).

[0369] <10> The production method described in any one of <7> to <9> includes the selection process of selecting citrus plants having the following traits (1) and (2).

[0370] (1) The fruit has a sugar content of over 13 Brix%, a good taste, and...

[0371] (2) The fruit weighs more than 200g.

[0372] <11> also includes a method for producing citrus plants according to any one of <7> to <10>, which involves a process of asexually propagating the citrus plants.

[0373] <12> The mating parent (A) mentioned above is a variety obtained by hybridization of "Nanxiang" as the seed parent and "Tiancao" as the pollen parent. The genetic markers Bf0158-3 are of type AA, Bf0036-3 are of type AA, Tf0150-2 are of type BB, Tf0271-2 are of type AB, Tf0300-3 are of type AB, Tf0419-2 are of type AB, Tf0420-2 are of type AA, Tf0318-2 are of type BB, Tf0293-4 are of type AA, A10302-2 are of type AA, Mf0097-2 are of type AB, Mf0090-2 are of type AB, Gn0073-2 are of type AA, and Gn0048-2 is of type AB. The method of producing citrus plants described in any one of <7> to <11>.

[0374] <13> Citrus plants, wherein the genotypes of the genetic markers in the citrus plants are:

[0375] Bf0158-3 is type AB.

[0376] Bf0036-3 is type AA.

[0377] Tf0150-2 is a BB type.

[0378] Tf0271-2 is type AA.

[0379] Tf0300-3 is type AB.

[0380] Tf0419-2 is type AB.

[0381] Tf0420-2 is type AA.

[0382] Tf0318-2 is of type BB.

[0383] Tf0293-4 is type AA.

[0384] Al0302-2 is type AB.

[0385] Mf0097-2 is type AB.

[0386] Mf0090-2 is type AB.

[0387] Gn0073-2 is type AA, and

[0388] Gn0048-2 is of type AA.

[0389] <14> A hybrid variety or a citrus plant described in <13> that is hybridized with the mating parent (A) shown below as the seed parent (mother parent) and the mating parent (B) as the pollen parent (father parent).

[0390] Cross-parent (A): Varieties obtained by crossing the citrus variety "Nanxiang" as the seed parent (female parent) and the citrus variety "Tencho" as the pollen parent (male parent).

[0391] mating parent (B): Citrus variety "Southwest Light".

[0392] <15> The fruit of the citrus plant described in <13> or <14> weighs more than 200g.

[0393] <16> Citrus plants described in any one of <13> to <15> that possess the following traits (1), (2), (3) and (4):

[0394] (1) The result obtained by hybridization using the parent (A) as the female parent and the parent (B) as the male parent, as shown below.

[0395] Cross-parent (A): Varieties obtained by crossing the citrus variety "Nanxiang" as the seed parent (female parent) and the citrus variety "Tencho" as the pollen parent (male parent).

[0396] mating parent (B): Citrus variety "Southwest Light"

[0397] (2) The fruit has a sugar content of over 13 Brix and a good taste.

[0398] (3) The fruit weighs more than 200g, and,

[0399] (4) Can use the genetic markers 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 to identify other citrus varieties containing the aforementioned mating parent (A) and mating parent (B).

[0400] <17> also has at least one of the following traits (5) and (6) of the citrus plants described in <16>:

[0401] (5) It can produce seedless fruit, and

[0402] (6) No floating skin fruit occurs.

[0403] The mating parent (A) mentioned in <18> is a variety obtained by hybridization of "Nanxiang" as the seed parent and "Tiancao" as the pollen parent, and the genetic markers Bf0158-3 is AA type, Bf0036-3 is AA type, Tf0150-2 is BB type, Tf0271-2 is AB type, Tf0300-3 is AB type, Tf0419-2 is AB type, Tf0420-2 is AA type, Tf0318-2 is BB type, Tf0293-4 is AA type, A10302-2 is AA type, Mf0097-2 is AB type, Mf0090-2 is AB type, Gn0073-2 is AA type, and Gn0048-2 is AB type, and the citrus plant mentioned in any one of <14> to <17> is AB type.

[0404] <19> A method for identifying any one of <13> to <18> citrus plants, comprising:

[0405] The process of amplifying regions containing genetic markers of citrus plant DNA using primer sets, and

[0406] The process of identifying the citrus plant based on the base polymorphism of the amplified DNA region.

[0407] The genetic markers are Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0408] The citrus plant described in any one of <20>, <13> to <18> is in the form of a seedling or scion.

[0409] The fruit of any one of the citrus plants described in <21>, <13> to <18>.

[0410] <22> Processed products of citrus plants or fruits of citrus plants obtained from any of the citrus plants described in <13> to <18> or the citrus plants described in <21>.

[0411] The citrus plant or processed product of the fruit of the citrus plant mentioned in <23> and <22> is at least one of the raw materials selected from juice, syrup soaking, jelly, powder processing of snacks, etc.

[0412] <2-1> Methods for producing citrus products, including:

[0413] The hybridization process of using the mating parent (A) as the female parent and the mating parent (B) as the male parent, as shown below, and...

[0414] Crossparent (A): Varieties obtained by crossing the citrus variety "Nanxiang" and the citrus variety "Amakusa".

[0415] mating parent (B): Citrus variety "Southwest Light"

[0416] A marker selection process is used to identify candidate citrus plants that are different from mating parents (A) and mating parents (B) on the genotype map of the genetic markers by using at least one of the following genetic markers: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0419-2, Tf0420-2, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2, from citrus plants or their progeny obtained by the hybridization process.

[0417] <2-2> In the marker selection process, a combination of at least one genetic marker selected from Bf0158-3 and Gn0048-2 and at least one genetic marker selected from Bf0036-3, Tf0150-2, Tf0419-2, Tf0420-2, Mf0097-2, Mf0090-2 and Gn0073-2, and at least one genetic marker selected from Tf0271-2 and Al0302-2, is used to select candidate citrus plants that are different from the mating parents (A) and (B) on the genotype map of the genetic markers, as described in <2-1>.

[0418] <2-3> The genetic markers selected are genotypes Bf0158-3 (AB type), Bf0036-3 (AA type), Tf0150-2 (BB type), Tf0271-2 (AA type), Tf0419-2 (AB type), Tf0420-2 (AA type), Al0302-2 (AB type), Mf0097-2 (AB type), Mf0090-2 (AB type), Gn0073-2 (AA type), and Gn0048-2 (AA type) as candidate citrus plants according to the method described in <2-1> or <2-2>.

[0419] <2-4> is a production method described in any one of <2-1> to <2-3>, which includes the selection process of selecting citrus plants having the following traits (1) and (2).

[0420] (1) The fruit has a sugar content of over 13 Brix%, a good taste, and...

[0421] (2) The fruit weighs more than 200g.

[0422] <2-5> also includes the method for producing citrus plants according to any one of <2-1> to <2-4>, which involves the asexual propagation process of the citrus plants.

[0423] <2-6> Citrus plants, which are citrus plants with the following characteristics (1), (2), (3) and (4):

[0424] (1) The result obtained by hybridization using the parent (A) as the female parent and the parent (B) as the male parent, as shown below.

[0425] Crossparent (A): Varieties obtained by crossing the citrus variety "Nanxiang" and the citrus variety "Amakusa".

[0426] mating parent (B): Citrus variety "Southwest Light"

[0427] (2) The fruit has a sugar content of over 13 Brix and a good taste.

[0428] (3) The fruit weighs more than 200g, and,

[0429] (4) Can be identified with other citrus varieties containing the mating parent (A) and mating parent (B) by using at least one of the genetic markers of Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2 and Gn0048-2.

[0430] <2-7> also includes citrus plants described in <2-6> that possess at least one of the following traits (5) and (6):

[0431] (5) It can produce seedless fruit, and

[0432] (6) No floating skin fruit occurs.

[0433] The genotypes of the genetic markers mentioned in <2-8> are Bf0158-3 (AB type), Bf0036-3 (AA type), Tf0150-2 (BB type), Tf0271-2 (AA type), Tf0300-3 (AB type), Tf0419-2 (AB type), Tf0420-2 (AA type), Tf0318-2 (BB type), Tf0293-4 (AA type), A10302-2 (AB type), Mf0097-2 (AB type), Mf0090-2 (AB type), Gn0073-2 (AA type), and Gn0048-2 (AA type) of the citrus plants described in <2-6> or <2-7>.

[0434] <2-9> A method for identifying any one of the citrus plants described in <2-6> to <2-8>, comprising:

[0435] The procedure of amplifying regions containing CAPS markers in the DNA of citrus plants using primer sets, and

[0436] This includes a process for identifying the citrus plant based on the polymorphism of the bases in the amplified DNA region.

[0437] 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, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

[0438] <2-10> Citrus plants or their fruits obtained by any one of the methods described in <2-1> to <2-5>.

[0439] The citrus plant described in any one of <2-11>, <2-6> to <2-8>, or the citrus plant or its fruit described in <10>, in the form of a seedling or scion.

[0440] <2-12> Processed products made using any one of the citrus plants described in <2-6> to <2-8> or the citrus plant or its fruit described in <2-10>.

[0441] This invention is not limited to the various embodiments described, and various modifications can be made within the scope shown in the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of this invention.

[0442]

Example

[0443] [1. Production of the Citrus System]

[0444] (origin)

[0445] (1) Breeding method: hybridization breeding method

[0446] (2) Parents: "System A" × "Light of the Southwest"

[0447] System A is a hybrid of "Nanxiang" and "Amakusa".

[0448] The superior "System A," characterized by early maturity, good flavor, relatively large fruit size, and high fruit set, was used as the female parent (seed parent). The mid-maturing, flavorful, aromatic, early-maturing, easy-to-peel, and low-seeded "Southwest Light," with its high sugar content and excellent flavor, was used as the male parent (pollen parent) for hybridization. The pedigree is shown in [reference needed]. Figure 1 .

[0449] [Cultivation Process]

[0450] In May 2008, at the Kuchinotsu Base of the Citrus Research Institute (now the Kuchinotsu Experimental Site of the Kyushu-Okinawa Agricultural Research Center of the National Agricultural Research and Development Institute), a hybridization system was developed with the goal of cultivating a mid-ripening citrus variety that matures from late December to mid-January, has high sugar content, good flavor, and large fruit characteristics. In October 2009, grafting onto trifoliate orange rootstock was undertaken to promote fruit production. First fruiting occurred in 2012. Following an investigation into fruit quality, and to confirm its superior quality, it was selected as an excellent individual in 2013. KN55 was used in the 12th Citrus System Adaptability and Characteristic Testing Trial starting in April 2017, with regional adaptability studies conducted at 27 experimental sites ranging from Chiba Prefecture in the east to Kagoshima Prefecture in the south. The results, presented at the August 2023 Symposium on the Results of the Same Trial (Evergreen Fruit Trees), concluded that it was suitable as a candidate for a new variety.

[0451] [Selection Process]

[0452] The selection criteria during the selection process are established through the following procedure.

[0453] Post-hoc evaluation of results (2012–2015)

[0454] 1. Large fruits with an average weight exceeding 200g

[0455] 2. It matures in January (with an acid content of approximately 1.0–1.2% by December).

[0456] 3. Average sugar content of 12 Brix% or higher

[0457] 4. Number of seeds: "None" to "Few"

[0458] Based on the selection criteria in 1. to 4., preliminary selection was carried out in 2013, and the selected plants were grafted and replanted.

[0459] 6. Continuous results and yield.

[0460] 7. Evaluation of physiological barriers on the tree (peel problems such as peeling and cracking).

[0461] 8. Observation of the severity of disease occurrence

[0462] Based on the selection criteria in sections 6 to 8, the first selection was conducted in 2017, and the selected plants continued to grow further.

[0463] 9. System adaptability testing (regional adaptability evaluation)

[0464] Based on the selection criteria in section 9, a second selection was conducted in 2023, and the selected plants were designated as "KN55".

[0465] A photo of the fruit of “KN55” is shown. Figure 2 Additionally, a photo of the tree trunk labeled "KN55" is shown. Figure 3 .

[0466] [2. Characteristic evaluation of various traits in the breeding site]

[0467] Next, the traits of the obtained varieties will be evaluated.

[0468] The evaluation methods for traits were based on the Agricultural, Forestry and Fisheries Plant Species Classification Review Standards (Classification: Other Citrus) (Ministry of Agriculture, Forestry and Fisheries) and the Methods for Investigating Adaptability and Characteristic Tests of Breeding Systems (National Research Institute of Agriculture and Food Science, 2007). The following traits are based on trial plantings in Japan. The traits described and the traits recorded in the examples are evaluated based on the Classification: Other Citrus (Citrus L.) Review Standards issued by the Ministry of Agriculture, Forestry and Fisheries of Japan (the standards published by the Ministry of Agriculture, Forestry and Fisheries at the time of trial planting) and the Methods for Investigating Adaptability and Characteristic Tests of Breeding Systems (National Research Institute of Agriculture and Food Science, 2007).

[0469] (Features Summary)

[0470] (1) The tree vigor is "medium" and the branches are open. The density of the branches is "dense". There are many thorns.

[0471] (2) The fruit weighs approximately 250g, with an orange peel. The smoothness of the peel is moderate, and the thickness is as thin as 2.1mm. Peeling is moderate. There is almost no loose peel. The juice vesicle membrane is soft, and the pulp content is as high as 85.8%. In a survey conducted in early January, the sugar content of the juice was as high as 13 Brix% or more, and the acid content was below 1.0%, resulting in a rich flavor. The ripening period is in early January. Under natural pollination conditions in open-field cultivation, there are almost no intact seeds, making seedless fruit production possible.

[0472] (3) In cultivation without heating facilities, it is possible to produce high-quality fruits with a sugar content of more than 13 Brix% and an acid content of approximately 1.0% or less by late December, which can be marketed within the year.

[0473] Table 6: Tree and fruit characteristics of "KN55"

[0474]

[0475] The characteristic survey was conducted over a two-year period from 2023 to 2024, and the average value is shown.

[0476] Record the number of thorns on the branches and their grade values ​​and occurrence rates.

[0477]

[0478] Record the quantity of seedless fruits and their grade values ​​and occurrence rates.

[0479] The tiered data for the number of complete seeds is represented as follows: No seeds: 0 seeds, Few seeds: 1-2 seeds, Medium seeds: 3-5 seeds, More than 5 seeds per fruit.

[0480] Table 7: Fruit characteristics of "Citrus KN55" in unheated cultivation

[0481]

[0482] The characteristic survey was conducted over a three-year period from 2020 to 2022, showing its average value or range (Miyazaki Hinami over a two-year period from 2020 to 2021).

[0483] A summary of the characteristics of “KN55” is shown in Table 8 below.

[0484] Table 8

[0485]

[0486] -: Not investigated

[0487] (Points to note regarding suitable planting sites and cultivation)

[0488] (1) In the system adaptability test to date, high sugar content fruits have been produced in all test sites, and it is considered to have a wide range of adaptability to soil and meteorological conditions.

[0489] (2) For the production of high sugar content fruits, there is no need to consider the necessity of water stress, and it is suitable for a wide range of orchard conditions.

[0490] (3) Because the peel is thin, while paying attention to the handling of the fruit at harvest, thorns should be thoroughly removed during the management of young trees.

[0491] (4) In order to avoid the occurrence of fruit peel obstacles and meteorological disasters such as cold damage during the cold season, and from the point of view of preventing the occurrence of canker disease, it is preferable to explore facility cultivation.

[0492] 2. Identification of citrus plants using CAPS markers

[0493] Verify whether the CAPS marker can be used to identify "KN55".

[0494] Using the CAPS marker disclosed in "DNA Variety Identification Technology of Fruits of 24 Citrus Varieties Using CAPS Markers (Non-Patent Document 1)," the reported CAPS marker was used to verify whether "KN55" could be identified with other citrus plants.

[0495] The experimental method was performed according to the manual in Non-Patent Literature 1. Specifically, the outer layer of the orange peel (exocarp) was cut from fresh fruit, and a DNA solution was obtained according to the manual in Non-Patent Literature 1. For each CAPS marker, PCR reactions were performed on the obtained DNA solution using the forward and reverse primers described in Table 1 to obtain amplification products. The obtained amplification products were treated with the restriction endonucleases described in Table 1 to prepare restriction endonuclease reaction solutions. The restriction endonuclease reaction solutions were applied to a 2% agarose gel, and polymorphism was confirmed by electrophoresis.

[0496]

result

[0497] The results of the polymorphism confirmed by electrophoresis are shown in Figures 5-7 Additionally, in Figure 4 Genotypes displaying the CAPS marker "KN55".

[0498] It was learned that the genotype of the CAPS marker “KN55” can be distinguished from all 24 varieties of the target species identified by this marker, and can be identified from the mating parents (Tables 2-5).

[0499] As mentioned above, using the CAPS marker, "KN55" can be identified from other varieties.

[0500] [Potential for Industrial Applications]

[0501] This invention can be used in the fields of agriculture and plant breeding.

Claims

1. Methods for processing citrus plants, including: The procedure for obtaining hybrid varieties by crossing mating parent (A) as the seed parent (maternal parent) and mating parent (B) as the pollen parent (paternal parent) as shown below: Cross-parent (A): Varieties obtained by crossing the citrus variety "Nanxiang" as the seed parent (female parent) and the citrus variety "Tencho" as the pollen parent (male parent). mating parent (B): Citrus variety "Southwest Light", and The procedure for selecting plants with the following genotypes for the aforementioned hybrid varieties or their offspring: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2: Bf0158-3 is type AB. Bf0036-3 is type AA. Tf0150-2 is a BB type. Tf0271-2 is type AA. Tf0300-3 is type AB. Tf0419-2 is type AB. Tf0420-2 is type AA. Tf0318-2 is of type BB. Tf0293-4 is type AA. Al0302-2 is type AB. Mf0097-2 is type AB. Mf0090-2 is type AB. Gn0073-2 is type AA, and Gn0048-2 is of type AA.

2. The method of production according to claim 1 further includes a second selection step of selecting citrus plants with the trait of having fruit weighing more than 200g.

3. The method of production according to claim 1 or 2, further comprising a second selection step of selecting citrus plants having the following traits (1) and (2). (1) The sugar content of the fruit is above 13 Brix%, and, (2) The fruit weighs more than 200g.

4. The method for producing citrus plants according to claim 1 or 2, further comprising a propagation step of causing the citrus plants to reproduce asexually.

5. Methods for processing citrus plants, including: The hybridization process using the mating parent (A) as the female parent and the mating parent (B) as the male parent, as shown below: Cross-parent (A): Varieties obtained by crossing the citrus variety "Nanxiang" as the seed parent (female parent) and the citrus variety "Tencho" as the pollen parent (male parent). mating parent (B): Citrus variety "Southwest Light", and A marker selection process is used to identify candidate citrus plants that are different from mating parents (A) and mating parents (B) on the genotype map of the genetic markers by using at least one of the following genetic markers: Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0419-2, Tf0420-2, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2, from citrus plants or their progeny obtained by the hybridization process.

6. The method of preparation according to claim 5, wherein in the marker selection step, a combination of at least one genetic marker selected from Bf0158-3 and Gn0048-2 and at least one genetic marker selected from Bf0036-3, Tf0150-2, Tf0419-2, Tf0420-2, Mf0097-2, Mf0090-2 and Gn0073-2, and at least one genetic marker selected from Tf0271-2 and A10302-2 is used to select candidate citrus plants that are different from the mating parents (A) and (B) on the genotype map of the genetic marker.

7. The method of preparation according to claim 6, wherein the system for selecting candidate citrus plants has the following genotypes for the genetic markers: Bf0158-3 is AB type, Bf0036-3 is AA type, Tf0150-2 is BB type, Tf0271-2 is AA type, Tf0419-2 is AB type, Tf0420-2 is AA type, Al0302-2 is AB type, Mf0097-2 is AB type, Mf0090-2 is AB type, Gn0073-2 is AA type, and Gn0048-2 is AA type.

8. The method of production according to any one of claims 5 to 7, comprising a selection step of selecting citrus plants having the following traits (1) and (2). (1) The sugar content of the fruit is above 13 Brix%, and, (2) The fruit weighs more than 200g.

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

10. Citrus plants, which are hybrid varieties or offspring plants of citrus plants that are crossed with the mating parent (A) as the seed parent (maternal parent) and the mating parent (B) as the pollen parent (paternal parent) as shown below: Cross-parent (A): Varieties obtained by crossing the citrus variety "Nanxiang" as the seed parent (female parent) and the citrus variety "Tencho" as the pollen parent (male parent). mating parent (B): Citrus variety "Southwest Light" The genotypes of the genetic markers in the citrus plants mentioned above are: Bf0158-3 is type AB. Bf0036-3 is type AA. Tf0150-2 is a BB type. Tf0271-2 is type AA. Tf0300-3 is type AB. Tf0419-2 is type AB. Tf0420-2 is type AA. Tf0318-2 is of type BB. Tf0293-4 is type AA. Al0302-2 is type AB. Mf0097-2 is type AB. Mf0090-2 is type AB. Gn0073-2 is type AA, and Gn0048-2 is of type AA.

11. The citrus plant of claim 10, wherein the fruit weighs more than 200g.

12. The citrus plant of claim 10, having the following characteristics (1), (2), (3) and (4): (1) The following is obtained by crossing parent (A) as the female parent and parent (B) as the male parent: Cross-parent (A): Varieties obtained by crossing the citrus variety "Nanxiang" as the seed parent (female parent) and the citrus variety "Tencho" as the pollen parent (male parent). mating parent (B): Citrus variety "Southwest Light" (2) The sugar content of the fruit is above 13 Brix%. (3) The fruit weighs more than 200g, and, (4) Can use the genetic markers 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 to identify other citrus varieties containing the aforementioned mating parent (A) and mating parent (B).

13. The citrus plant of claim 12, further comprising at least one of the following traits (5) and (6): (5) It can produce seedless fruit, and (6) No floating skin fruit occurs.

14. A method for identifying the citrus plant of claim 10, comprising: The process of amplifying regions containing genetic markers of citrus plant DNA using primer sets, and The process of identifying the citrus plant based on the base polymorphism of the amplified DNA region. The genetic markers mentioned are Bf0158-3, Bf0036-3, Tf0150-2, Tf0271-2, Tf0300-3, Tf0419-2, Tf0420-2, Tf0318-2, Tf0293-4, A10302-2, Mf0097-2, Mf0090-2, Gn0073-2, and Gn0048-2.

15. The citrus plant of claim 10, wherein it is in the form of a seedling or a scion.

16. The fruit of the citrus plant as claimed in claim 10.

17. A citrus plant or a processed product of the fruit of a citrus plant obtained from the citrus plant of claim 10 or the citrus plant of claim 16, wherein the citrus plant is a raw material selected from at least one of the following: juice, pulp, or juice containing juice and pulp, syrup-impregnated pulp, dried fruit, jelly, or powdered pastries.

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