Method for producing new varieties of kasuri of eustoma

Abstract

Provided is a method for clarifying the traits and genotype of "kasuri flower", the genotype of which has not been clear and the traits of which has been vaguely defined so far, and for producing new varieties of Lisianthus with various flower shapes and / or flower colors including kasuri flower, by utilizing the heredity pattern of Lisianthus including the genotypes of yellow flower, white flower, octuple, double, single, and ornamental traits. This method for producing new varieties of Lisianthus uses a genotype corresponding to a flower color trait and / or a flower shape trait of Lisianthus, wherein the genotype is (a) a genotype BXBX of a complex allele involved in the expression of the traits of fully colored flowers, dark kasuri flowers, and light kasuri flowers, respectively (BX is a complex allele represented by BM, BD, and BL, of which BMBM indicates homozygous for fully colored flowers, BMBD (or BDBM), BMBL (or BMBL) indicates heterozygous for fully colored flowers, BDBD indicates homozygous type for dark kasuri flowers, BDBL (or BLBD) indicates heterozygous for dark kasuri flowers, and BLBL indicates homozygous for light kasuri flowers).

Description

A method for producing new varieties of Eustoma

[0001] The present invention relates to a method for producing a new Kasuri type lisianthus cultivar using a genotype corresponding to flower color traits and / or flower shape traits of lisianthus.

[0002] Anthocyanins, a type of flavonoid compound, are water-soluble glycosides that produce colors such as purple and red in plants. They are widely distributed in flowers, fruits, and leaves, and release sugars and the aglycone anthocyanidin upon acid hydrolysis (Non-Patent Document 1). Anthocyanidins include pelargonidin (Pgn), which has one hydroxyl group attached to the 4' position of its B ring, exhibiting an orange to vermilion color; cyanidin (Cyn), which has two hydroxyl groups attached to the 3', 4' positions of its B ring, exhibiting a red to crimson color; and delphinidin (Dpn), which has three hydroxyl groups attached to the 3', 4', and 5' positions of its B ring, exhibiting a purple color. Various flower (fruit) colors are expressed by combining these anthocyanidins (Non-Patent Document 2). Patent Document 1 describes the inheritance of pelargonidin (Pgn), cyanidin (Cyn), and delphinidin (Dpn), which express the flower color of lisianthus. The genotypes related to the inheritance of pigment biosynthesis include four multiple alleles that control the inheritance of flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3',5'H), which are involved in the hydroxylation of the B ring of anthocyanidin precursors; Ｔ , H Ｆ , H Ｄ , H Ｏ has been discovered, and a "flower color genotype crossing method for Lisianthus" has been established, which allows for the creation of new flower colors without causing mutations through genetic recombination or radiation exposure experiments. Ｚ In Patent Document 3, a method for producing a flowering plant by adding the above five multiple alleles (H) derived from the pollen parent gamete and the seed parent gamete to the genotype of the corolla traits of double and marginated flowers has been reported. Ｔ , H Ｆ , H Ｚ , H Ｄ , H ＯA method for predicting the flower color of a flower before blooming based on the correlation between the combination of markers (A, B, C, D, E, E, F ... Furthermore, in Patent Document 5, the traits of all of these various "Kasuribana" varieties are classified as recessive homozygous genotype bb, but "Kasuribana" varieties that are said to have the bb type have various traits, such as dark kasuri flowers and light kasuri flowers, and the genotypes of "Kasuribana" are unclear and ambiguous, such as the genotypes of dark kasuri flowers and light kasuri flowers, and the genotype of the progeny when dark kasuri flowers and light kasuri flowers are crossed. As a result, it has not been possible to freely produce Kasuri flowers. Even today, Eustoma boasts an annual cut flower production of 100 million heads, and the wholesale price per head has been relatively high at 200 yen for the past 10 years, and it has been traded steadily. Further demand is expected both domestically and internationally, so a new F of Eustoma with a new "Kasuri trait" is being developed. １ There is a need for methods of cultivar creation.

[0003] JP 2004-236516 A: "Method for crossbreeding genotypes of flower color in lisianthus" JP 4314241 A: "Method for creating flowering plants" JP 5039967 A: "Method for predicting flower color" WO 2010 / 143749 A: "Method for selecting lisianthus that produce white flowers" JP 6153213 A: "Method for creating new lisianthus varieties"

[0004] Takao Murakami, "Anthocyanin Derivatives," Structure and Chemistry of Natural Products, Hirokawa Shoten, September 1984, pp. 170-172 Toshio Honda and Norio Saito, "The Science of Flower Colors," Modern Chemistry, Tokyo Kagaku Dojin, May 1998, pp. 25-32

[0005] The present invention aims to clarify the trait and genotype of "kasuri flower," which has previously been unclear in terms of genotype and ambiguously defined trait, and to provide a method for producing new lisianthus varieties with a variety of flower shapes and / or flower colors, including kasuri flowers, by utilizing the genetic patterns of lisianthus, including the genotypes of yellow flowers, white flowers, double flowers, double flowers, single flowers, and edge-ringed traits. In order to solve the above-mentioned problems, the inventors focused on the kasuri traits, which had not been elucidated until now, and repeatedly carried out selfing and reciprocal crossing of lisianthus to clarify the traits, their inheritance, and the relationship between these traits and the corresponding genotypes, and investigated the segregation of their genes, resulting in the following findings: (i) The traits of fully colored flowers / dark kasuri flowers / light kasuri flowers are B Ｍ / B Ｄ / B Ｌ It is controlled by the law of multiple allele inheritance by the combination of three alleles, Ｍ B Ｍ Homozygous or heterozygous B Ｍ B Ｄ , B Ｍ B Ｌ (or B Ｄ B Ｍ , B Ｌ B Ｍ (ii) In the case of an individual having the genotype of (i) above, the traits of (ii) above and (iii) above are expressed as fully colored flowers; Ｍ / B Ｄ / B Ｌ It is controlled by the law of multiple allele inheritance by the combination of three alleles, Ｄ B Ｄ Homozygous or heterozygous B Ｄ B Ｌ (or B Ｌ B Ｄ (iii) In the case of an individual having the genotype of (I), dark kasuri flowers are expressed; (iii) the traits of all colored flowers / dark kasuri flowers / light kasuri flowers are B Ｍ / BＤ / B Ｌ It is controlled by the law of multiple allele inheritance by the combination of three alleles, Ｌ B Ｌ In the case of individuals having a homozygous genotype of the above, pale kasuri flowers are expressed. The present invention was completed based on the above findings. That is, the present invention encompasses the following inventions: (1) A method for producing a new variety of lisianthus using a genotype corresponding to a flower color trait and / or a flower shape trait of lisianthus, wherein the genotype is: (a) genotype B of multiple alleles involved in the expression of each trait of fully colored flowers, dark kasuri flowers, and pale kasuri flowers. Ｘ B Ｘ (Here, B Ｘ is B Ｍ , B Ｄ , B Ｌ A multiple allele represented by B Ｍ B Ｍ is homozygous for fully colored flowers, B Ｍ B Ｄ (or B Ｄ B Ｍ ), and B Ｍ B Ｌ (or B Ｌ B Ｍ ) is a heterozygous type with fully colored flowers, B Ｄ B Ｄ is a homozygous dark kasuri flower, B Ｄ B Ｌ (or B Ｌ B Ｄ ) is a heterotype with dark kasuri flowers, B Ｌ B Ｌ (b) a genotype H of multiple alleles that control the hydroxylation of the B ring of the pigment precursor in flavonoid biosynthesis and are involved in the synthesis of pelargonidin (Pgn), cyanidin (Cyn), or delphinidin (Dpn), which are the main anthocyanidin pigments of lisianthus; Ｘ H Ｘ (Here, H Ｘ Is, H Ｔ , H Ｆ , H Ｄ , H Ｚ , H Ｏ, or H Ｅ (c) the genotypes Pg / pg, Cy / cy, and Dp / dp of pelargonidin (Pgn), cyanidin (Cyn), or delphinidin (Dpn), which are the main anthocyanidin pigments in lisianthus; (d) the genotype Ans / ans for colored flowers / white flowers; (e) the genotype Y that determines the combination of yellow and white flowers. Ｘ Y Ｘ (where Y Ｘ Is Y Ｃ , Y Ｓ , Y Ｗ A compound allele represented by Y Ｃ Y Ｃ is homozygous for yellow flowers, Y Ｃ Y Ｓ (or Y Ｓ Y Ｃ ) is a heterozygous yellow flower, Y Ｃ Y Ｗ (or Y Ｗ Y Ｃ ) is a heterozygous yellow flower, Y Ｗ Y Ｗ is a homozygous white flower, Y Ｓ Y Ｗ (or Y Ｗ Y Ｓ ) is a heterozygous white flower, Y Ｓ Y Ｓ indicates a homozygous yellow flower type; (f) genotype D, which determines the combination of double, double, and single types. Ｘ D Ｘ (where D Ｘ is D Ｄ , D Ｓ , D Ｗ A multiple allele represented by D Ｄ D Ｄ is a homozygous double flower, D Ｄ D Ｓ (or D Ｓ D Ｄ ) is a hetero double flower type, D Ｓ D Ｓ is a homozygous single flower, D Ｄ D Ｗ (or D Ｗ D Ｄ ) is a heterozygous double flower, D Ｗ DＳ (or D Ｓ D Ｗ ) is a single-flowered heterotype, D Ｗ D Ｗ (g) a genotype E / e of edge-ringed flowers / full-colored flowers; and (g) a genotype E / e of edge-ringed flowers / full-colored flowers. (3) The method according to (1), wherein the genotype is B Ｘ B Ｘ ・D Ｘ D Ｘ ・E / e・Ans / ans・H Ｘ H Ｘ ・Pg / pg・Cy / cy・Dp / dp・Y Ｘ Y Ｘ (4) The method according to (2), wherein the F of Eustoma is １ Variety, or F １ (5) A method for producing a new lisianthus variety, comprising determining a combination of multiple alleles based on the following quick reference table A, in which gametes of the pollen parent are represented as rows and gametes of the seed parent are represented as columns, to produce a lisianthus with the desired fully colored flowers, dark kasuri flowers, or light kasuri flowers. (6) F of lisianthus produced by the method according to any one of (1) to (3). １ Variety or F １ (7) Eustoma seed parent lines and Eustoma pollen parent lines for producing Eustoma varieties. １ Variety or F １ A seed parent line and a pollen parent line of Lisianthus for producing a variety, the genotypes of which are: (a) genotype B of multiple alleles involved in the expression of the traits of fully colored flowers, dark kasuri flowers, and light kasuri flowers, Ｘ B Ｘ (Here, B Ｘ is B Ｍ , B Ｄ , B Ｌ A multiple allele represented by B Ｍ BＭ is homozygous for fully colored flowers, B Ｍ B Ｄ (or B Ｄ B Ｍ ), and B Ｍ B Ｌ (or B Ｌ B Ｍ ) is a heterozygous type with fully colored flowers, B Ｄ B Ｄ is a homozygous dark kasuri flower, B Ｄ B Ｌ (or B Ｌ B Ｄ ) is a heterotype with dark kasuri flowers, B Ｌ B Ｌ indicates a homozygous type with pale kasuri flowers. Ｘ B Ｘ The above-mentioned Eustoma F １ Variety or F １ (8) A seed parent line and a pollen parent line of Lisianthus for producing a variety. (b) A genotype H of a multi-allele that controls the hydroxylation of the B ring of a pigment precursor in flavonoid biosynthesis and is involved in the synthesis of pelargonidin (Pgn), cyanidin (Cyn), or delphinidin (Dpn), which are the major anthocyanidin pigments of Lisianthus. Ｘ H Ｘ (Here, H Ｘ Is, H Ｔ , H Ｆ , H Ｄ , H Ｚ , H Ｏ , or H Ｅ (c) the genotypes Pg / pg, Cy / cy, and Dp / dp of pelargonidin (Pgn), cyanidin (Cyn), or delphinidin (Dpn), which are the main anthocyanidin pigments in lisianthus; (d) the genotype Ans / ans for colored flowers / white flowers; (e) the genotype Y that determines the combination of yellow and white flowers. Ｘ Y Ｘ (where Y Ｘ Is Y Ｃ , Y Ｓ , Y Ｗ A compound allele represented by Y Ｃ YＣ is homozygous for yellow flowers, Y Ｃ Y Ｓ (or Y Ｓ Y Ｃ ) is a heterozygous yellow flower, Y Ｃ Y Ｗ (or Y Ｗ Y Ｃ ) is a heterozygous yellow flower, Y Ｗ Y Ｗ is a homozygous white flower, Y Ｓ Y Ｗ (or Y Ｗ Y Ｓ ) is a heterozygous white flower, Y Ｓ Y Ｓ indicates a homozygous yellow flower type; (f) genotype D, which determines the combination of double, double, and single types. Ｘ D Ｘ (where D Ｘ is D Ｄ , D Ｓ , D Ｗ A multiple allele represented by D Ｄ D Ｄ is a homozygous double flower, D Ｄ D Ｓ (or D Ｓ D Ｄ ) is a hetero double flower type, D Ｓ D Ｓ is a homozygous single flower, D Ｄ D Ｗ (or D Ｗ D Ｄ ) is a heterozygous double flower, D Ｗ D Ｓ (or D Ｓ D Ｗ ) is a single-flowered heterotype, D Ｗ D Ｗ (g) the genotype E / e of the marginate / full colored flowers; Ｘ B Ｘ ・D Ｘ D Ｘ ・E / e・Ans / ans・H Ｘ H Ｘ ・Pg / pg・Cy / cy・Dp / dp・Y Ｘ Y Ｘ The eustoma F according to (7) １Variety or F １ (9) Genotype B of multiple alleles involved in the expression of the traits of fully colored flowers, dark kasuri flowers, and light kasuri flowers. Ｘ B Ｘ is homozygous B Ｌ B Ｌ The combination of genotypes relating to anthocyanidin synthase is AnsAns or Ansans, and the multiple alleles relating to the pigment phenotype are H Ｄ H Ｔ (or H Ｔ H Ｄ A blue lisianthus characterized by a PgpgCyCyDpDp (or pgPgCyCyDpDp) pigment phenotype and a flower color clearly recognizable as blue by the naked eye under sunlight. The present invention provides a method for producing new Kasuri-type lisianthus cultivars with a variety of flower shapes and / or flower colors, particularly blue-flowered lisianthus, based on the genetic pattern of lisianthus, including the newly elucidated genotype for the "kasuri flower" trait, as well as genotypes for yellow, white, double, single, and marginated flower traits. The method of the present invention enables the production of lisianthus with a variety of flower colors and / or flower shapes that are even more attractive for ornamental use. This application claims priority to Japanese Patent Application No. 2024-071330, filed April 25, 2024, and includes the contents of the specification of that patent application.

[0006] FIG. 1 shows a tree diagram based on cluster analysis. FIG. 2 shows seven lines classified into cluster 8 by cluster analysis. FIG. 3 shows eight lines classified into cluster 7 by cluster analysis. FIG. 4 shows ten lines classified into cluster 9 by cluster analysis. FIG. 5 shows ten lines classified into cluster 5 by cluster analysis. FIG. 6 shows 11 lines classified into cluster 6 by cluster analysis. FIG. 7A shows the distribution of C* (chroma) on the vertical axis and L* (lightness) on the horizontal axis for purple fully colored flowers, dark kasuri flowers, and light kasuri flowers. FIG. 7B shows the distribution of C* (chroma) on the vertical axis and L* (lightness) on the horizontal axis for red / pink fully colored flowers, dark kasuri flowers, and light kasuri flowers. FIG. 8 shows the lines for which petal cells were observed. 8A shows the fully colored flower line (B Ｍ B Ｌ ), and Figure 8B is a dark Kasuri flower line (B Ｄ B Ｌ ), Figure 8C shows a pale Kasuri flower system (B Ｌ B Ｌ ) are shown. Figure 9 shows the results of observation of petal cells under an optical microscope. Ｍ B Ｌ ). Figure 9B shows the dark Kasuri flower line (B Ｄ B Ｌ ), Figure 9C shows a pale Kasuri flower system (B Ｌ B Ｌ ) are shown. Figure 10 shows the results of observation of petal cells using a stereomicroscope. Ｍ B Ｌ ), and Fig. 10B shows a dark Kasuri flower line (B Ｄ B Ｌ ), and Fig. 10C shows a pale Kasuri flower system (B Ｌ B Ｌ ) is shown. Figure 11A shows a purple fully colored flower line (B Ｍ B Ｍ ), and Fig. 11B is a pale purple, light-spotted flower line (B Ｌ B Ｌ ), and Figure 11C shows F of fully colored purple flowers obtained by crossing these two lines. １ Strain (B Ｍ B Ｌ ) is shown. Figure 12A shows a red fully colored flower line (B Ｍ B Ｍ), and Fig. 12B is a pale pink, pale kasuri flower line (B Ｌ B Ｌ ), and Figure 12C shows F of fully red colored flowers obtained by crossing these two lines. １ Strain (B Ｍ B Ｌ ) is shown. Figure 13A shows a deep purple Kasuri flower line (B Ｄ B Ｄ ), and Fig. 13B is a pale purple, light-spotted flower line (B Ｌ B Ｌ ), and Figure 13C shows F of deep purple kasuri flowers obtained by crossing these two lines. １ Strain (B Ｄ B Ｌ ) is shown. Figure 14A shows a deep pink Kasuri flower line (B Ｄ B Ｄ ), and Fig. 14B is a pale pink, pale kasuri flower line (B Ｌ B Ｌ ), and Figure 14C shows the F of deep pink kasuri flowers obtained by crossing these two lines. １ Strain (B Ｄ B Ｌ ) is shown. Figure 15A shows a red fully colored flower line (B Ｍ B Ｌ ), and Fig. 15B is a pale yellow Kasuri flower line (B Ｌ B Ｌ 15C and 15D show the lines obtained by crossing these two lines, and FIG. 15C shows F １ Strain (B Ｍ B Ｌ ), and Fig. 15D is a pale pink, pale kasuri flower F １ Strain (B Ｌ B Ｌ ) is shown. Figure 16A shows a pale reddish yellow pale kasuri flower line (eeB Ｌ B Ｌ ), and Fig. 16B is a white pale kasuri flower line (EeB Ｌ B Ｌ 16C and 16D show the lines crossed between these two lines, and Fig. 16C shows the F １ System (eeB Ｌ B Ｌ ), Fig. 16D is a pale purple bordered, pale kasuri flower F １ System (EeB Ｌ B Ｌ ) is shown. Figure 17A shows a red edged flower line (EEBＭ B Ｍ ), and Fig. 17B shows a pale purple Kasuri flower line (eeB Ｌ B Ｌ ), and Figure 17C shows F of purple marginate flowers obtained by crossing these two lines. １ System (EeB Ｍ B Ｌ ) is shown. Figure 18A shows a red edged flower line (EEB Ｍ B Ｍ ), and Fig. 18B shows a pale yellow Kasuri flower line (eeB Ｌ B Ｌ ), and Figure 18C shows F of red marginate flowers obtained by crossing these two lines. １ System (EeB Ｍ B Ｌ ) Figure 19A shows the purple fully colored line (B Ｍ B Ｍ ), Figure 19B is a pale yellow Kasuri flower line (B Ｌ B Ｌ ), and Figure 19C shows F of fully colored purple flowers obtained by crossing these two lines. １ Strain (B Ｍ B Ｌ ) is shown. Figure 20A shows a purple fully colored flower line (B Ｍ B Ｍ ), and Fig. 20B is a pale purple, light-spotted flower line (B Ｌ B Ｌ ), and Figure 20C shows F of fully colored purple flowers obtained by crossing these two lines. １ Strain (B Ｍ B Ｌ ) is shown. Figure 21A shows a pale reddish yellow pale kasuri flower line (B Ｌ B Ｌ ), and Fig. 21B is a white, pale-flowered variety (B Ｌ B Ｌ 21C and 21D show the lines separated by crossing these two lines. Fig. 21C shows F １ Strain (B Ｌ B Ｌ ), and Fig. 21D is a white, pale-flowered F １ Strain (B Ｌ B Ｌ ) is shown. Figure 22A shows a pale reddish yellow pale kasuri flower line (B Ｌ B Ｌ ), and Fig. 22B is a white, pale-flowered variety (B Ｌ B Ｌ22C and 22D show the lines separated by crossing these two lines. Fig. 22C shows F １ Strain (B Ｌ B Ｌ ), Fig. 22D is a pale blue, pale kasuri flower (double), F １ Strain (B Ｌ B Ｌ ) is shown. Figure 23A shows a line with a deep white kasuri flower (B Ｄ B Ｌ ), and Fig. 23B shows a deep red Kasuri flower line (B Ｄ B Ｌ 23C and 23D show lines separated by crossing these two lines, and Fig. 23C shows F １ Strain (B Ｄ -), Fig. 23D is a pale red Kasuri flower (double) F １ Strain (B Ｌ B Ｌ ) is shown. Figure 24A shows a deep red Kasuri flower line (B Ｄ B Ｌ ), and Fig. 24B is a deep red Kasuri flower line (B Ｄ B Ｄ ), and Figure 24C shows a line separated by crossing these two lines, which is F with deep red kasuri flowers (double). １ Strain (B Ｄ −).

[0007] The method for producing a new Kasuri-type Eustoma cultivar of the present invention is characterized by using a genotype corresponding to the flower shape trait and / or flower color trait of Eustoma. 1. Definitions The meanings of terms and symbols used in the present invention are explained below. (Eustoma and line, and Kasuri-type Eustoma) Eustoma of the present invention refers to the parts of Eustoma that contain anthocyanins, such as the petals, sepals, bracts, perianth, pericarp, seed coat, and petiole (hereinafter abbreviated as petals, etc.), as well as the whole Eustoma. Furthermore, Kasuri-type Eustoma refers to a population in which less anthocyanins are observed in the vacuoles of the epidermal cells of the petals than in fully colored lines. A "line" refers to the F of Eustoma. １ Varieties, and F １This invention also includes individuals of seed parent lines and pollen parent lines of eustoma for producing varieties. The present invention also includes a group of Kasuri-type eustoma lines, registered under the registered trademark (standard characters) "Okutamayo" (pronounced "Oiyo", English name: Kagoshima University Elegant Eustoma Flowers) under Trademark Registration No. 6490744 (registration date: December 23, 2021). (B Ｘ B Ｘ ) B Ｘ B Ｘ is composed of three multiple alleles, namely B Ｍ , B Ｄ , and B Ｌ In the table, fully colored flowers refer to flowers in which the colored patterns on the petals are distributed almost uniformly, and kasuri flowers refer to flowers in which the colored patterns are distributed faded, as if sprayed with a spray bottle. B Ｍ The alleles of B control biochemical enzyme systems and genes that accumulate relatively large amounts of anthocyanins in the vacuoles of petal and other flower-forming cells, resulting in the expression of "fully colored flowers." Specifically, when the flower color of the petals is measured with a spectrophotometer, the C* (chroma) in the Lab color system is in the range of 49.46±8.37 for purple flower systems and 34.47±11.92 for red flower systems, the L* (lightness) is in the range of 35.99±7.68 for purple flower systems and 57.50±16.53 for red flower systems, and the h (hue angle) is in the range of 317.76±2.03 for purple flower systems and 352.27±7.41 for red flower systems. Ｍ The final allele determination is based on the other two alleles (B Ｄ and B Ｌ ) by cross-breeding test １ and the creation of F １ was self-fertilized, and the progeny F ２ By separating B Ｍ The allele of B is determined. ＤThe allele of B controls biochemical enzyme systems and genes that accumulate relatively less anthocyanin in the vacuoles of petal and other expressing cells compared to "fully colored flowers," resulting in the expression of "dark kasuri flowers." Specifically, when the flower color of the petals is measured with a spectrophotometer, the C* (chroma) in the Lab color system is in the range of 37.45±10.82 for purple flower systems and 23.15±4.69 for red flower systems, the L* (lightness) is in the range of 50.87±10.45 for purple flower systems and 71.14±7.76 for red flower systems, and the h (hue angle) is in the range of 319.32±10.81 for purple flower systems and 4.38±3.83 for red flower systems. Ｄ The final allele determination is based on the other two alleles (B Ｍ and B Ｌ ) by cross-breeding test １ and the creation of F １ was self-fertilized, and the progeny F ２ By separating B Ｄ The allele of B is determined. Ｌ The allele of B controls biochemical enzyme systems and genes that accumulate even less anthocyanin in the vacuoles of the petals and other flower cells compared to "dark kasuri flowers," resulting in the expression of "light kasuri flowers." Specifically, when the flower color of the petals is measured with a spectrophotometer, the C* (chroma) in the Lab color system is in the range of 8.48±4.19 for purple flower systems and 8.18±1.70 for red flower systems, the L* (lightness) is in the range of approximately 80.60±2.88 for purple flower systems and 87.25±0.94 for red flower systems, and the h (hue angle) is in the range of 321.53±4.29 for purple flower systems and 31.33±20.31 for red flower systems. Ｌ The final allele determination is based on the other two alleles (B Ｍ and B Ｄ ) by cross-breeding test １ and the creation of F １ was self-fertilized, and the progeny F ２ By separating B Ｌ The allele of is determined. Ｘ H Ｘ ) H Ｘ H Ｘis a set of six multi-alleles shown in the pathway formula below, namely H Ｔ , H Ｆ , H Ｄ , H Ｚ , H Ｏ , and H Ｅ The six alleles may be represented by other abbreviated notations such as T, F, D, Z, O, E, etc. Precursor compounds for anthocyanidin biosynthesis in lisianthus include naringenin, eriodictyol, pentahydroxyflavanone, dihydrokaempferol, dihydroquercetin, and dihydromyricetin. Ｔ Alleles of H control the biochemical conversion of naringenin to eriodictyol and dihydrokaempferol to dihydroquercetin. Ｆ The H allele is not involved in the hydroxylation of the B ring, as it produces naringenin and dihydrokaempferol. Ｄ The alleles of H control the biochemical conversion of naringenin to pentahydroxyflavanone and dihydrokaempferol to dihydromyricetin, and are characterized by the complete conversion of the substrates to pentahydroxyflavanone or dihydromyricetin. Ｚ The allele of controls the biochemical conversion of naringenin to pentahydroxyflavanone and dihydrokaempferol to dihydromyricetin. This allele is characterized by the complete conversion of the substrates to eriodictyol and dihydroquercetin, which are then further converted to pentahydroxyflavanone and dihydromyricetin. Therefore, H Ｚ The allele of Ｆ H paired with the allele of Ｚ H Ｆ In the heterozygous genotype, the intermediates eriodictyol and dihydroquercetin are taken as substrates, resulting in the production of four precursor compounds with two to three hydroxyl groups on the B ring (eriodictyol, pentahydroxyflavanone, dihydroquercetin, and dihydromyricetin). ＯAlleles of H control all biochemical conversions of naringenin to eriodictyol and pentahydroxyflavanone, and dihydrokaempferol to dihydroquercetin and dihydromyricetin. Ｅ The alleles are derived from wild species and are characterized by controlling the biochemical conversion of naringenin to pentahydroxyflavanone and dihydrokaempferol to dihydromyricetin, completely converting the substrate to pentahydroxyflavanone or dihydromyricetin. (Pg / pg, Cy / cy, Dp / dp) Pg / pg, Cy / cy, and Dp / dp represent the presence of alleles in the above pathway that control the expression of dihydroflavonol reductase (DFR), which is involved in the biosynthesis of the major anthocyanidin pigments pelargonidin (Pgn), cyanidin (Cyn), and delphinidin (Dpn). (Ans / ans) Ans / ans is the colored flower / white flower genotype, with colored flowers corresponding to the dominant type Ans and white flowers corresponding to the recessive type ans. Colored flowers are flowers that are colored by the expression of anthocyanin pigments, and white flowers are flowers that are white or yellow without color due to the non-expression of anthocyanin pigments. AnsAns (dominant homozygote) indicates a line with the traits of colored flowers, Ansans (dominant heterozygote) indicates a line with the traits of colored flowers, and ansans (recessive homozygote) indicates a line with the traits of white flowers. When the genotype is AnsAns (dominant homozygote) or Ansans (dominant heterozygote), these two genotypes are collectively referred to as Ans-. (D Ｘ D Ｘ ) D Ｘ D Ｘ is the genotype that determines the combination of double, double, and single flower types, and D Ｄ , D Ｗ , D Ｓ The combination of these three opposing genotypes results in the double, double, and single traits. Ｄ D Ｄ is a homozygous double flower, D Ｄ D Ｓ (or D Ｓ D Ｄ ) is a hetero double flower type, D Ｓ D Ｓ is a homozygous single flower, DＤ D Ｗ (or D Ｗ D Ｄ ) is a heterozygous double flower, D Ｗ D Ｓ (or D Ｓ D Ｗ ) is a single-flowered heterotype, D Ｗ D Ｗ indicates a homozygous type (wild type) for single flowers. (E / e) E / e is the genotype of edged / fully colored flowers, with edged flowers corresponding to the dominant type E and fully colored flowers corresponding to the recessive type e. An edged flower is a flower whose petal edges are bordered in a color different from the background, and fully colored flowers are flowers with no edged petals and are a single color. EE (dominant homozygous type) indicates a line with the traits of edged flowers, Ee (dominant heterozygous type) indicates a line with the traits of edged flowers, and ee (recessive homozygous type) indicates a line with the traits of fully colored flowers. (Y Ｘ Y Ｘ ) Y Ｘ Y Ｘ is the genotype that determines the combination of yellow and white flowers, and Y Ｃ , Y Ｓ , Y Ｗ The traits of yellow and white flowers correspond to the combination of the genotypes of the three alleles involved in the biosynthesis of carotenoid pigments in Eustoma. Ｃ Y Ｃ is homozygous for yellow flowers, Y Ｃ Y Ｓ (or Y Ｓ Y Ｃ ) is a heterozygous yellow flower, Y Ｃ Y Ｗ (or Y Ｗ Y Ｃ ) is a heterozygous yellow flower, Y Ｗ Y Ｗ is a homozygous white flower, Y Ｓ Y Ｗ (or Y Ｗ Y Ｓ ) is a heterozygous white flower, Y Ｓ Y Ｓindicates a line with homozygous traits for yellow flowers. (Pigment Phenotype) In the present invention, when the combination of genotypes related to anthocyanidin synthase, which is involved in the production of precursor compounds for anthocyanidin biosynthesis in lisianthus, is AnsAns (dominant homozygous) or Ansans (dominant heterozygous) (these two genotypes are collectively referred to as Ans-), the relationship between the pigment phenotype of lisianthus petals and the resulting flower color is shown below. PgnCynDpn type: reddish purple, red, purple-red, pale red, pink PgnCyn type: red, deep red, pale red, pink CynDpn type: pale purple, purple-red, purple, blue-purple Pgn type: red, pale red, pink, whitish-red, cream, white Cyn type: red, pale red, pink, whitish-red Dpn type: purple None type: white Note that PgnDpn type strains cannot be obtained in the present invention. The Dpn type pigment phenotype contains the methylated anthocyanidins malvidin (Mv) and petunidin (Pt). The Cyn type pigment phenotype contains the methylated anthocyanidin peonidin (Pn). ("-" and "--") In the present invention, a genotype marked with "-" means that it is dominantly controlled by the genotype marked immediately before it, and a genotype marked with "--" means that any genotype, whether dominant or recessive, or multi-allele, that controls the target trait can be used. (Blue Eustoma) In the present invention, "blue Eustoma" refers to a genotype B of the Kasuri flower described above. Ｍ , B Ｄ , B combination is homozygous B Ｌ B Ｌ The pale kasuri flower is a flower having a genotype combination of AnsAns (or Ansans) relating to anthocyanidin synthase, and a multiple allele genotype relating to a pigment phenotype is H Ｄ H Ｔ (or H Ｔ H Ｄ) type, the pigment phenotype is PgpgCyCyDpDp (or pgPgCyCyDpDp), and the flower color is clearly recognizable as blue by the naked eye under sunlight. More specifically, "blue lisianthus" in the present invention refers to a lineage group in which, when flower color is measured using a spectrophotometer, the L* (lightness) in the Lab color system is 81.3 or more, the C* (chroma) is around 6.0, the h (hue angle) is 325±1, and the b* / a* is in the range of -0.6 to -0.8. 2. Method for Producing a New Kasuri-Type Variety of Lisianthus 2-1. Genotype B Ｘ B Ｘ (inheritance of full colored flowers / dark kasuri flowers / light kasuri flowers) Ｍ , B Ｄ , and B Ｌ The combination of the homozygous or heterozygous genotypes is selected from the group consisting of: Ｍ B Ｍ If the combination is heterozygous, the flowers will be fully colored. Ｍ B Ｄ (or B Ｄ B Ｍ ), and B Ｍ B Ｌ (or B Ｌ B Ｍ ) results in fully colored flowers. Ｄ B Ｄ If the combination is heterozygous, the flower will be dark. Ｄ B Ｌ (or B Ｌ B Ｄ ) will result in a dark kasuri flower. Ｌ B Ｌ If the combination is homozygous, it will be a pale kasuri flower. Ｌ B Ｌ A pale kasuri flower having a genotype combination for anthocyanidin synthase of AnsAns (or Ansans), and a multiple allele genotype for a pigment phenotype of H Ｄ HＴ (or H Ｔ H Ｄ ) type and the pigment phenotype is PgpgCyCyDpDp (or pgPgCyCyDpDp) type, the flower color will be a "blue eustoma" that can be clearly confirmed as "blue" with the naked eye under sunlight. 2-2. Genotype B Ｘ B Ｘ ・Ans / ans・H Ｘ H Ｘ ・Pg / pg・Cy / cy・Dp / dp・Y Ｘ Y Ｘ (inheritance of white flowers, yellow flowers, and kasuri) <B Ｍ -Ansans H Ｘ H Ｘ Pg / pg Cy / cy Dp / dp> All lines with the Ans- genotype are colored lines based on anthocyanin pigments, but among lines with the Ans- genotype, the genotype is Ans-H. Ｆ H Ｆ Only when the phenotype is pgpgCyCyDpDp, the flowers will be white. Ｘ B Ｘ When shown as B Ｘ B Ｘ ・Ans / ans・H Ｘ H Ｘ Among the lines having the genotypes Pg / pg Cy / cy Dp / dp, if the genotype combination of anthocyanidin synthase (Ans) involved in the production of precursor compounds in anthocyanidin biosynthesis is ansans (recessive homozygote), that is, B Ｘ B Ｘ ・Ansans・H Ｘ H Ｘ If you have the Pg / pg Cy / cy Dp / dp genotype, regardless of the latent status of the Pg / pg Cy / cy Dp / dp genotype at the dihydroflavonol reductase (DFR) enzyme level, and Ｘ H Ｘ The genotype combination is 6 H Ｔ , H Ｆ , H Ｄ , H Ｚ , H Ｏ , HＥ Regardless of the genotype, all produce white flowers that lack anthocyanin synthesis. Ｓ Y Ｓ , +ansansY Ｓ Y Ｗ , +ansansY Ｗ Y Ｗ > (+ is B Ｄ B Ｄ , B Ｄ B Ｌ , B Ｌ B Ｄ , B Ｌ B Ｌ The combination of genotypes of anthocyanidin synthase (Ans) involved in the production of precursor compounds of anthocyanidin biosynthesis is ansans (recessive homozygote), and the combination of genotypes related to the kasuri trait is homozygote B. Ｄ B Ｄ , heterozygous B Ｄ B Ｌ (or B Ｌ B Ｄ ), homozygous B Ｌ B Ｌ In this case, the genotype for white / yellow flowers (dominant Y) corresponds to the petal traits of yellow flowers that are colored by the expression of carotenoid pigments, or white flowers that are colorless due to the absence of carotenoid pigments. Ｗ / recessive type Y Ｓ ) combination, + ansansY Ｗ Y Ｗ The line with the genotype is white flowered, + ansansY Ｓ Y Ｗ The line with the genotype is white flowered, + ansansY Ｓ Y Ｓ Lines having the genotype of Y have yellow flowers. Ｃ Y Ｃ , --Y Ｃ Y Ｓ , --Y Ｃ Y Ｗ > Genotype B of fully colored flowers / dark kasuri flowers / light kasuri flowers Ｘ B ＸRegardless of the superiority or inferiority of the colored flower / white flower genotype Ans / ans, Y Ｃ The genotype of -Y is a line that has a gene that preferentially expresses yellow flowers. Ｃ Y Ｃ is homozygous for yellow flowers, -Y Ｃ Y Ｓ is a heterozygous yellow flower, --Y Ｃ Y Ｗ indicates the heterozygous trait of yellow flowers. From the inheritance pattern of the white-strain flowers, the white-strain flowers (None pigment phenotype) are Ans-H Ｆ H Ｆ Genotype of pgpgCyCyDpDp, B Ｍ -ansansH Ｘ H Ｘ - CyCyDpDp genotype, + ansansH Ｘ H Ｘ --CyCyDpDpY Ｗ Y Ｗ The genotype of the individual is one of the three types of genotypes. Ｍ -Ans-H Ｘ H Ｘ --CyCyDpDpY Ｃ -> The carotenoid pigment phenotype of Eustoma, in which colored flowers synthesize anthocyanin pigments, is B Ｍ -Ans-H Ｘ H Ｘ --CyCyDpDpY Ｃ Y Ｃ , B Ｍ -Ans-H Ｘ H Ｘ --CyCyDpDpY Ｃ Y Ｓ , B Ｍ -Ans-H Ｘ H Ｘ --CyCyDpDpY Ｃ Y Ｗ These three genotypes are collectively referred to as B-Ans-H. Ｘ H Ｘ --CyCyDpDpY Ｃ -) and produces yellow flowers. Ｍ -Ans-H Ｘ HＸ -CyCyDpDpY Ｃ - In the anthocyanin biosynthesis multi-allelic type "H Ｘ H Ｘ The partial genotype of "--" is H Ｏ When Pg- is contained, the flowers are bicolored (two-color) with reddish purple and yellow (red-yellow-purple), H Ｄ , H Ｚ , H Ｅ If it contains purple and yellow bicolor (two-color) flowers (yellow-purple), H Ｔ In the case of red and yellow bicolor (two-color) flowers (red yellow [orange] color), H Ｆ In the case of Pg-, the flowers will be bicolor (two-color) red and yellow (red-yellow [orange]). Ｘ H Ｘ --CyCyDpDpY Ｓ Y Ｓ > Flowers of a line of carotenoid pigment phenotypes of Eustoma that do not synthesize anthocyanin pigments are +ansansH Ｘ H Ｘ --CyCyDpDpY Ｓ Y Ｓ Genotype B has the genotype B and produces yellow flowers. Ｘ B Ｘ ・D Ｘ D Ｘ ・E / e・Ans / ans・H Ｘ H Ｘ ・Pg / pg・Cy / cy・Dp / dp・Y Ｘ Y Ｘ (genetics of major anthocyanin pigments, flower shape [double / double / single], pedicel [maroon / full colored], white flowers, yellow flowers) The genotype combination of anthocyanidin synthase (Ans), which is involved in the production of precursor compounds for anthocyanidin biosynthesis in lisianthus, is AnsAns (dominant homozygous) or Ansans (dominant heterozygous) (hereinafter, the two genotypes AnsAns and Ansans are collectively referred to as Ans-), and the H of multiple alleles is Ｘ H Ｘ The genotype combination is H Ｔ H Ｄ , H Ｔ H Ｅ , HＴ H Ｚ and H Ｏ −(H Ｏ - means H Ｏ H Ｔ , H Ｏ H Ｆ , H Ｏ H Ｄ , H Ｏ H Ｅ , H Ｏ H Ｚ , H Ｏ H Ｏ In the case of the six combinations shown below), six precursor compounds (naringenin, eriodictyol, pentahydroxyflavanone, dihydrokaempferol, dihydroquercetin, dihydromyricetin) having 1 to 3 hydroxyl groups on the B ring are produced; Ｔ H Ｆ , H Ｔ H Ｔ In the case of (I), four precursor compounds having one and two hydroxyl groups on the B ring (naringenieriodictyol, dihydrokaempferol, dihydroquercetin) are produced; Ｆ H Ｆ In the case of (I), two precursor compounds (naringenin and dihydrokaempferol) having one hydroxyl group on the B ring are produced; Ｄ H Ｆ , H Ｄ H Ｄ , H Ｄ H Ｚ , H Ｅ H Ｆ , H Ｅ H Ｅ , H Ｅ H Ｚ , H Ｅ H Ｄ and H Ｚ H Ｚ In the case of (I), two precursor compounds (pentahydroxyflavanone and dihydromyricetin) having three hydroxyl groups on the B ring are produced; Ｚ H ＦIn this case, four precursor compounds (eriodictyol, pentahydroxyflavanone, dihydroquercetin, dihydromyricetin) with 2-3 hydroxyl groups on the B ring are produced. In addition, because there is a Pg / pg locus at the dihydroflavonol reductase (DFR) enzyme level, the precursor compounds for anthocyanidin biosynthesis are formed, and when a latent homozygote (pgpg) is formed, Pgn is not biosynthesized even if naringenin and dihydrokaempferol are produced as precursor compounds. In addition, when the Ans- and multiple allele combination is H, Ｄ H Ｄ Type, H Ｄ H Ｆ Type, H Ｄ H Ｚ Type, H Ｚ H Ｆ Type, H Ｚ H Ｚ Type, H Ｅ H Ｔ Type, H Ｅ H Ｆ Type, H Ｅ H Ｄ Type, H Ｅ H Ｚ Type, H Ｅ H Ｏ Type, H Ｅ H Ｅ In the case of the +D type, even if the genotype is such that Pg / pg is the dominant type (PgPg or Pgpg), Pgn is not biosynthesized. Ｄ D Ｄ EeAnsansH Ｄ H Ｄ PgpgCyCyDpDpY Ｃ Y Ｗ The line having this genotype has double flowers with a marginal edge, and the colored tip of the marginal edge is purple, and the petals other than the marginal edge are yellow. Ｄ D Ｗ eeB Ｍ -AnsansH Ｔ H Ｆ PgpgCyCyDpDpY Ｃ Y Ｗ The line having the genotype +D has fully colored double flowers, and the flower color is orange, a mixture of red and yellow. Ｄ DＳ EEansansH Ｏ H Ｏ PgpgCyCyDpDpY Ｓ Y Ｗ The line having this genotype is a line that has a dominant homozygous genotype of the edge ring genotype EE, but since there is no coloring by anthocyanin, the edge ring trait cannot be observed in appearance. 3. Quick reference table used in the method for producing a new Kasuri type variety of Eustoma The method for producing a new Kasuri type variety of Eustoma of the present invention is based on the F １ Varieties, and F １ The present invention can be used to create seed parent lines and pollen parent lines of Lisianthus for producing new varieties. The following quick reference tables A (Table 4), B, and C (Table 5) used in the method for producing new Kasuri-type Lisianthus varieties of the present invention determine genotype crossing combinations that produce flower colors, with the gametes of the pollen parents as rows and the gametes of the seed parents as columns, and also display the combinations of multiple alleles, as well as the pigment phenotypes and flower colors corresponding to the combinations of multiple alleles. Quick reference table A shows the combinations of three multiple alleles, namely B Ｍ , B Ｄ , and B Ｌ and are expressed by a combined homozygous or heterozygous genotype. More specifically, the combination shown in the following quick reference table A is a homozygous B Ｍ B Ｍ If the combination is heterozygous, the flowers will be fully colored. Ｍ B Ｄ (or B Ｄ B Ｍ ), and B Ｍ B Ｌ (or B Ｌ B Ｍ ) results in fully colored flowers. Ｄ B Ｄ If the combination is heterozygous, the flower will be dark. Ｄ B Ｌ (or B Ｌ B Ｄ ) will result in a dark kasuri flower.Ｌ B Ｌ If the expression is 0, the color will be pale kasuri flower. Quick reference table B is a combination table when the locus represented by Pg / pg, Cy / cy, and Dp / dp is expressed as PgPgCyCyDpDp or PgpgCyCyDpDp, and quick reference table C is a combination table when the locus represented by Pg / pg, Cy / cy, and Dp / dp is expressed as pgpgCyCyDpDp. For example, if the locus is PgPgCyCyDpDp and one multiple allele H Ｅ and another multi-allele H Ｅ is fertilized, and the combination is H Ｅ H Ｅ In this case, it is possible to quickly determine that the pigment phenotype is Dpn from Table B. Thus, the following Tables A to C used in the method for producing a new Kasuri type variety of Eustoma of the present invention determine the genotype cross combinations that produce the flower color and / or flower shape, and the gametes of the pollen parent are arranged as rows, the gametes of the seed parent are arranged as columns, and the genotypes containing the multiple alleles (B Ｘ B Ｘ +Ans / ans+Y Ｘ Y Ｘ or B Ｘ B Ｘ +D Ｘ D Ｘ +E / e+Ans / ans) and the flower color and / or flower shape corresponding to the genotype combination. The method for producing new Kasuri-type Eustoma cultivars of the present invention involves extracting anthocyanins from colored parts of Eustoma flowers, such as petals, sepals, perianth, bracts, and pericarp, using 50% aqueous acetic acid or 50% acetic acid-methanol (a 10-50% acetic acid concentration is also acceptable, and 0.5-2 N hydrochloric acid can be used instead of acetic acid), hydrolyzing the extracted anthocyanidins, and analyzing the anthocyanidin-containing hydrolysate using high-performance liquid chromatography (HPLC) or other methods. The genotypes of the progeny obtained through repeated selfing and crossing are determined to be dominant homozygotes, dominant heterozygotes, or recessive homozygotes, and various flower colors and / or flower shapes can be freely produced from these genotypes.

[0008] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. (Reference Example) Method for Determining Genotype: Petals of lisianthus were collected, and the colored portions of the petals of fully colored or marginated types (including single, double, and double flowers) were removed and precisely weighed. Anthocyanin pigments were extracted by adding an acidic solvent such as 0.5 to 2 N hydrochloric acid aqueous solution to a test tube. Extraction was performed using a method described in the literature (Uddin, et al.: J. Japan. Soc. Hort. Sci., 71:40-47, 2002; Wang, et al.: J. Plant Res., 114:213-221, 2001; Naotaka Matsuzoe et al.: Horticultural Science, 68:138-145, 1999). The extract was filtered through a cotton plug, and the filtrate was heated to 95-100°C for hydrolysis to obtain a solution containing 1-6 anthocyanidins. Hydrolysis was performed using a method described in the literature (Uddin, et al.: J. Japan. Soc. Hort. Sci., 71:40-47, 2002). After the reaction, the solution was filtered through a membrane filter, and the filtrate was analyzed using an HPLC system. The HPLC analysis conditions and analytical equipment used were the same as those described in the literature (Uddin, et al.: J. Japan. Soc. Hort. Sci., 71:40-47, 2002). From the HPLC chromatogram, the peak area of ​​each of the three anthocyanidins, namely, pelargonidin (Pgn), cyanidin (Cyn), and delphinidin (Dpn), was calculated as the occupied area, and the total peak area of ​​pelargonidin (Pgn), cyanidin (Cyn), and delphinidin (Dpn) was set as 100%. The pigment genotype of the flower was determined from the resulting unique peaks for anthocyanidins. (Example 1) Method for Differentiating Fully Colored Flowers / Dark-Smoke Flowers / Light-Smoke Flowers by Cluster Analysis (Phenotype Definition 1 of Fully Colored Flowers / Dark-Smoke Flowers / Light-Smoke Flowers) To determine the differentiation of lisianthus flowers into fully colored flowers / dark-smoke flowers / light-smoke flowers, flower color was measured using a spectrophotometer, and the measurement results were subjected to cluster analysis based on principal factor analysis to differentiate the phenotypes. The method and results are as follows.Using a spectrophotometer (COLOR CHECKER NR-11, Nippon Denshoku Industries Co., Ltd.), L* (lightness), a* (chromaticity), and b* (chromaticity) were measured, and h (hue angle) was calculated from the L*, a*, and b* values ​​using the following formula 1, and C* (chroma) and b* / a* were calculated using the following formula 2. Based on the six data points (L*, a*, b*, h, C*, b* / a*), principal factor analysis and cluster analysis using Ward's minimum variance method were performed using the statistical analysis software SAS® SYSTEM 9.4 (Figure 1). Flower color measurements were performed on the center of the lisianthus petals (the part determined visually as the center). For the edge-of-lisianthus flowers, the color was measured on the darkest colored part of the petal. Color measurements were performed on 46 individuals from 44 different petals (Figures 2–6): six lines of fully colored purple flowers (except for 'COV307', which includes two individuals from two flowers, including edge-of-lisianthus), seven lines of dark purple-spotted flowers (except for 'MY71', which includes two individuals from two flowers), ten lines of light purple-spotted flowers (including edge-of-lisianthus), seven lines of fully colored red-pink flowers (including edge-of-lisianthus), eight lines of dark red-pink-spotted flowers, and six lines of light red-pink-spotted flowers. The cluster analysis dendrogram (Figure 1) and photographs of flowers of the strains in the dendrogram (Figures 2 to 6) are shown. For the 25 purple lisianthus plants, seven strains (eight plants) with fully colored flowers and three strains with dark kasuri flowers were mixed in Cluster 6 (Figure 6), while the five strains with dark kasuri flowers were classified in Cluster 5 (Figure 5). Furthermore, all 10 strains with light kasuri flowers were classified in Cluster 9 (Figure 4). Thus, the fully colored flowers were clearly clustered against the dark kasuri flowers. For the 21 red-pink lisianthus plants, the four strains with fully colored flowers were classified in Cluster 5 (Figure 5), all eight strains with dark kasuri flowers in Cluster 7 (Figure 3), and all six strains with light kasuri flowers in Cluster 8 (Figure 2). This cluster analysis revealed a clear distinction between fully colored flowers, dark kasuri flowers, and light kasuri flowers. The results of the main factor analysis are shown in Table 6 below. As shown in Table 6, factor Z １ ~Z ６ The cumulative contribution rate of the six factors up to factor Z was 100%. １ is 70.91%, factor Z ２ gives a contribution rate of 20.27%, and Z１ and Z ２ Since the cumulative contribution rate of exceeded 90%, this Z １ and Z ２ It was found that the above two factors clearly distinguish between fully colored flowers, dark kasuri flowers, and light kasuri flowers. The results of the factor patterns are shown in Table 7 below. As shown in Table 7, factor Z １ The factors that gave a strong positive correlation to the L* (lightness) and chromaticity b* were. In other words, in the flower color of the fully colored flower / dark kasuri flower / light kasuri flower phenotype, as the L* value increases, the b* value also increases. On the other hand, factor Z １ The C* (saturation) and chromaticity a* showed a strong negative correlation. In other words, for the flower color phenotypes of all colored flowers, dark kasuri flowers, and light kasuri flowers, a decrease in the C* value corresponds to a decrease in the a* value. Furthermore, for the flower color phenotypes of all colored flowers, dark kasuri flowers, and light kasuri flowers, a relationship was found in which an increase in the L* (b*) value corresponds to a decrease in the C* (a*) value. Next, analysis of variance was performed on C*, L*, and h, followed by multiple comparisons using the Tukey-Kramer test for each flower color. Specifically, using all 46 individuals that underwent cluster analysis, analysis of variance was performed on C*, L*, and h for each phenotype and flower color, followed by multiple comparisons using the Tukey-Kramer test. The results are shown in Table 8 below (the data in Table 8 shows data for 45 individuals excluding TK39D). The analysis of variance revealed significant differences at the 1% level for C*, L*, and h for each flower color in the red-pink lineage. Furthermore, multiple comparisons revealed significant differences between the phenotypes of the purple and red-pink lisianthus. This analysis revealed a clear distinction between fully colored flowers, dark kasuri flowers, and light kasuri flowers. Next, scatter plots of the purple and red-pink lineages, with C* on the vertical axis and L* on the horizontal axis, are shown in Figure 7A (purple lineage) and Figure 7B (red-pink lineage). It can be seen that for both lines, fully colored flowers, dark kasuri flowers, and light kasuri flowers are roughly distributed into three categories. These results demonstrate that fully colored flowers, dark kasuri flowers, and light kasuri flowers can be distinguished using color system components for purple lisianthus and red-pink lisianthus. (Example 2) Microscopic Observation Method for Fully Colored Flowers / Dark Kasuri Flowers / Light Kasuri Flowers (Definition 2 of Fully Colored Flowers / Dark Kasuri Flowers / Light Kasuri Flowers) Three cultivars were used for microscopic observation: the fully colored purple line 'MY69', the dark purple Kasuri flower line 'MY7', and the light purple Kasuri flower line 'MY49' (Figure 8). Images of flowers from the three lines observed are shown in Figure 8. For observation of petal cross sections, petals were harvested, petal sections were prepared, solidified with 4% agar, cut with a knife, and observed under an optical microscope. The cell surfaces on the adaxial and abaxial sides of the petals were also observed. Tweezers were used to peel off the epidermal cell surfaces on the adaxial and abaxial sides. Images of the pigmented cells from each of the three lines observed are shown in Figures 9 and 10. In the petal cross-sections of the fully pigmented flower line 'MY69' (Figures 9A and 10A), anthocyanin pigmentation was observed in almost all epidermal cells on both the adaxial and abaxial sides, with relatively more pigmentation observed on the adaxial side. Furthermore, when the adaxial and abaxial sides were peeled and examined, pigmentation was confirmed in almost all epidermal cells, and the presence of anthocyanin aggregates present as small globules in vacuoles was also confirmed. In contrast, in the petals of the dark-spotted flower line 'MY71' (Figures 9B and 10B), pigmentation was confirmed on both the adaxial and abaxial sides, but there was a clear distinction between pigmented and unpigmented cells in the peeled epidermal cells on both the adaxial and abaxial sides. Furthermore, the presence or absence of pigmentation was confirmed to be mottled.As shown above, the streaky coloration of the Kasuri trait was visible to the naked eye and also confirmed under a stereomicroscope (Figure 9). This suggests that the mottled or absent coloration is the characteristic of the Kasuri trait. In the petals of the pale Kasuri flower line 'MY49' (Figures 9C and 10C), faint coloration was observed on the adaxial and abaxial sides. However, unlike 'MY69' and 'MY71', the detached epidermal cells on the adaxial and abaxial sides did not show obvious visible coloration. However, some cells were faintly colored and others were not. In summary, the fully colored line 'MY69' displayed coloration in almost all epidermal cells (adaxial and abaxial), the dark Kasuri flower line 'MY71' displayed mottled coloration, and the pale Kasuri flower line 'MY49' displayed both mottled and faint coloration. In other words, the reduction in pigmented cells is the morphological mechanism exhibited by the kasuri trait, and the pale kasuri trait exhibits pale coloring due to low accumulation of anthocyanins compared to the dark kasuri trait. The results of this Example 2 and Example 1 are shown together in the graphs of Figures 7A and 7B. Even when the plots for fully colored flowers and dark kasuri flowers overlap on the graph, it is clear that the two traits, fully colored flowers and dark kasuri flowers, can be distinguished visually. Furthermore, the pale kasuri flowers are clearly different from the two traits, fully colored flowers and dark kasuri flowers, and it is clear that the pale kasuri flower trait can be distinguished visually from the two traits, fully colored flowers and dark kasuri flowers. Below, we will explain the inheritance mode of fully colored flowers / dark kasuri flowers / pale kasuri flowers: new multiple allele group B. Ｘ B Ｘ [Three new multiple alleles, namely B Ｍ , B Ｄ , and B Ｌ (Example 3) Cross between a line with fully colored flowers (homozygous genotype) and a line with pale kasuri flowers (homozygous genotype) B Ｍ B Ｍ A line of fully colored flowers (line number COV70, seed parent) having the genotype of (Figure 11A) and B Ｌ B ＬA pale-flowered line (line number KD24, pollen parent) having the genotype of B Ｍ B Ｌ F having the genotype １ A line (line number MY67) (FIG. 11C) was produced. All 36 individuals of MY67 observed were fully colored flowers. All genotypes of line number COV70 were B Ｍ B Ｍ D Ｓ D Ｓ eeAnsAnsH Ｄ H Ｄ pgpgCyCyDpDpY Ｗ Y Ｗ The overall genotype of line number KD24 is B Ｌ B Ｌ D Ｓ D Ｓ eeAnsAnsH Ｄ H Ｄ pgpgCyCyDpDpY Ｗ Y Ｗ Therefore, F １ The genotype of the strain (strain number MY67) was B Ｍ B Ｌ D Ｓ D Ｓ eeAnsAnsH Ｄ H Ｄ pgpgCyCyDpDpY Ｗ Y Ｗ It can be attributed to B. Ｍ B Ｍ A line with fully colored flowers (line number COV84) (FIG. 12A) having the genotype of B Ｌ B Ｌ A thin-flowered line (line number KD29) ​​having the genotype of B Ｍ B Ｌ All 36 individuals of MY72 observed were fully colored flowers. The entire genotype of line COV84 was Ｍ B Ｍ D Ｄ D Ｄ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y ＷThe overall genotype of line number KD29 is B Ｌ B Ｌ D Ｓ D Ｓ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ Therefore, F １ The genotype of the strain (strain number MY72) was B Ｍ B Ｌ D Ｄ D Ｓ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ As mentioned above, from these two crossing experiments, it was found that the pale-flowered variety (genotype B Ｌ ) is a fully colored flower (genotype B Ｍ ) and heterozygous genotype B Ｍ B Ｌ It can be seen that fully colored flowers are obtained in this case. (Example 4) Crossing between a line with dark kasuri flowers (homozygous genotype) and a line with light kasuri flowers (homozygous genotype) B Ｄ B Ｄ A line with a dark kasuri flower (line number COV32) having the genotype of (Figure 13A) and B Ｌ B Ｌ A thin-flowered line (line number KD29) ​​having the genotype of B Ｄ B Ｌ F having the genotype １ A line (line number MY71) (FIG. 13C) was produced. All 36 individuals of MY71 observed had dark kasuri flowers. All genotypes of line number COV32 were B Ｄ B Ｄ D Ｓ D Ｓ eeAnsAnsH Ｄ H Ｄ pgpgCyCyDpDpY Ｗ Y Ｗ The overall genotype of line number KD29 is B Ｌ B Ｌ D Ｓ D Ｓ eeAnsAnsHＤ H Ｄ pgpgCyCyDpDpY Ｗ Y Ｗ Therefore, F １ The genotype of the strain (strain number MY71) was B Ｄ B Ｌ D Ｓ D Ｓ eeAnsAnsH Ｄ H Ｄ pgpgCyCyDpDpY Ｗ Y Ｗ It can be attributed to B. Ｄ B Ｄ A line with a dark kasuri flower (line number COV37) having the genotype of (Figure 14A) and B Ｌ B Ｌ A thin-flowered line (line number KD29) ​​having the genotype of B Ｄ B Ｌ F having the genotype １ A line (line number MY73) (FIG. 14C) was produced. All 33 individuals of MY73 observed had dark kasuri flowers. All genotypes of line number COV37 were B Ｄ B Ｄ D Ｓ D Ｓ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ The overall genotype of line number KD29 is B Ｌ B Ｌ D Ｓ D Ｓ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ Therefore, F １ The genotype of the strain (strain number MY73) was B Ｄ B Ｌ D Ｓ D Ｓ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ From the above, it can be attributed to the pale kasuri flower (genotype BＬ ) is a dark kasuri flower (B Ｄ ) is a recessive trait, and genotype B Ｄ B Ｌ It can be seen that the flowers become darker in color. (Example 5) Line with fully colored flowers (heterogeneous genotype B Ｍ B Ｌ ) and a thin Kasuri flower line (homo genotype): Testcross B Ｍ B Ｌ A heterozygous line with fully colored flowers (line number COV153) (FIG. 15A) and B Ｌ B Ｌ As a result of crossing the genotype of the light-spotted kasuri line (SR101) (FIG. 15B), １ The line segregated into fully colored flowers (Fig. 15C) and lightly kasuri flowers (Fig. 15D), with the ratio of segregating individuals being 9:8. The genotype of the fully colored flowers (Fig. 15C) was B Ｍ B Ｌ A heterozygous genotype is assumed, and the genotype of the pale-flowered variety (Fig. 15D) is B Ｌ B Ｌ Since the genotype of the fully colored flower (B Ｍ B Ｌ ): Light Kasuri flower (B) Ｌ B Ｌ Based on the theoretical value of a 1:1 segregation ratio, a chi-squared test was performed on the number of segregating individuals of 9:8, resulting in a chi-squared value of 0.059, giving a matching probability of 80.8%. As a result, it can be seen that the assumed genotypes of COV153 and SR101 are correct. The entire genotype of lineage number COV153 is B Ｍ B Ｌ D Ｄ D Ｄ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ The overall genotype of line number SR101 is B Ｌ B Ｌ D Ｓ D Ｓ eeansansH Ｔ H Ｔ PgPgCyCyDpDpY Ｓ Y ＳTherefore, F １ All genotypes of all colored flowers of the line (Fig. 15C) were B Ｍ B Ｌ D Ｄ D Ｓ eeAnsansH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｓ and F １ The whole genotype of the line with pale kasuri flowers (Fig. 15D) was B Ｌ B Ｌ D Ｄ D Ｓ eeAnsansH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｓ (Example 6) A line of white, pale-flowered kasuri (white-flowered kasuri strain with homozygous genotype B Ｌ B Ｌ ) and a line with pale reddish yellow and pale kasuri flowers (homozygous genotype B Ｌ B Ｌ ) Crossing between: Testcross B Ｌ B Ｌ A pale reddish-yellow, pale kasuri flower line (line number TK392) having the genotype of (Figure 16A) and B Ｌ B Ｌ As a result of crossing the white, pale-flowered line (BF1) (Fig. 16B) with the genotype １ The line segregated into pale purple, pale kasuri flowers (Fig. 16C) and pale purple, pale kasuri flowers with an edged border (Fig. 16D), with a segregation ratio of 43:41. The white, pale kasuri flower line (BF1) (Fig. 16B) is a line with the heterozygous genotype Ee for the edged border. Therefore, TK392 (Fig. 16A) is considered to have the homozygous genotype ee without edged borders, and F １ The pale-flowered (Fig. 16C) strain is considered to be a homozygous ee genotype, and the pale-margined (Fig. 16D) strain is considered to be a heterozygous ee genotype. Assuming that the segregation ratio of ee to Ee is a theoretical value of 1:1, χ ２ As a result of the test, χ ２ The value was 0.048, giving a matching probability of 82.7%. From this result, the entire genotype of line number TK392 was B ＬB Ｌ D Ｓ D Ｓ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｓ Y Ｓ The overall genotype of line number BF1 is B Ｌ B Ｌ D Ｄ D Ｓ EeansansH Ｄ H Ｄ pgpgCyCyDpDpY Ｗ Y Ｗ Therefore, F １ The whole genotype of the line with pale kasuri flowers (Fig. 16C) was B Ｌ B Ｌ D Ｄ D Ｓ eeAnsansH Ｔ H Ｄ PgpgCyCyDpDpY Ｓ Y Ｗ and F １ The genotypes of the line with pale-margined Kasuri flowers (Fig. 16D) were all B Ｌ B Ｌ D Ｄ D Ｓ EeAnsansH Ｔ H Ｄ PgpgCyCyDpDpY Ｗ Y Ｓ (Example 7) Line of marginal flowers (homozygous genotype B Ｍ B Ｍ ) and a pale-flowered strain (homozygous genotype B Ｌ B Ｌ ) hybridization EEB Ｍ B Ｍ A line of a periwinkle flower (line number TK39D) having the genotype of (Figure 17A) and eeB Ｌ B Ｌ A pale-flowered line (line number KD29) ​​having the genotype of EeB was crossed with a line (FIG. 17B) having the genotype of EeB. Ｍ B Ｌ F having the genotype １ A line (line number MY77) (FIG. 17C) was produced. All 36 individuals of MY77 observed had normal marginal flowers. All genotypes of line number TK39D were BＭ B Ｍ D Ｓ D Ｓ EEAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ The overall genotype of lineage number KD29 is B Ｌ B Ｌ D Ｓ D Ｓ eeAnsAnsH Ｄ H Ｄ pgpgCyCyDpDpY Ｗ Y Ｗ Therefore, F １ The genotype of the strain (strain number MY77) was B Ｍ B Ｌ D Ｓ D Ｓ EeAnsAnsH Ｔ H Ｄ PgpgCyCyDpDpY Ｗ Y Ｗ EEB Ｍ B Ｍ A line of a periwinkle flower (line number TK39D) having the genotype of (Figure 18A) and eeB Ｌ B Ｌ A pale-flowered line (line number KD23) having the genotype of EeB was crossed with a line (FIG. 18B) having the genotype of EeB. Ｍ B Ｌ F having the genotype １ A line (line number MY84) (FIG. 18C) was produced. All 32 individuals of MY84 observed had normal marginal flowers. All genotypes of line number TK39D were B Ｍ B Ｍ D Ｓ D Ｓ EEAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ The overall genotype of line number KD23 is B Ｌ B Ｌ D Ｓ D Ｓ eeansansH Ｔ H Ｔ PgPgCyCyDpDpY Ｓ YＳ Therefore, F １ The genotype of the strain (strain number MY84) was B Ｍ B Ｌ D Ｓ D Ｓ EeAnsansH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｓ (Example 8) Eustoma wild species line (homozygous genotype B Ｍ B Ｍ ) and a pale-flowered strain (homozygous genotype B Ｌ B Ｌ ) cross breeding B Ｍ B Ｍ A wild-type, fully colored flower line (line number AZ1814) having the genotype of (Figure 19A) and B Ｌ B Ｌ A thin-flowered line (line number SR101) having the genotype of B Ｍ B Ｌ F having the genotype １ A line (line number MY81) (FIG. 19C) was produced. All 36 individuals of MY81 observed were fully colored flowers. All genotypes of line number AZ1814 were B Ｍ B Ｍ D Ｓ D Ｓ eeAnsAnsH Ｅ H Ｅ pgpgCyCyDpDpY Ｗ Y Ｗ The overall genotype of line number SR101 is B Ｌ B Ｌ D Ｓ D Ｓ eeansansH Ｔ H Ｔ PgPgCyCyDpDpY Ｓ Y Ｓ Therefore, F １ The genotype of the strain (strain number MY81) was B Ｍ B Ｌ D Ｓ D Ｓ eeAnsansH Ｅ H Ｔ pgPgCyCyDpDpY Ｗ YＳ It can be attributed to B. Ｍ B Ｍ A wild-type, fully colored flower line (line number BC2) (FIG. 20A) having the genotype of Ｌ B Ｌ A thin-flowered line (line number KD25) having the genotype of B Ｍ B Ｌ F having the genotype １ A line (line number MY107) (FIG. 20C) was produced. All 36 individuals of MY107 observed were fully colored flowers. All genotypes of line number BC2 were B Ｍ B Ｍ D Ｓ D Ｓ eeAnsAnsH Ｅ H Ｅ pgpgCyCyDpDpY Ｗ Y Ｗ The overall genotype of line number KD25 is B Ｌ B Ｌ D Ｓ D Ｓ eeansansH Ｄ H Ｄ pgpgCyCyDpDpY Ｗ Y Ｗ Therefore, F １ The genotype of the strain (strain number MY107) was B Ｍ B Ｌ D Ｓ D Ｓ eeAnsansH Ｅ H Ｄ pgpgCyCyDpDpY Ｗ Y Ｗ The clusters can be attributed to the following. (Example 9) Results of flower color measurements for each line used in the cluster analysis of Example 1: "Oku Gyokuyo's Blue Eustoma" with blue flowers. The flower colors of each line used in the cluster analysis (clusters 8, 7, 9, 5, and 6) were measured for L* (lightness), a* (chromaticity), and b* (chromaticity) using a spectrophotometer (COLOR CHECKER NR-11, Nippon Denshoku Industries Co., Ltd.) as described in Example 1, and six data points, L*, a*, b*, h, and C* b* / a*, were compiled. The color measurement results for cluster 8 (Figure 2) are shown in Table 9. The colorimetric results for cluster 7 (FIG. 3) are shown in Table 10. The colorimetric results for cluster 9 (FIG. 4) are shown in Table 11. The colorimetry results for Cluster 9 (Figure 4) showed that the lineages within Cluster 9 (Figure 4), particularly those with lineage numbers KD28LBP and QM2LBP, appeared "blue" to the naked eye under sunlight, and therefore these lineages were designated "Okutamayo's Blue Eustoma" with blue flower color. Based on the colorimetry results for KD28LBP and QM2LBP, lineages with an L* (lightness) of 81.3 or higher, a C* (chroma) of approximately 6.0, an h (hue angle) of 325±1, and a b* / a* in the range of -0.6 to -0.8 can be designated "blue Eustoma." The colorimetry results for Cluster 5 (Figure 5) are shown in Table 12. The colorimetric results for Cluster 6 (FIG. 6) are shown in Table 13. (Example 10) Example of production of "blue lisianthus" B Ｌ B Ｌ A pale reddish-yellow, pale kasuri flower line (line number SR101L104) (FIG. 21A) having the genotype of B Ｌ B Ｌ A white pale-flowered line (line number SR21HA45) (FIG. 21B) having the genotype of Ｌ B Ｌ F of pale blue pale kasuri flowers having the genotype １ line (line number TK15QM2) (Fig. 21C) and a white pale-flowered F １ A line (line number TK44QM2) (Figure 21D) was produced. The observed population of TK15QM2 (Figure 21C) and TK44QM2 (Figure 21D) segregated at a ratio of 12:8. The overall genotype of line number SR101L104 (Figure 21A) was B Ｌ B Ｌ D Ｓ D Ｓ eeAnsansH Ｔ H Ｔ PgPgCyCyDpDpY Ｓ Y Ｓ The overall genotype of line number SR21HA45 (Figure 21B) is B Ｌ B Ｌ D Ｓ D Ｓ eeansansHＤ H Ｄ pgpgCyCyDpDpY Ｗ Y Ｗ Therefore, the pale blue and pale kasuri flower F １ The genotype of the strain (strain number TK15QM2) (Fig. 21C) is B Ｌ B Ｌ D Ｓ D Ｓ eeAnsansH Ｔ H Ｄ PgpgCyCyDpDpY Ｓ Y Ｗ , white pale kasuri flower F １ The genotype of the line (line number TK44QM2) (Figure 21D) was B Ｌ B Ｌ D Ｓ D Ｓ eeansansH Ｔ H Ｄ PgpgCyCyDpDpY Ｓ Y Ｗ It can be attributed to B. Ｌ B Ｌ A pale reddish-yellow, pale kasuri flower line (line number SR100L104) having the genotype of (Figure 22A) and B Ｌ B Ｌ A white, pale-flowered line (line number SR21HA45) (FIG. 22B) having the genotype of Ｌ B Ｌ F of pale blue pale kasuri flowers having the genotype １ line (line number TK8QM4) (Fig. 22C) and a pale blue, pale kasuri flower F １ A line (line number TK45QM4) (Figure 22D) was produced. TK8QM4 (Figure 22C) and TK45QM4 (Figure 22D) segregated into single and double. The overall genotype of line number SR100L104 (Figure 22A) was B Ｌ B Ｌ D Ｄ D Ｓ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｓ Y Ｓ The overall genotype of line number SR21HA45 (Figure 22B) is B Ｌ B Ｌ DＳ D Ｓ eeansansH Ｄ H Ｄ pgpgCyCyDpDpY Ｗ Y Ｗ Therefore, the pale blue Kasuri flower F １ The genotype of the strain (strain number TK8QM4) (Fig. 22C) is B Ｌ B Ｌ D Ｓ D Ｓ eeAnsansH Ｔ H Ｄ PgpgCyCyDpDpY Ｓ Y Ｗ , pale blue kasuri flower F １ The genotype of the line (line number TK45QM4) (Figure 22D) was B Ｌ B Ｌ D Ｄ D Ｓ eeAnsansH Ｔ H Ｄ PgpgCyCyDpDpY Ｓ Y Ｗ Thus, the genotype of the doublets of line number SR100L104 (FIG. 22A) can be assigned to, for example, D Ｄ D Ｄ If the homozygote is F, when crossed with line number SR21HA45 (Figure 22B), all progeny will be F １ Since the traits of the line (line number TK45QM4) (Figure 22D) are the same as those of the line, it is possible to produce blue lisianthus by using this crossing method. In other words, "blue lisianthus" is a genotype B of multiple alleles involved in the expression of the traits of fully colored flowers, dark kasuri flowers, and light kasuri flowers. Ｘ B Ｘ is homozygous B Ｌ B Ｌ The pale kasuri flower is a flower having a genotype combination of AnsAns (or Ansans) relating to anthocyanidin synthase, and a multiple allele genotype relating to a pigment phenotype is H Ｄ H Ｔ (or H Ｔ H Ｄ) type, the pigment phenotype is PgpgCyCyDpDp (or pgPgCyCyDpDp) type, and the flower color is clearly recognizable as blue to the naked eye under sunlight. (Example 11) Example of crossing between heterozygotes: Crossing between lines (heterozygotes) with dark kasuri flowers Ｄ B Ｌ A line with deep white kasuri flowers (line number MAR43) having the genotype of Ｄ B Ｌ As a result, the progeny segregated into a line with deep red kasuri flowers (line number GAI2A) (Fig. 23C) and a line with pale red kasuri flowers (line number GAI2B) (Fig. 23D) at a ratio of 13:5 (approximately 3:1). The overall genotype of line number MAR43 was B Ｄ B Ｌ D Ｓ D Ｓ eeansansH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ The overall genotype of line number MAR33 is B Ｄ B Ｌ D Ｄ D Ｄ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ Therefore, F １ The genotype of the line (line number GAI2A) (Figure 23C) was B Ｄ -D Ｓ D Ｄ eeansAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ and F １ The genotype of the line (line number GAI2B) (Figure 23D) was B Ｌ B Ｌ D Ｓ D Ｄ eeansAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ YＷ (Example 12) Deep red Kasuri flower F １ Example of strain creation: Example of crossbreeding between heterozygous and homozygous genotypes B Ｄ B Ｌ A line with deep red kasuri flowers (line number MAR33) (Figure 24A) having a heterozygous genotype of B Ｄ B Ｄ A line with a deep red kasuri flower (line number MAR32) (Figure 24B) having a homozygous genotype of B Ｄ - genotype F １ A line (line number GAI3) (Figure 24C) was produced. All 20 individuals of GAI3 observed had deep red kasuri flowers. All genotypes of line number MAR33 were B Ｄ B Ｌ D Ｄ D Ｄ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ The overall genotype of line number MAR32 is B Ｄ B Ｄ D Ｓ D Ｓ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ Therefore, F １ The genotype of the strain (strain number GAI3) is B Ｄ -D Ｄ D Ｓ eeAnsAnsH Ｔ H Ｔ PgPgCyCyDpDpY Ｗ Y Ｗ From the above examples, it is clear that the method of producing a new Kasuri type lisianthus cultivar, in particular "Blue Lisianthus," using a genotype corresponding to the flower color trait and / or flower shape trait of the lisianthus of the present invention is an excellent breeding and variety improvement technique.

[0009] The present invention can be used in the fields of horticulture, agriculture, and agribusiness, and by using the method of the present invention, it is possible to create ornamental lisianthus plants with more attractive and diverse flower colors and / or flower shapes. All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

1. A method for producing a new variety of lisianthus using a genotype corresponding to a flower color trait and / or a flower shape trait of lisianthus, wherein the genotype is: (a) genotype B of multiple alleles involved in the expression of each of the traits of fully colored flowers, dark kasuri flowers, and light kasuri flowers; Ｘ B Ｘ (Here, B Ｘ is B Ｍ , B Ｄ , B Ｌ A multiple allele represented by B Ｍ B Ｍ is homozygous for fully colored flowers, B Ｍ B Ｄ (or B Ｄ B Ｍ ), and B Ｍ B Ｌ (or B Ｌ B Ｍ ) is a heterozygous type with fully colored flowers, B Ｄ B Ｄ is a homozygous dark kasuri flower, B Ｄ B Ｌ (or B Ｌ B Ｄ ) is a heterotype with dark kasuri flowers, B Ｌ B Ｌ indicates a homozygous type with pale kasuri flowers.) 2. The genotype further includes the following genotypes: (b) a genotype H of multiple alleles that control the hydroxylation of the B ring of the pigment precursor in flavonoid biosynthesis and are involved in the synthesis of pelargonidin (Pgn), cyanidin (Cyn), or delphinidin (Dpn), which are the main anthocyanidin pigments in lisianthus; Ｘ H Ｘ (Here, H Ｘ Is, H Ｔ , H Ｆ , H Ｄ , H Ｚ , H Ｏ , or H Ｅ (c) the genotypes Pg / pg, Cy / cy, and Dp / dp of pelargonidin (Pgn), cyanidin (Cyn), or delphinidin (Dpn), which are the main anthocyanidin pigments in lisianthus; (d) the genotype Ans / ans for colored flowers / white flowers; (e) the genotype Y that determines the combination of yellow and white flowers. Ｘ Y Ｘ (where Y Ｘ Is Y Ｃ , Y Ｓ , Y Ｗ A compound allele represented by Y Ｃ Y Ｃ is homozygous for yellow flowers, Y Ｃ Y Ｓ (or Y Ｓ Y Ｃ ) is a heterozygous yellow flower, Y Ｃ Y Ｗ (or Y Ｗ Y Ｃ ) is a heterozygous yellow flower, Y Ｗ Y Ｗ is a homozygous white flower, Y Ｓ Y Ｗ (or Y Ｗ Y Ｓ ) is a heterozygous white flower, Y Ｓ Y Ｓ indicates a homozygous yellow flower type; (f) genotype D, which determines the combination of double, double, and single types. Ｘ D Ｘ (where D Ｘ is D Ｄ , D Ｓ , D Ｗ A multiple allele represented by D Ｄ D Ｄ is a homozygous double flower, D Ｄ D Ｓ (or D Ｓ D Ｄ ) is a hetero double flower type, D Ｓ D Ｓ is a homozygous single flower, D Ｄ D Ｗ (or D Ｗ D Ｄ ) is a heterozygous double flower, D Ｗ D Ｓ (or D Ｓ D Ｗ ) is a single-flowered heterotype, D Ｗ D Ｗ (g) a genotype E / e of edge-ringed flowers / fully colored flowers; and (g) a genotype E / e of edge-ringed flowers / fully colored flowers.

3. The genotype is B Ｘ B Ｘ ・D Ｘ D Ｘ ・E / e・Ans / ans・H Ｘ H Ｘ ・Pg / pg・Cy / cy・Dp / dp・Y Ｘ Y Ｘ The method of claim 2, wherein 4. Eustoma F １ Variety, or F １ The method according to any one of claims 1 to 3, for producing a seed parent line of eustoma and a pollen parent line of eustoma for producing a variety.

5. A method for producing new varieties of lisianthus, in which the combinations of multiple alleles are determined based on the quick reference table A below, in which the gametes of the pollen parent are the rows and the gametes of the seed parent are the columns, to produce the desired lisianthus with fully colored flowers / dark kasuri flowers / light kasuri flowers.

6. Eustoma F produced by the method according to any one of claims 1 to 3 １ Variety or F １ Eustoma seed parent lines and Eustoma pollen parent lines for cultivar development.

7. Eustoma F having a genotype corresponding to the flower color trait and / or flower shape trait of Eustoma １ Variety or F １ A seed parent line and a pollen parent line of Lisianthus for producing a variety, the genotypes of which are: (a) genotype B of multiple alleles involved in the expression of the traits of fully colored flowers, dark kasuri flowers, and light kasuri flowers, Ｘ B Ｘ (Here, B Ｘ is B Ｍ , B Ｄ , B Ｌ A multiple allele represented by B Ｍ B Ｍ is homozygous for fully colored flowers, B Ｍ B Ｄ (or B Ｄ B Ｍ ), and B Ｍ B Ｌ (or B Ｌ B Ｍ ) is a heterozygous type with fully colored flowers, B Ｄ B Ｄ is a homozygous dark kasuri flower, B Ｄ B Ｌ (or B Ｌ B Ｄ ) is a heterotype with dark kasuri flowers, B Ｌ B Ｌ indicates a homozygous type with pale kasuri flowers. Ｘ B Ｘ The above-mentioned Eustoma F １ Variety or F １ Eustoma seed parent lines and Eustoma pollen parent lines for cultivar development.

8. Additionally, the following genotypes: (b) Genotype H of multiple alleles that control the hydroxylation of the B ring of the pigment precursor in flavonoid biosynthesis and are involved in the synthesis of pelargonidin (Pgn), cyanidin (Cyn), or delphinidin (Dpn), which are the major anthocyanidin pigments in lisianthus. Ｘ H Ｘ (Here, H Ｘ Is, H Ｔ , H Ｆ , H Ｄ , H Ｚ , H Ｏ , or H Ｅ (c) the genotypes Pg / pg, Cy / cy, and Dp / dp of pelargonidin (Pgn), cyanidin (Cyn), or delphinidin (Dpn), which are the main anthocyanidin pigments in lisianthus; (d) the genotype Ans / ans for colored flowers / white flowers; (e) the genotype Y that determines the combination of yellow and white flowers. Ｘ Y Ｘ (where Y Ｘ Is Y Ｃ , Y Ｓ , Y Ｗ A compound allele represented by Y Ｃ Y Ｃ is homozygous for yellow flowers, Y Ｃ Y Ｓ (or Y Ｓ Y Ｃ ) is a heterozygous yellow flower, Y Ｃ Y Ｗ (or Y Ｗ Y Ｃ ) is a heterozygous yellow flower, Y Ｗ Y Ｗ is a homozygous white flower, Y Ｓ Y Ｗ (or Y Ｗ Y Ｓ ) is a heterozygous white flower, Y Ｓ Y Ｓ indicates a homozygous yellow flower type; (f) genotype D, which determines the combination of double, double, and single types. Ｘ D Ｘ (where D Ｘ is D Ｄ , D Ｓ , D Ｗ A multiple allele represented by D Ｄ D Ｄ is a homozygous double flower, D Ｄ D Ｓ (or D Ｓ D Ｄ ) is a hetero double flower type, D Ｓ D Ｓ is a homozygous single flower, D Ｄ D Ｗ (or D Ｗ D Ｄ ) is a heterozygous double flower, D Ｗ D Ｓ (or D Ｓ D Ｗ ) is a single-flowered heterotype, D Ｗ D Ｗ (g) the genotype E / e of the marginate / full colored flowers; Ｘ B Ｘ ・D Ｘ D Ｘ ・E / e・Ans / ans・H Ｘ H Ｘ ・Pg / pg・Cy / cy・Dp / dp・Y Ｘ Y Ｘ The eustoma of claim 7, comprising: １ Variety or F １ Eustoma seed parent lines and Eustoma pollen parent lines for cultivar development.

9. Genotype B of the multiple alleles involved in the expression of the traits of fully colored flowers, dark kasuri flowers, and light kasuri flowers Ｘ B Ｘ is homozygous B Ｌ B Ｌ The combination of genotypes relating to anthocyanidin synthase is AnsAns or Ansans, and the multiple alleles relating to the pigment phenotype are H Ｄ H Ｔ (or H Ｔ H Ｄ ) type, the pigment phenotype is PgpgCyCyDpDp (or pgPgCyCyDpDp) type, and the flower color can be clearly recognized as blue by the naked eye under sunlight.

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