JP5039967B2 - Flower color prediction method - Google Patents

Description

  The present invention relates to a gene for flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′H) playing an important role in flower color expression of flower buds, and a method for predicting and modifying flower color using this gene. . More specifically, the present invention relates to a multi-allele comprising F3 ′, 5′H gene structure and / or a compound that can be used in breeding methods of inherited flower color genotype mating methods involved in flavonoid biosynthesis. The present invention relates to a method for predicting flower color of a flower bud before flowering using a F3 ′, 5′H gene in which a retrotransposon gene is inserted into an allele or a method for modifying the flower color.

  Research on the expression gene of flower color expression has made a great leap in about 20 years from the first half of the 1980s to the second half of the 1990s. Flavonoid biosynthesis is well known as a biosynthetic pathway of plants involved in flower color expression. Among the flavonoids that are metabolites of this pathway, flavonol glycoside pigments that express a yellow color and anthocyanin pigments that are important for the expression of flower colors such as red, purple, and blue are more universal than other pigments. Exists.

  Concerning research on flower color expression genes of corn, snapdragon and petunia, structural genes encoding enzymes of anthocyanin biosynthesis, such as chalcone synthase (CHS), chalcone isomerase (CHI), flavone 3-hydroxylase (F3H) There are reports of flavonoid 3′-hydroxylase (F3′H), flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′H) and the like. There is also a report of a gene locus that regulates anthocyanin biosynthesis, such as CHS, DFR, 3GT, and ANS (Non-Patent Document 1).

  Regarding the flower color expression gene of Eustoma grandiflorum, there is a report comparing the F3′5′H gene (F3′5′-H cDNA) encoded by messenger RNA (mNA) with similar genes of gentian, petunia and eggplant (Non-patent literature) 2). This gene has been shown to be involved in the biosynthesis of delphinidin that expresses purple flower color.

  There is a report that F3'5'-H cDNA and TG1 were isolated from Eustoma russelianum using F3'5'-H cDNA and AK14 isolated from Petunia. Furthermore, there is a report in which TG1 and AK14 are inserted into petunia and tobacco and the flower color expression of the transformed plant is investigated (Non-patent Document 3). Furthermore, F3'5'-H cDNA, Ka1, isolated from Campanula medium, was inserted into tobacco, and the flower color expression of the transformed plant was investigated, and this gene turned the flower color to blue using genetic engineering. There is a report that it is the best genetic resource that can be modified (Non-Patent Document 4).

  There is a report on cytochrome P450-dependent flavonoid hydroxylase obtained from Catharanthus roseus. Moreover, as a result of conducting screening (heterologous screening) for F3′5′-H cDNA in periwinkle using CYP75 Hf1 cDNA obtained from petunia as a probe, 68% to 78% of flavonoid hydroxylase obtained from both were obtained. It has been reported that there is homology (Non-patent Document 5).

  Regarding the flower color polymorphism of the morning glory, it is described that the mutation of the morning glory genetic phenotype is usually the result of the transposon being inserted into the gene, and most of which is altered. In addition, according to Mendel's law that the genotype that determines the flower color phenotype of morning glory is controlled by four loci, among them, there is a description that the A / a locus is upper dominant (epistasis) ( Non-patent document 6).

  Regarding the research on genes related to flower color expression of eustoma, chalcone synthase (CHS), chalcone isomerase (CHI), flavone 3-hydroxylase (F3H), flavonoid 3 ′, 5′-hydroxylase (F3 ′) 5′H), dihydroflavonol reductase (DFR), and anthocyanin synthase (ANS) have been reported to synthesize cDNA from mRNA and analyze their structures (Non-patent Document 7). According to this document, it is described that the F3′5′H cDNA of Eustoma grandiflorum has a length of 1892 bp, and the synthesizeable flavonoid 3 ′, 5′-hydroxylase protein consists of a 510 amino acid sequence. .

  For the study of flavonoid 3 ′, 5′-hydroxylase (F3′5′H) involved in flower color expression of periwinkle (Vinca major), F3′5′H gene cloned from periwinkle by PCR method was isolated, It was named VmFH1. This gene has a translation sequence or putative gene region (open reading frame, ORF) base sequence of 1518 bp, and consists of two exons of 885 bp and 633 bp. In addition, since this gene encodes 506 amino acids and can biosynthesize delphinidin, there is a description that it is a first-stage study for obtaining a blue flower modified plant of a lily family in the future (non-patent literature) 8).

  Regarding the study of anthocyanin biosynthetic enzyme gene and its regulatory gene involved in fruit color expression of grape (Vitis vinifera), grape skin color is expressed in VbmybA1 gene, which is a myb-related gene, and varieties “Italy” and “Ruby Okuyama” As a result of analyzing the structure of the clone gene of “VvmybA1”, it was found that the VvmybA1 gene contained a retrotransposon called Gret1 (Grapevine retrotransposon 1), which was inserted into the upstream part encoding the VvmybA1 gene. There is a description that a white-like skin color appeared naturally from a wild-type purple skin color (Non-patent Document 9). In addition, there is a description that the gene sequences of four flavonoid 3′-hydroxylase (F3′H) DNAs were clarified from the grape cultivar “Cabernet Sauvignon” (Non-patent Document 10). . The four genes are referred to as F3′h1, F3′h2, F3′h3, and F3′h4, respectively, and two introns are inserted at the same positions, and the description is similar to morning glory, soybean, and Arabidopsis. (Non-Patent Document 10).

  There is a description that a transposon-like factor has been inserted into the gene of anthocyanidin 3-glucoside-rhamnosyltransferase (RT), a petunia cultivar. The transposon-like factor is referred to as dTph3s, and it is described that a red flower color can be expressed by being inserted into the RT gene at 1181 bp. Furthermore, this transposon insertion is unstable and can be easily removed from the inserted RT gene. After the withdrawal, the RT gene has a footprint, and the RT gene with the footprint has a transposon-like factor dTph3s. There is a description that it is more useful for breeding red flowers than the inserted RT gene (Non-patent Document 11).

  Further, two transposon-like factors dTph9 capable of causing mutations in the third exon (exon) of the Hf1 gene with respect to the dominant gene Hf1 gene and the recessive gene hf1 encoding F3′5′H of petunia. As a result, there is a report that the genes into which rTph1 and rTph1 are inserted are hf1 which is a recessive gene (Non-patent Document 12).

  Patent Document 1 discloses a genotyping method in which a long DNA fragment in which a transposon is inserted into a gene, a state in which a transposon is inserted into a gene, or a normal medium-length DNA fragment and a transposon foot It is described that the length of a short DNA fragment corresponding to a print can be analyzed by electrophoresis, and the genotype can be determined by distinguishing these by a combination thereof.

  In Patent Document 2, regarding a novel transposon-like factor of wheat such as rye, the transposon-like factor is useful as a probe or primer that contributes to molecular breeding of a useful resource plant, and further as a tool for developing a chromosomal marker. There is a description.

  Patent Document 3 describes rice transposon-like DNA and its utilization method. This transposon-like DNA was found as a DNA fragment consisting of 391 bp in the microsatellite in the process of analyzing the microsatellite consisting of (TA / AT) repeat sequences present in the promoter region of the rice phytochrome A gene. It was.

  Patent Document 4 describes a retrotransposon, a DNA fragment having promoter activity, and use thereof.

  Patent Document 5 relates to a gene disruption method using a retrotransposon of tobacco, a step of introducing the retrotransposon into the plant, and redifferentiation by culturing the plant into which the retrotransposon has been introduced. A method is described that includes the step of creating.

  Patent Document 6 describes a protein having flavonoid 3 ', 5'-hydroxylase activity and a nucleic acid encoding the same.

  In Patent Document 7, regarding gene sequences encoding flavonoid biosynthesis and use thereof, flavonoid 3′-hydroxylase (F3′H), or related proteins, and pigment expression of plants and plant organs by using them are disclosed. It is described to control.

Patent Document 8 describes a flower color genotype mating method. In particular, focusing on the inheritance of flavonoid 3′-hydroxylase (F3′H) and flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′H), etc., regarding hydroxylation of the B ring in flavonoid biosynthesis, As a result of examining the segregation of heredity, the inheritance of the enzyme systems of dihydroflavonol reductase (DFR) and anthocyanidin synthase (AS) involved in the biosynthesis of pelargonidin (Pgn), cyanidin (Cyn), delphinidin (Dpn) is respectively Pg / In addition to being regulated by pg, Cy / cy, and Dp / dp genes, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′H) Finds the law that the inheritance of is controlled by four multiple alleles, As a result, it is possible to freely create the flower color from the pigment genotype of the floret without using a method of causing mutation by gene recombination, irradiation with radiation, etc. The enzyme reaction system of flavonoid 3′-hydroxylase (F3′H) and flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′H) involved in hydroxylation includes H ^T , H ^F , H ^D , There are 4 double alleles of H 2 ^O , these are 3 ′ hydroxylation, 5 ′ hydroxylation, 3 ′, 5 ′ hydroxylation, and 3 ′ and 3 ′, 5 ′ It is described that the hydroxylation is controlled and the combination of these determines the pigment phenotype and the flower color phenotype.

JP 2004-201582 A JP-A-2005-73561 JP-A-11-206374 JP 2002-291473 A International Publication WO00 / 71699 Pamphlet JP2000-23686 US Patent Application Publication No. 2005081264 International Publication WO2004 / 103065 Pamphlet Holton, T.W. A. And Cornish, E .; C. "Genetics and Biochemistry of Anthocyanin Biosynthesis", The Plant Cell, 1995, Vol. 7, pp. 1071-1083. Nielson, K.M. M.M. And Podivinsky, E .; , "CDNA Cloning and Endogenous Expression of a Flavonoid 3'5'-Hydroxylase from Petals of Lithiantus (Eustoma grandiflorum), p. 29, p. 29, p. Shimada, Y. et al. Et al., "Expression of Chimeric P450 Genes Encoding Flavonoid-3 ', 5'-Hydroxylase in Transgenic Tobacco and Petunia Plants, FEBS Letters, Vol. 241-245. Okinawa, Y .; "Selective Accumulation of Delphindin Derivatives in Tobacco Using a Putative Flavonoid 3 ', 5'-Hydroxylase cDNA from Campandium", Bioscientific 3rd, Biosci. Kaltenbach, M.M. “Flavonoid Hydroxylas from Catalanthus roseus: cDNA, Heterologous Expression, Enzyme Properties and Cell-Type Special Expression in 19th, 19th, 19th, 19th, 19th, 19th. Clegg, M.M. T.A. And Durbin, M .; L. "Flower Color Variation: A Model for the Experimental of Evolution", Proceeding of the National Academy of Science, Vol. Noda et al., “Regulation of Gene Expression Involved in Flavonol and Anthocyanin Biosynthesis duration Petroleum in Ps. 305, Eustoma Grand. Mori, S.M. "Heterologous Expression of the Flavoroid 3 ', 5'-Hydroxylase Gene of Vinca major Alters Flower Color in Transgenic Petrihyd. Kobayashi, S .; , “Association of VvmybA1 Gene Expression with Anthocyanin Production in Grain 6 (Span-Color Mutants et al., Vol. Jeong, S.J. T.A. "Expression of the Flavoroid 3'-Hydroxylase and Flavonoid 3 ', 5'-Hydroxylase Genes and Flavonoid Composition in Grapes (page 170, Vis. Nakajima, T .; "Insertion and Exclusion of a Transposable Element Governs the Red Floral Phenotype in Commercial Petunias", Theoretical and Applied Genetics, Vol. 110, 2005, No. 3, 2005. Matsubara, K .; Et al., “Two Novel Transposable Elements in a Cytochrome P450 Gene Governor Anthocyanin Biosynthesis of Commercial Petunias”, Gene, 2005, 358, 121-126.

  Among the above-mentioned patent documents, a gene in which a transposon and / or a retrotransposon has been inserted into a gene, and a gene that has left a footprint in the state in which a transposon has been inserted into the gene have been revealed. Although it has been clarified that various expression of plants can be artificially controlled by inserting transposon and / or retrotransposon, the relationship between the biallelic gene involved in flower color expression and transposon and / or retrotransposon is described. It was also known to analyze the length of these DNA fragments by electrophoresis and distinguish them by their combination to determine the genotype, but have their genotype Determining what flower color / fruit color the individual and / or line has Could not Rukoto.

  Furthermore, in this connection, it is ambiguous how the genotype possessing the gene into which the transposon and / or retrotransposon is inserted is inherited to the progeny by the genotype breeding method related to flower color, and there is a problem when it is put to practical use. is there. The inheritance of petunia flower pigments represented by Hf1 / hf1 described in Non-Patent Document 12 should basically follow Mendel's laws of inheritance, but there are two or more types of hf1 genotypes. Therefore, since there is an ambiguous point in the separation of the progeny, it has not been put into practical use. Furthermore, the color of the flower is accurately measured using the CIELab color coordinate system, etc., to determine what kind of flower color the individual has a genotype having a gene into which a transposon and / or retrotransposon is inserted.・ There is also a problem that digitization and inheritance were not sufficient.

  If the genotypes of biallelic genes related to flower color expression of the florets are structurally elucidated and the correlation between these genotypes and flower color is clarified, which individuals are inherited after judging their genotypes Such flower color can be easily predicted to be expressed to the present and / or progeny, and such genotype may be used as a genetic indicator (marker) for predicting or testing flower color. As a result, it is possible to easily predict the flower color of the present and / or progeny inherited as a value on the coordinate axis of the CIELab color system, etc., by simply determining at an early stage without flowering for the biallelic type. It is thought that it is possible to know the flower color accurately at an early stage without greeting.

  In addition, if the relationship between the genotype possessing a gene into which a new transposon and / or retrotransposon is inserted and the flower color is clear, the flower color is bred more accurately as a value on the coordinate axis of the CIELab color system or the like. May be used as a technology.

  The present inventors have clarified gene structures of five biallelic types related to flower color expression, clarified their structures and flower color inheritance inherited in the present generation and / or progeny, CIELab color system, etc. The color of the flower is accurately measured and converted into a numerical value, the relationship between the pigment genotype and the flower color inheritance is clarified, and a practical flower color modification method for the production of a new flower color of the flower bud and / or the flower color It is an object of the present invention to provide a practical flower color prediction method that can test a gene at an early stage without flowering, and then assign a multi-allele type and know the inherited flower color.

In order to solve the above-mentioned problems, the inventors of the present invention have five biallelic types related to flower color expression, that is, a gene comprising any one of H ^T , H ^F , H ^D , H ^Z , and H ^O or a combination thereof. Using flower varieties having types, flower colors were measured and quantified by the CIELab color coordinate system, their pigment phenotypes were analyzed, and their genotypes were assigned. Furthermore, for each of the five assigned multiallelic types, H ^T , H ^F , H ^D , H ^Z , H ^O , the flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) gene As a result, the five biallelic types H ^T , H ^F , H ^D , H ^Z , H ^O are the three flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) genes. This time, we found that it can be classified into groups.

SUMMARY OF THE INVENTION In summary, the present invention has the following features.
(1) Pollen parent gametes and seed parents of an enzyme reaction system of a floret containing a flavonoid 3 ′, 5′-hydroxylase involved in hydroxylation of the B ring of the flavonoid biosynthesis pigment precursor of the pathway formula shown in FIG. A combination of gamete-derived biallelic genes H ^T , H ^F , H ^Z , H ^D and H 2 ^O based on the flavonoid 3 ′, 5′-hydroxylase gene corresponding to the multi-allelic gene Based on the correlation between the pollen parent gamete and the seed parent gamete and the flower color for the genotype H ^X H ^X · Pg / pg · Cy / cy · Dp / dp before flowering a method of predicting the flower color of flowers, where multiple alleles H ^T is to control the hydroxide of 3 'position with respect to hydroxylation of the B ring in a precursor of the flavonoid biosynthetic, pelargonidin (Pgn) And cyanidin (Cy ) Is a gene involved in the biosynthesis of, multiple alleles H ^F controls the hydroxide of 5 'position with respect to hydroxylation of the B ring in a precursor of the flavonoid biosynthesis, biosynthesis of pelargonidin (Pgn) a gene involved, multiple alleles H ^D or H ^Z is 3 with respect to hydroxylation of the B ring in a precursor of the flavonoid biosynthetic ', 5' position of the controls hydroxide, delphinidin (Dpn) raw A gene involved in synthesis, and the biallelic H 2 ^O controls the hydroxylation of the 3 ′ and 5 ′ positions with respect to hydroxylation of the B ring in the precursor of the flavonoid biosynthesis, and the production of delphinidin (Dpn) The method, which is a gene involved in synthesis.

(2) The method according to (1), wherein the combination of multiple alleles is determined by electrophoresis.
(3) The biallelic H ^T or H ^F has a nucleotide sequence of SEQ ID NO: 1, a complementary sequence thereof, or 80% identity with those sequences or hybridizes under stringent conditions with these sequences. The method according to (1) or (2), which is a DNA containing a base sequence for soybean.

(4) hybridizing multiple alleles H ^Z or H ^D is the nucleotide sequence of SEQ ID NO: 2, its complementary sequence, or or with the sequences under stringent conditions with those sequences with 80% or more identity The method according to (1) or (2), which is a DNA containing a base sequence for soybean.

(5) A base in which the biallelic H 2 ^O has a nucleotide sequence of SEQ ID NO: 3, a complementary sequence thereof, or a sequence having a identity of 80% or more, or hybridizes with these sequences under stringent conditions The method according to (1) or (2), which is DNA containing a sequence.

(6) The method according to any one of (1) to (5), wherein the florets are selected from dicotyledonous plants or monocotyledonous plants.
(7) The method according to (6), wherein the groom is eustoma.

(8) The base sequence of SEQ ID NO: 1, derived from the biallelic H ^T or H ^F , its complementary sequence, or a sequence having at least 80% identity or hybridizing under stringent conditions with these sequences DNA containing a base sequence for soybean or a fragment of 15 bases or more thereof.

(9) including multiple alleles H ^Z or H ^D SEQ ID NO: hybridizing nucleotide sequence 2 base sequence or or their sequence under stringent conditions with those sequences 80% or more identity from The base sequence of the retrotransposon of SEQ ID NO: 4 or a base sequence that has 60% or more identity with those sequences or hybridizes with these sequences under stringent conditions is inserted into the first exon sequence of DNA. The DNA according to (8) or a fragment of 15 or more bases thereof.

  (10) A retrotransposon having a nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having 60% or more identity with those sequences or hybridizing with these sequences under stringent conditions, or a fragment of 15 or more nucleotides thereof.

(11) The base sequence of SEQ ID NO: 3 derived from the biallelic H 2 ^O , its complementary sequence, or a base which has 80% or more identity with those sequences or hybridizes with these sequences under stringent conditions DNA containing a sequence or a fragment of 15 or more bases thereof.

(12) A kit for predicting the flower color of a flower bud before flowering, the following DNA (a) to (c) or a fragment thereof:
(A) the base sequence of SEQ ID NO: 1, derived from the biallelic H ^T or H ^F , its complementary sequence, or those sequences having 80% or more identity or hybridizing under stringent conditions DNA containing a base sequence for soybean, or a fragment of 15 or more bases of the DNA,
(B) multiple alleles H ^Z or H ^D nucleotide sequence of SEQ ID NO: 2 from its complementary sequence, or or hybridization with those sequences under stringent conditions with those sequences with 80% or more identity DNA containing a base sequence for soybean, or a fragment of 15 or more bases of the DNA, and
(C) the base sequence of SEQ ID NO: 3 derived from the biallelic H 2 ^O , its complementary sequence, or a base having 80% or more identity with these sequences or hybridizing with these sequences under stringent conditions DNA containing a sequence, or a fragment of 15 or more bases of the DNA,
The kit comprising at least one of the above as a probe or primer.

(13) The kit according to (12), wherein the probe is labeled.
(14) The kit according to (12) or (13), further comprising a quick reference table showing a correlation between the combination of the biallelic genes and the flower color.

(15) floriculture multiple alleles H ^Z, H ^D or exon portions of both the base sequence or or their sequence under stringent conditions with those sequences with 60% identity of SEQ ID NO: 4 A method for modifying the flower color of a floret, comprising inserting a retrotransposon comprising a base sequence that hybridizes with the above.

(16) The method according to (15), wherein the exon is a first exon.
(17) flower color modification of lose the function of multiple alleles ^{H D} or ^{H Z,} caused by acquiring the function of the newly multiple alleles ^{H T} or ^{H F,} according to (15) or (16) Method.

  (18) The method according to any one of (15) to (17), wherein the florets are selected from dicotyledonous plants or monocotyledonous plants.

(19) Pollen parent gametes and seed parents of an enzyme reaction system of a floret containing a flavonoid 3 ′, 5′-hydroxylase involved in hydroxylation of the B ring of the flavonoid biosynthesis pigment precursor of the pathway formula shown in FIG. A game chart showing a correlation between a combination of a gamete-derived multi-allele H ^T , H ^F , H ^Z , H ^D and H 2 ^O and flower color, wherein the multi-allele H ^T is the flavonoid biosynthesis precursor of B ring in substance to control the 3'-position hydroxide respect hydroxide, a gene involved in the biosynthesis of pelargonidin (Pgn) and cyanidin (Cyn), multiple alleles H ^F is the flavonoid biosynthetic precursor of B ring in substance to control the 5 'position hydroxide respect hydroxide, a gene involved in the biosynthesis of pelargonidin (Pgn), multiple alleles H ^D or H ^Z is of the flavonoid biosynthetic B ring in precursor 3 with respect hydroxide ', 5' position of the controls hydroxide, a gene involved in the biosynthesis of delphinidin (Dpn), as well as multiple alleles H ^O is the B ring in the precursor of the flavonoid biosynthetic The quick reference table, which is a gene that controls hydroxylation at the 3 ′ and 5 ′ positions and is involved in delphinidin (Dpn) biosynthesis.
(20) The quick reference table according to (19), which is any of the following.

Definitions Terms used herein have the following definitions.
“A flavonoid 3 ′, 5′-hydroxylase gene corresponding to a multi-allele” refers to a plurality of genetic factors involved in floron flavonoid 3 ′, 5 ′ hydroxylation and present in a multi-allelic locus. Means part of the element.

  “Flower bud” refers to angiosperms, particularly dicotyledonous and monocotyledonous plants, having flowers containing flavonoids.

  The “flower color genotype mating method” refers to the hydroxylation of the B ring of a precursor in flavonoid biosynthesis in the genotype breeding method for flavonoid anthocyanidin pigments, ie, pelargonidin (Pgn), cyanidin (Cyn) and delphinidin (Dpn). A method for freely producing flower colors by arbitrarily combining five multi-alleles related to.

  “Pollen parent gametes” refers to male gametes. On the other hand, the “seed parent gamete” refers to a female gamete.

  “% Identity” refers to a sequence comparison (alignment) with the DNA sequence of the target biallelic gene, with or without a gap, and the number of identical bases relative to the total number of bases in the target DNA sequence. The percentage (%). Sequence comparison can usually be performed using algorithms such as BLAST, FASTA (eg, SF Altschul et al., J. Mol. Biol. 215: 403-410 (1990); WR Pearson). S. Lipman, Proc. Natl. Acad. Sci. USA 85: 2444-2448 (1988)).

  “Stringent conditions” are conditions that allow hybridization to the DNA sequence of the subject multiallele, for example, 3 to 5 × SSC, 0.1 to 1% at 30 to 50 ° C. This refers to conditions consisting of hybridization in SDS for 1-15 hours, followed by washing in, for example, 0.1-2 × SSC, 0.1-1% SDS at room temperature to 70 ° C. The DNA that can hybridize to the DNA sequence of the subject multiallele under such conditions is, for example, a mutant or homologue of the biallelic. Here, the homolog refers to a cognate multiallele of a plant or plant variety different from the target plant.

  As a result of elucidating the structure of five multialleles related to flower color expression according to the present invention, it is easy to determine what flower color an individual who inherits after expressing their genotype will express to the present generation and / or progeny In addition, the flower color can be predicted at an early stage without reaching flowering. This makes it possible to create a quick reference table showing the relationship between the polyallelic combination between the pollen parent gamete and the seed parent gamete and the flower color, which can be used conveniently for the prediction of flower color.

  Furthermore, as a result of the discovery of a novel retrotransposon according to the present invention and the elucidation of the genotype possessing the gene into which it was inserted, the relationship between the five multialleles and flower color became clear, and It was possible to modify the flower color, particularly red, through retrotransposon insertion.

As described in part in Biallelic Patent Document 8, the present inventors have found that the flower color genotype is related to the flavonoid biosynthesis of pathway formula (I) shown in FIG.
In FIG. 4, H ^T , H ^F , H ^Z , H ^D and H 2 ^O represent a multi-allele involved in hydroxylation of the B ring at the precursor of flavonoid biosynthesis, where the multi-allele H ^T but by controlling the hydroxide of 3 'position with respect to hydroxylation of the B ring in the precursor is a gene involved in the biosynthesis of pelargonidin (Pgn) and cyanidin (Cyn), multiple alleles H ^F is drive front controls hydroxide 5 'position with respect to hydroxylation of the B ring of a material, a gene involved in the biosynthesis of pelargonidin (Pgn), multiple alleles H ^D or H ^Z is, the B ring in the precursor Is a gene that regulates hydroxylation at the 3 ′ and 5 ′ positions and is involved in delphinidin (Dpn) biosynthesis, and the biallelic H 2 ^O is related to hydroxylation of the B ring in the precursor Delphinidin by controlling hydroxylation at 3 'and 5' positions (Dpn) is a gene involved in biosynthesis.

  Furthermore, in FIG. 4, for the inheritance of pelargonidin (Pgn), cyanidin (Cyn), and delphinidin (Dpn), which are the main anthocyanidin pigments involved in flower color expression of the florets, the genetic loci of Pgn, Cyn, and Dpn pigments are respectively Pg / pg, It was shown as Cy / cy and Dp / dp. Inheritance of the enzyme systems of dihydroflavonol reductase (DFR) and anthocyanidin synthase (AS) involved in the biosynthesis of Pgn, Cyn, and Dpn is regulated by genes of Pg / pg, Cy / cy, and Dp / dp, respectively ( Patent Document 8).

According to the present invention, the five multialleles H ^T , H ^F , H ^Z , H ^D and H 2 ^O are converted into three flavonoids 3 ′, 5′-hydroxylase (F3 ′) controlled by the biallelic gene. , based on the 5'-H) gene cluster, ^{H T} and ^H F, can be classified into ^{H Z} and ^{H D} and ^{H O.}

The genotype of the flavonoid 3′-hydroxylase (F3′H) and flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′H) enzyme reaction system involved in hydroxylation of the B ring of the flavonoid dye precursor , As indicated above, indicated by five multialleles of H ^T , H ^F , H ^D , H ^Z , H 2 ^O , and selecting two of the symbols Pg / pg (ie, a combination of PgPg, Pgpg, pgpg) Select one of the symbols), select two of the Cy / cy symbols (ie, select one of the Cycy, Cycy, cycy combination symbols), and select two of the Dp / dp symbols. (Ie, select one of the combination symbols DpDp, Dpdp, dpdp) and select two of the symbols H ^T , H ^F , H ^D , H ^Z , H ^O ( That is, H ^T H ^T , H ^T H ^{F, H T H D, H} T H Z, H T H O, H F H F, H D H F, H Z H F, H O H F, H D H D, H D H Z, H D H ^{O 1} , H ^Z H 2 ^O , H 2 ^{O 2 O} , and one of the combination symbols of H 2 H 2 ^{O 2} ), and the genotype (pigment genotype) that determines flower color is D / d · E / e · H ^X H ^X · Pg / pg Can be expressed as Cy / cy · Dp / dp.

  The three genes of the present invention extract DNA from every part of a plant body such as a floret tissue, for example, roots, leaves, stems, seeds, etc., and perform polymerase chain reaction (PCR) using primers to obtain amplification products. It can be isolated and then obtained by sequencing. That is, PCR is performed using the 15 to 30 bp oligonucleotides facing each other as primers and DNA extracted from the floret tissue as a template. By this PCR, a DNA fragment in a region sandwiched between the primers is amplified.

In this way, the multiple alleles ^{H D} and ^{H Z} comprises nucleotide sequence of 3115bp (SEQ ID NO: 2), the multiple alleles ^{H O} comprises nucleotide sequence of 2498bp (SEQ ID NO: 3), multiple alleles H ^T or ^{H F} comprises nucleotide sequence of 8525bp (SEQ ID NO: 1), retrotransposon like element rTeg1 comprising the nucleotide sequence of SEQ ID NO: 4 in the sequence (retrotransposon Eustoma grandiflorum 1) can contain were found (Fig. 2 reference).

Specifically, flavonoid 3 of multiple alleles type ^{H D} or ^{H Z} ', 5'-hydroxylase (F3', 5'-H) structural gene is a nucleotide sequence of 3115bp of SEQ ID NO: 2, This structure has an intron of 1129 bp between two exons consisting of 986 bp and 1000 bp.

The structure of the F3 ′, 5′-H gene of the biallelic H 2 ^O is the 2498 bp base sequence shown in SEQ ID NO: 3, and has a 512 bp intron between two exons consisting of 986 bp and 1000 bp. This is the structure.

Furthermore, F3 of multiple alleles type ^{H T} or ^{H F} ', the structure of the 5'-H gene is a nucleotide sequence of 8525bp of SEQ ID NO: 1, of 1129bp between two exons consisting of 991bp and 1000bp This structure has an intron, and a 5405 bp retrotransposon is inserted between 491 bp and 500 bp of the first exon of 991 bp.

Flower Color Prediction Method According to the present invention, a method for predicting flower color of a flower bud before flowering is provided. In this method, a combination of the above-mentioned multi-alleles H ^T , H ^F , H ^Z , H ^D and H 2 ^O between a pollen parent and a seed parent gamete is converted into a flavonoid 3 ′, 5′-hydroxylase gene of the multi-allele. Between the allele combination between pollen parent and seed parent gamete and flower color for genotype H ^X H ^X · Pg / pg · Cy / cy · Dp / dp And predicting the flower color of the flower bud before flowering.

  In the present invention, florets are flowers, fruits, seeds, and leaves containing flavonoids, that is, flowering plant gates (Anthophyta) having petals, sepals, buds, flower coats, pericarps, seed coats, petiole containing flavonoids, and the like. ) Angiospermae, which includes dicotyledonae and monocotyledonae.

  Examples of flower buds of the dicotyledonous plant, Sympetalae, include, but are not limited to, for example, Campanulatae (Compositae), Styridaceae, Goodeniaceae ), Campanulacae), Cucurbitales (Cucurbitaceae), Rubials (Dipacaceae), Cepidae (Raeaceae) )), Tubiflorae (Acanthaceae), Lentiaceae (Lent) bulariaceae, Gesneriaceae, Martyniaceae, Pedialaceae, Bignaceae, Scrophularaceae, Scrophulaceae , Boraginaceae, Hydrophyllaceae, Polymonaceae, Convolvuleae, Contacetaceae, Ascaceae, Ascepiaceae aceae), Oleaceae (Oleaceae)), Plumbaginales (Plumbaginaceae), Primales (Pleumaceae), Pleumaceae (Pleumaceae) (Ericales) (Ericaceae), Pyrolaceae), Diapensiaes (Diapensiaaceae).

  Examples of flower buds of dicotyledonous leaflets (Archichlamydeae) include, but are not limited to, Myrtiflorae (Onaglaceae), Melanomataceae, Myrtacear , Combretaceae, Punicaceae, Lythraceaae, Geraidae, Thymelaeaceae, Parietalesceae, Betaceae Cistaceae, Violaceae, Camellia (Came) liaceae), Malvales (Malvaeaae, Elaeocarpaceae), Rhamnales (Vitaceae), Rhamedaceae (Rhamaneceae) (Balsaminaceae), Hippocastanaceae, Mapleaceae (Aceraceae), Asteraceae (Celastraceae), Aceraceaeaceae (A), Aceraceae (Aceraceae), Gourdaceae (A) Polygalaceae), Rutaceae Rutaceae, Linaceae, Geraniaceae, Oxalilidaceae), Rosales (Leguminosae), Rosaceae, Spiraceae (Hacemaceae) ), Saxifragagaceae, Crassulaceae), Sarraceniales (Sarracenaceae), Nepenthaesae, Nepentaceae, ), Clevisaceae (Ca pparidaceae), Papaveraceae), Ranuncules (Lauraceae), Berberidaceae, Ranunceaceae, Launceaceae (Lanecaceae) ), Magnoliaceae), Centrospermae (Caryophyllaceae), Nyctagineaceae (Polygones) (Polyaceae), Polyaceae (Polyaceae) Moraceae)) Myricales) (bayberry family (Myricaceae)), and the like.

  Examples of monocotyledonous flower buds include, but are not limited to, for example, Orchidales (Orchidaceae), Ginger (Citaminea) (Cannaceae), Zingiberaceae, (Musaceae)), Lilyflorae (Iridaceae), Amaryllidaceae, Lilyaceae), Commelinales (Comellaceae), Ponidaceae (Ponteaceae) Family (Bromeliaceae), Araceae (Araceae).

  In the present invention, the combination of the above five biallelic types is determined from the flowering tissue before flowering based on the three flavonoid 3 ', 5'-hydroxylase genes.

In order to obtain the above three kinds of flavonoid 3 ′, 5′-hydroxylase genes, a first primer corresponding to a 5 ′ → 3 ′ direction base sequence sandwiching the insertion site of the retrotransposon-like factor rTeg1 and 3 ′ → PCR can be performed using the second primer corresponding to the 5′-oriented base sequence. The primer for that is the following, for example.
First primer:
5′-CTCAGACCCTCTGTTACCTCAGTCAG-3 ′ (SEQ ID NO: 5)
Second primer:
3′-AGAAACTGAACTCTCATCC-5 ′ (SEQ ID NO: 6)

  The amplified DNA can be separated by electrophoresis and detected as a band on the electrophoresis gel. The band on the gel can be isolated from the gel, and the isolated DNA fragment can be determined for the entire base sequence according to a commonly used base sequencing method such as primer walking by a cycle sequencing method. When the base sequence of a DNA fragment is determined, if a transposon-like DNA is inserted into the fragment, the transposon sequence and insertion position are also determined.

  For example, when DNA extracted from Eustoma varieties Asuka Beni is used as a template, the above-described oligonucleotide is used as a primer, PCR is performed, and the PCR product is subjected to electrophoresis on an agarose gel, about An 8 kbp DNA fragment is obtained. When this DNA fragment was extracted from an agarose gel and the entire base sequence was determined by primer walking by the cycle sequencing method, it was found that a transposon-like DNA consisting of the 8525 bp base sequence shown in SEQ ID NO: 1 was contained.

  For example, when DNA extracted from Eustoma variety Royal Violet was used, PCR was performed using the oligonucleotides shown above as primers, and the PCR product was subjected to electrophoresis on an agarose gel, about 2.5 kbp DNA Fragments are obtained. When this DNA fragment was extracted from an agarose gel and the entire base sequence was determined by primer walking by the cycle sequencing method, it was found that DNA consisting of the 2498 bp base sequence shown in SEQ ID NO: 3 was contained.

  For example, when DNA extracted from Eustoma variety mellow lavender is used, PCR is performed using the oligonucleotides shown above as primers, and the PCR product is electrophoresed on an agarose gel, it is about 3 kbp. A DNA fragment is obtained. When this DNA fragment was extracted from an agarose gel and the entire base sequence was determined by primer walking by the cycle sequencing method, it was found that DNA consisting of the 3115 bp base sequence shown in SEQ ID NO: 2 was contained.

  Therefore, according to the present invention, the three types of flavonoid 3 ', 5'-hydroxylase genes that determine the above-described biallelic types are as follows.

(1) represents the multiple alleles type H ^T, involved in the biosynthesis of controlling the hydroxide of 3 'position with respect to hydroxylation of the B ring in a precursor of the flavonoid biosynthetic pelargonidin (Pgn) and cyanidin (Cyn) DNA comprising a nucleotide sequence of SEQ ID NO: 1, a complementary sequence thereof, or a nucleotide sequence having 80% or more identity with those sequences or hybridizing under stringent conditions with these sequences Or
Representing the multiple alleles type H ^F, controls the hydroxide of 5 'position with respect to hydroxylation of the B ring in a precursor of the flavonoid biosynthesis, a DNA involved in the biosynthesis of pelargonidin (Pgn), sequence A DNA comprising the nucleotide sequence of No. 1, a complementary sequence thereof, or a nucleotide sequence having 80% or more identity with these sequences or hybridizing with these sequences under stringent conditions.

(2) Control of hydroxylation at the 3 'and 5' positions with respect to hydroxylation of the B ring in the precursor of the flavonoid biosynthesis, which represents the biallelic H ^D or H ^Z, for biosynthesis of delphinidin (Dpn) DNA involved, comprising the nucleotide sequence of SEQ ID NO: 2, its complementary sequence, or a nucleotide sequence having 80% or more identity with these sequences or hybridizing under stringent conditions with these sequences DNA.

(3) Regulates hydroxylation at the 3 ′ and 5 ′ positions with respect to hydroxylation of the B ring in the precursor of the flavonoid biosynthesis, which represents a biallelic type H 2 ^O , and participates in biosynthesis of delphinidin (Dpn) A DNA comprising a base sequence of SEQ ID NO: 3, a complementary sequence thereof, or a base sequence having 80% or more identity with those sequences or hybridizing under stringent conditions with these sequences.

  In the DNAs of (1) to (3) above, the DNA has the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or its complementary under the condition that it has an activity related to the flavonoid biosynthesis. Also included is DNA having a sequence identity of usually 60% or more, preferably 70% or more, 75% or more, more preferably 80% or more, 85% or more, more preferably 90% or more, 95% or more, or 98% or more. Is done.

  Such DNA also includes DNA comprising a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or its complementary sequence. The stringent conditions are as defined above, that is, not particularly limited to the following, but for example, at 30-50 ° C., 3-5 × SSC, 0.1-1% SDS for 1-15 hours And subsequent washing in, for example, 0.1 to 2 × SSC, 0.1 to 1% SDS at room temperature to 70 ° C. Here, 1 × SSC is an aqueous solution (pH 7.2) containing 150 mM sodium chloride and 15 mM sodium citrate. Other examples of stringent conditions are described in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, the disclosure of which can be used in the present invention.

  DNA having 80% identity or more, or DNA that hybridizes under stringent conditions is a mutant of the base sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or its complementary sequence, It includes homologs of different plants or plant varieties.

  In the method of the present invention, DNA is extracted from an arbitrary site such as a flower bud tissue such as a leaf, a root, or a stem before flowering. If necessary, the DNA is amplified by a PCR method, and the DNA is size-separated. The size of DNA is, for example, 8525 bp (SEQ ID NO: 1), 3115 bp (SEQ ID NO: 2), and 2498 bp (SEQ ID NO: 3) in the case of Eustoma, which are Ef3, Ef2, and Ef1 in Example 6 or FIG. 3 described later, respectively. It is called. These specific DNA sizes are merely examples, and if the types of plants are different, their sizes may be different. In the present invention, the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 are used. Alternatively, any DNA size is acceptable as long as it is similar in primary structure and activity to its complementary sequence.

  In the extraction of DNA, the floret tissue is cut into fine particles, and this is homogenized in an appropriate buffer, and then the total DNA is extracted by a known DNA extraction method such as the phenol method. For this purpose, a commercially available DNA extraction kit may be used, and an example of such a kit is a Plant Genomics DNA Mini kit (Biogene).

  For example, electrophoresis or Southern hybridization can be used to size-separate the target DNA from the total DNA. In the case of electrophoresis, the gel is usually preferably an agarose gel.

  When the target DNA is detected by electrophoresis, the target DNA is preferably amplified in advance by the PCR method. Since the primers for PCR have the same sequences in the 5 ′ terminal sequence and the 3 ′ terminal sequence of SEQ ID NOs: 1, 2, and 3, the same forward primer and reverse primer are designed and prepared, Can be used. Depending on the plant species, primer sequences designed from the nucleotide sequences of SEQ ID NOs: 1, 2 and 3 may not be used due to differences in the sequences of the 5 ′ end or 3 ′ end, for example. In such a case, DNA is extracted from the tissue of the plant species, PCR amplified using random primers prepared in consideration of the identity with the nucleotide sequences of SEQ ID NOs: 1, 2, and 3, and the amplified DNA An appropriate primer sequence can be determined through determining the base sequence for. Alternatively, the relevant sequences of various plant species can be searched for the target DNA sequence via a sequence data bank such as GenBank, and the primer sequence can be determined based on the obtained sequence. The size of the primer is, for example, 15 to 50 bases, preferably 17 to 25 bases.

PCR conditions are not limited as long as the target DNA of interest can be amplified. For example, denaturation at 94 to 95 ° C. for 10 seconds to 1 minute, annealing at 50 to 65 ° C. for 10 seconds to 1 minute, and annealing at 72 ° C. for 30 seconds to This includes 10 cycles of extension reaction as one cycle and 20 to 50 cycle reactions. In PCR, a thermostable reverse transcriptase (for example, Taq polymerase), dNTP (where N is A, T, G and C), a buffer containing Mg ²⁺ or the like is used.

  When the target DNA is detected by Southern hybridization, the total DNA extracted from the plant tissue is subjected to size separation by electrophoresis as described above, then treated with alkali, transferred to a membrane such as nylon, and a labeled probe is used. To detect the target DNA. The probe is at least one of the following DNAs (1) to (3) or a fragment thereof.

(1) The base sequence of SEQ ID NO: 1, derived from the biallelic H ^T or H ^F , its complementary sequence, or a sequence having at least 80% identity or hybridizing under stringent conditions with these sequences DNA containing a base sequence for soybean, or a fragment of 15 or more bases of the DNA.

(2) multiple alleles H ^Z or H ^D nucleotide sequence of SEQ ID NO: 2 from its complementary sequence, or or hybridization with those sequences under stringent conditions with those sequences with 80% or more identity DNA containing a base sequence for soybean, or a fragment of 15 or more bases of the DNA.

(3) The base sequence of SEQ ID NO: 3 derived from the biallelic H 2 ^O , its complementary sequence, or a base that has 80% or more identity with these sequences, or hybridizes with these sequences under stringent conditions DNA containing a sequence or a fragment of 15 or more bases of the DNA.

  The size of the probe is 15 bases or more, for example, 20 to 200 bases, 25 to 100 bases, 50 to 80 bases, and the like. The labeling of the probe is performed with, for example, a fluorescent labeling agent or a radioisotope.

  In the present invention, it is possible to know five biallelic flavonoid 3 ′, 5′-hydroxylase genes involved in hydroxylation of the B ring of the precursor compound for flavonoid biosynthesis of the anthocyanidins by the above-described method. Is possible.

If multiple allelic forms of ^{H T H} T, 4 kinds of precursor hydroxy groups in the B ring has one and two (naringenin (naringenin), Elio decrease-thiol (eriodictyol), dihydro kaempferol (dihydrokaempferol), dihydro In the case of H ^F H ^F , two precursors having one B-ring hydroxyl group (naringenin, dihydrokaempferol) are generated, and H ^T H ^F In this case, six kinds of precursors (naringenin, eriodictyl), pentahydroxyflavanone (pentent) having 1 to 3 hydroxyl groups of B ring (B-ring) It produces hydroflavone, dihydrokaempferol, dihydroquercetin, dihydromyricetin, but these flavonoids 3 ′, 5′-hydroxylase (F3 ′, 5′-hydroxylase (F3 ′, 5′-hydroxylase) As a result, a gene having a base sequence of 8525 bp shown in SEQ ID NO: 1 is detected.

When the biallelic type is H ^D H ^D , two precursors having three B-ring hydroxyl groups (pentahydroxyflavanone, dihydromyricetin) are generated, and H ^D H ^Z , H ^In the case of ^Z H ^Z , two precursors having three B-ring hydroxyl groups (pentahydroxyflavanone, dihydromyricetin) are produced, but these flavonoids 3 ′, 5′-hydroxylation are produced. As the enzyme (F3 ′, 5′-H) gene, a gene having a base sequence of 3115 bp shown in SEQ ID NO: 2 is detected.

When the biallelic type is H 2 ^{O 2} H 2 ^O , 6 precursors having 1 to 3 hydroxyl groups in the B ring (naringenin, eriodictylol, pentahydroxyflavanone) ), Dihydrokaempferol, dihydroquercetin, dihydromyricetin), as flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) genes, A gene having a base sequence of 2498 bp shown in SEQ ID NO: 3 is detected.

That is, the gene of the nucleotide sequence of 8525bp of SEQ ID NO: 1, alleles of ^{H T} related to the control of (allele), therefore, naringenin Elio decrease-tee-ol from (naringenin) (eriodictyol) and dihydro kaempferol (dihydrokaempferol) It relates to the control of biochemical transformation from to dihydroquercetin. Further, the gene of the nucleotide sequence of 8525bp is also related to the control of the alleles ^{H F,} therefore, pentahydroxy flavanone (pentahydroxyflavanone) from Elio decrease-tee-ol (eriodictyol) and dihydroquercetin (dihydroquercetin) from dihydromyricetin (Dihydromyricetin ) Related to the control of biochemical conversion to.

Further, the gene of the nucleotide sequence of 3115bp of SEQ ID NO: 2 is related to the control of the alleles of ^{H D} and / or ^{H Z,} therefore, pentahydroxy flavanone (pentahydroxyflavanone) from naringenin (naringenin) and dihydro kaempferol (dihydrokaempferol ) To the control of biochemical conversion from dihydromyricetin.

Furthermore, the gene of the base sequence of 2498 bp shown in SEQ ID NO: 3 is involved in the regulation of the ^HO allele, and therefore, from naringenin to eriodictyol and pentahydroxyflavanone as well as dihydro It is involved in the control of all biochemical conversions of dihydrokaempherol to dihydroquercetin and dihydromyricetin.

  The biosynthetic pathway of Pgn, Cyn and Dpn including the structure of the above precursor is described in Takao Murakami, Structure and chemistry of natural products, pages 155 to 185, Yodogawa Shoten, 1984.

In the present invention, for example, a dye genotype lisianthus petal for (Pigment ^genotype), if a dye genotype H ^T H ^T Pg-CyCyDpDp PgnCyn type dye phenotype ^{(PgnCyn-pigment phenotype), H} T H F Pg for phenotype PgnCynDpn type genotype of -CyCyDpDp, and ^H F ^H F Pg-CyCyDpDp in genotype when phenotype Pgn type, any flavonoid 3 ', 5'-hydroxylase (F3' The 5′-H) gene is also a gene having a base sequence of 8525 bp shown in SEQ ID NO: 1. In the notation of pigment genotype, “-” indicates that it is dominantly controlled by the gene (gene and / or genotype) indicated immediately before, and that any gene can be used. means.

For ^{example, H Z H Z --CyCyDpDp, H} D H Z --CyCyDpDp, H D H D If a dye genotype --CyCyDpDp of Dpn type pigment phenotype, any flavonoid 3 ', 5'-hydroxylase The enzyme (F3 ′, 5′-H) gene is also a gene having a base sequence of 3115 bp shown in SEQ ID NO: 2. In the notation of pigment genotype, “-” means that any gene (gene and / or genotype) can be used.

For example, in the case of the pigment phenotype of H ^O H ^O pgpgCyCyDpDp and the pigment phenotype of CynDpn, and the pigment genotype of H ^O H ^O Pg-CyCyDpDp and the pigment phenotype of PgnCynDpn, any flavonoid 3 ′, 5 ′ -The hydroxylase (F3 ', 5'-H) gene is also a gene having a base sequence of 2498 bp shown in SEQ ID NO: 3.

In the present invention, the heterozygous genotype can be identified from the flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) gene. For ^example, if the pigment phenotype of PgnCynDpn type a dye genotype H ^T H O Pg-CyCyDpDp, flavonoid 3 ', 5'-hydroxylase (F3', 5'-H) gene are shown in SEQ ID NO: 1 A gene having a base sequence of 8525 bp and a gene having a base sequence of 2498 bp shown in SEQ ID NO: 3 are detected. For ^example, if the pigment phenotype of PgnCynDpn type a dye genotype H ^T H D Pg-CyCyDpDp, flavonoid 3 ', 5'-hydroxylase (F3', 5'-H) gene are shown in SEQ ID NO: 1 A gene having a base sequence of 8525 bp and a gene having a base sequence of 3115 bp shown in SEQ ID NO: 2 are detected.

  In anthocyanidins, coloration is determined by different hydroxylation of the B ring in the structure. That is, pelargonidin (Pgn) having one hydroxyl group at the 4 ′ position of the B ring exhibits orange to vermilion red, and cyanidin (Cyn) having two hydroxyl groups at the 3 ′ and 4 ′ positions of the B ring is red to deep. Delphinidin (Dpn), which has a red color and has three hydroxyl groups at the 3 ′, 4 ′, and 5 ′ positions of the B ring, exhibits a reddish purple to purple color, and when these coexist, it expresses various flower colors (Toshio Honda) Et al., Contemporary Chemistry, 25-32, Tokyo Chemical Doujin, 1998).

  In the present invention, for example, if the Eustoma flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) gene has a base sequence of 8525 bp shown in SEQ ID NO: 1, reddish purple, red, Deep red, purple red, light red, pink, white red, and white flowers can be obtained. Here, the white color is attributed to the fact that it does not contain any anthocyanidins. If the flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) gene of Eustoma grandiflorum is a gene having a base sequence of 3115 bp shown in SEQ ID NO: 2, light purple, purple red, purple, blue-violet You can get flowers. If the Eustoma flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) gene has the 2498 bp base sequence shown in SEQ ID NO: 3, red-purple, red, purple-red flowers are obtained. be able to.

As flowers other than lisianthus, for example, it is possible to obtain sweet pea (sweet pea, Lathyrus odoratus) for dye genotypes ^petals, PgnCyn type in H ^T H ^T Pg-CyCyDpDp the (PgnCyn phenotype). ^{H T H F pgpgCyCyDpDp, H T} H D pgpgCyCyDpDp and CynDpn type in ^H O ^{-pgpgCyCyDpDp} (CynDpn phenotype) can be obtained. It can be obtained Cyn-type (Cyn phenotype) in H ^T H ^T pgpgCyCyDpDp. H ^D H ^F --CyCyDpDp and ^H D ^H D Dpn types in --CyCyDpDp (Dpn phenotype) can be obtained. White flowers can be obtained with H ^F H ^F pgpgCyCyDpDp. In the pigment genotype, “-” and “-” are as defined above. The Dpn type (Dpn phenotype) pigment phenotype includes methylated anthocyanidin, malvidin (Mv) and petunidin (Ptn), both of which are Dpn type (Dpn). Phenotype) of the pigment phenotype. Furthermore, the dye phenotype of Cyn type (Cyn phenotype) includes peonidin (Pn), which is a methylated anthocyanidin, and this is included in the dye phenotype of Cyn type (Cyn phenotype).

Furthermore, for example, it can be obtained for dye genotype of azalea and rhododendron ^petal, Cyn type in H ^T H ^T pgpgCyCyDpDp the (Cyn phenotype). ^{H T H F pgpgCyCyDpDp, H T} H O pgpgCyCyDpDp, can be obtained CynDpn type (CynDpn phenotype) in H ^O H ^O pgpgCyCyDpDp. White flowers can be obtained with H ^F H ^F pgpgCyCyDpDp. Note that the PgnDpn type (PgnDpn phenotype) cannot be obtained. As a characteristic of the pigment genotype of azalea petals, a recessive homozygous gene locus related to the expression of dihydroflavonol reductase (DFR) or anthocyanidin synthase (AS) involved in biosynthesis of Pgn pigment (Pgn phenotype) ( pgpg), Pgn dye is not generated. Dpn type (Dpn phenotype) pigment phenotypes include methylated anthocyanidins, malvidin (Mv) and petunidin (Pt), both of which are included in the Dpn type (Dpn phenotype) pigment phenotype. The Further, the dye phenotype of Cyn type (Cyn phenotype) includes peonidin (Pn) which is a methylated anthocyanidin, and these are included in the dye phenotype of Cyn type (Cyn phenotype).

  The flower color of the flower buds is anthocyanins using 50% (v / v) aqueous acetic acid solution or 50% (v / v) methanolic-acetic acid from colored parts such as petals or sepals, flower coats, buds, and peels. (The concentration of acetic acid may be 10 to 50%, and 0.5 to 2 N hydrochloric acid may be used in place of acetic acid), and this is hydrolyzed with hydrochloric acid to obtain anthocyanidin. Can be measured by analyzing various anthocyanidins using high performance liquid chromatography (HPLC) or the like (for example, Uddin et al., Japan Soc. Hort. Sci., 71: 40-47, 2002).

  Further, for progeny genotypes obtained by repeated self-pollination and crossing, dominant homozygote, dominant heterozygote, recessive homozygote, etc. are determined. Identify flower color, pigment genotype and three flavonoid 3 ', 5'-hydroxylase (F3', 5'-H) genes. Based on this, it is possible to create a quick reference table showing a correlation between the color allele type combination between the pollen parent gamete and the seed parent gamete and the flower color (for example, see Example 7 (6) described later). Examples of the quick reference table include a quick reference table (Table 6 and Table 7 described later) for predicting the flower color of eustoma.

  As for the flower color, the color of the flower can be accurately measured and converted into a numerical value by using a CIELab color coordinate system.

Therefore, three flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) genes of florets are measured, and five biallelic types H ^T , H ^F , H ^Z among the gametes. By determining the combination of H ^D and H 2 ^O , it is possible to predict the flower color of a flower bud before flowering using the quick reference table as described above.

Kit and DNA
The present invention further provides a kit for predicting the flower color of a flower bud before flowering.
The kit includes the following DNAs (1) to (3) or fragments thereof:
(1) The base sequence of SEQ ID NO: 1, derived from the biallelic H ^T or H ^F , its complementary sequence, or a sequence having at least 80% identity or hybridizing under stringent conditions with these sequences DNA containing a base sequence for soybean, or a fragment of 15 or more bases of the DNA,
(2) multiple alleles H ^Z or H ^D nucleotide sequence of SEQ ID NO: 2 from its complementary sequence, or or hybridization with those sequences under stringent conditions with those sequences with 80% or more identity DNA containing a base sequence for soybean, or a fragment of 15 or more bases of the DNA, and
(3) The base sequence of SEQ ID NO: 3 derived from the biallelic H 2 ^O , its complementary sequence, or a base that has 80% or more identity with these sequences, or hybridizes with these sequences under stringent conditions DNA containing a sequence, or a fragment of 15 or more bases of the DNA,
At least one of them as a probe or primer.

The size of the primer is, for example, 15 to 50 bases, preferably 17 to 25 bases.
The size of the probe is 15 bases or more, for example, 20 to 200 bases, 25 to 100 bases, 50 to 80 bases, and the like.

The probe is preferably labeled with a labeling agent such as a fluorescent labeling agent or a radioisotope. Fluorescent labeling agents include, for example, rhodamine, fluoresamine, dansyl, derivatives thereof, Cy dyes, and the like, while radioisotopes include, for example, ³² P, ³⁵ S, and the like.

  The kit of the present invention may further include a quick reference table showing a correlation between the combination of the above-mentioned biallelic types and the flower color. The kit may further contain an enzyme or buffer for PCR, a reagent or buffer for hybridization, and the like.

The kit of the present invention can be subjected to Southern hybridization using a probe, and can be used for biallelic type testing and flower color prediction.
The present invention further provides the following DNA or a fragment thereof.

(4) The base sequence of SEQ ID NO: 1, derived from the biallelic H ^T or H ^F , its complementary sequence, or those sequences having 80% or more identity or hybridizing under stringent conditions with these sequences DNA containing a base sequence for soybean, or a fragment thereof having 15 bases or more.

The DNA is multiple alleles H ^Z or H ^D SEQ ID NO: 2 nucleotide sequence or hybridize to nucleotide sequences in their sequences or their sequence under stringent conditions with a 80% or more identity from The base sequence of the retrotransposon of SEQ ID NO: 4 or a base sequence that has 60% or more identity with these sequences or hybridizes with these sequences under stringent conditions is inserted into the first exon sequence of the DNA that it contains DNA.

  (5) The base sequence of SEQ ID NO: 4 or a retrotransposon having a nucleotide sequence having 60% or more identity with those sequences or hybridizing with these sequences under stringent conditions, or a fragment thereof having 15 or more nucleotides .

(6) multiple alleles H ^Z or H ^D nucleotide sequence of SEQ ID NO: 2 from its complementary sequence, or or hybridization with those sequences under stringent conditions with those sequences with 80% or more identity DNA containing a base sequence for soybean, or a fragment thereof having 15 bases or more.

(7) The base sequence of SEQ ID NO: 3 derived from the biallelic H 2 ^O , its complementary sequence, or a base having 80% or more identity with these sequences or hybridizing under stringent conditions with these sequences DNA containing the sequence, or a fragment thereof having 15 bases or more.

  The DNAs of the above (4) to (7) or fragments thereof of the present invention can also be used to detect the chromosomes of florets such as eustoma by performing fluorescence in situ hybridization (FISH) using them as probes. is there. In addition, it can be used as a probe and / or primer to obtain a homolog corresponding to DNA having the nucleotide sequence of SEQ ID NO: 1, 2 or 3 of the present invention from various plant species, and for chromosome mapping and the like. Application is also possible. Furthermore, when PCR is performed as a primer, various DNA fragments are amplified from the genome. Since such fragments can be handled in the same manner as chromosomes, chromosome mapping from DNA polymorphisms is possible. Therefore, by using such a primer, it becomes possible to develop a new chromosomal marker or genetic marker. Furthermore, artificial trait modification can be achieved by using the above-described DNA for techniques such as gene recombination.

Retrotransposon present inventors, as described above, in the nucleotide sequence of multiple alleles H ^T or H ^F shown in SEQ ID NO: 1, retrotransposon in its first exon; to include (RTeg1 SEQ ID NO: 4) (FIG. 2). It also has been found that the sequence before the retrotransposon sequences are inserted are multiple alleles H ^D or H ^Z.

  The retrotransposon of the present invention is usually 60% or more, preferably 70% or more, 75% or more, more preferably 80% or more, 85% or more, more preferably 90% or more, 95% or more with the nucleotide sequence of SEQ ID NO: 4. Alternatively, DNA having retrotransposon activity having 98% or more identity is also included. Such DNA includes mutants among Eustoma varieties, homologs of closely related species of Gentianaceae, and the like.

  Here, “retrotransposon activity” refers to an activity that alters or inactivates the function of a gene when inserted into another gene.

Other genes retrotransposon of the present invention, by inserting the multiple alleles H ^D or H ^Z particular, it is possible to alter the function of that gene. For example, it is possible to change the purple flower color to red by inserting a retrotransposon into the flavonoid 3 ′, 5′-hydroxylase (F3′5′H) gene.

That is, the present invention further, floriculture multiple alleles H ^Z, to H ^D, or exon portions of both the base sequence or having or their sequence those sequences 60% identity or more to SEQ ID NO: 4 strings Provided is a method for modifying flower color of a floret comprising inserting a retrotransposon including a base sequence that hybridizes under a gentle condition.
In the present invention, the exon is preferably a first exon.

Modification of flower color, for insertion retrotransposon, the function of multiple alleles H ^D or H ^Z is lost caused by newly the function of multiple alleles H ^T or H ^F is obtained instead.

  The florets that can be used in the present invention include dicotyledonous plants and monocotyledonous plants as exemplified above.

  Transformation with the transposon of the present invention can be carried out using a general transformation method for dicotyledonous plants and monocotyledonous plants. As the vector, a binary vector (for example, pBI101, pBI121, etc.) is preferably used. In this vector, the base sequence of SEQ ID NO: 4 or the sequence thereof has 60% or more identity or their sequence between the left border (LB) and the right border (RB) of the T-DNA region And a retrotransposon sequence containing a base sequence that hybridizes under stringent conditions. The vectors further include selection markers such as kanamycin resistance gene for bacterial selection, neomycin resistance gene, selection markers such as neomycin phosphotransferase II (NPTII) and dihydrofolate reductase for selection in plants, cauliflower mosaic virus (CaMV). ) Promoters such as promoters can be included. Transformation can be performed by the Agrobacterium method (including the use of acetosyringone), particle gun method, microinjection method, electroporation method and the like. Examples of Agrobacterium include Agrobacterium tumefaciens (eg, EHA105, EHA101, etc.). Thereby, the retrotransposon sequence is integrated into the plant genome by homologous recombination. Transformation is performed on callus, multi-buds, etc., and is regenerated to plant bodies through a normal plant culture method. Regarding genetic engineering of plants, for example, translation by Matsuhashi Michio et al., Watson Recombinant DNA Molecular Biology, 2nd edition, Maruzen, 1994; supervised by Isao Shimamoto, plant cell engineering series 15, experimental protocol for model plants, From genetic methods to genome analysis, descriptions such as Shujunsha can be used.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the scope of the present invention is not limited to these examples.

[Example 1] Confirmation of F3 ′ and 5′-H DNA of biallelic genes H ^T , H ^F , H ^Z , H ^D and H 2 ^O from leaves of Eustoma grandiflorum Plant Genomic DNA Mini from 5 leaves of Eustoma grandiflorum DNA was extracted using a kit (Biogene). The following two primer sets were designed in such a way that the retrotransposon insertion site is sandwiched between them.
Primer set R:
GTACTTAAAGGTAGGCAGCTGTGGATACCTCCCTATAGACGAGAAGCC (SEQ ID NO: 7)
Primer set L:
GTACTTAAAGGTAGGCAGCTGTGGCCCTCTTGCTGCTATGGTCACTC (SEQ ID NO: 8)
PCR was performed using the GeneAmp9700 system (ABI). In 20 μl of the reaction solution, 10 ng DNA, 0.5 units TAKARA EXTaq HS (Takara Bio Inc.), 0.2 mM dNTPs, 2 μl 10 × EXTaq buffer, 0.4 μM primer are contained. PCR conditions were denaturation at 95 ° C. for 10 seconds, annealing at 62 ° C. for 30 seconds, and extension reaction at 72 ° C. for 3 minutes as one cycle for 45 cycles. After the reaction, the band was confirmed by electrophoresis on 1% agarose gel S (Nippon Gene). The result is shown in FIG. H ^T and ^{H F} are similar band of about 8 kb, ^{H Z} and ^{H D} are similar band of about 3 kb, ^{H O} It can be seen that the band for the 2.5 kb (Fig. 1). In FIG. 1, the band detected using the primer set R is the right (R), and the band detected using the primer set L is the left band (L).

EXAMPLE 2] F3 involved from the leaves of Eustoma in multiple alleles H ^T and H ^F ', 5'-H DNA structure confirmed Example 1 results in, the lines delphinidin in the petals does not exist in all strains A band of about 8 kb indicating retrotransposon insertion was observed. On the other hand, in the strain in which delphinidin is present, there are cases where only a 2 to 2.5 kb band is present, and both a 2 to 2.5 kb band and an 8 kb band are present. The sequence of these bands was determined by the direct sequence method. Specifically, DNA was extracted from the obtained band using QIAEXII gel-purification kits (Qiagen). The extracted fragment DNA was subjected to determination of the base sequence by the primer walking method. The sequence reaction was performed using Big Dye Terminator Ver. The cycle sequence according to 3 was adopted. In 20 μl of the reaction solution, 10-100 ng DNA, 2 μl Big Dye Terminator Ver. 3 premixes, 1.5 μl 10 × Taq buffer (Nippon Gene), 100 ng DNA, 3.2 pm primer. The conditions of the cycle sequence reaction were 25 cycles, with one cycle consisting of denaturation at 96 ° C. for 10 seconds, annealing at 50 ° C. for 5 seconds, and extension reaction at 60 ° C. for 4 minutes. After the reaction, the reaction product was purified by ethanol / sodium acetate precipitation, and requested to the DNA sequence analysis service of Kagoshima University Frontier Science Research Promotion Center (Kagoshima) to determine a sequence of about 500 bp from the end. Primers were designed based on the determined sequence, and sequencing by cycle sequencing was performed again. Finally, the full-length sequence was determined.

The result is shown in FIG. Involving multiple alleles ^{H T} and ^{H F} F3 ', the structure of the 5'-H DNA, it is understood that the gene of the nucleotide sequence of 8525bp of SEQ ID NO: 1. Further, it can be seen that the retrotransposon has a 5405 bp nucleotide sequence shown in SEQ ID NO: 4 (FIG. 2).

[Example 3] from the leaves of Eustoma involving multiple alleles ^{H Z} and ^{H D} F3 ', in the same manner as confirmed in Example 1 of the structure of 5'-H DNA, DNA is extracted from the system with a ^{H D} homo In addition, primers (5′-GGCCGCATTCTTACCAAGAT-3 ′ (SEQ ID NO: 9) and 5′-) sandwiching the entire translation region of the Eustoma flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) gene. An oligonucleotide having the DNA sequence of TAGAAACATGGACGAACTCC-3 ′ (SEQ ID NO: 10) was designed. PCR was performed in the same manner as in Examples 1 and 2, DNA was extracted from the electrophoresis band, and the base sequence was determined.

The result is shown in FIG. Involving multiple alleles ^{H Z} and ^{H D} F3 ', the structure of the 5'-H DNA, it is understood that the gene of the nucleotide sequence of 3115bp of SEQ ID NO: 2 (Figure 2).

[Example 4] Confirmation of structure of F3 ′, 5′-H DNA related to biallelic H 2 ^O from leaves of Eustoma DNA DNA was extracted from a strain having H 2 ^O homozygous by the same method as in Example 3. The DNA was extracted from the electrophoresis band and the nucleotide sequence was determined. The result is shown in FIG. It can be seen that the structure of F3 ′, 5′-H DNA related to the biallelic H 2 ^O is a gene having a base sequence of 2498 bp shown in SEQ ID NO: 3 (FIG. 2).

[Example 5] Structure of protein translated by F3 ′, 5′-H DNA of biallelic genes H ^T , H ^F , H ^Z , H ^D , H 2 ^O (1) Involved in multi-allelic genes H ^T , H ^F , F3 'of the nucleotide sequence of 8525bp of SEQ ID NO: 1, 5'-H DNA are protein structure multiple alleles ^H T to translate, related to ^{H F,} amino acids predicted from the nucleotide sequence of 8525bp of SEQ ID NO: 1 As a result of examination of the sequence, it was found that the following two proteins were translated, one of which has 136 amino acid sequences and the other of which has 386 amino acid sequences. .
MAVGNGVLLLHIAASMLFFHVQKLVQYLWMNSNRHRLPPGPIGWPVLGALRLLGTMPHVALANMAKYKYPVMVMYLKVGSCGLAVASTPEAAKAFLKTLDMNFSSNRPPNGATHLAYNALCDMVFFADYGPLC array
MKNVMFPSNKLSNIHILGGKALQGWEEVRKKELGYMLYAMAESGRHGQPVVVSEMLTYAMANMLGQVMLSKRVFGSQGSESNEFKDMVVELMTVAGYFNIGDFIPSIAWMDLQGIQGGMKRLHKKFDALLTRLLEEHTASAHERKGSPDFLDFVVANGDNSEGERLQTVNIKALLLNMFTAGTDTSSSVIEWALAELLKNPIILRRAQEEMDGVIGRDRRFLEADISKLPYLQAICKEAFRKHPSTPLNLPRIASQACEVNGHYIPKGTRLSVNIWAIGRDPSVWENPNEFNPDRFLERKNAKIDPRGNDFELIPFGAGRRICAGTRLGILLV YILGTLVHSFVWELPSSVIELNMDESFGLALQKAVPLAAMVTPRLPLHIYSP (SEQ ID NO: 12)

(2) multiple alleles ^H Z, related to ^{H D,} the nucleotide sequence of 3115bp of SEQ ID NO: 2 F3 ', 5'-H structural multiple alleles of protein DNA translates ^H Z, related to ^{H D,} sequence As a result of examining the amino acid sequence predicted from the base sequence of 3115 bp shown in No. 2, it was found that a protein having the following 510 amino acid sequences was translated. The amino acid sequence structure of this protein matched that of Non-Patent Document 7.
MAVGNGVLLHIAASLMLFFHVQKLVQYLWMNSRRHRLPPGPIGWPVLGALRLLGTMPHVALANMAKKYGPVMYLKVGSCGLAVASTPEAAKAFLKTLDMNFSNRPPNAGATHLAYNAQDMVFADYGPRWKLLRKLSNIHILGGKALQGWEEVRKKELGYMLYAMAESGRHGQPVVVSEMLTYAMANMLGQVMLSKRVFGSQGSESNEFKDMVVELMTVAGYFNIGDFIPSIAWMDLQGIQGGMKRLHKKFDALLTRLLEEHTASAHERKGSPDFLDFVVANGDNSEGERLQTVNIKALLLNMFTAGTDTSSSVIEWALAELLKNPIILRRAQE MDGVIGRDRRFLEADISKLPYLQAICKEAFRKHPSTPLNLPRIASQACEVNGHYIPKGTRLSVNIWAIGRDPSVWENPNEFNPDRFLERKNAKIDPRGNDFELIPFGAGRRICAGTRLGILLVEYILGTLVHSFVWELPSSVIELNMDESFGLALQKAVPLAAMVTPRLPLHIYSP (SEQ ID NO: 13)

(3) Structure of the protein translated by F3 ′, 5′-H DNA of the 2498 bp nucleotide sequence shown in SEQ ID NO: 3 related to the biallelic H 2 ^O 2498 bp shown in SEQ ID NO: 3 related to the double allele H 2 ^O As a result of examining the amino acid sequence predicted from the base sequence, it was found that a protein having the following 510 amino acid sequences was translated.
MAVGNGVLLHIAASLMLFFHVQKLVQYLWMNSRRHRLPPGPIGWPVLGALPLLGTMPHVALANMAKKYGPVMYLKVGSCGLAVASTPEAAKAFLKTLDMNFSNRPPNAGATHLAYNAQDMVFADYGPRWKLLRKLSNIHILGGKALQGWEEVRKKELGYMLYAMAESGRHGQPVVVSEMLTYAMANMLGQVMLSKRVFGSQGSESNEFKDMVVELMTVAGYFNIGDFIPSIAWMDLQGIQGGMKRLHKKFDALLTRLLEEHTASAHERKGSPDFLDFVVANRDNSEGERLHTVNIKALLLNMFTAGTDTSSSVIEWALAELLKNPIILKRAQE MDGVIGRDRRFLEADISKLPYLQAICKEAFRKHPSTPLNLPRIASQACEVNGHYIPKGTRLSVNIWAIGRDPSLWENPNEFNPDRFLERKNAKIDPRGNDFELIPFGAGRRICAGTRLGILLVEYILGTLVHSFDWELPSSVIELNMDEPFGLALQKAVPLAAMVTPRLPLHIYCP (SEQ ID NO: 14)

(4) Structure of the protein translated by the retrotransposon DNA (rTeg1) having the base sequence of 5405 bp shown in SEQ ID NO: 4 As a result of examination of the amino acid sequence predicted from the base sequence of 5405 bp shown in SEQ ID NO: 4, the following 1591 It was found to translate a protein having a single amino acid sequence.
MSETSPNPQEPKTESSVHTTPGDLALQVAQILKDSLGSTPSQSITLPENLNVAVKLTGNNYSLWSRIIYRAILGRGRQYHLTGTPPPPLPTDPRFSRWEQDDNSVFTWILQNVDASMINNVSRYPTAKALWDGLALTYGSRGDSLQVFDLHRKANNIRQGDDTLEACWNNLQDIWVSIDTLDTNPMKCPEDISLYNQKMQEFRLYQFLTAVSDRFETEKKELLKRTPLPNVEAAFFEFKRAESQAGLIKHGPSEQISSLGIGQGLTVKPATGKGRGKGTDRSMNTIRSNNASSRMDKSNLLCEHCGKKGHSREKCFQIHGYPEWWEGKRITTG GKTAAARTPGEGVSSNEAQMTGESREEEEQNRENPRAQGNSAAAGMGLGENPSPNPPFKSIFTPHAPHFSVLSPDPLSSSDIIFPSPSSLNFSPPSPQSSHRANMVQHTSEFTDQPHTFPISRLDTHTSPKYPFSPQPPPGLSHHIRVTPSQWIFDCGATDTMTFDPTDLLTQSPPLKSHVETASGDTIPVQASGPITITPDITLQNCLLVPSLSTKLLSISQLTKALDCVVLMYPSFCLLQDIRTQAIIGRGTENGGLYYVDAVVQHGSSNLATGTVTRQIWLWHRRLGHPSFSYLQKLFPTLFSRTLPPLTCDTCLRAKQPRATYRSNNT VNKVFSLIHSDVWGPSPHSTPCGFKYFVIFVDDCSRMTWLYILRHKSEVATKFVEFYHMIHTQYSSTIQILRTDNGGEYFAGALQQFFRDNGIIHQTTCPDTPEQNGVAERKNRTLLEMTRALMLESSVPRFLWPEAVSTATYLSNRLPTVTLNHQTPLDVLASQTLIPSLLTLPPKVFGCTVFIHISKTHRDKLDPCAEKCVFVGYASTQKGYRCYNPRTRQIHVTLNCVFLETEFFFGTHPRSQGEIATDGYLDWLPNLSWSVADPTRQVVEPACTIAPSDNMDSIPVPTNDIPRENVLQQVNESIHDDTGSPVPFSSPPFVPNTTLDDSL TTSIAIPDPSSELDHTNPCVKEQGRTLPPRKTRGVPPDRYSPTKIARATLYPVSTSRKNLGHAAKAFFTQICSEKIPRTVDEACSQSNWRDAMIMEMDALNKNNTWERCQLPPGKRTVGCRWVFTVKYKADGTIERHKARLVAKGYTQTYGVDYSETFSPVARIDTIRVLFAIAATENWPLHQFDVKNAFLHGTLKEEVFMDPPPGFSKEFLPGQVCKLNRALYGLKQSPRAWFGRFTQAMKKDGYRQSNADHTLFIKRQGSLVTCLIIYVDDMIITGNDANEISRLRDYLFTQFEMKDLGGLKYFLGIEVLRSAEGIYISQRKYILDLLTEV LLDAKPADTPMVQNHKLDIVQGAASADREQYQRLVGKLIYLSHTRPDIAYAVGVVSQFMHSPQKHHLEAVFRIMRYLKGTPGRGLLFKNNGHLNIEAYTDADWAGSQIDRRSTSGYFTLVGGNVVTWRSKKQKVVALSSAEAEYRGIVKGVSEVLWIRKLLQELGFPVTDPTCLMCDNKASISISENPVQHDRTKHVEIDRHFVKEKIEDGIIALPHVRSEDQLADILTKAVNGRIFEFILRKLNIVDPTIQLEGEC (SEQ ID NO: 15)

[Example 6] Confirmation of F3 ', 5'-H DNA of heterozygous biallelic genes from Eustoma leaves F3', 5'-H DNA genes from four Eustoma leaves with purple and pink flower colors Extracted. DNA extraction, PCR, and electrophoresis followed the method of Example 1. The primer of FIG. 1 (R) in Example 1 was used as a PCR primer.

The result is shown in FIG. H ^{T, H} D, respectively a ^{H O} in individuals with homo, nucleotide sequence of 8525bp of SEQ ID NO: 1 is ^{H T} homo, nucleotide sequence of 3115bp of SEQ ID NO: 2 is ^{H D} homo, SEQ ID NO: in ^{H O} homo Bands Ef3, Ef2, and Ef1 corresponding to the 2498 bp base sequence shown in FIG. The band electrophoresis system having a hetero-type multiple alleles of H ^T and ^{H D,} and the nucleotide sequence of 8525bp of SEQ ID NO: 1, each band Ef3 corresponding to the nucleotide sequence of 3115bp of SEQ ID NO: 2 and Ef2 are shown, whereas, electrophoretic bands of lines with multiple alleles of H ^T and H ^O in heterozygous includes a nucleotide sequence of 8525bp of SEQ ID NO: 1 corresponds to the nucleotide sequence of 2498bp of SEQ ID NO: 3 It can be seen that each band Ef3 and Ef1 is shown (FIG. 3). M and 1K indicate markers.

[Example 7] Pigmentary phenotype, pigment genotype assignment, flower colorimetry and detection of F3 ', 5'-H DNA band of Eustoma grandiflorum (1) Investigation of commercial varieties of Eustoma cultivar' Asuka Maihime ',' Azuma The flower color of the cultivar 'King of Snow' is measured using the CIELab color system using the flower color genotype mating method described in Patent Document 8, and the pigment of anthocyanidin using high performance liquid chromatography The phenotype was analyzed. Further, the flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) DNA band was measured in the same manner as in the above Examples. The results are shown in Table 1.

Both the 'Asuka no Maihime' and 'Azuma's makeup' in the ring systems were both of which the pigment phenotype was PgnCyn type and both detected the band Ef3 corresponding to the 8525 bp base sequence shown in SEQ ID NO: 1. it is understood pigment genotype is ddEeH ^T H ^T Pg-CyCyDpDp. 'Asuka Maihime' and 'Azuma's makeup' both showed red flower color.

  Moreover, the cover ring 'Asuka Maihime' has a relatively bright flower color with a lightness of 35.1 and a relatively dark color with a saturation of 63.1. The pigment angle was 1.5 degrees and the color was in the red direction. 'Azuma's makeup' is a bright flower color with a lightness of 65.3 and a saturation of 35.3. The pigment angle was -1.0 degrees and the color was in the red direction.

For the cultivar 2 of “King of Snow”, the pigment phenotype was Pgn type and the band Ef3 corresponding to the 8525 bp nucleotide sequence shown in SEQ ID NO: 1 was detected. it is found that DdeeH ^F H ^F Pg-CyCyDpDp.

On the other hand, for the cultivar 9 and 11 of the eight-fold 'King of Snow', the pigment phenotype was the Dpn type and the band Ef2 corresponding to the 3115 bp base sequence shown in SEQ ID NO: 2 was detected. , It can be seen that the pigment genotype is DdeeH ^D H ^D Pg-CyCyDpDp.

In addition, for the cultivar 10 of the eight-fold 'King of Snow', the pigment phenotype was Dpn type, and corresponds to the base sequence of 3115 bp shown in SEQ ID NO: 2 and the base sequence of 8525 bp shown in SEQ ID NO: 1. since the detection of the band Ef2 and Ef3 simultaneously to, it is understood pigment genotype is DdeeH ^D H ^F Pg-CyCyDpDp.

  'King of Snow' has a lightness in the range of 88 to 92 and is very bright, and the saturation is in the range of 4 to 10 and has an almost white flower color. Further, the pigment angle was in the range of 93 to 106 degrees, and the color was in the yellow direction.

Thus, in the past, it was not possible to discriminate between heterozygous and homozygous by the flower color genotype mating method in the cultivar group 9 to 11 of the eight-fold 'King of Snow', but the DNA band was detected. By doing so, it is possible to discriminate that the varieties 9 and 11 are H ^D H ^D homotypes and the Cultivar 10 is H ^D H ^F heterotypes. These four 'King of Snow' varieties showed a creamy "white" color due to the low pigment accumulation.

(2) Investigation of S1 progeny progeny S ₁ of Eustoma grandiflorum: Horticultural varieties' Asuka Soft Breeze (ASY17 strain) ',' Acropolis White (AW3 strain) ',' Candy Orchid (CAO24 strain) ',' Papion Rose Pink ( selfed varieties PRP5 strains) ', the progeny S _1, using a flower genotype breeding method described in Patent Document 8, to measure the flower color using the CIELab color coordinate system, using high performance liquid chromatography The pigment phenotype of anthocyanidins was analyzed. Further, the flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) DNA band was measured in the same manner as in the above Example. The results are shown in Table 2.

Inbred posterity ASY17-13 system of _{S 1} 'of ASCA breeze' detects two bands of Ef2 and Ef3 (indicates magenta), ASY17-22 lines and bands were detected (red) of Ef3. As a result, the dye genotype 'breeze Asuka' is found to be ddeeH ^T H ^D Pg-CyCyDpDp. The ASY17-13 line of the self-breeding progeny S1 of “Asuka Soft Breeze” was very vivid (85.2) and had a red-purple flower color (-21.7). On the other hand, the ASY17-22 line was slightly bright (40.3) and a red flower color (-1.3).

Inbred posterity S ₁ 'of Acropolis White' is similar to the separation of the flower color AW3-8 system, dye genotype from the detection of the bands Ef2, 'Acropolis White' is DDE-H ^D H It can be seen that ^D-- CyCyDpDp. 'Acropolis White' varieties showed a creamy “white” color due to low pigment accumulation. As a result of CIELab measurement, the AW3-8 line of S1 progeny S1 of “Acropolis White” showed very bright (92.5) and flower color (107.0) in the yellow direction.

Inbred posterity S ₁ 'of candy Orchid' from the flower color was separated and analyzed CAO24 system of the three. As a result, CAO24-5 (showing creamy “white”) and CAO24-6 (red flower) detected the Ef3 band, while CAO24-20 (purple red flower) detected the Ef1 band. from the dye genotype 'candy Orchid' it is found to be ddeeH ^T H ^O Pg-CyCyDpDp. Since the progeny S1 of “Candy Orchid” was separated in flower color, the three CAO24 lines were analyzed. As a result of CIELab measurement, CAO24-5 has a yellow color (104.0) and a very bright color (90.6), and CAO24-6 has a red color (-3.5) and slightly brighter. Color (23.1), CAO24-20 was a slightly brighter color (30.1), and a reddish purple color (-27.1).

'Papillon Rose Pink' inbred posterity S ₁ of the were analyzed its three PRP5 system from that flower color is separated. As a result, PRP5-2 (red flower) and PRP5-15 (red flower) detected the Ef3 band, while PRP5-1 (red purple flower) detected the Ef1 band. it can be seen dyes genotype is ddeeH ^T H ^T Pg-CyCyDpDp. “Papion Rose Pink” PRP5-2 and PRP5-15 are bright (61.3, 55.6) and slightly bright (39.7, 49.0), both in the red direction. It was flower color (-3.2, -5.6). On the other hand, the brightness and vividness (58.4, 44.6) of PRP5-1 were the same as those of the former two, but the hue showed a reddish purple color (-14.7).

(3) Investigation of Eustoma wild-type hybrids Eustoma grandiflorum Arizona strain (AZ3 (Dp) 7), Eustoma grandiflorum Texas strain (TX34) self-breeding progenies S _{2 to} S ₃ Flower color was measured using the CIELab color system using the flower color genotyping method described, and the anthocyanidin pigment phenotype was analyzed using high performance liquid chromatography. Further, the flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) DNA band was measured in the same manner as in the above Examples. The results are shown in Table 3.

The flower color of Eustoma grandiflorum Arizona strain AZ3 (Dp) 7-6 was purple red, and an Ef2 band was detected. Since pigment phenotype were Dpn type, it can be seen Eustoma grandiflorum Arizona line AZ3 (Dp) 7-6 dye genotype is ddeeH ^D H ^D --CyCyDpDp. The flower color of the Arizona line AZ3 (Dp) 7-6 was very dark (26.7), the vividness was very vivid (82.7), and the hue was a flower color (-23.1) in the direction of magenta.

The five lines of the Eastoma grandiflorum Texas line TX34 were all red flowers and were Ef3 bands. Since pigment phenotype were PgnCyn type, it can be seen all Eustoma grandiflorum 5 strains dye genotype Texas strain TX34 is ddeeH ^T H ^T PgPgCyCyDpDp. The five lines of the Texas line TX34 are bright in the range of values from 67 to 85, the vividness is in the range of values from 14 to 38, which is not so vivid or slightly bright, and the hue is from -7 to The range of 4 degrees, therefore, the five hues were generally in the red direction.

(4) Investigation of inbred progeny S ₁ of Eustoma wild type hybrids Eustoma wild type hybrid: For the inbred progeny S ₂ of the Eastoma grandiflorum Texas strain (TX61), the flower color genotype mating method described in Patent Document 8 is used. The flower color was measured using the CIELab color system and the pigment phenotype of anthocyanidins was analyzed using high performance liquid chromatography. Further, the flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) DNA band was measured in the same manner as in the above Examples. The results are shown in Table 4.

TX61-2 was a purple-red flower color and was an Ef1 band. Since pigment phenotype were CynDpn type, it can be seen that dye genotype TX61-2 is ddeeH ^O H ^O pgpgCyCyDpDp.

TX61-1 is a purple-red flower color, and two bands of Ef1 and Ef3 were detected. Since pigment phenotype were CynDpn type, it can be seen that dye genotype TX61-1 is ddeeH ^O H ^T pgpgCyCyDpDp.

TX61-8 and TX61-11 are reddish purple flowers, and two bands of Ef1 and Ef3 were detected. Since the pigment phenotype was the PgnCynDpn type, it can be seen that the pigment genotypes of TX61-8 and TX61-11 are ddeE H ^{O T} Pg-CyCyDpDp.

The TX61-3, TX61-5, TX61-6, TX61-9, and TX61-15 lines were all red flowers and had an Ef3 band. Since pigment phenotype were CynDpn type, it can be seen that dye genotype TX61-2 is ddeeH ^O H ^O pgpgCyCyDpDp. In the TX61 line, those in which the biallelic type possesses H 2 ^O shows a hue angle (h) of approximately purple to purple-red (a value of −23 ° to −34 °), and the homologous type of the biallelic type is HT. What was a type | mold showed the hue angle (value of -1 degree-4 degree | times) of substantially red.

(5) Lisianthus investigation horticultural varieties of inbred posterity _{S 3} of the commercial varieties 'Royal violet (RV10CR41 system)', 'Asuka of dancer (AM1A system)', selfed the '粧(AY2A9 system) of Azuma', progeny for S _3, using the flower genotype breeding method described in Patent Document 8, to measure the flower color using the CIELab color system, were analyzed pigment phenotype anthocyanidins using high performance liquid chromatography. Further, the flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) DNA band was measured in the same manner as in the above Examples. The results are shown in Table 5.

Two lines of RV10CR41 of "Royal Violet" were reddish purple (h is -23.5 degrees) to purple-red (h is -29.7 degrees), both of which were Ef2 bands. Since pigment phenotype were Dpn type, it can be seen that two systems of dyes genotype RV10CR41 is ddeeH ^D H ^D --CyCyDpDp.

All three lines of AM1A of “Asuka Maihime” were red (h was in the range of −2.2 to 2.8 degrees) and were Ef3 bands. Since pigment phenotype were PgnCyn type, it can be seen three systems of dyes genotype AM1A is ^{ddE-H T H T Pg-} CyCyDpDp.

The two AY2A9 lines of “Azuma no makeup” were both red (h is −1.3 and −4.3 degrees) flower color, and were Ef3 bands. Since pigment phenotype were PgnCyn type, it AY2A9-12 and two systems of dyes genotype AY2A9-19 are each, ddeeH ^T H ^T Pg-CyCyDpDp and ^{ddE-H T H T Pg-} CyCyDpDp I understand.

(6) Preparation of a quick reference table Taking Eustoma grandiflorum as an example, three bands corresponding to flower color and flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) gene from the combination of each multiallele, Ef1 Tables 6 and 7 show quick reference tables for knowing Ef2, Ef3. The rows show the pollen parent gametes, and the columns show the seed parent gametes. Table 6 is a combination table when the loci represented by Pg / pg, Cy / cy and Dp / dp are represented by PgPgCyCyDpDp or PgpgCyCyDpDp, and Table 7 shows Pg / pg, Cy / cy and Dp / dp. It is a combination table | surface when the locus shown by is represented by pgpgCyCyDpDp.

For example, one of multiple alleles ^{H T} and another multiple alleles ^{H D} is fertilized, if the combination becomes ^H T ^{H D,} the pigment phenotype from Table 6 is PgnCynDpn, the flower color It is purple-red and, therefore, the band corresponding to the flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) gene is quickly found from this chart that Ef2 and Ef3 are two bands. be able to.

On the other hand, when the bands corresponding to the flavonoid 3 ′, 5′-hydroxylase (F3 ′, 5′-H) gene are Ef2 and Ef3, the flower color is purple, and the pigment phenotype is the Dpn type Thus, it can be quickly known from this chart that the combination of the biallelic types is either H ^F H ^D or H ^D H ^F.

  This time, we have clarified the structure of the five multiallelic genes described in the Flower Color Genotype Mating Method. Moreover, their structure and flower color inheritance inherited in the present generation and / or progeny were clarified. Furthermore, the color of the flower was accurately measured and converted into a numerical value using the CIELab color system, and the relationship between the pigment genotype, the flower color inheritance, and the gene could be clarified. Based on these findings, the present invention assigns a biallelic genotype to a practical flower color modification method for producing a new flower color of a flower bud and / or after early testing of a gene without floret flowering. In addition, the present invention provides an excellent practical flower color prediction method capable of knowing the inherited flower color.

The electropherogram showing the characteristics of the F3 ′, 5′-H DNA bands of each multi-allele H ^T , H ^F , H ^Z , H ^D , H 2 ^O is shown. Multiple alleles ^{H T, H F, H Z} , H D, F3 obtained from ^{H O} ', shows a diagram of the structure schematizes the 5'-H DNA gene. The combination of multiple alleles of heterozygous, ^{H T} and ^{H D,} and show electropherograms showing the F3 ', 5'-H DNA bands ^{H T} and ^{H O.} The pathway diagram of floronoid flavonoid biosynthesis is shown.

SEQ ID NO: 5 Description of artificial sequence: Primer SEQ ID NO: 6 Description of artificial sequence: Primer SEQ ID NO: 7 Description of artificial sequence: Primer SEQ ID NO: 8 Description of artificial sequence: Primer SEQ ID NO: 9 Description of artificial sequence: Primer SEQ ID NO: 10 Artificial sequence Description: Primer

Claims (10)

Pollen parent gametes and seeds of an enzyme reaction system of a floret containing a flavonoid 3 ′, 5′-hydroxylase involved in hydroxylation of the B ring of the flavonoid biosynthesis pigment precursor of the pathway formula shown in FIG. 4 of the specification A combination of the parent gamete-derived biallelic H ^T , H ^F , H ^Z , H ^D and H 2 ^O using the electrophoresis, the flavonoid 3 ′, 5′-hydroxylase gene corresponding to the biallelic Genotype H ^X H ^X · Pg / pg (ie, PgPg, Pgpg or pgpg) · Cy / cy (ie, CyCy, Cycy or cycy) · Dp / dp (i.e., DPDP, DPDP or DPDP) pollen parent gametes and combinations and flower color and flower color correlation in the previous flowering based floriculture of multiple alleles between seed parent gametes for A prediction method,
Here, the biallelic H ^T regulates hydroxylation at the 3 ′ position with respect to hydroxylation of the B ring in the precursor of the flavonoid biosynthesis, and is involved in the biosynthesis of pelargonidin (Pgn) and cyanidin (Cyn). a gene, a gene that multiple alleles H ^F controls the hydroxide of 5 'position with respect to hydroxylation of the B ring in a precursor of the flavonoid biosynthesis involved in the biosynthesis of pelargonidin (Pgn), double allele H ^D or H ^Z is 3 with respect to hydroxylation of the B ring in a precursor of the flavonoid biosynthetic ', 5' position of the controls hydroxide, a gene involved in the biosynthesis of delphinidin (Dpn) In addition, the biallelic H 2 ^O is a gene involved in delphinidin (Dpn) biosynthesis by controlling hydroxylation at the 3 ′ and 5 ′ positions with respect to hydroxylation of the B ring in the flavonoid biosynthesis precursor. , In beauty,
(A) The double allele H ^T or H ^F is the same as the 8525 bp nucleotide sequence of SEQ ID NO: 1, or the same as the protein encoded by the nucleotide sequence of SEQ ID NO: 1 with 95% identity or more nucleotide sequence encoding a protein having the activity, a DNA containing, and corresponding to the DNA indicates Ef3 band (electrophoresis), (b) multiple alleles ^{H Z} or ^{H D} is of SEQ ID NO: 2 of 3115bp A DNA comprising a base sequence or a base sequence encoding a protein having 95% or more identity with the sequence and having the same activity as the protein encoded by the base sequence of SEQ ID NO: 2; and the DNA It shows the corresponding Ef2 band (electrophoresis) in, (c) multiple alleles ^{H O} is the nucleotide sequence of 2498bp of SEQ ID NO: 3, or a sequence 95% A DNA comprising a base sequence encoding a protein having identity and having the same activity as the protein encoded by the base sequence of SEQ ID NO: 3, and showing an Ef1 band (electrophoresis) corresponding to the DNA As well as
Combined with flower color of the multiple alleles between pollen parent gametes and seed parent gametes, table (in the table below, Ef1 indicates electrophoresis bands representing H ^O homo, Ef2 a is H ^D or H ^Z homo- shows the electrophoresis band representing, and Ef3 have a correlation shown in.) showing the electrophoresis bands representing H ^F or H ^T homo,
And said method.

The method according to claim 1 , wherein the florets are selected from dicotyledonous plants or monocotyledonous plants. The method according to claim 2 , wherein the flower bud is eustoma. Multiple alleles H ^T or H ^F SEQ ID NO: 1 nucleotide sequence derived from, or a protein having the same activity as the protein encoded by the nucleotide sequence of the chromatic vital said SEQ ID NO: 1 sequence and 95% identity thereof A DNA comprising a coding base sequence or a complementary base sequence thereof, wherein the biallelic H ^T controls the hydroxylation at the 3 ′ position with respect to hydroxylation of the B ring in the precursor of the flavonoid biosynthesis and a gene involved in the biosynthesis of pelargonidin (Pgn) and cyanidin (Cyn), a hydroxide of 5 'position plurality allele H ^F is with respect to hydroxylation of the B ring in a precursor of the flavonoid biosynthetic The DNA, which is a gene that regulates and participates in the biosynthesis of pelargonidin (Pgn) . The DNA is in the first exon sequence of DNA comprising the nucleotide sequence of SEQ ID NO: 2 from multiple alleles H ^Z or H ^D, the nucleotide sequence of the retrotransposon SEQ ID NO: 4, or the same sequence of 95% or more thereof nucleotide sequence encoding a protein having the same activity as the protein encoded for by gender nucleotide sequence of the chromatic vital said SEQ ID NO: 4, a is inserted DNA, although the plurality allele H ^D or H ^Z is the The DNA according to claim 4 , which is a gene that controls hydroxylation at the 3 'and 5' positions with respect to hydroxylation of the B ring in a precursor of flavonoid biosynthesis and is involved in delphinidin (Dpn) biosynthesis . Nucleotide sequence of SEQ ID NO: 4, or retrotransposon comprising a nucleotide sequence, which encodes a protein having the same activity as the protein encoded by the sequence 95% or more identity with the nucleotide sequence of the chromatic vital said SEQ ID NO: 4. Nucleotide sequence of multiple alleles H ^O from SEQ ID NO: 3, or bases which encodes a protein having the same activity as the protein encoded by the nucleotide sequence of the chromatic vital said SEQ ID NO: 3 sequence 95% or more identity that A DNA comprising a sequence, or a complementary base sequence thereof, wherein the biallelic H 2 ^O controls hydroxylation at the 3 ′ and 5 ′ positions with respect to hydroxylation of the B ring in the precursor of the flavonoid biosynthesis. The DNA, which is a gene involved in biosynthesis of delphinidin (Dpn) . A kit for predicting flower color of a flower bud before flowering by the method according to any one of claims 1 to 3 , comprising the following DNAs (1) to (3):
(1) having multiple alleles H ^T or H ^F derived from SEQ ID NO: 1 nucleotide sequence or the same activity as the protein encoded by the sequence and nucleotide sequence of the chromatic vital said SEQ ID NO: 1 95% or more identity DNA comprising a base sequence encoding a protein or a complementary base sequence thereof,
(2) having multiple alleles H ^Z or H ^D derived SEQ ID NO: 2 nucleotide sequence or the same activity as the protein encoded by the sequence 95% or more identity with the nucleotide sequence of the chromatic vital said SEQ ID NO: 2 DNA containing a base sequence encoding a protein , or a complementary base sequence thereof, and
(3) encodes a protein having the same activity as the protein encoded by the nucleotide sequence of multiple alleles H ^O derived from SEQ ID NO: 3 in the nucleotide sequence or chromatic vital said SEQ ID NO: 3 sequence 95% or more identity that DNA comprising a base sequence to be complementary, or a complementary base sequence thereof,
As a probe or gene marker. The kit according to claim 8 , wherein the probe is labeled. The following is a quick reference table showing the correlation between the combination of the above-mentioned alleles between the pollen parent gamete and the seed parent gamete and the flower color (Ef1 is an electrophoresis band in which Ef1 represents ^HO homozygote) are shown, Ef2 indicates electrophoresis bands representing ^{H D} or ^{H Z} homo- and Ef3 further comprises.) showing the electrophoresis bands representing ^{H F} or ^{H T} homo kit according to claim 8 or 9 .

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