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US007973216B2

(12) Patent (10) Patent N0.: US 7,973,216 B2 Espley et al. (45) Date of Patent: Jul. 5, 2011

(54) COMPOSITIONS AND METHODS FOR 6,037,522 A 3/2000 Dong et al. MODULATING PIGMENT PRODUCTION IN 6,074,877 A 6/2000 D’Halluin et al. 2004/0034888 A1 2/2004 Liu et al. FOREIGN PATENT DOCUMENTS (75) Inventors: Richard Espley, Auckland (NZ); Roger W0 WO 01/59103 8/2001 Hellens, Auckland (NZ); Andrew C. W0 WO 02/00894 1/2002 Allan, Auckland (NZ) W0 W0 02/055658 7/2002 W0 W0 03/084312 10/2003 W0 WO 2004/096994 11/2004 (73) Assignee: The New Zealand Institute for W0 WO 2005/001050 1/2005 and food Research Limited, Auckland (NZ) OTHER PUBLICATIONS Bovy et al. (Plant Cell, 14:2509-2526, Published 2002).* ( * ) Notice: Subject to any disclaimer, the term of this Wells (Biochemistry 29:8509-8517, 1990).* patent is extended or adjusted under 35 Guo et al. (PNAS, 101: 9205-9210, 2004).* U.S.C. 154(b) by 0 days. Keskin et al. (Protein Science, 13: 1043-1055, 2004).* Thornton et al. (Nature structural Biology, structural genomics (21) Appl. No.: 12/065,251 supplement, Nov. 2000).* Ngo et al., (The Protein Folding Problem and Tertiary Structure (22) PCT Filed: Aug.30, 2006 Prediction, K. MerZ., and S. Le Grand (eds.) pp. 492-495,1994).* Doerks et al., (TIG, 14:248-250, 1998).* (86) PCT No.: PCT/NZ2006/000221 Smith et al. (Nature Biotechnology, 15:1222-1223, 1997).* Bork et al. (TIG, 12:425-427, 1996).* § 371 (0X1), Vom Endt et al. (Phytochemistry 61:107-114, 2002).* (2), (4) Date: Jul. 16, 2008 Korban et al. (NCBI, GenBank Sequence Accession No. CV628545, Published Oct. 25, 2004).* (87) PCT Pub. No.: WO2007/027105 Accession No. AJ554700, Jan. 5, 2004, “Gerbera Hybrid cv. ‘Terra Regina’ mRNA for MYBlO Protein,” Eloma et al. PCT Pub. Date: Mar. 8, 2007 Accession No. Q8L5P3, Oct. 1, 2002, “SubName: FuIIIMyb-Re lated Transcription Factor VlMYBAl-l,” Kobayashi et al. (65) Prior Publication Data GenBank Accession No. DQ267896, Jul. 21, 2006, Malus X US 2011/0072539 A1 Mar. 24, 2011 domestica RedField MYB 1 0a mRNA, Partial cds, Espley et al. (30) Foreign Application Priority Data GenBank Accession No. DQ267897, Jul. 21, 2006, “Malus X domestica Cultivar Pacicic MEYBlOa mRNA, Partial cds,” Espley et al. Aug. 30, 2005 (NZ) ...... 542110 GenBank Accession No. DQ267898, Jul. 21, 2006, “Mauls X domestica Cultivar Granny Smith NYBlOa mRNA, Partial cds,” (51) Int. Cl. Allan et al. A01H 5/00 (2006.01) GenBank Accession No. DQ886415, Nov. 18, 2006, “Malus X C12N 15/82 (2006.01) domestica MYB Transcription Factor (MYBl) Gene, MYB1-2 C07H 21/00 (2006.01) allele, Promoter Region and Complete cds,” Takos et al. (52) US. Cl. 800/295; 800/278; 800/298; 435/320.1; GenBank Accession No. DQ267900, Jul. 21 2006, “Malus X 435/419; 435/468; 536/232; 536/23.6 domestica Cultivar Royal Gala MYB9 mRNA, Complete cds,” Allan et al. (58) Field of Classi?cation Search ...... None GenBank Accession No. AF336284, Mar. 15, 2001, “Gossypium See application ?le for complete search history. hirsutum GHMYB36 (ghmyb36) mRNA, Complete cds,” MatZ et al. GenBank Accession No. DQ074463, Jan. 24, 2006, Malus X (56) References Cited domestica MYB11 mRNA, Complete cds, Hellens et al. U.S. PATENT DOCUMENTS (Continued) 4,795,855 A 1/1989 Fillatti et al. 5,004,863 A 4/1991 Umbeck Primary Examiner * Vinod Kumar 5,159,135 A 10/1992 Umbeck (74) Attorney, Agent, or Firm * Greenlee Sullivan PC 5,177,010 A 1/1993 Goldman et al. 5,187,073 A 2/1993 Goldman et al. (57) ABSTRACT 5,188,958 A 2/1993 Moloney et al. 5,416,011 A 5/1995 Hinchee et al. This invention relates to polynucleotides encoding novel 5,463,174 A 10/1995 Moloney et al. transcription factors and to the encoded transcription factors, 5,563,455 A 10/1996 Cheng that are capable of regulating anthocyanin production in 5,569,834 A 10/1996 Hinchee et al. 5,591,616 A 1/1997 Hiei et al. plants. The invention also relates to constructs and vectors 5,750,871 A 5/1998 Moloney et al. comprising the polynucleotides, and to host cells, plant cells 5,792,935 A 8/1998 ArntZen et al. and plants transformed With the polynucleotides, constructs 5,824,877 A 10/1998 Hinchee et al. and vectors. The invention also relates to methods of produc 5,846,797 A 12/1998 Strickland ing plants With altered anthocyanin production and plants by 5,952,543 A 9/1999 FirooZabady et al. 5,968,830 A 10/1999 Dan et al. the methods. 5,981,840 A 11/1999 Zhao et al. 6,020,539 A 2/2000 Goldman et al. 16 Claims, 11 Drawing Sheets US 7,973,216 B2 Page 2

OTHER PUBLICATIONS Heim et al. (2003) “The Basic Helix-Loop-Helix Transcription Fac tor Family in Plants: A Genome-Wide Study of Protein Structure and GenBank Accession No. CN 938023, Jun. 7, 2004. Functional Diversity,” Mol. Biol. Evol. 20:735-747. GenBank Accession No. CN 934367, Jun. 7, 2004. Hofmann et al. (1999) “The PROSITE Database, it’s Status in 1999,” GenBank Accession No. AF 117267, Apr. 20, 1999, “Malus Nuc. Acids. Res. 27(1):215-219. domestica UDP Glucose: 3-0-glucsyl Tranferase Holton et al. (Jul. 1995) “Genetics and Biochemistry of Anthocyanin (UFGT1) mRNA, Complete cds,” Lee et al. Biosynthesis,” Plant Cell 7: 1071-1083. GenBankAccession No. AF 117269, Apr. 20, 1999, Malus domestica Anthocyanidin Synthase (ANS) mRNA, Complete cds, Lee et al. Honda et al. (2002) “Anthocyanin Biosynthetic Genes are Coordi GenBank Accession No. AY 227729, Mar. 27, 2003, Malus X nately Expressed During Red Coloration in Skin,” Plant domestica Cultivar Weirouge dihydro?avonol 4-Reductase mRNA, Physiol. Biochem. 40:955-962. Complete cds, Fischer et al. International Search Report, Corresponding to International Appli GenBank Accession No. CN 491664, Apr. 27, 2004, Korban et al. cation No. PCT/NZ2006/000221, Mailed Feb. 22, 2007. GenBank Accession No. CN 946541, Jun. 7, 2004, Beuning et al. Jin et al. (1999) “Multifunctionality and Diversity within the Plant GenBank Accession No. CN 944824, Jun. 7, 2004, Beuning et al. MYB-Gene Family,” Plant Mol. Biol. 41:577-585. GenBank Accession No. AF325123, Feb. 7, 2001, “Arabidopsis Jouvenot et al. (2003) “Targeted Regulation of Imprinted Genes by thaliana Production of Anthocyanin Pigment 1 Protein (PAP1) Gene, Synthetic Zinc-Finger Transcription Factors,” Gene Ther. 10:513 Complete cds,” BorovitZ et al. 522. GenBank Accession No. AF 146702, May 1, 2000, Petunia X hybrida An2 Protein (an2) mRNA, an2-V26 allele, Complete cds, Quattroc Kim et al. (2003) “Molecular Cloning and Analysis of Anthocyanin chio et al. Biosynthesis Genes Preferentially Expressed in Apple Skin,” Plant SwissProt Accession No. Q6V7VO Jul. 5, 2004, “Anthcyanin 1,” Sci. 165:403-413. Mathews et al. Kobayashi et al. (2002) “Myb-Related Geens of the Kyoho Grape SwissProt Accession No. Q9ATD3, Jun. 1, 2001, “GHMYB36,” (J/ltis labruscana) Regulate Anthocyanin Biosynthesis,” Planta MatZ et al. 215:924-933. SwissProtAccession No. Q9ATD5 Jun. 1,2001, “GHMYB10,” MatZ Kubo (Jul. 1999) “Anthocyanless2, a Homeobox Gene Affecting et al. Anthocyanin Distribution and Root Development in Arabidopsism” SwissProt Accession No. Q6QP46, Jul. 5, 2004, “Anthocyanin Plant Cell. 11:1217-1226. Biocynthesis Regulatory Protein P11iB73,” Swigonova et al. Lancaster J. (1992) “Regulation of Skin Color in ,” Crit Rec. Abbott et al. (Mar. 2002) “Simultaneous Suppression of Multiple Genes by Single Transgenes. Down-Regulation of Three Unrelated Plant Sci. 10:487-502. Lignin Biosynthetic Genes in Tobacco,” Plant Physiol. 128(3):844 Mathews et al. (Aug. 2003) “Ativation Tagging in Tomato Identi?es 853. a Transcriptional Regulator of Anthocyanin Biosynthesis, Modi?ca Aharoni et al. (2001) “The Strawberry FaMYBl Transcription Factor tion, and Transport,” The Plant Cell 15:1689-1703. Suppresses Anthocyanin and Flavonol Accumulation in Transgenic Mehrtens et al. (Jun. 2005) “The Arabidopsis Transcription Factor Tobacco,” Plant]. 28: 319-332. MYB12 is a Flavonol-Speci?c Regulator of Phenylpropanoid Altschul et al. (1997) “Gapped BLAST and PSI-BLAST: A New Biosynthesis,” Plant Phys. 138: 1083-1096. Generation of Protein Database Search Programs,” Nucleic Acids Mol et al. (1996) “Signal Perception, Transduction, and Gene Res. 25(17):3389-3402. Expression Involved in Anthocyanin Biosynthesis,” Crit. Rev. Plant. Bairoch et al. (1994) “PROSITE: Recent Developments,” Nuc. Acids Sci. 15(5-6):525-557. Res. 22(17):3583-3589. Napoli et al. (Apr. 1990) “Introduction of a Chimeric Chalone Bolton et al. (1962) “A General Method for the Isolation of RNA Complementary to DNA,”Proc. Nat. Acad Sci. USA 48: 1390-1397. Synthase Gene into Petunia Results in Reversible Co-Suppression of BorevitZ et al. (Dec. 2000) “Activation Tagging Identi?es a Con Homologous In Trans,” Plant Cell 2:279-289. served MYB Regulator of Phenylprooanoid Biosynthesis,” Plant Nesi et al. (Sep. 2001) “The Arabidopsis T 12 Gene Encodes an R2R3 Cell 12: 2383-2394. MYB Domain Protein that Acts as a Key Determinant for Boss et al. (1996) “Expression of Anthocyanin Biosynthesis Pathway Proanthocyanidin Accumulation in Developing Seed,” Plant Cell Aenes in Red and White Grapes,” Plant Mol. Biol. 32: 56fr569. 13:2099-2114. Bovy et al, (Oct. 2002) “High-Flavonol Tomatoes Resulting from the Page R. (1996) “TreeView: An Application to Display Phylogenetic Heterologous Expression of the Maize Transcription Factor Genes Trees on Personal Computers,” Comput. Applic. Biosci. 12(4):357 LC and CI,” The Plant Cell, 14: 2509-2526. 358. Broun, P (2005) “Transcriptional Control of Flavonoid Bio synthesis: PiaZZa et al. (Mar. 2002) “Members of the c1/pII Regulatory Gene a Complex Network of Conserved Regulators Involved in Multiple Family Mediate the Response of Maize Aleurone and Mesocotyl to Aspects of Differentiation in Arabidopsis,” Curr. Opin. Plant. Biol. 8: Different Light Qualities and Cytokinins,” Plant Phys. 128:1077 272-279. 1086. de Carvalho Niebel et al. (Mar. 1995) “Post Transcriptional Cosup Quattrocchio et al. (1998) “Analysis of bHLH and MYB Domain pression of [5-1,3-Glucanase Genes Does NotAffect Accumulation of Proteins: -Speci?c Regulatory Differences are Caused by Transgene Nuclear mRNA,” Plant Cell 7:347-358. Divergent Evolution of Taraet Anthocyanin Genes,” Plant J. Elomaa et al. (Dec. 2003) “Activation of Anthocyanin Biosynthesis in Gerbera hybrida (Asteraceae) Suggests Conserved Proteinprotein 13(4):475-488. and Protein-Promoter Interactions Between the Anciently Diverged Quattrocchio et al. (Aug. 1999) “Molecular Analysis of the anthocyanin2 Gene of Petunia and its Role in the Evolution of Monocots and ,” Plant Phys. Biochem. 133: 1831-1842. Espley et al. (2007) “Red Colouration in Apple is Due to the Color,” Plant Cell 11:1433-1444. Activity of the MYB Transription Factor, MdMYB10,” Plant J. Saito et al. (2002) “Biochemistry and Molecular Biology of the 49:414-427. Late-Stage of Biosynthesis of Anthocyanin: Lessons from Perilla Falquet et al. (2002) “The PROSITE Database, it’s Status in 2002,” frutescens as a Model Plant,” New Phytologist 155:9-23. Nuc. Acids Res. 30(1):235-238. Schwinn et al. (2004) “,” In; Davies, K.M. ed, Plant Pig Giesen et al. (Nov 1, 1998) “A Formula for Thermal Stability (Tm) ments and their Manipulation, vol. 14. Blackwell Oxford, pp. Prediction of PNNDNA Duplexes,” Nuc. Acids Res. 26(21):5004 92-149. 5006. Stracke et al. (2001) “The R2R3-MYB Gene Family in Arabidopsis Grotewold et al. (Dec. 5, 2000) “Identi?cation of the Residues in the thaliana,” Curr". Opin. Plant Biol. 4:447-456. Myb Domain of Maize C1 that Specify the Interaction with the bHLH Supplementary European Search Report, Corresponding to Euro Cofactor R,” Proc. Nat. Acad. Sci. USA 97: 13579-13584. pean Application No. EP 06 78 4027, Completed Sep. 23, 2008. US 7,973,216 B2 Page 3

Triqlia et a1. (1998) “A Procedure for in vitro Ampli?cation of DNA Goff, et al.Transactivation of Anthocyanin Biosynthetic Genes Fol Segments that Lie Outside the Boundaries of Known Sequences,” lowing Transfer of B Regulatory Genes Into Maize Tissues, The Nuc. Acids Res. l6(l6):8l86. EMBO Journal, 1990, pp. 2517-2522, vol. 9, No. 8, Oxford Univer Walker et a1. (Jul. 1999) “The Transparent Testa Glabrai Locus, sity Press. Which Regulates Trichome Differentiation and Anthocyanin Biosynthesis in Arabidopsis, Encodes a WD40 Repeat Protein,” Plant Cell 11:1337-1350. * cited by examiner

US. Patent Jul. 5, 2011 Sheet 2 0f 11 US 7,973,216 B2 U.S. Patent Jul. 5, 2011 Sheet 3 0f 11 US 7,973,216 B2

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US. Patent Jul. 5, 2011 Sheet 9 0f 11 US 7,973,216 B2

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AtMYB Co E] Md Ms Pb Pc Pcf Ppr Ppy Ps Pav Pd Mg Pdm 75 (Quince) (loquat) apple crab pear pear ( (peach) (pear (Japanese (sweet (almond) (medlar) (European apple YALI ) Nashi) plum) cherry) plum) AIMYB75 100 37 37 36 36 37 36 40 4O 37 4O 40 44 38 41 C0 100 81 89 89 92 92 70 75 92 7O 71 66 72 72 (Quince) El (loqual) 100 77 77 78 79 69 72 78 68 69 65 71 70 '(Vld I) 100 99 92 93 69 73 92 69 69 66 74 69 appe M850?) 100 94 94 69 73 94 69 69 66 74 69 appe $2539“ 100 98 70 74 100 69 70 65 74 70 P0 (pear) 100 70 75 98 7O 70 65 74 71 P01‘ 100 83 70 97 94 81 86 80 (cherry plum) F’Pr 100 74 83 84 82 78 93 (peach) Ppy (Pear 100 69 70 65 74 70 Nashi) PS 100 92 82 86 80 (Japanese plum) Pav 100 86 91 82 (sweet cherry) Pd 100 79 79 (almond) M9 100 77 (medlar) Pdm 100 (European plum) US. Patent Jul. 5, 2011 Sheet 10 0111 US 7,973,216 B2

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m0 35S-MdMYB1U 0 Control 5 s13"91o11121s141s1e171a1s2o Retention Time (minutes) US 7,973,216 B2 1 2 COMPOSITIONS AND METHODS FOR ity and correlated pigmentation increases in immature fruit MODULATING PIGMENT PRODUCTION IN and then again at ripening Which appears to depend on the PLANTS cultivar. Studies shoW that there is highly speci?c regulation of CROSS-REFERENCE TO RELATED genes in the anthocyanin pathWay by speci?c binding of APPLICATIONS transcription factors (TFs) as complexes With promoter ele ments (Holton and Cornish, 1995, Plant Cell 7, 1071-1083). This application is a United States national stage applica This regulation may also extend to non-pathWay genes such tion under 35 USC §371 of International Application No. as anthocyanin transport proteins. PCT/NZ2006/000221, ?led Aug. 31, 2006, Which claims MYB TFs have been shoWn to play an important role in bene?t of NeW Zealand Patent Application No. 542110 ?led transcriptional regulation of anthocyanins. Plant MYBs have Aug. 30, 2005; both of Which are hereby incorporated by been implicated in controlling pathWays as diverse as second reference in their entireties to the extent not inconsistent With ary metabolism (including the anthocyanin pathWay), devel the disclosure herein. opment, signal transduction and disease resistance (Jin and Martin, 1999, Plant Mol Biol, 41, 577-585). They are char TECHNICAL FIELD acterised by a structurally conserved DNA binding domain consisting of single or multiple imperfect repeats; those asso The present invention is in the ?eld of pigment develop ciated With the anthocyanin pathWay tend to the tWo-repeat ment in plants. (R2R3) class. Regulation can also be speci?c to discreet 20 groups of genes, either early or late in the anthocyanin bio BACKGROUND ART synthetic pathWay. In the ofperilla, Perilla?’uilescens, TF-driven regulation has been observed in virtually all stages The accumulation of anthocyanin pigments is an important of anthocyanin biosynthesis from CHS to the resultant antho determinant of fruit quality. Pigments provide essential cul cyanin protein transport genes Whilst in grape, I/nis vinifera, tivar differentiation for consumers and are implicated in the 25 speci?c regulation by MybA is restricted to the end-point of health attributes of apple fruit (Boyer and Liu, 2004). protein production (UFGT). Anthocyanin pigments belong to the diverse group of ubiq There are approximately 140 R3 MYB TFs inArabidopsis, uitous secondary metabolites, collectively knoWn as ?a divided into 24 sub groups (Stracke et al. 2001, Current vonoids. In plants, ?avonoids are implicated in numerous Opinion in Plant Biology, 4, 447-556). The Production of biological functions, including defence, Whilst the pigmented 30 Anthocyanin Pigment 1 (PAP1) MYB (BorevitZ et al., 2000, anthocyanin compounds in particular play a vital physiologi Plant Cell, 12, 2383-2394) falls into subgroup 10 (When the cal role as attractants in plant/animal interactions. phylogeny of Stracke et al., 2001 is used) and demonstrates a The predominant precursors for all ?avonoids, including high degree of amino acid conservation With other knoWn anthocyanins, are malonyl-CoA and p-coumaroyl-CoA. anthocyanin regulators. When PAP1 Was overexpressed in From these precursors the enzyme chalcone synthase (CHS) 35 transgenic Arabidopsis this led to up-regulation of a number forms chalcone, the ?rst committed step toWards anthocyanin of genes in the anthocyanin biosynthesis pathWay from PAL production and the establishment of the C 15 backbone. Chal to CHS and DFR (BorevitZ et al., 2000, Plant Cell, 12, 2383 cone is then isomerised by chalcone isomerase (CHI) to pro 2394; Tohge et al., 2005, Plant Journal, 42, 218-235). duce chalcone and from there a hydroxylation step In general MYBs interact closely With basic Helix Loop via ?avanone 3[3-hydroxylase (F3H) converts naringenin to 40 Helix TFs (bHLH), and this has been extensively studied in dihydro?avonol. Reduction of dihydro?avonon by dihy relation to the production of ?avonoids (Mol et al., 1996; dro?avolon 4-reductase (DFR) produces leucoanthocyanin Winkel-Shirley, 2001). Examples include the maiZe ZmC Which is converted into the coloured compound anthocyan MYB and ZmB bHLH and the petunia AN2 MYB and AN1/ indin by leucoanthocyanidin dioxygenase (LDOX) Whilst the JAF13 bHLHs (Goffet al., 1992 Genes Dev, 6, 864-875; Mol ?nal glycosylation step is mediated by uridin diphosphate 45 et al., 1998, Trends in Plant Science, 3, 212-217). Evidently (UDP)-glucose:?avonoid 3-0-glucosyltransferase (UFGT). there is a degree of conservation, in different species, for this The difference in anthocyanin colour can be due to a number co-ordination. HoWever, a MYB-bHLH partnership is not of factors including the molecular structure and the type and alWays necessary. Results from the overexpression of PAP1 number of hydroxyl groups, sugars and acids attached and the suggested that, like the MaiZe P MYB (GroteWold et al., 2000 cellular environment such as pH or ultrastructure. Of the 50 Proc Natl Acad Sci USA, 97, 13579-13584) and Arabidopsis many anthocyanin pigments it is cyanidin, in the form of MYB12 (Mehrtens et al., 2005 Plant Physiology, 138, 1083 cyanidin 3-0-galactoside, Which is primarily responsible for 1096), PAP1 did not require an over-expressed bHLH co the red colouration in apple skin and the enZymes in this regulator to drive a massive increase in anthocyanin produc biosynthetic pathWay for apple have been Well described tion. HoWever, further studies shoWed that PAP1 does interact (Kim et al., 2003, Plant Science 165, 403-413; Honda et al., 55 closely With bHLHs leading to stronger promoter (DFR) acti 2002, Plant Physiology and Biochemistry 40, 955-962). It has vation in in vivo assays (Zimmermann et al., 2004 Plant J, 40, long been ob served that anthocyanins are elevated in response 22-34). More recently, integrated transcriptome and metabo to particular environmental, developmental and pathogenic lome analysis of PAP1 over-expressing lines con?rmed PAP1 stimuli. Research into apple fruit has demonstrated both the upregulates the bHLH TT8 (At4g09820) by 18-fold (Tohge et environmental and developmental regulation of anthocyanin 60 al., 2005, Plant J, 42, 218-235). This dependency on a co accumulation. Pigment biosynthesis can be induced When regulator is linked to a small number of amino acid changes in fruit are subjected to White light, or more signi?cantly, UV the highly conserved R2R3 binding domain as evident in the light, a phenomenon also observed in other species. Further comparison betWeen the bHLH independent maiZe P and the more, anthocyanin levels can be elevated by cold temperature bHLH dependent maiZe C1 MYBs, and is suf?cient to direct storage of the fruit. There is evidence for the coordinate 65 activation of distinct sets of target genes (GroteWold et al., induction of anthocyanin enZymes in a developmental man 2000, Proc Natl Acad Sci USA, 97, 13579-13584). In this ner in apple fruit With pronounced anthocyanin enzyme activ study substitution of just six amino acids from the R2R3 US 7,973,216 B2 3 4 domain of C1 into the corresponding positions in P1 resulted c) the complement of the sequence of a) in a mutant With bHLH-dependent behaviour similar to C1. d) the complement of the sequence of b) More recently it Was suggested that this may be a key mecha e) a sequence, of at least 15 nucleotides in length, capable of nism Which permits MYBs to discriminate betWeen target hybridising to the sequence of genes (Hernandez et al., 2004, J. Biol. CHem, 279, 48205 a) under stringent conditions. 48213). These key amino acids are marked on FIG. 1. In In a further embodiment the polypeptide has at least 65% contrast to PAP1, FaMYBl, represses anthocyanin biosyn identity to the amino acid sequence of SEQ ID NO: 1. Pref thesis during the late development of strawberry fruit. Despite erably polypeptide has the amino acid sequence of SEQ ID this alternative role FaMYBl shares homology With activa NO: 1. tion MYBs and can interact With (activation) bHLHs such as In a further embodiment the polypeptide has at least 65% the Petunia AN1 and JAF13 (Aharoni etal., 2001, Plant], 28, identity to the amino acid sequence of SEQ ID NO: 2. Pref 319-332). Despite key residues being the same for PAP-like erably the polypeptide has the amino acid sequence of SEQ activators and FaMYB-like repressors, activators tend to fall ID NO: 2. in subgroup 10 While repressors fall in subgroup 17 (accord In a further embodiment the polypeptide has at least 65% ing to Stracke et al.). identity to the amino acid sequence of SEQ ID NO: 3. Pref An additional level of anthocyanin regulation involves a erably the polypeptide has the amino acid sequence of SEQ separate class of proteins, containing WD40 domains, Which ID NO: 3. form complexes With MYB and bHLH proteins (as revieWed In a further embodiment the polypeptide has at least 65% in Ramsay and Glover, 2005, Trends in Plant Science, 10, identity to the amino acid sequence of SEQ ID NO: 4. Pref 63-70). Examples include an11 in petunia (de Vetten et al., 20 erably the polypeptide has the amino acid sequence of SEQ 1997 Genes Dev, 11, 1422-1434) and TTG1 in Arabidopsis ID NO: 4. (Walker et al., 1999, Plant Cell, 11, 1337-1350). The tran In a further embodiment the sequence in a) has at least 70% scriptional control of anthocyanins may be further compli identity to the sequence of any one of SEQ ID NO: 5-8, 22-47 cated by tissue speci?c regulation (Kubo et al., 1999, Plant and 102. Preferably the sequence in a) has at least 70% Cell, 11, 1217-1226) and possibly different layers of regula 25 identity to the coding sequence of any one of SEQ ID NO: tion dependent on stimuli such as cold, light and developmen 5-8, 22-47 and 102. tal cues (Davuluri et al., 2005, Nature Biotechnology, 23, In a further embodiment the sequence in a) has at least 70% 890-895). identity to the sequence of SEQ ID NO: 5. Preferably the Although studies into the activation and repression of sequence in a) has at least 70% identity to the coding anthocyanin synthesis in apple fruit have shoWn developmen 30 sequence of SEQ ID NO: 5. More preferably the sequence in tal and environmental regulation, to date transcription factors a) has the sequence of SEQ ID NO: 5. More preferably the regulating anthocyanin synthesis have not been identi?ed in sequence in a) has the coding sequence of SEQ ID NO: 5. this species or any other fruit. The control of antho In a further embodiment the sequence in a) has at least 70% cyanin accumulation in apple is a key question in understand identity to the sequence of SEQ ID NO: 6. Preferably the ing and manipulating fruit colour. Identi?cation of the factors 35 sequence in a) has at least 70% identity to the coding that exert this control provides tools for moderating the extent sequence of SEQ ID NO: 6. More preferably the sequence in and distribution of anthocyanin-derived pigmentation in fruit a) has the sequence of SEQ ID NO: 6. More preferably the tissue. sequence in a) has the coding sequence of SEQ ID NO: 6. It is therefore an object of the invention to provide tran In a further embodiment the sequence in a) has at least 70% scription factor sequences Which regulate anthocyanin pro 40 identity to the sequence of SEQ ID NO: 7. Preferably the duction in apple species and/ or at least to provide the public sequence in a) has at least 70% identity to the coding With a useful choice. sequence of SEQ ID NO: 7. More preferably the sequence in a) has the sequence of SEQ ID NO: 7. More preferably the SUMMARY OF THE INVENTION sequence in a) has the coding sequence of SEQ ID NO: 7. 45 In a further embodiment the sequence in a) has at least 70% In the ?rst aspect the invention provides an isolated poly identity to the sequence of SEQ ID NO: 8. Preferably the nucleotide comprising sequence in a) has at least 70% identity to the coding a) a sequence encoding a polypeptide With any one of the sequence of SEQ ID NO: 8. More preferably the sequence in amino acid sequences of SEQ ID NO: 1-4 and 9-21 or a a) has the sequence of SEQ ID NO: 8. More preferably the variant thereof, Wherein the polypeptide or variant thereof 50 sequence in a) has the coding sequence of SEQ ID NO: 8. is a transcription factor capable of regulating anthocyanin In a further aspect the invention provides an isolated poly production in a plant; nucleotide comprising: b) a fragment, of at least 15 nucleotides in length, of the a) a sequence With at least 70% identity to any one of the sequence of a); nucleotide sequences of SEQ ID NO: 5-8, 22-47 and 102, c) the complement of the sequence of a) 55 Wherein the sequence encodes a transcription factor d) the complement of the sequence of b) capable of regulating anthocyanin production in a plant; e) a sequence, of at least 15 nucleotides in length, capable of b) a fragment, of at least 15 nucleotides in length, of the hybridising to the sequence of sequence of a); a) under stringent conditions. c) the complement of the sequence of a) In one embodiment the isolated polynucleotide comprises 60 d) the complement of the sequence of b) a) a sequence encoding a polypeptide With at least 65% iden e) a sequence, of at least 15 nucleotides in length, capable of tity to any one of the amino acid sequences of SEQ ID NO: hybridising to the sequence of 1-4 and 9-21, Wherein the polypeptide is a transcription a) under stringent conditions. factor capable of regulating anthocyanin production in a In one embodiment the sequence in a) has at least 70% plant; 65 identity to the sequence of SEQ ID NO: 5. Preferably the b) a fragment, of at least 15 nucleotides in length, of the sequence in a) has at least 70% identity to the coding sequence of a); sequence of SEQ ID NO: 5. More preferably the sequence in US 7,973,216 B2 5 6 a) has the sequence of SEQ ID NO: 5. More preferably the In a further aspect the invention provides an isolated poly sequence in a) has the coding sequence of SEQ ID NO: 5. nucleotide comprising: In one embodiment the sequence in a) has at least 70% a) a sequence encoding a polypeptide variant any one of the identity to the sequence of SEQ ID NO: 6. Preferably the amino acid sequences of SEQ ID NO: 1-4 and 9-21, sequence in a) has at least 70% identity to the coding Wherein the polypeptide is a transcription factor capable of sequence of SEQ ID NO: 6. More preferably the sequence in regulating anthocyanin production in a plant, and Wherein a) has the sequence of SEQ ID NO: 6. More preferably the the polypeptide comprises the sequence of SEQ ID NO: sequence in a) has the coding sequence of SEQ ID NO: 6. 101; In one embodiment the sequence in a) has at least 70% b) a fragment, of at least 15 nucleotides in length, of the identity to the sequence of SEQ ID NO: 7. Preferably the sequence of a); sequence in a) has at least 70% identity to the coding c) the complement of the sequence of a) sequence of SEQ ID NO: 7. More preferably the sequence in a) has the sequence of SEQ ID NO: 7. More preferably the d) the complement of the sequence of b) sequence in a) has the coding sequence of SEQ ID NO: 7. e) a sequence, of at least 15 nucleotides in length, capable of In one embodiment the sequence in a) has at least 70% hybridising to the sequence of identity to the sequence of SEQ ID NO: 8. Preferably the a) under stringent conditions. sequence in a) has at least 70% identity to the coding Preferably the variant polypeptide is derived from a sequence of SEQ ID NO: 8. More preferably the sequence in species. a) has the sequence of SEQ ID NO: 8. More preferably the In a further aspect the invention provides an isolated sequence in a) has the coding sequence of SEQ ID NO: 8. 20 polypeptide comprising: In the further aspect the invention provides an isolated a) a sequence With at least 65% identity to an amino acid polynucleotide having at least 70% sequence identity to a sequence selected from any one of SEQ ID NO: 1-4 and nucleotide sequence that encodes a polypeptide comprising 9-21, Wherein the polypeptide is a transcription factor an amino acid sequence selected from any one of SEQ ID NO: capable of regulating anthocyanin production in a plant; or 1 to 4 and 9 to 21, Wherein the polynucleotide encodes a 25 b) a fragment, of at least 5 amino acids in length, of the transcription factor capable of regulating anthocyanin pro sequence of a) duction in a plant. In one embodiment the sequence in a) has at least 65% In one embodiment the isolated polynucleotide has at least sequence identity to the amino acid sequence of SEQ ID NO: 70% sequence identity to a nucleotide sequence that encodes 1. Preferably the sequence in a) has the sequence of SEQ ID a polypeptide comprising the amino acid sequence of SEQ ID 30 NO: 1. NO.1. In one embodiment the sequence in a) has at least 65% In a further embodiment the nucleotide sequence com sequence identity to the amino acid sequence of SEQ ID NO: prises the nucleotide sequence of SEQ ID NO: 5. Preferably the nucleotide sequence comprises the coding sequence from 2. Preferably the sequence in a) has the sequence of SEQ ID NO: 2. SEQ ID NO: 5. 35 In a further aspect the invention providing an isolated poly In one embodiment the sequence in a) has at least 65% nucleotide comprising sequence identity to the amino acid sequence of SEQ ID NO: a) a sequence encoding a polypeptide With at least 65% iden 3. Preferably the sequence in a) has the sequence of SEQ ID tity to any one of the amino acid sequences of SEQ ID NO: NO: 3. 1-4 and 9-21, Wherein the polypeptide is a transcription 40 In one embodiment the sequence in a) has at least 65% factor capable of regulating the promoter of a gene in the sequence identity to the amino acid sequence of SEQ ID NO: anthocyanin bio synthetic pathWay; 4. Preferably the sequence in a) has the sequence of SEQ ID b) a fragment, of at least 15 nucleotides in length, of the NO: 4. sequence of a); In a further aspect the invention provides a polynucleotide c) the complement of the sequence of a) 45 encoding a polypeptide of the invention. d) the complement of the sequence of b) In a further aspect the invention provides an antibody e) a sequence, of at least 15 nucleotides in length, capable of raised against a polypeptide of the invention. hybridising to the sequence of In a further aspect the invention provides a genetic con a) under stringent conditions. struct comprising a polynucleotide of any one of the inven In a further aspect the invention provides an isolated poly 50 tion. nucleotide comprising: In a further aspect the invention provides a host cell com a) a sequence With at least 70% identity to any one of the prising a genetic construct of the invention. nucleotide sequences of SEQ ID NO: 5-8, 22-47 and 102, In a further aspect the invention provides a host cell geneti Wherein the sequence encodes a transcription factor cally modi?ed to express a polynucleotide of any one of the capable of regulating the promoter of a gene in the antho 55 cyanin bio synthetic pathWay; invention. b) a fragment, of at least 15 nucleotides in length, of the In a further aspect the invention provides a plant cell com sequence of a); prising the genetic construct of the invention. c) the complement of the sequence of a) In a further aspect the invention provides a plant cell d) the complement of the sequence of b) 60 genetically modi?ed to express a polynucleotide of the inven e) a sequence, of at least 15 nucleotides in length, capable of tion. hybridising to the sequence of In a further aspect the invention provides a plant Which a) under stringent conditions. comprises the plant cell of the invention. In one embodiment the gene to be regulated encodes dihy In a further aspect the invention provides a method for dro?avolon 4-reductase (DER). 65 producing a polypeptide of the invention, the method com In an alternative embodiment the gene to be regulated prising the step of culturing a host cell comprising an a encodes chalcone synthase (CHS). genetic construct of the invention. US 7,973,216 B2 7 8 In a further aspect the invention provides a plant cell or Preferably the transcription factors and variants of the plant With altered anthocyanin production, the method com invention, that are capable of regulating anthocyanin produc prising the step of transformation of a plant cell or plant With tion in plants, are capable of regulating the production of the a genetic construct including: anthocyanins selected from the group including but not lim a) at least one polynucleotide encoding of a polypeptide of the ited to: cyanidin-3-glucoside, cyanidin-3-0-rutinoside, cya invention; nadin-3-glucoside and cyanadin-3-pentoside. b) at least one polynucleotide comprising a fragment, of at Preferably the plants or plant cells With altered production least 15 nucleotides in length, of the polynucleotide of a); of anthocyanins, produced by or selected by the methods of or the invention, are altered in production of anthocyanins c) at least one polynucleotide comprising a complement, of at selected from the group including but not limited to: cyana least 15 nucleotides in length, of the polynucleotide of a). din-3-glucosidase, cyaniding-3-0-rutinoside, cyanadin-3 In a further aspect the invention provides a method of producing a plant cell or plant With altered anthocyanin pro glucoside and cyanadin-3 -pentoside. duction, the method comprising the step of transforming a The polynucleotides and polynucleotide variants, of the plant cell or plant With a genetic construct including: invention may be derived from any species or may be pro a) at least one of the polynucleotides of any one of the inven duced by recombinant or synthetic means. tion; In one embodiment the polynucleotide or variant, is b) at least one polynucleotide comprising a fragment, of at derived from a plant species. least 15 nucleotides in length, of the polynucleotide of a), In a further embodiment the polynucleotide or variant, is or 20 derived from a gymnosperm plant species. c) at least one polynucleotide comprising a complement, of at In a further embodiment the polynucleotide or variant, is least 15 nucleotides in length, of the polynucleotide of a) derived from an angiosperm plant species. d) at least one polynucleotide capable of hybridising under In a further embodiment the polynucleotide or variant, is stringent conditions to the polynucleotide of a) or b). derived from a from dicotyledonous plant species. In one embodiment of the method, the construct is 25 The polypeptides and polypeptide variants of the invention designed to express a pair of transcription factors, and the may be derived from any species, or may be produced by construct comprises: recombinant or synthetic means. i) a polynucleotide sequence encoding a MYB transcription In one embodiment the polypeptides or variants of the factor With at least 65% identity to the amino acid sequence invention are derived from plant species. ofany one of SEQ ID NO: 1,2 and 9 to 21; and 30 In a further embodiment the polypeptides or variants of the ii) a polynucleotide sequence encoding a bHLH transcription invention are derived from gymnosperm plant species. factor With at least 65% identity to the amino acid sequence In a further embodiment the polypeptides or variants of the of SEQ ID NO: 1 or 2. invention are derived from angiosperm plant species. In a further embodiment the polynucleotide sequence in i) In a further embodiment the polypeptides or variants of the has at least 70% sequence identity to the nucleotide sequence 35 invention are derived from dicotyledonous plant species. of any one of SEQ ID NO: 5, 6, 22 to 27 and 102; and the In a further embodiment polypeptide or variant is derived polynucleotide sequence in ii) has at least 70% sequence from a monocotyledonous plant species. identity to the nucleotide sequence of SEQ ID NO: 7 or 8. The plant cells and plants of the invention may be from any In a further embodiment the polynucleotide sequence in i) species. has at least 70% sequence identity to the nucleotide sequence 40 In one embodiment the plants cells and plants of the inven of any one of SEQ ID NO: 5, 6, 22 to 27 and 102; and the tion are from gymnosperm species. coding sequence in ii) has at least 70% sequence identity to In a further embodiment the plants cells and plants of the the coding sequence of SEQ ID NO: 7 or 8. invention are from angiosperm species. In a further aspect the invention provides a plant produced In a further embodiment the plants cells and plants of the by the method of the invention. 45 invention are from dicotyledonous species. In a further aspect the invention provides a method for In a further embodiment the plants cells and plants of the selecting a plant altered in anthocyanin production, the invention are from monocotyledonous species. method comprising testing of a plant for altered expression of Preferred plant species (for the polynucleotide and vari a polynucleotide of the invention. ants, polypeptides and variants and plant cells and plants of In a further aspect the invention provides a method for 50 the invention) include fruit plant species selected from a selecting a plant altered in anthocyanin production, the group comprising but not limited to the folloWing genera: method comprising testing of a plant for altered expression of Malus, Pyrus Prunis, , Rosa, Fragaria, Aclinidia, a polypeptide of the invention. Cydonia, Citrus, and Vaccinium. In a further aspect the invention provides a plant selected Particularly preferred fruit plant species are: Malus domes by the method of the invention. 55 Zica, Aclidinia deliciosa, A. chinensis, A. erianlha, A. argula In a further aspect the invention provides a method for and hybrids of the four Aclinidia species, Prunis persica selecting a plant cell or plant that has been transformed, the Pyrus L., Rubus, Rosa, and Fragaria. method comprising the steps Preferred plants (for the polynucleotide and variants, a) transforming a plant cell or plant With a polynucleotide or polypeptides and variants and plant cells and plants of the polypeptide of the invention capable of regulating antho 60 invention) also include vegetable plant species selected from cyanin production in a plant; a group comprising but not limited to the folloWing genera: b) expressing the polynucleotide or polypeptide in the plant Brassica, Lycopersicon and Solanum, cell or plant; and Particularly preferred vegetable plant species are: Lycoper c) selecting a plant cell or plant With increased anthocyanin sicon esculenlum and Solanum luberosum pigmentation relative to other plant cells or plants, the 65 Preferred plants (for the polynucleotide and variants, increased anthocyanin pigmentation indicating that the polypeptides and variants and plant cells and plants of the plant cell or plant has been transformed. invention) also include crop plant species selected from a US 7,973,216 B2 10 group comprising but not limited to the following genera: lucida, nanluckelensis, Amelanchier pumila, Glycine, Zea, Hordeum and Oryza. Amelanchier quinZi-marli, Amelanchier sanguinea, Amelan Particularly preferred crop plant species include Glycine chier slolonifera, Amelanchier ulahensis, Amelanchier wie max, Zea mays and Oryza saliva. gandii, Amelanchier X neglecla, Amelanchier barlramiana X Preferred plants (for the polynucleotide and Variants, Amelanchier sp. rdemala’, Amelanchier sp. rdentala’, polypeptides and Variants and plant cells and plants of the Amelanchier sp. rerecla ’, Amelanchier sp. rerecZa’XAmelan invention) also include those of the Rosaceae family. chier laevis, Amelanchier sp. rserolina ’, Aria alnifolia, Aro Preferred Rosaceae genera include Exochorda, Maddenia, nia prunifolia, Chaenomeles calhayensis, Chaenomeles spe Oeinleria, Osmaronia, Prinsepia, , Maloideae, ci0sa, Chamaemespilus alpina, Cormus domeslica, Amelanchier, Aria, Aronia, Chaenomeles, Chamaemespilus, Coloneasler apiculalus, Coloneasler lacleus, Coloneasler Cormus, Coloneasler, Cralaegus Osmaronia, Prinsepia, Pru pannosus, Cralaegus azarolus, Cralaegus columbiana, Cra nus, Maloideae, Amelanchier, Aria, Aronia, Chaenomeles, Zaegus crus-galli, Ciralaegus curvisepala, Cralaegus laevi Chamaemespilus, Cormus, Coloneasler, Cralaegu, Cydonia, gala, Cralaegus mollis, Cralaegus monogyna, Cialaegus Dicholomanlhes, Docynia, Docyniopsis, Eriobolrya, Eriolo niga, Cralaegus rivularis, Cralaegus sinaica, Cydonia bus, Heleromeles, Kageneckia, Lindleya, Malacomeles, oblonga, Dicholomanlhes Zrislaniicarpa, Docynia delavayi, Malus, Mespilus, Osleomeles, Peraphyllum, Pholinia, Docyniopsis Zschonoskii, Eriobolrya japonica, Eriobolrya Pseudocydonia, Pyracanlha, Pyrus, Rhaphiolepis, Sorbus, prinoides, Eriolobus lrilobalus, Heleromeles arbulifolia, Slranvaesia, Torminalis, Vauquelinia, , , Kageneckia anguslifolia, Kageneckia oblonga, Lindleya Acomaslylis, , , Aphanes, Aremonia, mespiloides, Malacomeles denliculala, Malus anguslifolia, Bencomia, Chamaebalia, Cli?'brlia, Coluria, Cowania, Dali 20 Malus asialica, Malus baccala, Malus coronaria, Malus barda, Dendriopolerium, Dryas, Duchesnea, Erylhrocoma, doumeri, Malus?orenlina, Malus?oribunda, Malus fusca, Fallugia, Filipendula, Fragaria, , Hagenia, Horkelia, Malus halliana, Malus honanensis, Malus hupehensis, Malus Ivesia, Kerria, Leucosidea, Marcelella, Margyricarpus, ioensis, Malus kansuensis, Malus mandshurica, Malus Novosieversia, Oncoslylus, , Polenlilla, Rosa, micromalus, Malus niedzwelzkyana, Malus ombrophilia, Rubus, , Sarcopolerium, Sibbaldia, Sieversia, 25 Malus orienlalis, Malus prallii, Malus prunifolia, Malus Taihangia, Telraglochin, Waldsleinia, Rosaceae incerlae pumila, Malus sargenlii, Malus sieboldii, Malus sieversii, sedis, Adenosloma, Aruncus, Cercocarpus, Chamaebaliaria, Malus sylveslris, Malus Zoringoides, Malus lransiloria, Chamaerhodos, Gillenia, Holodiscus, Lyonolhamnus, Neil Malus lrilobala, Malus Zschonoskii, Malus X domeslica, lia, Neviusia, , Purshia, Rhodolypos, Sorbaria, Malus X domeslica X Malus sieversii, Malus X domeslica X and Slephanandra. 30 Pyrus communis, Malus xiaojinensis, Malus yunnanensis, Preferred Rosaceae species include Exochorda giraldii, Malus sp., Mespilus germanica, Osleomeles anlhyllidifolia, Exochorda racemosa, Exochorda, Exochorda giraldii, Exo Osteomeles schwerinae, Peraphyllum ramosissimum, Pho chorda racemosa, Exochorda serralifolia, Maddenia hypo Zinia?’aseri, Pholinia pyrifolia, Pholinia serrulala, Pholinia leuca, Oemleria cerasiformis, Osmaronia cerasiformis, Prin villosa, Pseudocydonia sinensis, Pyracanlha coccinea, Pyra sepia sinensis, Prinsepia uni?ora, Prunus alleghaniensis, 35 canlha forluneana, Pyrus calleryana, Pyrus caucasica, , Prunus andersonii, Prunus anguslifolia, Pyrus communis, Pyrus elaeagrifolia, Pyrus hybrid cullivar, Prunus apelala, Prunus argenlea, , Prunus Pyruspyrifolia, Pyrus salicifolia, Pyrus ussuriensis, Pyrus X avium, Prunus bi?’ons, Prunus briganlina, Prunus bucha brelschneideri, Rhaphiolepis indica, Sorbus americana, Sor rica, Prunus buergeriana, Prunus campanulala, Prunus bus aria, Sorbus aucuparia, Sorbus cal ifornica, Sorbus com caroliniana, , Prunus cerasus, Prunus 40 mixla, Sorbus hupehensis, Sorbus scopulina, Sorbus sibirica, choreiana, Prunus cocomilia, Prunus cyclamina, Prunus Sorbus lorminalis, Slranvaesia davidiana, Torminalis clusii, davidiana, Prunus debilis, Prunus domeslica, Prunus dulcis, Vauquelinia californica, Vauquelinia corymbosa, Acaena Prunus emarginala, Prunusfasciculala, Prunusferganensis, anserinifolia, Acaena argenlea, Acaena caesiiglauca, Prunusfordiana, Prunus?’einonlii, Prunusfrulicosa, Prunus Acaena cylindrislachya, Acaena digilala, Acaena echinala, geniculala, Prunus glandulosa, Prunus gracilis, Prunus 45 Acaena elongala, Acaena eupaloria, Acaena ?ssislipula, grayana, Prunus horlulana, Prunus ilicifolia, , Acaena inermis, Acaena laevigala, Acaena lalebrosa, Prunus jacquemonlii, Prunus japonica, Prunus kuramica, Acaena lucida, Acaena macrocephala, Acaena magellanica, Prunus laurocerasus, Prunus leveilleana, Prunus lusilanica, Acaena masafuerana, Acaena monlana, Acaena mulli?da, Prunus maackii, Prunus mahaleb, Prunus mandshurica, Pru Acaena novaezelandiae, Acaena ovalifolia, Acaena pinnali nus marilima, Prunus maximowiczii, Prunus mexicana, Pru 50 ?da, Acaena splendens, Acaena subincisa, Acaena X anse nus microcarpa, Prunus mira, Prunus mume, Prunus munso rovina, Acomaslylis elala, Acomaslylis r0ssii, Acomaslylis niana, Prunus nigra, , Prunus padus, sikkimensis, Agrimonia eupaloria, Agrimonia nipponica, Prunus pensylvanica, Prunus persica, Prunus pelunnikowii, Agrimonia parvi?ora, Agrimonia pilosa, Alchemilla alpina, Prunus proslrala, Prunus pseudocerasus, Prunus pumila, Alchemilla erylhropoda, Alchem illa japonica, Alchemilla , , Prunus sargenlii, Prunus 55 mollis, Alchemilla vulgaris, Aphanes arvensis, Aremonia sellowii, Prunus serolina, Prunus serrulala, , agrimonioides, Bencomia brachyslachya, Bencomia cau Prunus simonii, Prunus spinosa, Prunus spinulosa, Prunus dala, Bencomia exslipulala, Bencomia sphaerocarpa, subcordala, Prunus subhirlella, Prunus Zakesimensis, Pru Chamaebalia f0li0l0sa, Cliforlia burmeana, Cli?'brlia nus Zenella, Prunus Zexana, Prunus lomenlosa, Prunus cuneala, Cli?'brlia denlala, Cli?'brlia graminea, Cli?'brlia Zschonoskii, Prunus umbellala, Prunus verecunda, Prunus 60 helerophylla, Cliforlia nilidula, Cliforlia odorala, Cli?'brlia virginiana, Prunus webbii, Prunus X yedoensis, Prunus Zip ruscifolia, Cli?'brlia sericea, Coluria elegans, Coluria peliana, Prunus sp. BSP-2004-l, Prunus sp. BSP-2004-2, geoides, Cowania slansburiana, Dalibarda repens, Dendrio Prunus sp. EB-2002, , Amelanchier poleium menendezii, Dendriopolerium pulidoi, Dryas drum arborea, Amelanchier asialica, Amelanchier barlramiana, mondii, Dryas oclopelala, Duchesnea chrysanlha, Duch Amelanchier canadensis, Amelanchier cusickii, Amelanchier 65 esnea indica, Erylhrocoma Zri?'ora, Fallugia paradoxa, fernaldii, Amelanchier ?orida, Amelanchier humilis, Filipendula mullijuga Filipendula purpurea, Filipendula Amelanchier inlermedia, Amelanchier laevis, Amelanchier ulmaria, Filipendula vulgaris, Fragaria chiloensis, Fragaria US 7,973,216 B2 11 12 dalloniana, Fragaria gracilis, Fragaria grandi?ora, Fia minusculus, Rubus moorei, Rubus mullibraclealus, Rubus garia iinumae, Fragaria moschala, Fragaria nilgerrensis, neomexicanus, Rubus nepalensis, Rubus nessensis, Rubus Fragaria nipponica, Fragaria nubicola, Fragaria orienlalis, nivalis, , Rubus nubigenus, Rubus occidenlalis, Fragaria penlaphylla, Fragaria vesca, Fragaria virginiana, Rubus odoralus, Rubus palmalus, Rubus parvi?orus, Rubus Fragaria viridis, Fragaria X ananassa, Fragaria sp. CFRA parvifolius, Rubusparvus, Rubuspeclinellus, Rubaspedalus, 538, Fragaria sp., Geum andicola, Geum borisi, Geum bul Rubaspedemonlanus, Rubuspensilvanicus, Rubusphoenico garicum, Geum callhifolium, Geum chiloense, Geum genicu lasius, Rubus piclicaulis, Rubus pubescens, Rubus rigidus, lalum, Geum helerocarpum, Geum macrophyllum, Geum Rubus robuslus, Rubus roseus, Rubus rosifolius, Rubus sanc monlanum, Geum replans, Geum rivale, Geum scho?eldii, Zus, Rubus sapidus, Rubus saxalilis, Rubus selosus, Rubus Geum speciosum, Geum urbanum, Geum vernum, Geum sp. speclabilis, Rubus sulcalus, Rubus Zephrodes, Rubus trian rChase 2507 K’, Hagenia abyssinica, Horkelia cuneala, Hor keliafusca, Ivesia gordoni, Kerriajaponica, Leucosidea seri Zhus, Rubus tricolor, Rubus Zri?dus, Rubus Zrilobus, Rubus cea, Marcelella maderensis, Marcelella moquiniana, Margy Zrivialis, Rubus ulmifolius, Rubus ursinus, Rubus urlicifolius, ricarpus pinnalus, Margyricarpus selosus, Novosieversia Rubus vigorosus, Rubus sp. JPM-2004, Sanguisorba albi glacialis, Oncoslylus cockaynei, Oncoslylus leiospermus, ?ora, Sanguisorba alpina, Sanguisorba ancislroides, San Polylepis auslralis, Polylepis besseri, Polylepis crisZa-galli, guisorba annua, Sanguisorba canadensis, Sanguisorba?li Polylepis hieronymi, Polylepis incana, Polylepis lanuginosa, formis, Sanguisorba hakusanensis, Sanguisorbajaponensis, Polylepis mullijuga, Polylepis neglecla, Polylepis paula, Sanguisorba minor, Sanguisorba oblusa, Sanguisorba o?ici Polylepis pepei, Polylepis quadrijuga, , nalis, Sanguisorba parvi?ora, Sanguisorba slipulala, San Polylepis reliculala, Polylepis rugulosa, Polylepis sericea, 20 guisorba Zenuifolia, Sarcopolerium spinosum, Sibbaldia Polylepis subsericans, Polylepis Zarapacana, Polylepis procumbens, Sieversia penlapelala, Sieversia pusilla, Zomenlella, Polylepis weberbaueri, Polenlilla anserina, Taihangia rupeslris, Telraglochin crislalum, Waldsleinia Polenlilla argula, Polenlilla bifurca, Polenlilla chinensis, fragarioides, Waldsleinia geoides, Adenoslomafasciculalum, Polenlilla dickinsii, Polenlilla erecla, Polenlillafragarioides, Adenosloma sparsifolium, Aruncus dioicus, Cercocarpus Polenlilla frulicosa, Polenlilla indica, Polenlilla micranlha, 25 beluloides, Cercocarpus ledifolius, Chamaebaliaria millefo Polenlilla mulli?da, Polenlilla nivea, Polenlilla norvegica, lium, Chamaerhodos erecla, Gillenia slipulala, Gillenia Zri Polenlilla paluslris, Polenlilla peduncularis, Polenlilla rep foliala, , Holodiscus microphyllus, Zans, Polenlilla salesoviana, Polenlilla slenophylla, Poten Lyonolhamnus ?oribundus, Neillia a?inis, Neillia gracilis, Zilla Zridenlala, Rosa abielina, Rosa abyssinica, Rosa acicu Neillia sinensis, Neillia sparsi?ora, Neillia Zhibelica, Neillia laris, Rosa agreslis, Rosa alba, Rosa alba X Rosa 30 Zhyrsi?ora, Neillia uekii, Neviusia alabamensis, Physocarpus corymbifera, Rosa allaica, Rosa arkansana, Rosa arvensis, allernans, Physocarpus amurensis, Physocaipus capilalus, , Rosa beggeriana, Rosa blanda, Rosa brac Physocarpus malvaceus, Physocapus monogynus, Physocar Zeala, Rosa brunonii, Rosa caesia, Rosa californica, Rosa pus opulifolius, Purshia Zridenlala, Rhodolypos scandens, canina, , , Rosa cinnamomea, Sorbaria arborea, Sorbaria sorbifolia, Spiraea belulifolia, Rosa columnifera, Rosa corymbifera, Rosa cymosa, Rosa 35 Spiraea canloniensis, Spiraea densi?ora, Spiraea japonica, davurica, , Rosa ecae, Rosa eglanleria, Rosa Spiraea nipponica, Spiraea X vanhoullei, Spiraea sp. Stepha elliplica, Rosa fedlschenkoana, Rosa foelida, Rosa foliolosa, nandra chinensis, Slephanandra incisa and Slephanandra Rosa gallica, Rosa gallica X Rosa dumelorum, Rosa Zanakae. giganlea, , Rosa helenae, Rosa henryi, Rosa Particularly preferred Rosaceae genera include: Malus, hugonis, Rosa hybrid cultiVar, Rosa inodora, Rosa jundzillii, 40 Pyrus, Cydonia, Prunus, Eriobolrya, and Mespilus. Rosa laevigala, Rosa laxa, Rosa luciae, , Rosa Particularly preferred Rosaceae species include: Malus marrelii, Rosa maximowicziana, Rosa micranlha, Rosa mol domeslica, Malus sylveslris, Pyrus communis, Pyrus pyrifo lis, Rosa monlana, Rosa moschala, Rosa moyesii, Rosa mulli lia, Pyrus brelschneideri, Cydonia oblonga, Prunus salicina, bracleala, Rosa mulli?ora, Rosa nilida, Rosa odorala, Rosa Prunus cerasifera, Prunus persica, Eriobolrya japonica, paluslris, , , Rosa phoenicia, 45 Prunus dulcis, Prunus avium, Mespilus germanica and Pru Rosa plalyacanlha, , Rosa pseudoscabriuscula, nus domeslica. Rosa roxburghii, , Rosa rugosa, Rosa sam More particularly preferred Rosaceae genera include bucina, Rosa sempervirens, Rosa sericea, Rosa serlala, Rosa Malus and Prunus seligera, Rosa sherardii, Rosa sicula, Rosa spinosissima, Particularly preferred Rosaceae species include Malus Rosa slellala, Rosa slylosa, Rosa subcanina, Rosa subcol 50 domeslica and Prunus cerasifera. lina, Rosa su?‘ulla, Rosa Zomenlella, Rosa Zomenlosa, Rosa The term “plant” is intended to include a Whole plant, any Zunquinensis, , Rosa virginiana, Rosa wichurana, part of a plant, propagules and progeny of a plant. Rosa willmolliae, Rosa woodsii; Rosa X damascena, Rosa X The term ‘propagule’ means any part of a plant that may be forluniana, Rosa X macranlha, Rosa xanlhina, Rosa sp. used in reproduction or propagation, either seXual or aseXual, Rubus alceifolius, , Rubus alpinus, 55 including seeds and cuttings. Rubus amphidasys, Rubus arclicus, Rubus argulus, Rubus assamensis, Rubus auslralis, Rubus bifrons, Rubus caesius, DETAILED DESCRIPTION Rubus caesius X Rubus idaeus, Rubus canadensis, Rubus canescens, Rubus caucasicus, Rubus chamaemorus, Rubus In this speci?cation Where reference has been made to corchorifolius, Rubus cralaegifolius, Rubus cuneifolius, 60 patent speci?cations, other eXtemal documents, or other Rubus deliciosus, Rubus divaricalus, Rubus elliplicus, sources of information, this is generally for the purpose of Rubus?agellaris, Rubus frulicosus, Rubus geoides, Rubus providing a conteXt for discussing the features of the inven glabralus, Rubus glaucus, Rubus gunnianus, Rubus hawaien tion. Unless speci?cally stated otherWise, reference to such sis, X Rubus rosifolius, Rubus hispidus, eXtemal documents is not to be construed as an admission that Rubus hochslelleroruni, Rubus humulifolius, Rubus idaeus, 65 such documents, or such sources of information, in any juris Rubus lamberlianus, Rubus lasiococcus, , diction, are prior art, or form part of the common general Rubus linealus, , Rubus maximiformis, Rubus knowledge in the art.