Mildew Resistance Gene in Kalanchoe

Abstract

Provided herein are proteins capable of providing a mildew resistant phenotype, or mildew resistance, to plants belonging to the Kalanchoe genus. Also provided herein are nucleic acid sequences, or cDNA sequences, and genes encoding the present proteins. Further provided herein are methods using the proteins, nucleic acid sequences and genes for selecting mildew resistant Kalanchoe plants, such as Oidium kalanchoeae resistant Kalanchoe plants, and to generate mildew resistant Kalanchoe plants, such as Oidium kalanchoeae resistant Kalanchoe plants, comprising the present proteins, mRNA forms of the present cDNAs or the present genes.

Claims

1.-18. (canceled)

19. A protein comprising the sequence of SEQ ID NO: 1, wherein the sequence comprises one or more amino acid substitutions, deletions, or insertions, and wherein said one or more amino acid substitutions, deletions, or insertions provides mildew resistance in a Kalanchoe plant as compared to a Kalanchoe plant comprising a protein having the sequence of SEQ ID NO: 1.

20. The protein of claim 19, wherein the one or more amino acid substitutions, deletions or insertions are present in one or more of amino acids 1 to 50 of SEQ ID NO: 1.

21. The protein of claim 19, wherein the one or more amino acid substitutions are a substitution at positions 22 and/or 23 of SEQ ID NO: 1.

22. The protein according to claim 19, wherein the one or more amino acid substations are a substitution of the amino acid threonine at position 22 and/or at position 23 of SEQ ID NO: 1.

23. The protein according to claim 19, wherein the one or more amino acid substations are an amino acid substitution of threonine to alanine at position 22 and/or at position 23 of SEQ ID NO: 1.

24. The protein according to claim 19, wherein the protein comprises the sequence of SEQ ID NO: 2.

25. The protein according to claim 19, wherein the protein comprises a motif having the sequence of SEQ ID NO: 7.

26. The protein according to claim 19, wherein the one or more amino acid substitutions, deletions, or insertions provides resistance against the mildew pathogen Oidium kalanchoeae.

27. A nucleic acid encoding the protein of claim 19.

28. The nucleic acid according to claim 27, wherein the nucleic acid comprises the sequence of SEQ ID NO: 3, and wherein the sequence comprises a nucleic acid substitution at position 64 and/or at position 67 of SEQ ID NO: 3.

29. The nucleic acid according to claim 27, wherein said nucleic acid comprises the sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

30. A method for obtaining a Kalanchoe plant that is resistant to mildew, comprising introducing one or more substitutions, deletions, or insertions in a nucleic acid having the sequence of SEQ ID NO: 3 into a Kalanchoe plant, wherein the mutation results in one or more amino acid substitutions, deletions, or insertions in a protein having the sequence of SEQ ID NO: 1 and resistance to mildew.

31. The method according to claim 30, wherein the method comprises introducing a nucleic acid having the sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 into the Kalanchoe plant.

32. The method according to claim 30, wherein the Kalanchoe plant is selected from the group consisting of Kalanchoe adelae, Kalanchoe arborescens, Kalanchoe beauverdii, Kalanchoe beharensis, Kalanchoe bentii, Kalanchoe blossfeldiana, Kalanchoe bouvetii, Kalanchoe bracteata, Kalanchoe campanulata, Kalanchoe crenata, Kalanchoe crundallii, Kalanchoe daigremontiana, Kalanchoe delagoensis, Kalanchoe dinklagei, Kalanchoe eriophylla, Kalanchoe farinacea, Kalanchoe fedtschenkoi, Kalanchoe figuereidoi, Kalanchoe flammea, Kalanchoe gastonis, Kalanchoe glaucescens, Kalanchoe gracilipes, Kalanchoe grandidieri, Kalanchoe grandiflora, Kalanchoe hildebrantii, Kalanchoe jongmansii, Kalanchoe kewensis, Kalanchoe laciniata, Kalanchoe laetivirens, Kalanchoe lateritia, Kalanchoe laxiflora, Kalanchoe linearifolia, Kalanchoe longiflora, Kalanchoe luciae, Kalanchoe macrochlamys, Kalanchoe manginii, Kalanchoe marnieriana, Kalanchoe marmorata, Kalanchoe millottii, Kalanchoe miniata, Kalanchoe nyikae, Kalanchoe obtusa, Kalanchoe orgyalis, Kalanchoe peltata, Kalanchoe petitiana, Kalanchoe pinnata, Kalanchoe porphyrocalyx, Kalanchoe prolifera, Kalanchoe pubescens, Kalanchoe pumila, Kalanchoe quartiniana, Kalanchoe rhombopilosa, Kalanchoe robusta, Kalanchoe rolandi, Kalanchoe rosei, Kalanchoe rotundifolia, Kalanchoe schizophylla, Kalanchoe serrata, Kalanchoe sexangularis, Kalanchoe streptantha, Kalanchoe suarezensis, Kalanchoe synsepala, Kalanchoe synsepala f. dissecta, Kalanchoe thyrsiflora, Kalanchoe tomentosa, Kalanchoe tubiflora, Kalanchoe uniflora, Kalanchoe velutina, Kalanchoe viguieri, and hybrids thereof.

33. The method according to claim 30, wherein the resistance to mildew comprises resistance to the mildew pathogen Oidium kalanchoeae.

34. A Kalanchoe plant produced from the method according to claim 30, wherein the plant comprises the one or more amino acid substitutions, deletions, or insertions in SEQ ID NO: 1 and exhibits resistance to mildew.

35. A seed, tissue, or plant part of the Kalanchoe plant according to claim 34, wherein the seed, tissue, or plant part comprises the one or more amino acid substitutions, deletions, or insertions in SEQ ID NO: 1.

36. A Kalanchoe plant comprising a protein comprising the sequence of SEQ ID NO: 2 and exhibiting resistance to mildew.

37. The Kalanchoe plant according to claim 36, wherein the Kalanchoe plant is selected from the group consisting of Kalanchoe adelae, Kalanchoe arborescens, Kalanchoe beauverdii, Kalanchoe beharensis, Kalanchoe bentii, Kalanchoe blossfeldiana, Kalanchoe bouvetii, Kalanchoe bracteata, Kalanchoe campanulata, Kalanchoe crenata, Kalanchoe crundallii, Kalanchoe daigremontiana, Kalanchoe delagoensis, Kalanchoe dinklagei, Kalanchoe eriophylla, Kalanchoe farinacea, Kalanchoe fedtschenkoi, Kalanchoe figuereidoi, Kalanchoe flammea, Kalanchoe gastonis, Kalanchoe glaucescens, Kalanchoe gracilipes, Kalanchoe grandidieri, Kalanchoe grandiflora, Kalanchoe hildebrantii, Kalanchoe jongmansii, Kalanchoe kewensis, Kalanchoe laciniata, Kalanchoe laetivirens, Kalanchoe lateritia, Kalanchoe laxiflora, Kalanchoe linearifolia, Kalanchoe longiflora, Kalanchoe luciae, Kalanchoe macrochlamys, Kalanchoe manginii, Kalanchoe marnieriana, Kalanchoe marmorata, Kalanchoe millottii, Kalanchoe miniata, Kalanchoe nyikae, Kalanchoe obtusa, Kalanchoe orgyalis, Kalanchoe peltata, Kalanchoe petitiana, Kalanchoe pinnata, Kalanchoe porphyrocalyx, Kalanchoe prolifera, Kalanchoe pubescens, Kalanchoe pumila, Kalanchoe quartiniana, Kalanchoe rhombopilosa, Kalanchoe robusta, Kalanchoe rolandi, Kalanchoe rosei, Kalanchoe rotundifolia, Kalanchoe schizophylla, Kalanchoe serrata, Kalanchoe sexangularis, Kalanchoe streptantha, Kalanchoe suarezensis, Kalanchoe synsepala, Kalanchoe synsepala f. dissecta, Kalanchoe thyrsiflora, Kalanchoe tomentosa, Kalanchoe tubiflora, Kalanchoe uniflora, Kalanchoe velutina, Kalanchoe viguieri, and hybrids thereof.

38. A seed, tissue, or plant part of the Kalanchoe plant according to claim 36, wherein the seed, tissue, or plant part comprises SEQ ID NO: 2.

Description

[0028] The present invention will be further detailed in the example below of especially preferred embodiments of the present invention. In the example, reference is made to figures wherein:

[0029] FIG. 1: shows an alignment of a thioesterase protein according to the present invention providing mildew resistance (upper sequence) and the thioesterase protein of SEQ ID No. 1 (bottom sequence). The consensus sequence between the two proteins is shown in the middle line;

[0030] FIG. 2: shows a boxplot of the present mildew resistance in relation to the nucleotide sequences found at position at positions 64 (SNP_14) and 67 (SNP_17) of SEQ ID No. 3 in homozygous and heterozygous form. For position 67, the non-coding nucleotides are shown, i.e. C/C; T/C and T/T instead of the coding nucleotides G/G; A/G; A/A, respectively.

EXAMPLE

[0031] An Oidium kalanchoeae mildew resistant Kalanchoe blossfeldiana parent plant of which representative seeds were deposited at NCIMB Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom under deposit number NCIMB 42379 on Mar. 2, 2015, was crossed with a mildew susceptible Kalanchoe blossfeldiana plant and a segregating progeny was obtained. Progeny was assayed for Oidium kalanchoeae resistance by infecting plants of approximately 4 weeks old with Oidium kalanchoeae. Subsequently, the infected plants were grown for approximately 4 weeks under the following conditions: [0032] temperature: day 20 C./night 18 C.; [0033] relative humidity: day 60%/night 80%; [0034] day length: 20 hours (artificial light with an intensity between 1000 and 2000 lux).
Then the plants were visually scored for mildew susceptibility/resistance on a scale of 1 to 9 whereby 1 denoted completely mildew susceptible and 9 completely mildew resistant (no visible infection).

[0035] Analysis of the progeny showed that the observed mildew resistant phenotype correlated with two nucleotide substitutions at positions 64 and 67 of the coding sequence of a gene encoding a thioesterase protein or thioesterase-like protein. As depicted in FIG. 1, both nucleotide substitutions resulted in amino acid substitutions in the N-terminal region of the thioesterase protein or thioesterase-like protein.

[0036] The analysis of the mildew phenotype in the above segregating population is summarized in Table 1 below and FIG. 2.

TABLE-US-00001 TABLE 1 Analysis of a segregating population of the present mildew resistance phenotype wherein 1 denotes minimal mildew resistance or the absence thereof and 9 denotes maximal mildew resistance. Plant Phenotype score SNP_14 SNP_17_RC Ka-SxM-100 1.00 A/A T/T Ka-SxM-084 1.00 A/A T/T Ka-SxM-024 1.00 A/A T/T Ka-SxM-064 1.00 A/A T/T Ka-SxM-073 1.17 A/A T/T Ka-SxM-126 1.17 A/A T/T Ka-SxM-004 1.17 A/A T/T Ka-SxM-086 1.17 A/A T/T Ka-SxM-079 1.17 A/A T/T Ka-SxM-026 1.33 A/A T/T Ka-SxM-020 1.33 A/A T/T Ka-SxM-136 1.33 A/A T/T Ka-SxM-069 1.33 A/A T/T Ka-SxM-078 1.33 A/A T/T Ka-SxM-087 1.33 A/A T/T Ka-SxM-072 1.33 A/A T/T Ka-SxM-027 1.50 A/A T/T Ka-SxM-081 1.83 A/A T/C Ka-SxM-012 1.83 A/A T/T Ka-SxM-062 1.83 A/A T/T Ka-SxM-044 2.00 A/A T/T Ka-SxM-052 2.17 A/A T/T Ka-SxM-082 2.33 A/A T/T Ka-SxM-092 2.50 A/A T/T Ka-SxM-061 2.50 A/A T/T Ka-SxM-075 2.83 A/A T/T Ka-SxM-050 3.00 A/A T/T Ka-SxM-090 3.00 A/A T/T Ka-SxM-053 3.00 A/A T/T Ka-SxM-113 3.00 A/A T/T Ka-SxM-008 3.00 A/A T/T Ka-SxM-080 3.00 A/A T/T Ka-SxM-028 3.17 A/A T/T Ka-SxM-013 3.50 A/A T/T Ka-SxM-037 3.50 A/A T/T Ka-SxM-022 4.00 A/A T/T Ka-SxM-068 4.83 A/A T/T Ka-SxM-046 4.83 G/G C/C Ka-SxM-030 5.17 A/A T/T Ka-SxM-014 5.33 A/A T/T Ka-SxM-106 5.50 A/A T/T Ka-SxM-060 5.67 Undetermined Undetermined Ka-SxM-066 5.83 G/A T/C Ka-SxM-114 6.00 G/A T/C Ka-SxM-009 6.33 A/A T/T Ka-SxM-041 6.33 G/G C/C Ka-SxM-038 6.83 G/G C/C Ka-SxM-065 7.00 G/A T/C Ka-SxM-067 7.00 G/A T/C Ka-SxM-029 7.00 G/A T/C Ka-SxM-007 7.00 G/A T/C Ka-SxM-049 7.17 G/A T/C Ka-SxM-051 7.17 G/A T/C Ka-SxM-005 7.17 G/G C/C Ka-SxM-040 7.17 G/A T/C Ka-SxM-033 7.33 G/A T/C Ka-SxM-074 7.33 G/G C/C Ka-SxM-019 7.33 G/A T/C Ka-SxM-094 7.33 G/A T/C Ka-SxM-139 7.33 A/A T/T Ka-SxM-056 7.33 G/G C/C Ka-SxM-042 7.50 G/A T/C Ka-SxM-138 7.50 G/A T/C Ka-SxM-077 7.50 G/G C/C Ka-SxM-054 7.50 G/A T/C Ka-SxM-001 7.67 G/G C/C Ka-SxM-018 7.67 G/A T/C Ka-SxM-034 7.67 Undetermined T/C Ka-SxM-071 7.67 A/A T/T Ka-SxM-057 7.83 G/G C/C Ka-SxM-058 7.83 G/G Undetermined Ka-SxM-036 7.83 G/G C/C Ka-SxM-045 7.83 G/G C/C Ka-SxM-117 7.83 G/A T/C Ka-SxM-117 7.83 G/A T/C Ka-SxM-017 8.00 G/A T/C Ka-SxM-089 8.00 G/A T/C Ka-SxM-003 8.00 G/A T/C Ka-SxM-043 8.00 G/A T/C Ka-SxM-076 8.00 G/A T/C Ka-SxM-023 8.00 G/A T/C Ka-SxM-031 8.17 G/G C/C Ka-SxM-035 8.33 G/A T/C Ka-SxM-032 8.33 G/A T/C Ka-SxM-048 8.50 G/A T/C