Compositions and Methods for Manipulating the Development of Plants

Abstract

The invention provides a methods and materials for producing and selecting plants with at least one dwarfing-associated phenotype. The methods and materials relate to altering the expression, or activity, of an ARF3 poypeptide in the plant, and selecting plants with altered the expression, or activity, of an ARF3 poypeptide. The invention also provides plants produced or selected by the methods. The methods also involve crossing plants of the invention with other plants to produce further plants with at least one dwarfing-associated phenotype.

Claims

1-47. (canceled)

48. A method for producing a plant with at least one dwarfing-associated phenotype the method comprising altering the expression, or activity, of an ARF3 poypeptide in the plant, wherein the dwarfing-associated phenotype is selected from: i) one of the following phenotypes in the plant: a) altered auxin transport, b) slower auxin transport, c) reduced apical dominance, d) an altered xylem/phloem ratio, e) an increased number of phloem elements, f) smaller phloem elements, g) thicker bark, h) a bushier habit, i) reduced root mass, and ii) competence to induce one of the following phenotypes in a scion grafted on to the plant: j) reduced vigour, k) less vegetative growth, l) earlier termination of shoot growth, m) earlier competence to flower, n) precocity, o) earlier phase change, p) smaller canopy, q) reduced stem circumference, r) reduced branch diameter, s) fewer sylleptic branches, t) shorter sylleptic branches, u) more axillary flowers, v) an earlier teminating primary axis, w) earlier teminating secondary axes, and x) shorter intenode length y) reduced scion mass.

49. The method of claim 48 comprising increasing the expression of the ARF3 poypeptide in the plant.

50. The method of claim 48 comprising transforming the plant to express the ARF3 poypeptide in the plant.

51. The method of claim 50 comprising transforming the plant with polynucleotide encoding the ARF3 polypeptide.

52. The method of claim 51 wherein polynucleotide is operably linked to a heterologous promoter.

53. The method of claim 48 comprising modifying the sequence of an endogenous polynucleotide encoding the ARF3 polypeptide in the plant.

54. The method of claim 53 wherein modifying the endogenous polynucleotide alters the activity of the ARF3 polypeptide in the plant to induce the dwarfing-associated phenotype.

55. The method of claim 48 wherein the dwarfing-associated phenotype in the plant is at least one of reduced apical dominance, a bushier habit, an altered xylem/phloem ratio, an increased number of phloem elements and reduced root mass.

56. The method of claim 48 wherein the dwarfing-associated phenotype is the competence to induce at least one of: reduced vigour, less vegetative growth, earlier termination of shoot growth, a smaller canopy, reduced stem circumference, and reduced scion mass, in a scion grafted on to the plant.

57. The method of claim 48 wherein the method includes the step of grafting a scion on to a plant produced by the method.

58. A method for producing a plant with at least one dwarfing-associated phenotype selected from: j) reduced vigour, k) less vegetative growth l) earlier termination of shoot growth m) earlier competence to flower n) precocity o) earlier phase change p) smaller canopy, q) reduced stem circumference r) reduced branch diameter s) fewer sylleptic branches t) shorter sylleptic branches u) more axillary flowers v) an earlier teminating primary axis, w) earlier teminating secondary axes, x) shorter intenode length y) reduced scion mass the method comprising the steps: A. providing a plant with altered the expression or activity of a ARF3 poypeptide produced by the method of claim 48, B. grafting a scion onto the plant in A wherein at least one of j) to y) is exhibited in the scion grafted on to the plant in A.

59. The method of claim 58 wherein the phenotype exhibited in the scion is at least one of: reduced vigour, less vegetative growth, earlier termination of shoot growth, a smaller canopy, reduced stem circumference, and reduced scion mass.

60. The method of claim 48 in which the ARF3 polypeptide has a sequence with at least 70% identity to any one of SEQ ID NO:1 to 11, 28 and 29.

61. The method of claim 60 in which the ARF3 polypeptide has a sequence with at least 70% identity to SEQ ID NO:1 or 28 (MdARF3).

62. The method of claim 60 in which the ARF3 polypeptide comprises a Leucine residue at the position corresponding amino acid residue 72 in SEQ ID NO:1 or 28 (MdARF3).

63. The method of claim 60 in which the ARF3 polypeptide comprises the sequence of SEQ ID NO:2 or 29 (M9 MdARF3)

64. The method of claim 48 in which the alteration results in expression of an ARF3 polypeptide with a Leucine residue at a position corresponding the amino acid residue 72 in SEQ ID NO:1 or 28 (MdARF3).

65. A construct, cell or plant comprising a polynucleotide encoding an ARF3 polypeptide, or a fragment or variant thereof with ARF3 activity, comprising a Leucine residue at a position corresponding the amino acid residue 72 in SEQ ID NO:1 or 28 (MdARF3).

66. The construct, cell or plant of claim 65 wherein the ARF3 polypeptide comprises at least 70% identity to SEQ ID NO:2 or 29 (MdARF3).

67. The construct, cell or plant of claim 65 wherein the polypeptide comprises the sequence of SEQ ID NO:2 or 29 (M9 MdARF3).

68. The construct, cell or plant of claim 65 wherein the polynucleotide has at least 70% identity to at least one of SEQ ID NO:14 and 15.

69. An isolated polynucleotide comprising the sequence with at least 70% identity to at least one of SEQ ID NO:14 or 15, or a fragment thereof, encoding a polypeptide with ARF3 activity.

70. An isolated ARF3 polypeptide encoded by the polynucleotide of claim 65, or a fragment thereof with ARF3 activity, comprising a Leucine residue at a position corresponding the amino acid residue 72 in SEQ ID NO:1 or 28.

71. A construct comprising the polynucleotide of claim 65 operably linked to a heterologous promoter.

72. A construct comprising the polynucleotide of claim 69 operably linked to a heterologous promoter.

73. A cell plant, plant part, propagule or progeny comprising the polynucleotide of claim 65.

74. A cell plant, plant part, propagule or progeny comprising the polynucleotide of claim 69.

75. A method for identifying a plant with a genotype indicative of at least one dwarfing-associated phenotype, the method comprising testing a plant for at least one of: a) altered expression of at least one ARF3 polypeptide, b) altered expression of at least one ARF3 polynucleotide, c) presence of a marker associated with altered expression of at least one ARF3 polypeptide, d) presence of a marker associated with altered expression of at least one ARF3 polynucleotide, e) presence of a marker associated with altered activity of at least one ARF3 polypeptide, wherein presence of any of A) to E) indicates that the plant has at least one dwarfingassociated phenotype, and wherein the dwarfing-associated phenotype is selected from: i) one of the following phenotypes in the plant: a) altered auxin transport, b) slower auxin transport, c) reduced apical dominance, d) an altered xylem/phloem ratio, e) an increased number of phloem elements, f) smaller phloem elements, g) thicker bark, h) a bushier habit, i) reduced root mass, and ii) competence to induce one of the following phenotypes in a scion grafted on to the plant: j) reduced vigour, k) less vegetative growth, l) earlier termination of shoot growth, m) earlier competence to flower, n) precocity, o) earlier phase change, p) smaller canopy, q) reduced stem circumference, r) reduced branch diameter, s) fewer sylleptic branches, t) shorter sylleptic branches, u) more axillary flowers, v) an earlier teminating primary axis, w) earlier teminating secondary axes, and x) shorter intenode length y) reduced scion mass.

76. The method of claim 75 in which the marker associated with altered activity of at least one ARF3 polypeptide is presence of a Leucine residue at a position corresponding the amino acid residue 72 in SEQ ID NO:1 or 28 (MdARF3).

77. The method of claim 76 in which the method involves detection of a polynucleotide encoding the Leucine residue at a position corresponding the amino acid residue 72 in SEQ ID NO:1 or 28 (MdARF3).

78. The method of claim 75 including an additional step of at least one of: a) cultivating the identified plant, and b) breeding from the identified plant.

79. A method for producing a plant with at least one dwarfing-associated phenotype, the method comprising crossing a plant produced by a method of claim 48 with another plant, wherein the off-spring produced by the crossing is a plant with at least one dwarfing-associated phenotype.

80. A method of producing a plant with at least one dwarfing-associated phenotype selected from: j) reduced vigour, k) less vegetative growth, l) earlier termination of shoot growth, m) earlier competence to flower, n) precocity, o) earlier phase change, p) smaller canopy, q) reduced stem circumference, r) reduced branch diameter, s) fewer sylleptic branches, t) shorter sylleptic branches, u) more axillary flowers, v) an earlier teminating primary axis, w) earlier teminating secondary axes, x) shorter intenode length, y) reduced scion mass, the method comprising grafting a scion onto a plant produced by a method of claim 48.

81. The method of claim 80 in which the at least one dwarfing associated phenotype is exhibited in the grafted scion.

82. The method of claim 81 in which the grafted scion exhibits at least one of reduced vigour, less vegetative growth, earlier termination of shoot growth, a smaller canopy, reduced stem circumference and reduced scion mass.

83. A plant that has been altered from the wild type to include a Leucine residue at the position corresponding to amino acid residue 72 in SEQ ID NO: 1 or 28.

84. The plant of claim 83 that has at least one dwarfing associated phenotype selected from: i) one of the following phenotypes in the plant: a) altered auxin transport, b) slower auxin transport, c) reduced apical dominance, d) an altered xylem/phloem ratio, e) an increased number of phloem elements, f) smaller phloem elements, g) thicker bark, h) a bushier habit, i) reduced root mass, and ii) competence to induce one of the following phenotypes in a scion grafted on to the plant: j) reduced vigour, k) less vegetative growth, l) earlier termination of shoot growth, m) earlier competence to flower, n) precocity, o) earlier phase change, p) smaller canopy, q) reduced stem circumference, r) reduced branch diameter, s) fewer sylleptic branches, t) shorter sylleptic branches, u) more axillary flowers, v) an earlier teminating primary axis, w) earlier teminating secondary axes, and x) shorter intenode length y) reduced scion mass.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0559] FIG. 1 shows identification of the rootstock dwarfing loci, Dw1. a) Using a bulked segregant analysis, a major dwarfing locus (Dw1) from M9 was identified at the top of linkage group (LG) 5. The markers flanking Dw1 were NZraAM18-700 (developed by Plant & Food Research, not publically avaliable) and CH03a09 (publically available). b) A multi-trait QTL analysis identified Dw1 as having a very strong influence on rootstock induced dwarfing. The markers flanking Dw1 are Hi01c04a and CH03a09.

[0560] FIG. 2 shows genetic markers flanking o Dw1 according to the applicant, and that described by Fazio et al. a) Markers flanking our Dw1 are shown in red and extend from 4.72 Mb to 7.62 Mb. b) Markers flanking the Fazio et al Dw1 are shown in green. The distal marker CH05b06z is not mapped. c) The proximal marker CH05b06z maps elsewhere, and the distal most maps incorrectly. d) The distal marker C3843 does not map to LG5. Based on the markers that do map, this would place the Fazio et al Dw1 more distal than ours.

[0561] FIG. 3 shows recombinant Dwarf & Semi-Dwarf individuals narrow the genomic interval containing Dw1 to <1.1 Mb. Parents and progeny are listed along the left most column, phenotypes in the next column over, each the remaining columns are genotypes for genetic markers sequentially ordered along LG5. Pink indicates the M9 allele and green the R5 allele. Individuals highlighted in yellow are recombinant over the interval. Only dwarfed (D) and semi-dwarfed (SD) individuals are informative, as some intermediate (I) and vigorous (V) individuals carry Dw1.

[0562] FIG. 4 shows the number of trees in each flowering class and composition of classes by Dw1 and Dw2 genotype. Flowering was assessed by estimating the total number of flower clusters on each tree in the spring of year two, and placing them into quartiles relative to the most highly floral trees, ie, 1%-25%, 26-50%, 51-75%, 76-100%. Trees with no flowers were also recorded. Data is from 109 trees from the first population, replicate 1.

[0563] FIG. 5 shows the average year seven TCA of trees in each genotypic class. The number of individuals in each class is given in parentheses, error bars indicate standard error. Average TCAs were compared to the group with neither Dw1 nor Dw2 by ANOVA, asterisks indicate the means are significantly different with a p value of ?0.001. Data is from 303 trees from the second population.

[0564] FIG. 6 shows the composition of each phenotypic class by Dw1 and Dw2 genotype. Trees from both populations (449 trees in total) were visually assessed after seven years of growth and placed into one of five phenotypic classes, D=dwarf, SD=semi-dwarf, I=intermediate, V=vigorous, and VV=very vigorous.

[0565] FIG. 7 shows quantitative RT-PCR of ARF3. For each time point, RNA was isolated and analysed from vascular-enriched tissue from 4-6 separate biological replicates of each genotype. Error bars indicate standard error for biological replicates.

[0566] FIG. 8 shows an amino acid line up of ARF3 proteins from plants. ARF3 proteins have a highly conserved B3 DNA binding domain, an auxin response element and a tasi-ARF recognition site. M9 is heterozygous for a non-synonymous SNP that changes a conserved Serine/Proline to a Leucine (indicated by red box)

[0567] FIG. 9 shows a table demonstrating % similarity between ARF 3 proteins. Proteins were aligned using MUSCLE and the phylogenetic tree used to generate this table was constructed with PHYML, using JTT substitution model and 1,000 bootstrap interations

[0568] FIG. 10 shows over-expression of M9 ARF3 in petunia. a) Non-transformed and b-f) 355:M9 ARF3 flowers. Three independent lines showed incomplete petal fusion at the tube (b-c), irregular petal margins (d), and vascular patterning defects (e). (f) shows a close up of the abaxial (outside) of the flower, revealing incomplete petal fusion and vascular patterning defects.

[0569] FIG. 11 shows over-expression of M9 ARF3 in petunia. a) untransfomed and b) 35S:M9 ARF3 flower showing petaloid stamen that appear in two lines.

[0570] FIG. 12 shows over-expression of M9 ARF3 in tobacco. (a) un-transformed and (b-c) 35S: M9 ARF3. Vascular patterning defects were observed in several lines (arrows in b and c). One line showed an asymmetric leaf phenotype (arrowheads in c).

[0571] FIG. 13 shows the vascular patterning defects in the M9 ARF3 overexpression tobacco plants.

[0572] FIG. 14 shows M9 overexpression plants exhibiting reduced height, thick stems, shorter internodes and more axillary outgrowth compared to wild-type tobacco.

[0573] FIG. 15 shows floral phenotypes of 35S:ARF3 in tobacco. Extra petaloid organs are common (arrows in a, c, e) as well as patterning defects, irregular vascular patterning (arrows in a, b) and unfused tube (arrow in d).

[0574] FIG. 16 shows irregular vascular development in 35S:ARF3 in tobacco. Sections of (a) untransformed and (b-d) 35S: M9 ARF3 tobacco petioles. Tobacco has a co-lateral arrangement of xylem surrounded by phloem on both abaxial (AB) and adaxial (AD) sides. The M9 ARF3 over-expression lines show irregular vascular patterning, with more inner phloem cells (red arrows in b-d).

[0575] FIG. 17 shows a summary of Dw1 and Dw2 genotyping of rootstock accessions. SSR makers were used to genotype rootstock accessions for the presence of Dw1 and Dw2. A green square indicates the presence of a single allele of Dw1, yellow represents Dw2. The very dwarfing rootstock M27 is homozygous for Dw1, suggesting that Dw1 is a semi-dominant mutation.

[0576] FIG. 18 shows that a pear rootstock QTL maps to the same position as Dw1. a) A rootstock QTL affecting scion flowering, shoot growth and TCA (Trunk Cross-sectional Area) was detected on LG5, in the same position as Dw1. One major difference between the two QTLs, the pear QTL controlling early flowering is on the same position, but on the other chromosome, ie derived from the other parent. An HRM marker detecting the ARF3 SNP in apple was screened over the pear population. In b-d, individuals scored as AA were statistically different than siblings scored as AB for b) flowering, c) primary axis growth and d) TCA. *=p value<0.001, very significant.

[0577] FIG. 19 illustrates a grafting experiment to demonstrate effect on scion. Aillustrates that one apical meristem is allowed to grow out. Bshows the grafted non-transformed wild-type stem. Cshows thwe graft junction. Dshows the rootstock which can be 35S:Dw1 (M9 mutant allele), 35S:dw1 (M793 non-dwarf allele) or non-transformed (WT).

[0578] FIG. 20 shows the phenotypic characteristics of scions grafted onto 4 different rootstocks as indicated. Panel A (left side) shows shoot length of the grafted scion. Panel B (right side) shows days to flowering of the grafted scion. Values were compared to WT/WT by ANOVA, **=p-value<0.01, *=p-value<0.05.

[0579] FIG. 21 shows the phenotypic characteristics of scions grafted onto 4 different rootstocks as indicated. Panel A (left side) shows number of nodes on the grafted scion. Panel B (right side) shows Trunk Cross-sectional Area (TCA) of the grafted scion. Values were compared to WT/WT by ANOVA, **=p-value<0.01, *=p-value<0.05.

[0580] FIG. 22 shows the total scion dry weight of scions grafted onto 4 different rootstocks (same root stocks as in FIGS. 21 and 22). Values were compared to WT/WT by ANOVA, **=p-value<0.01, *=p-value<0.05.

[0581] FIG. 23 shows the total leaf area of scions grafted onto 4 different rootstocks (same root stocks as in FIGS. 21 and 22). Values were compared to WT/WT by ANOVA, *=p-value<0.05.

[0582] FIG. 24 shows tree dry weight accumulation during the first year of growth. Royal Gala scions were grafted to M793 (vigorous), M9 (dwarfing) or M27 (very dwarfing). At each time point, six composite trees of each rootstock genotype were severed at the graft junction, a) scion and b) rootstock were dried and weighed. Values were compared by ANOVA and the only significant differences detected between vigorous and dwarfing rootstocks was at the final time point (*=p-value<0.001). Error bars are SE.

[0583] FIG. 25 shows average primary and total lateral root length of two week old seedlings. Seedlings were germinated on media, grown for two weeks, then harvested for photography. Digital images were measured using Image J. Error bars are standard error.

BRIEF DESCRIPTION OF THE SEQUENCES

[0584]

TABLE-US-00004 SEQ ID NO: Sequence type Common name Species Reference 1 Polypeptide Apple Malus domestica MdARF3 2 Polypeptide Apple Malus domestica MdARF3 M9 3 Polypeptide Arabidopsis Arabidopsis thaliana ARF3/ETTIN 4 Polypeptide Bean Phaseolus vulgaris PvARF3 5 Polypeptide Tomato Solanum lycopersicum SIARF3 6 Polypeptide Mandarin orange Citrus clemantina CcARF3 7 Polypeptide Strawberry Frageria vesca FvARF3 8 Polypeptide Plum Prunus persica PpARF3 9 Polypeptide Pear Pyrus communis PcARF3 10 Polypeptide Poplar Populus tremula PtARF3 11 Polypeptide Grape Vitis vinefera VvARF3 12 Polynucleotide Apple Malus domestica MdARF3 (cDNA) 13 Polynucleotide Apple Malus domestica MdARF3 (gDNA) 14 Polynucleotide Apple Malus domestica MdARF3 M9(cDNA) 15 Polynucleotide Apple Malus domestica MdARF3 M9(gDNA) 16 Polynucleotide Arabidopsis Arabidopsis thaliana ARF3/ETTIN (cDNA) 17 Polynucleotide Arabidopsis Arabidopsis thaliana ARF3/ETTIN (gDNA) 18 Polynucleotide Bean Phaseolus vulgaris PvARF3 (cDNA) 19 Polynucleotide Tomato Lycopersicum esculentum LeARF3 (cDNA) 20 Polynucleotide Mandarin orange Citrus clemantina CcARF3 (cDNA) 21 Polynucleotide Strawberry Frageria vesca FvARF3 (cDNA) 22 Polynucleotide Plum Prunus persica PpARF3 (cDNA) 23 Polynucleotide Pear Pyrus communis PcARF3 (cDNA) 24 Polynucleotide Poplar Populus tremula PtARF3 (cDNA) 25 Polynucleotide Grape Vitis vinefera VvARF3 (cDNA) 26 Polynucleotide Apple Malus domestica Hi01C04 marker 27 Polynucleotide Apple Malus domestica Hi04A08 marker

EXAMPLES

[0585] The invention will now be illustrated with reference to the following non-limiting examples.

[0586] It is not the intention to limit the scope of the invention to the present example only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention.

Example 1: Refining the Genomic Region Containing the Dw1 Loci

BACKGROUND

[0587] In a previous QTL study, the closest genetic markers that defined Dw1 were Hi01c04 and Ch03a09 (FIG. 1), which are located at 4.72 and 7.62 Mb respectively on the reference golden delicious genome (Celton et al 2009). More recently Fazio and co-workers (Fazio et al 2014) found a more distal position for Dw1, between Hi22f12 (2.69 Mb) and Hi04a08 (5.15 Mb) (FIG. 2).

[0588] In the present work, the applicants developed genetic markers based on genomic sequence from the interval between 4.5 Mb and 7.2 Mb on linkage group 5 (LG5). By screening these markers over the parents and progeny of their rootstock population, the applicants were able to identify recombinants within this interval (i.e. had a chromosomal cross over between M9 and R5). Intermediate and vigorous recombinants were not informative, because some of the individuals carried Dw1. However, all dwarfed and semi-dwarfed individuals carried Dw1, so these recombinants were informative in defining the interval that contains Dw1. Based on four dwarfed and two semi-dwarfed recombinant individuals, the applicants were able to narrow the genomic interval containing Dw1 to a smaller region, between 4.75 Mb and 5.80 Mb (FIG. 3).

[0589] This region defines an interval of 1.05 Mb (5.80-4.75 Mb).

[0590] Although this is a smaller interval, this region could still contain over 100 genes. It is also possible that the genetic determinant of dwarfing at the Dw1 locus would be a micro RNA (miRNA) or other non-protein encoding gene. Furthermore, prior to the present application, there were no obvious candidate gene/s, or even classes of candidate genes that might be responsible for the dwarfing effect of the the Dw1 locus.

[0591] Dw1 has a More Significant Effect than Dw2 on Rootstock-Induced Dwarfing

[0592] To elucidate the relative contributions of Dw1 and Dw2 to dwarfing of the scion, the applicants examined three of the most robust phenotypes associated with dwarfing, i.e. early flowering (spring of year two), final TCA (year seven), and overall visual assessment (year seven) of scions grafted to rootstocks carrying various combinations of Dw1 and Dw2.

[0593] Early flowering was assessed in the spring of year two by estimating the number of floral clusters on 109 trees from the first population. The majority of the trees with the highest degree of flowering had been grafted onto rootstocks carrying both Dw1 and Dw2 (50%), or Dw1 alone (41.7%) (FIG. 4). Conversely, the trees with no flowers or the fewest flowers were predominantly grafted onto rootstocks carrying Dw2 alone (33.9%), or neither dwarfing locus (44.6%).

[0594] After seven years of growth, the TCA of 303 trees from the second population were measured. Trees grafted onto rootstocks carrying both Dw1 and Dw2 exhibited the lowest average TCA, only 23% of that of scions on rootstocks with neither loci. Rootstocks with Dw1 alone reduced scion TCA to 73% of those with neither rootstock loci. Surprisingly, trees grafted onto rootstocks with Dw2 alone had the highest TCA of all (FIG. 5).

[0595] As rootstock-induced dwarfing becomes more pronounced over successive growth cycles, an expert visual assessment of the whole tree phenotype after seven years provided an overall measure of scion vigour. When 449 grafted trees from both populations were compared, a clear trend relating rootstock genotype to phenotypic class was observed. All the dwarfed and semi-dwarfed trees were grafted onto rootstocks with Dw1 and Dw2 or Dw1 alone, whereas the vigorous and very vigorous trees had rootstocks carrying Dw2 alone, Dw1 alone, or neither locus (FIG. 6). Nearly 40% of the vigorous trees were on rootstocks carrying Dw2, indicating that this locus alone is not sufficient to dwarf the scion.

[0596] However in contrast to the recent work of Fazio et al (Fazio, Wan et al. 2014) the present study does indicate that the Dw1 loci can influence dwarfing alone (i.e. even in the absence of Dw2).

[0597] Other Dwarfing and Semi-Dwarfing Rootstocks Carry Dw1 and Dw2

[0598] Genetic markers linked to Dw1 and Dw2 were screened over 41 rootstock accessions that confer a range of effects on scion growth. The majority of dwarfing and semi-dwarfing rootstock accessions screened carried marker alleles linked to both Dw1 and Dw2 (Foster et al, 2015 and FIG. 17). This suggests that most apple dwarfing rootstocks have been derived from the same genetic source.

Example 2: A Pear Rootstock QTL Influencing Scion Size and Flowering

[0599] Pear does not have a true dwarfing rootstock, such as M9, although some rootstocks are known to influence scion size and flowering. A pear segregating rootstock population was generated by crossing Old Home to Louis Bon Jersey. The progeny were grafted with Cornice, and scions were phenotyped for 4 years. A QTL influencing scion size and flowering was identified at the top of LG5, in the exact location as Dw1 (FIG. 18, PFR, unpublished). No QTL corresponding to Dw2 was identified. Pear and apple are very closely related and show strong synteny of gene order along their orthologous chromosomes. This finding raises the exciting possibility that Dw1 predates the divergence of apple and pear and that the same gene may be influencing both the apple and pear QTL.

Example 3: Identification of ARF3 as a Candidate Gene for Dw1

[0600] The applicants found that there are approximately 168 annotated genes within the 1.1 Mb interval (unpublished). Based on expressed sequence ESTs from the Plant and Food proprietary Malus database (Newcomb, Crowhurst et al. 2006) and RNA seq experiments (unpublished), the applicants estimated the number of expressed genes is about 100.

[0601] The applicants identified an Auxin Response Factor 3 (ARF3) transcription factor gene present in the refined Dw1 interval, which they showed to be upregulated in M9 rootstock, as a candidate gene for the Dw1 QTL effect.

[0602] Many hypotheses to explain the mechanism of dwarfing rootstocks implicate auxin, but the genetic basis of any auxin effect is completely unknown. ARF3 is a member of a large family of Auxin Response Factors, transcription factors that activate or repress downstream genes in response to auxin. ARF3/ETTIN was first discovered as a gene required for normal patterning of floral organs in Arabidopsis (Sessions and Zambryski 1995; Sessions, Nemhauser et al. 1997). It was later discovered that ARF3 and the transcription factor KANADI mediate both auxin flow and organ polarity, which includes vascular patterning (Pekker, Alvarez et al. 2005; Izhakia and Bowman 2007; Kelley, Arreola et al. 2012). ARF3 also has a key role in promoting phase change (transition to flowering), increased ARF3 expression leads to earlier flowering, loss of ARF3 function delays flowering. (Fahlgren, Montgomery et al. 2006; Hunter, Willmann et al. 2006).

[0603] ARF3 is Up-Regulated in M9 and M27 Relative to Vigorous Rootstocks

[0604] The applicants used quantitative real time PCR (qRT-PCR) to compare ARF3 expression in vascular-enriched tissue from M9 and another dwarfing rootstock M27 with a vigorous rootstock, M793 (FIG. 7). ARF3 expression was about four times higher in M9 than M793 at all time points. In M27, ARF3 expression was 2-4 times higher levels than M793.

[0605] M9 has a Mutation in the ARF3 Gene

[0606] To identify any M9-specific DNA changes that might alter gene expression or function/activity the applicants performed genomic sequencing of M9. This revealed that the M9 MdARF3 (MDP000173151) carried a single nucleotide polymorphism (SNP) that changed a conserved Serine to a Leucine. FIG. 8 shows an amino acid line-up with the M9, the reference MdARF3 proteins and ARF3 proteins from a variety of plants. This SNP alter the function of the ARF3 protein.

[0607] The M9 ARF3 SNP as a Genetic Marker in Apple and Pear

[0608] To test if the SNP identified in the M9ARF3 segregates with dwarfing individuals, the applicants used primers that amplify the SNP in a High Resolution Melting (HRM) analysis over the entire M9?R5 rootstock population. The results showed clear segregation of a distinct melting curve with all individuals that were previously identified as having Dw1. The same marker was also tested on the pear rootstock population and showed clear segregation with one curve associated with high flowering individuals, another with low or no flowering trees.

Example 4: Transgenic Expression of ARF3 in Petunia and Tobacco

[0609] To test if the higher expression and/or the non-synonymous SNP in the M9 ARF3 cause phenotypes associated with dwarfing rootstocks, the applicants made transgenic lines of both tobacco and petunia that over-express either the M9 or the reference allele of ARF3. These are hence referred to as M9 ARF3 and wt ARF3 respectively. Petunia and tobacco were chosen as models because they are both amenable to grafting.

[0610] The applicants generated 10 independent lines expressing 35S: M9 ARF3, but the applicants were unable to recover 35S: wt ARF3 petunias. The applicants verified that the plants were expressing the construct by q-RT-PCR. Three independent lines of the 35S:M9 ARF3 had a floral phenotype, ranging from irregular petal margins, incomplete tube fusion, vascular defects, and petaloid stamens (FIGS. 10, 11). Microscopic analysis of the irregular petal margins revealed small patches of inverted petal polarity, which is consistent with the known function of ARF3 in adaxial-abaxial patterning.

[0611] The applicants generated 10 M9 ARF3 and 10 wt ARF3 over-expression lines in tobacco. The applicants verified that all T.sub.0 plants were expressing the construct. Preliminary analysis indicates that several of the plants exhibit irregular vascular patterning in the leaves (FIG. 13). Two plants have asymmetric leaves, with half of the blade missing entirely or double midveins (FIG. 12 b, c). The most extreme line of 35S: M9 ARF 3 (#6) is much shorter than wild-type with thick stems, and decreased apical dominance, creating a bushy phenotype (FIG. 14). The lines with the highest ARF3 expression flowered earlier than the others. Early flowering is also seen in dwarfed scions in apple. Many of the M9 and wt ARF3 plants have floral phenotypes. These include incomplete fusion of the tube, patterning defects, and extra petaloid organs (FIG. 15).

[0612] To examine the vascular patterning defects in more detail, petioles from untransformed and ARF3 over-expression plants were fixed, sectioned and stained with safranin fast green. FIG. 16 shows representative micrographs illustrating that 35S:M9 ARF3 plants have irregular vascular patterning, with more inner phloem cells, consistent with the similar phenotype seen in M9 apple rootstock.

[0613] Phenotypic analysis of the ARF3 over-expression tobacco plants, can also be carried out on plants produced from T.sub.1 seed.

[0614] Plants transformed to express ARF3 and M9 ARF3 can be phenotyped, as can scions grafted onto the transgenic, and control plants.

[0615] Such phenotyping can involve a detailed architectural analysis to document metamer initiation rate, the outgrowth and size of axillary brances, the size and node number of the primary shoot, and time to flowering.

[0616] Growth chambers can also be used to test if the transgenic plants have an altered sensitivity to long days or short days.

[0617] Further histological analysis can also be undertaken to compare vascular development between transgenic lines and untrasformed controls.

Example 5: Transgenic Expression of ARF3 in Apple

[0618] The constructs described in Example 4 above were transformed into apple, to further assess the phenotypic effect of higher expression and/or the non-synonymous SNP.

[0619] Plantlettes generated, can be tested to verify that ARF3 is over-expressed using qRT-PCR. Transgenic lines can be assessed for dwarfing-associated phenotypes by comparing the overall plant architecture (main axis hight, outgrowth of axillary branches, etc) with un-transformed controls. To examine any changes to the vasculature, tissue can be fixed, sectioned, stained and photographed on a microscope to compare with untransformed controls.

[0620] Once plantlettes have generated roots and are large enough, they can be grafted with un-transformed controls. Scions can be assessed for dwarfing-associated phenotypes by comparing the number of growth units on the primary and secondary axis, comparing the number and size of sylleptic and prolleptic shoots, and eventually the number of flowers.

Example 6: Transgenic Expression of ARF3 in Pear

[0621] The constructs described in Example 4 above were transformed into pear, to further assess the phenotypic effect of higher expression and/or the non-synonymous SNP.

[0622] Plantlettes generated, can be tested to verify that ARF3 is over-expressed using qRT-PCR. Transgenic lines can be assessed for dwarfing-associated phenotypes by comparing the overall plant architecture (main axis hight, outgrowth of axillary branches, etc) with un-transformed controls. To examine any changes to the vasculature, tissue can be fixed, sectioned, stained and photographed on a microscope to compare with untransformed controls.

[0623] Once plantlettes have generated roots and are large enough, they can be grafted with un-transformed controls. Scions can be assessed for dwarfing-associated phenotypes by comparing the number of growth units on the primary and secondary axis, comparing the number and size of sylleptic and prolleptic shoots, and eventually the number of flowers.

Example 7: Determine if the M9 SNP Alters Protein Function

[0624] Transient expression experiments in Nicotiana benthamiana (Martin, Kopperud et al. 2009), can be used to further assess the function of the non-synonomous SNP in the M9 ARF3. First an an auxin responsive reporter line, DR5:LUC (Ulmasov, Murfett et al. 1997) can be generated. This reporter will result in an enzyme that generates fluorescent compound in response to auxin.

[0625] The reporter construct can be co-expressed with either the M9 or wt ARF3 and the fluorescent compound measured after 1-3 days. These experiments can also be repeated with application of exogenous auxin to compare auxin sensitivity.

Example 8: Determine if Pear has Altered ARF3 Sequence and/or Expression

[0626] ARF3 expression in pear can be assessed by qRT-PCR to determine if dwarfish individuals from the pear rootstock population have higher expression of ARF3 than vigorous individuals. To determine if the same non-synonomous SNP exists dwarfish individuals, the pear ARF3 gene can be amplified and sequenced.

Example 9: Examination of the Phenotype of Apple Seedlings Genotyped for Dw1 and Dw2

[0627] Seedlings derived from controlled crosses can be genotyed for Dw1 and Dw2 to identify individuals that have zero, one or two copies of Dw1, and either zero or one copy of Dw2. ARF3 expression in apple seedlings and young trees can be assessed. Seedlings/trees can be measured for differences in metamer number of primary and secondary axes, the outgrowth of axillary shoots, and the time to flowering. Stem vascular development can also be assessed histologically.

Example 10: Tree Dry Weight Accumulation During the First Year of Growth

[0628] Royal Gala scions were grafted to M793 (vigorous), M9 (dwarfing) or M27 (very dwarfing). At each time point (60, 120, 180 and 300 days after bud break [DABB]), four to six composite trees of each rootstock genotype were severed at the graft junction. Scion and rootstock material was oven dried at 60? C. to a constant mass and weighed. Dry weights of scion include scion budwood, primary axis, sylleptic shoots and leaves, whilst dry weights of rootstock include roots and rootstock stem. Values were compared by ANOVA and the only significant differences detected between vigorous and dwarfing rootstocks was at the final time point (*=p-value<0.001). The results are shown in FIG. 24. Error bars are SE.

Example 11: Grafting Experiments

[0629] Methods of Grafting

[0630] Tobacco plants were grown in pots until plants had 10-15 leaves. In this experiment, all scions were wild-type tobacco, the rootstocks were wild-type, M9 ARF3 (2 independent lines, 2 and 6) and 35S: 793 (wt) ARF3 (line 4). We note M27 has the same ARF3 allele as M9, thus M27 contains the M9 allele of ARF3. In FIGS. 20 to 23, the M9 ARF3 rootstock lines are labelled M27 2-1 and M27 6-16 and the WT ARF3 rootstock line is labelled M793 4-3.

[0631] At the time of grafting, a horizontal cut was made through the rootstock stem at the very top of node 4-5. A V-shaped notch was cut vertically into the stem, 5-10 mm deep. The wild-type scion was cut from the base of the plant such that the base was approximately the diameter of the rootstock. Leaves and shoot tip were removed and a piece of stem containing 2 nodes (each with an axillary meristem) was cut into a wedge shape at the bottom end. The scion was inserted into the rootstock notch and the junction was secured with a small piece of parafilm. Plants were placed in a mist tent to recover. After one week, all leaves from the rootstock were removed. Once it became apparent that one or more axillary meristems of the scion was growing out, the other was removed.

[0632] The scion shoots were grown until the first flower was fully extended, this date was considered the flowering date. The time between grafting date and the flowering date is the days to flowering. Once plants had flowered, architectural data was collected from the scion. The shoot length and node number was measured from the axil to the uppermost leaf base, this does not include the original scion stem segment, only the shoot that grew from the axillary meristem. The scion shoot diameter was measured at the base of the shoot using an electronic calliper. Trunk circumference area (TCA) was calculated with the formula: (diameter/2).sup.2 and is given in mm.sup.2. The area of each leaf was measured with an electronic leaf scanner, total leaf area is the sum of all leaves on a plant and is given in cm.sup.2. The scions were dried and weighed to determine dry weight (gm). Each line was compared to WT/WT by one way-ANOVA to determine significant differences.

[0633] Results

[0634] As ungrafted plants, 35S: M9 ARF3 line 6, hereafter referred to as line 6, show the most extreme phenotype. 35S: M9 ARF3 line 2 (line 2) has the mildest phenotype and 35S:793 ARF3 line 4 is undistinguishable from wild-type.

[0635] Relative to the WT/WT homografts, the WT scions on line 6 rootstocks were significantly shorter (FIG. 20). Scions on line 2 and line 4 had slightly shorter lengths, but these were not significant.

[0636] Scions on all three transgenic rootstocks flowered slightly earlier than the WT/WT (FIG. 21).

[0637] Line 6 had significantly fewer nodes than WT/WT (FIG. 20).

[0638] Scions on both line 2 and line 6 had a smaller TCA than WT/WT. Line 6 was significantly different than WT/WT (FIG. 21).

[0639] Scions on line 2 and line 6 had a smaller dry weight than WT/WT. Line 6 was significantly different than WT/WT (FIG. 22).

[0640] Although lines 2, 6 and 4 had less total leaf area, only line 6 was significantly different from WT/WT (FIG. 23).

[0641] To our knowledge, there has been no report of dwarfing rootstocks causing smaller leaf size in scions.

[0642] Seedling Root Measurements

[0643] 35S: M9 ARF3, 35S: wt ARF3 and wild-type tobacco seeds were sterilized in 2% bleach for 30 minutes, rinsed in distilled H2O, 3?, for 10 minutes each, then plated on MS media containing Kanamycin (for the transgenic seeds) or just MS (wild-type). Two weeks after plating, seedlings were removed from the media, excess media was removed and seedlings were photographed on a grid using a stereo microscope equipped with a digital camera. Primary and lateral root length were measured from digital images using Image J, total lateral root length is the sum of all lateral root lengths. (see FIG. 25).

[0644] In terms of shoot length, node number, TCA, scion dry weight, and scion mass, the effect of line 6 on the scion appears to replicate the effect of the M9 dwarfing rootstock.

[0645] Summary of Data Shown in Transgenic Plants, and Grafted Scions.

[0646] The phenotypes shown in transgenic plants over-expressing M9 ARF1 or WT ARF1, and in WT plants grafted onto transgenic plants over-expressing M9 ARF1 or WT ARF1, in comparison to the known phenotypes in known root stock and dwarfed grafted scions are summarised in the tables below.

TABLE-US-00005 TABLE 2 Phenotypes shown in transgenic plants over-expressing M9 ARF1 or WT ARF1 Known dwarfing- associated phenotypes Shown in plants Shown in plants found in dwarfing over-expressing over-expressing rootstock plants M9 ARF1 WT ARF1 (previous data) (this study) (this study) bushier Yes No altered xylem/phloem ratio Yes No more phloem elements Yes No reduced apical dominance Yes No reduced root mass Yes Yes

TABLE-US-00006 TABLE 3 Phenotypes shown in WT plants grafted onto transgenic plants over-expressing M9 ARF1 or WT ARF1 Known dwarfing- Shown in WT scions Shown in WT scions associated phenotypes grafted on to root- grafted on to root- found in scions grafted stock plants over- stock plants over- onto dwarfing rootstock expressing M9 ARF1 expressing M9 ARF1 plants (previous data) (this study) (this study) reduced vigour Yes Yes less vegetative growth Yes Yes earlier termination of Yes Yes shoot growth smaller canopy Yes No reduced stem Yes No circumference reduced scion mass Yes No

[0647] Materials and Methods

[0648] Plant Material

[0649] A rootstock population derived from crosses between Malus?domestica Malling9 (M9) and Malus robusta 5 (R5) was used for QTL analysis. For the first population, 135 seedlings were planted in 1998 and grown as stoolbeds to produce multiple rooted stocks of each genotype. The rootstocks were cleft grafted with Braeburn scions, grown in the nursery for two years, then transplanted into the Plant & Food Research orchard (Havelock North, New Zealand) as described by Pilcher et al. (Pilcher, Celton et al. 2008) Replicates of the original 135 rootstocks were propagated in 2000 and planted in the orchard as one-year-old grafted trees. Of the replicated trees, 112 individuals from replicate two, and 57 individuals from replicate three were phenotyped for QTL analysis. The second population consisted of 350 seedlings, which were grafted as described above and planted in the orchard as one-year-old trees in 2004. From the second population, 81 individuals were evaluated for the QTL analysis and 314 survived until final phenotypic assessment in year seven. Trees were grown with in-row spacing of 1.5 m between trees and a double wire trellis as support, in a complete randomized block design. Scions grafted onto M9 and R5 were planted throughout as controls. Trees were not pruned, to allow full expression of the rootstock effects on scion growth. Once trees began fruiting, chemical thinning sprays were applied to avoid over-cropping and limb breakage.

[0650] Forty-one (41) apple rootstock accessions (Malus spp.) representing rootstock varieties used in major apple-growing regions in the world were used for pedigree analysis of Dw1 and Dw2.

[0651] Phenotypic Analysis

[0652] Rootstock effects on the development of Braeburn scions were assessed using multiple methods, over seven years, within the two populations. Table 1 presents the specific traits that were assessed for the QTL analysis in each population/replicate and the sample size phenotyped. Height, internode number, and average internode length of the scion were recorded at the end of the first year of growth after grafting (year one). Flowering was scored by estimating the total number of flower clusters on each tree in the spring of year two, and placing them into quartiles relative to the most highly floral trees, i.e., 1-25% had the fewest flowers, 75-100% had the most flowers. Trees without any flowers in year two were recorded as 0. Trunk Cross-sectional Area (TCA) was measured 20 cm above the graft junction at the end of each year from year two to year seven. From year two to year seven, the overall vigour of each tree was assessed annually by comparing trunk size, crown height and spread, branch density and vigour. For the QTL analysis, an overall dwarfing phenotype (DW %) was assigned in year seven, with 100%=very vigorous, 80%=vigorous, 60%=intermediate, 40%=semi-dwarfed, and 20%=dwarfed.

[0653] The 41 rootstocks accessions used for the pedigree analysis were classified according to their dwarfing effect in accordance with the literature and in-house Plant & Food Research professional expertise.

[0654] DNA Isolation and Genotyping of M9?R5 Rootstock Population and Rootstock Accessions

[0655] Total genomic DNA was extracted from leaves and quantified according to Gardiner et al. (Gardiner, Bassett et al. 1996) Leaf material was collected from 135 seedlings from the first M9?R5 population and 350 from the second population. Leaves of the rootstock accessions were collected from the Plant & Food Research germplasm collection in Havelock North, NZ, or from the USDA-ARS collection in Geneva, N.Y., USA.

[0656] For Dw1 and Dw2 genotyping of the entire population of M9?R5 rootstocks, polymerase chain reaction (PCR) products containing single nucleotide polymorphisms (SNP) were amplified on a LightCycler480 instrument (Roche Diagnostics) and screened using the High Resolution Melting (HRM) technique as described by Chagn? et al. (Chagn?, Gasic et al. 2008) Supplementary Table 1 lists the position of markers on the Golden Delicious genome (Velasco, Zharkikh et al. 2010) and primer sequence.

[0657] Markers detecting SSRs located on LG5 and LG11 were employed to genotype the 41 rootstock accessions. Hi01c04, Hi04a08, CH03a09 and CH02d08 were developed by Silfverberg-Dilworth et al. (Silfverberg-Dilworth, Matasci et al. 2006) and Liebhard et al. (Liebhard, Gianfranceschi et al. 2002) Two new SSR markers (MDP0000365711 and MDP00024370) located at the top of LG11 were developed using the Plant & Food Research Malus genome database (Newcomb, Crowhurst et al. 2006), with the programmes Sputnik and Primer3. The M13 sequence TGTAAAACGACGGCCAGT was added to the 5 end of the forward primer to enable the use of Schuelke's (Schuelke 2000) approach to fluorescent labelling. PCR reactions were performed and analysed on an ABI 3500 Genetic Analyzer (Applied Biosystems) as described by Hayden et al. (Hayden, Nguyen et al. 2008)

[0658] QTL Analysis

[0659] The parental genetic maps for M9 and R5 were constructed using a total of 316 loci amplified from 296 primer pairs as described in Celton et al. (Celton, Tustin et al. 2009) The maps span a total of 1,175.7 and 1,086.7 cM for M9 and R5 respectively. (Celton, Tustin et al. 2009) The linkage phase of the markers was determined using JoinMap? 3.0 (Kyazma, NL). QTL analysis was performed for all growth traits using MapQTL? 5 Software (Kyazma, NL). Traits evaluated over multiple years and replicates were analysed separately. Interval mapping (IM), followed by multiple QTL model (MQM) analysis using the best markers obtained by IM as co-factors, was used for normally quantitative traits. Only additive models were considered for the QTL analysis. The threshold for QTL genome-wide significance was calculated after 1,000 permutations. Kruskal-Wallis analysis was used for ordinal traits such as the estimated number of flower clusters and expert assessment of dwarfing.

[0660] RNA Purification

[0661] For RNA-seq, tissue was collected from the rootstock stem of two M.793 and two M.9 individuals in November (60 DABB, ?90 days after grafting). M.27 was not included in the RNA-seq experiment because suitable material was not available. For qRT-PCR expression analysis, 30 Royal Gala trees grafted onto M.9, M.27 and M.793 rootstocks were grown as previously described. Tissue was collected for RNA purification in November, January, March and July (60, 120, 180 and 300 DABB respectively). For each time point, four to six trees of each genotype were selected for uniform scion growth to minimize any effects due to differential tree size. RNA was pooled from four shoots from each of the rootstock accessions shown in FIG. 5. For all other experiments, RNA from each individual was extracted and analysed separately. For all collections, the outer bark was removed, vascular tissue was scraped off with a scalpel, and snap frozen in liquid nitrogen. Tissue was harvested between four and five hours after sunrise for all time points. Total RNA was isolated and cDNA generated as described in (Janssen et al. 2008). The quality and concentration of the RNA samples was assessed with an RNA Nano kit (Agilent) and only samples with a RIN value of 8 or higher were further analyzed by sequencing or qRT-PCR.

[0662] RNA Sequencing and Data Processing

[0663] RNA was sent to Axeq/Macrogen for library preparation and sequencing using an Illumina Hiseq 2000 instrument. Individual samples were run as a multiplexed sample on one lane to produce 100 nucleotide paired end sequence reads. The first 13 bases of all RNAseq reads were trimmed using an in-house perl script. Adapters were removed using fastq-mcf from the ea-utils package (Aronesty 2011) using a minimum read retention length of 50 and a minimum quality score threshold of 20. Quality score analysis was performed using fastqc (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) both before and after trimming. Trimmed reads were mapped to the reference using bowtie2 (Langmead and Salzberg 2012) using the following settings: -aend_to_endsensitive. SAM file to BAM file conversion was undertaken using samtools (Li et al. 2009). Raw read counts and reads per kilobase per million (RPKM) values were extracted from BAM files using the multicov option of bedtools (Quinlan and Hall 2010) and either an in-house R script or cufflinks (Trapnell et al. 2010). Apple homologues of Arabidopsis flowering genes were determined by BLASP value and tested by reciprocal BLASTP. Differentially expressed genes were selected using the Limma package (Smyth 2005) in BioConductor, genes were selected using an adjusted P value of <0.05 and fold change cutoff>6 (Smyth 2005).

[0664] Transformation of ARF3 into Plants

[0665] Primers were designed to amplify the MdARF3 gene, from from 100 bp upstream of the start codon to 50 bp 5 of the stop codon. Single products were amplified from cDNA derived from Royal Gala or M9 meristem enriched tissue. These products were cloned into an expression vector (pHEX), which uses the cauliflower mosaic virus (CaMV) 35S promoter to drive expression and contains the neomycin phoshotransferase II gene (NPTII) to confer kanamycin resistance. Agrobacterium tumefaciens strain GV3-101 transformed with either the Royal Gala (wt) or the M9 ARF3 was used to transform leaf discs from N. tabacum (Samsun), petunia (Mitchell) or apple transformation cell lines. Callus formation and regeneration of plantlettes are as described in (Kotoda and Wada 2005).

[0666] Histology

[0667] Stem and petiole sections were fixed overnight in FAA (3.7% Formaldehyde, 50% EtOH, 5% Acetic Acid), processed and embedded in paraffin as described in Ruzin (Ruzin 1999). Tissue was sectioned to 10?m on a rotary microtome, and slides were stained using a safranin/fast green procedure to distinguish xylem from phloem.

[0668] Grafting

[0669] Scions can be grafted onto rootstocks using cleft grafting or chip-budding depending on the material (Stoltz and Strang 1982; Webster and Wertheim 2003; Crasweller 2005).

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