Identification of the dwarfing gene Dw1

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1 Identification of the dwarfing gene Dw1 Toshi M. Foster, Jean-Marc Celton, David Chagné, and Susan E. Gardiner

3 Dwarfing rootstocks reduce scion vigour Less branching More flowers Earlier shoot termination RG/M.793 (vigorous) RG/M.9 (dwarfing) RG/M.27 (very dwarfing)

4 M.9 x R5 rootstock population grafted with Braeburn scions & phenotyped for > 7 years M.9 R5 Dwarf Semi-dwarf Intermediate Vigorous Very vigorous

5 2 major QTLs are responsible for most of the M.9 dwarfing effects 3 M.9 LG11 Hi07d11 NZraAG02 NZscAZ16AC15 NZmsDR NZsn NZmsCN892357b NZraAQ13(300) CH02d12 NZraAZ13(1150) NZmsCN892357b NZraAU15(900) CH02d08 Hi02c06 CH04a12 NH013a_b Hi02c06 NZraAU14 NB135a Hi04g11 NZmsPal8 NH045a NB135a CH04g07 NZmsEB NB121a NZmsCN CH04d07 NZmsEB NB105a NZmsEB NB118a CH04d07 R5 LG11 TCA-increase-5 th -6 th -Rep2 TCA-increase-4 th -5 th -Rep3 Average-internode-length Height-main-axis Node-number TCA-2 nd -year-after-grafting TCA-increase-6 th -7 th -Rep1 TCA-increase-5 th -6 th -Rep2 TCA-increase-5 th -6 th -Rep1 TCA-increase-4 th -5 th -Rep3 TCA-increase-4 th -5 th -Rep1 TCA-increase-3 rd -4 th -Rep1 TCA-increase-2 nd -7 th -Rep1 Number-flower-cluster DW% M.9 LG8 M.9 LG5 R5 LG5 NZraAV11(850) Hi01c04a NZmsEB Hi04a08 Hi01c04b NB115b Hi01c04a NZraAM18(700) Hi09b04 CH03a09 Hi04a08 NZraAP07 NB115b NZscAE16 CH03a09 NZscAI02 CH05e06 CH05e06 CH04g09 CH04g09 NZmsEB NZmsEB NH041aa NZmsEB NZmsEB NZmsEB NZraAE02 NZraAD02 NZmsEB NZsc11600 GD103 CH05f06 GD136 NZraAR10(1200) NZmsCN NZscAQ06 CN NZraAJ14 CH05f06 NZraAF07(500) CH04e03 NZraAQ06(850) NZraAR15(900) NZmsCN CH04e03 Average-internode-length TCA-2 nd -year-after-grafting TCA-increase-6 th -7 th -Rep1 TCA-increase-5 th -6 th -Rep3 TCA-increase-5 th -6 th -Rep2 TCA-increase-5 th -6 th -Rep1 TCA-increase-4 th -5 th -Rep3 TCA-increase-4 th -5 th -Rep2 TCA-increase-4 th -5 th -Rep1 TCA-increase-3 rd -4 th -Rep1 TCA-increase-2 nd -3 rd -Rep1 TCA-increase-2 nd -7 th -Rep1 Number-flower-cluster DW% Dw1 Dw2 CH01c06 Jean-Marc Celton

6 All Dwarf & Semi-dwarf progeny have Dw1, most have Dw1+ Dw2 300 Number of trees and genotype D SD I V VV neither DW1+DW2 just DW2 just DW1

7 Dw1 alone has some effect on dwarfing, Dw2 alone has no effect Year 7 TCA by genotype Mean TCA 2012 (cm 2 ) * * 0.0 DW1+DW2 DW 1 DW2 Neither (75) (76) (69) (83) * = p< 0.01

Most dwarfing and semi-dwarfing rootstocks have Dw1, or Dw1+Dw2 M2 M11 M12 Dw1 Dw2 M9 M8 Bud9 CG007 CG11 CG41 Mac9 Vineland1 M27 M26 JM7 CG179 CG202 CG210 CG935 M3 M4 M6 MM102 MM106

8 Most dwarfing and semi-dwarfing rootstocks have Dw1, or Dw1+Dw2 M2 M11 M12 Dw1 Dw2 M9 M8 Bud9 CG007 CG11 CG41 Mac9 Vineland1 M27 M26 JM7 CG179 CG202 CG210 CG935 M3 M4 M6 MM102 MM106 AR AR Bud Bemali M25 M793 MM104 MM105 MM108 MM111 Northern Spy M10 M13 M15 MM113 Mo84a R5 Very dwarfing Dwarfing Semi-dwarfing Intermediate Vigorous Very vigorous

9 Recombinant Dwarf & Semi-Dwarf individuals from the mapping population narrow interval containing Dw1 to <1.1Mb M27 VD AB AB AB BB AB BB AB BB BB AA AA AB AA AB BB AB M9 D AB AB AB AB AB AB AB AB AB ab AB AB AB AB AB AB R5 V BB AA BB BB BB BB BB AB AB AB AA BB AA AA BB BB AJ143 D AB AA AB AB BB AB AB AB AB AA AA BB AA AA BB AB BZ 787 D AB AA AB AB BB AB AB BB BB BB AB AB AB AB ab BB BZ844 D BB AB BB BB AB BB BB BB BB AB AA BB AA AA BB AB CA106 D AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB CA109 D AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB CA10A D AB AA AB AB BB AB AB BB BB ab AA BB AA AA BB AB CA10B D AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB CA119 D AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB CA120 D AB AA AB AB BB AB AB AB AB AA AA BB AA AA BB AB CA121 D AB AA AB AB BB AB AB AB AB AA AA BB AA AA BB AB CA124 D AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB CA125 D AB AA AB AB BB AB AB AB AB BB AB AB AB AB NC BB CA135 D AB AA AB AB BB AB AB BB BB ab AA BB AA AA BB AB CA20 D AB AA AB AB BB AB AB AB AB AA AA BB AA AA BB NC CA34 D AB AA AB AB BB AB AB AB AB AA AA BB AA AA BB BB CA49 D AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB CA65 D AB AA AB AB BB AB AB AB AB AA AA BB AA AA BB AB CA76 D AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB AJ104 SD AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB AJ120 SD AB AA AB AB BB AB AB AB AB AA AA BB AA AA BB ab BZ 794 SD AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB ab BZ813 SD AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB BZ897 SD AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB CA12 SD AB AA AB AB BB AB AB AB BB ab AA BB AA AA BB AB CA61 SD AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB CA15 I-SD AB AA AB AB BB AB AB AB AB aa AA BB AA AA BB AB CA152 I-SD AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB CA67 I-SD BB AB BB BB AB BB BB BB BB ab AA BB AA AA BB AB BZ995 I-SD AB AA AB AB BB AB AB AB AB AA AB AB AB AB AB BB CA149 I AB AA AB AB BB AB AB AB AB AA AA BB AA AA BB AB CA32 I AB AA ab AB BB AB AB BB BB ab AA BB AA AA BB AB CA41 I AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB CA54 I AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB CA64 I AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB CA69 I AB AA AB AB BB AB AB BB BB ab AA BB AA AA BB AB BZ827 V BB AB BB BB AB BB BB AB AB AA AA BB AA AA BB AB BZ944 V BB AB BB BB AB BB BB BB BB AB AA BB AA AA BB AB The genomic interval contains ~ 100 expressed genes CA148 V bb AB BB BB AB BB BB AB AB BB AB AB AB AB ab BB CA2 V BB AB BB BB AB BB BB AB AB BB AB AB AB AB NC BB CA28 V BB AB BB BB AB BB BB AA AA AB AB AB AB AB AB BB CA68 V AB AA AB AB BB BB BB AA AA AB AB AB AB AB AB BB CA85 V AB AA AB AB BB AB AB BB BB AB AA BB AA AA BB AB M.9 (dwarf) R5 (vig) Identify candidate genes based on predicted function qpcr of candidate genes Genomic sequencing of M9 to identify M9-specific mutations RNAseq of M9, M27 and M793 (vigorous)

10 Dw1 candidate gene is expressed significantly higher in dwarfing tissue relative to non-dwarfing *** *** Target/reference gene ratio *** *** *** *** M27 M9 0.0 NOV JAN MAR M793 ***p<0.001

11 M9 has a non-synonomous SNP mutation in Dw1 candidate gene Does this mutation change protein function and/or expression? Overexpression of Dw1 and dw1 (wt) in tobacco and apple Transient expression in N. benthiamiana with reporter construct

12 35S:Dw1 candidate gene in tobacco: shorter than wt shorter internodes thick stems reduced apical dominance abnormal vascular patterning 35S:Dw1 wt

13 wt 35S:Dw1 leaves: irregular margins, lobes wide, diffuse midvein irregular vein branching less leaf area M9 ARF3 WT 35S:Dw1

14 35S:Dw1 leaves and stems have more phloem relative to wt vascular patterning is disorganized inner phloem inner phloem outer phloem outer phloem wt 35S:Dw1

15 Grafting experiments to determine effect on the scion One axillary meristem allowed to grow out Untransformed (wt) stem grafted onto: Rootstock 1) 35S:Dw1 (M9 mutant allele) 2) 35S:dw1(M793 non-dwarf allele) 3) Untransformed (wt)

16 wt/35s:dw1 have shorter, thinner stems, reduced leaf area and flower earlier than wt/wt grafts rootstock Dw1 dw1 WT Sh Length (cm) Days to flowering 700 total leaf area 600 cm Dw1 dw1 WT Dw1 dw1 WT 10 5 Node # TCA (mm)2

17 RNA sequencing of dwarf (M9+M27) and vigorous tissue down in dwarf up in dwarf

18 SUMMARY We have narrowed Dw1 to a 1.1Mb region and have identified candidate genes within the interval Overexpression of Dw1 candidate gene in tobacco results in reduced plant height, less apical dominance and vascular patterning defects Grafting experiments suggest Dw1 overexpression in roots alters scion growth RNAseq indicates more gene down-regulation in dwarf tissue relative to vigorous

19 Merci pour votre attention! Apple Dwarfing Rootstock Jean-Marc Celton (INRA, Angers) David Chagné Sue Gardiner Stuart Tustin Bioinfomatics Ross Crowhurst Roy Story Robert Schaffer Transformation Kui Lin-Wang Sumathi Tomes

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