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Commit e77b9362 authored by Gesa Helmsorig's avatar Gesa Helmsorig
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edited growth conditions / readme

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**dissection of main shoot apices (MSAs) to score the reproductive development of plants** **dissection of main shoot apices (MSAs) to score the reproductive development of plants**
Scoring of the development according to: Scoring of the development according to the Waddington Stage (Waddington et al., 1983).
Waddington SR, Cartwright PM, Wall PC. A quantitative scale of spike initial and pistil development in barley and wheat. Ann Bot. 1983:51(1):119–130. https://doi.org/10.1093/oxfordjournals.aob.a086434
- assay "MSA_dissection" is applied to the plants **study overview**
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input: BW, BW(Ppd-H1), BW(eam7), BW(Ppd-H1,eam7) grown under SD and LD
-> assay: MSA_dissection
output: - Waddington Stage by DAE
- spikelet meristem number by W-Stage
- floret number by W-Stage
Results can be found in Figure 1C-E, Supplemental Figure S1 and Supplemental Table S1.
**References**
Waddington SR, Cartwright PM, Wall PC. A quantitative scale of spike initial and pistil development in barley and wheat. Ann Bot. 1983:51(1):119–130. https://doi.org/10.1093/oxfordjournals.aob.a086434
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**Biparental mapping to identify eam7**
The F2 generation of a cross between BW(*eam7*) and BW(*Ppd-H1*) was used to identify and reduced the introgression area of *eam7* and to reduce the number of candidate genes to a minimum. CAPS marker were designed based on the 50k Illumina Infinium iSelect SNP Array (Bayer et al. 2017).
**study overview**
F2 plants and parents
-> assay: biparental mapping
output: - CAPS marker results
- flowering time of F2 plants
- reduced introgression area
- target genes for eam7
Results can be found in Figure 4, and Supplemental Tables S2, S3, S5.
**References**
Bayer MM, Rapazote-Flores P, Ganal M, Hedley PE, Macaulay M, Plieske J, Ramsay L, Russell J, Shaw PD, Thomas W, et al. Development and evaluation of a barley 50k iSelect SNP array. Front Plant Sci. 2017:8:1792. https://doi.org/10.3389/fpls.2017.01792
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...@@ -4,10 +4,23 @@ ...@@ -4,10 +4,23 @@
Gene expression of CO1, ELF3, FT1, GI, LHY, LUX1, LWD1, PHYB, PHYC, Ppd-H1, PRR1, PRR59, PRR73, PRR95 and VRN1 were tested. Gene expression of CO1, ELF3, FT1, GI, LHY, LUX1, LWD1, PHYB, PHYC, Ppd-H1, PRR1, PRR59, PRR73, PRR95 and VRN1 were tested.
**The following assays were applied:**
1. leaf_sampling **study overview**
2. RNA_extraction
3. cDNA_synthesis input: 2-weeks old BW, BW(Ppd-H1), BW(eam7), BW(Ppd-H1,eam7) grown under SD
4. qRT-PCR
-> assay: leaf_sampling
output: frozen leaf tissue (3 biol. replicates)
-> assay: RNA_extraction
output: RNA samples
-> assay: cDNA_synthesis
output: cDNA samples
-> assay: qRT-PCR
output: gene expression data (2 techn. replicates)
Results can be found in Figure 3 and in Supplemental Figure S3E. Results can be found in Figure 3 and in Supplemental Figure S3E.
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...@@ -34,4 +34,11 @@ genotypes: ...@@ -34,4 +34,11 @@ genotypes:
ZT16 ZT16
ZT18 ZT18
ZT20 ZT20
ZT22 ZT22
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**References**
Druka A, Franckowiak J, Lundqvist U, Bonar N, Alexander J, Houston K, Radovic S, Shahinnia F, Vendramin V, Morgante M, et al. Genetic dissection of barley morphology and development. Plant Physiol. 2011:155(2):617–627. https://doi.org/10.1104/pp.110.166249
Gol L, Haraldsson EB, von Korff M. Ppd-H1 integrates drought stress signals to control spike development and flowering time in barley. J Exp Bot. 2021:72(1):122–136. https://doi.org/10.1093/jxb/eraa261
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**Test diurnal gene expression of circadian clock and flowering time genes in *lwd1* mutants and WT plants**
2-week old plants from the genotypes *lwd1-26*, *lwd1-390*, *lwd1-402*, and GP-fast were sampled at Zeitgeber Time (ZT) 0, 4, 8, 12, 16, and 20.
Gene expression of CO1, ELF3, FT1, LUX1, LWD1, PHYB, PHYC, Ppd-H1, and PRR1 were tested.
**study overview**
input: input: 2-weeks old lwd1-26, lwd1-390, lwd1-402 and GP-fast plants grown under SD.
-> assay: leaf_sampling
output: frozen leaf tissue (3 biol. replicates)
-> assay: RNA_extraction
output: RNA samples
-> assay: cDNA_synthesis
output: cDNA samples
-> assay: qRT-PCR
output: gene expression data (2 techn. replicates)
Results can be found in Figure 5D-L.
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**eam7 diurnal expression: plant material and growth conditions** **lwd1 mutants diurnal expression: plant material and growth conditions**
**plant material* **plant material*
...@@ -29,4 +29,10 @@ genotypes: ...@@ -29,4 +29,10 @@ genotypes:
ZT8 (lights are turned off) ZT8 (lights are turned off)
ZT12 ZT12
ZT16 ZT16
ZT20 ZT20
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**References**
Druka A, Franckowiak J, Lundqvist U, Bonar N, Alexander J, Houston K, Radovic S, Shahinnia F, Vendramin V, Morgante M, et al. Genetic dissection of barley morphology and development. Plant Physiol. 2011:155(2):617–627. https://doi.org/10.1104/pp.110.166249
Gol L, Haraldsson EB, von Korff M. Ppd-H1 integrates drought stress signals to control spike development and flowering time in barley. J Exp Bot. 2021:72(1):122–136. https://doi.org/10.1093/jxb/eraa261
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**Phenotype *eam7* plants under SD and LD conditions**
Grow plants under short-day (SD) and long-day (LD) conditions. Score flowering time and phenotypic traits at flowering and at harvest when plants are fully dry.
**study overview**
input: BW, BW(Ppd-H1), BW(eam7), BW(Ppd-H1,eam7) grown under SD and LD
-> assay: plant_phenotyping
output:
SD plants
- flowering time (in DAE)
- at flowering: plant height, leaf number, tiller number, flag leaf length/width
- at harvest (on main culm): spike length, floret and grain number, spike fertility
LD plants
- flowering time (in DAE)
- at flowering: plant height, leaf number, tiller number
- at harvest (on main culm): spike length, floret and grain number, spike fertility
Results can be found in Figure 1A and B, Figure 2, and Supplemental Figure S2.
**References**
Waddington SR, Cartwright PM, Wall PC. A quantitative scale of spike initial and pistil development in barley and wheat. Ann Bot. 1983:51(1):119–130. https://doi.org/10.1093/oxfordjournals.aob.a086434
**eam7 developmental expression: plant material and growth conditions** **Plant material and growth conditions**
**plant material* **plant material**
species: Hordeum vulgare species: Hordeum vulgare
genotypes:
genotypes:
- BW: Bowman, spring cultivar - BW: Bowman, spring cultivar
- BW(Ppd-H1): Ppd-H1 introgression line BW281 in BW background, Druka et al. (2011) - BW(Ppd-H1): Ppd-H1 introgression line BW281 in BW background, Druka et al. (2011)
- BW(eam7): eam7 introgression line BW281 in BW background, Druka et al. (2011) - BW(eam7): eam7 introgression line BW281 in BW background, Druka et al. (2011)
- BW(Ppd-H1, eam7): cross of the two introgression lines - BW(Ppd-H1, eam7): cross of the two introgression lines
**growth condtions** **growth condtions**
- short day (SD): 8h light / 16h dark, 20/16 °C, 60% humidity, ~ 250 PAR) - short day (SD): 8h light / 16h dark, 20/16 °C, 60% humidity, ~ 250 PAR)
- long day: (LD) 16h light / 8h dark, 20/16 °C, 60% humidity, ~ 250 PAR)
- QuickPot E24/10 trays (HerkuPlast Kubern GmbH, pot volume 480 cm^3) - QuickPot E24/10 trays (HerkuPlast Kubern GmbH, pot volume 480 cm^3)
- Einheitserde ED73 (Einheitserde Werkverband e.V.) with 7% sand and 4 g/L Osmocote Exact Hi.End 3-4M, 4th generation (ICL Group Ltd.) - Einheitserde ED73 (Einheitserde Werkverband e.V.) with 7% sand and 4 g/L Osmocote Exact Hi.End 3-4M, 4th generation (ICL Group Ltd.)
**sampling**
- always sample youngest, fully elongated leaf **References**
- sample at ZT 9 (1h after lights turned off, as FT1 expression is highest)
- sample genotypes at 19, 26, 33, 40, 47 and 59 DAE Druka A, Franckowiak J, Lundqvist U, Bonar N, Alexander J, Houston K, Radovic S, Shahinnia F, Vendramin V, Morgante M, et al. Genetic dissection of barley morphology and development. Plant Physiol. 2011:155(2):617–627. https://doi.org/10.1104/pp.110.166249
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**Phenotype *lwd1* mutants under SD conditions**
Grow *lwd* mutants and the wild-type GP-fast under short-day (SD) conditions. Score flowering time and phenotypic traits at flowering and at harvest when plants are fully dry.
**study overview**
input: lwd1-26, lwd1-390, lwd1-402, GP-fast (WT)
-> assay: plant_phenotyping
output:
- flowering time (in DAE)
- at flowering: plant height, leaf number, tiller number
- at harvest (on main culm): spike length, floret and grain number, spike fertility
Results can be found in Figure 5A-C and Supplemental Figure S7.
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**Plant material and growth conditions**
**plant material*
species: Hordeum vulgare
genotypes:
- GP-fast: WT, Ppd-H1 introgression line in Golden Promise background, lwd1 mutant background, Gol et al. (2021)
- lwd1-26: lwd1 mutant in GP-fast background, truncated protein of 26 aa
- lwd1-390: lwd1 mutant in GP-fast background, truncated protein of 390 aa
- lwd1-402: lwd1 mutant in GP-fast background, truncated protein of 402 aa
**growth condtions**
- short day (SD): 8h light / 16h dark, 20/16 °C, 60% humidity, ~ 250 PAR)
- QuickPot 96T trays (HerkuPlast Kubern GmbH, pot volume 75 cm^3)
- Einheitserde ED73 (Einheitserde Werkverband e.V.) with 7% sand and 4 g/L Osmocote Exact Hi.End 3-4M, 4th generation (ICL Group Ltd.)
**References**
Gol L, Haraldsson EB, von Korff M. Ppd-H1 integrates drought stress signals to control spike development and flowering time in barley. J Exp Bot. 2021:72(1):122–136. https://doi.org/10.1093/jxb/eraa261
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