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  • hhu-institute-of-plant-genetics/helmsorig-2024-eam7
  • ceplas/helmsorig-2024-eam7
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with 19 additions and 37 deletions
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## measure RNA concentration of samples
- dilute small amount of RNA sample 1:5 with DEPC water
- use 2 µl of dilution to measure the concentration with a Nanophotometer
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## Results: target gene identification for *eam7*
**Candidates related to the circadian clock:**
| MorexV3 gene ID | pident | evalue | Araport11 gene ID | Gene name | SNP positions | Changes in aa sequence |
| ------ | ------ | ------ | ------ | ------ | ------ | ------ |
| HORVU.MOREX.r3.6HG0592840 | 73.75 | 2.26E-40 | AT2G21660 | GR-RBP1 | None | None
| HORVU.MOREX.r3.6HG0583670 | 69.309 | 0 | AT1G12910 | LWD1 | 32 bp del (1219-1250) | A407F, N408S, K409F, L410* |
| HORVU.MOREX.r3.6HG0587680 | 66.082 | 0 | AT4G08920 | CRY1 | None | None |
| HORVU.MOREX.r3.6HG0595250 | 38.475 | 3.92E-106 | AT5G61380 | PRR1 | A642G | T1300C | T215A, S434P |
| HORVU.MOREX.r3.6HG0578070 | 55.367 | 0 | AT5G51200 | EDS4 | - | - |
| HORVU.MOREX.r3.6HG0589220 | 34.855 | 9.66E-46 | AT3G07640 | PBAC5 | - | - |
| HORVU.MOREX.r3.6HG0571470 | 36.735 | 1.3 | AT3G42170 | DAY-SLEEPER | - | - |
| HORVU.MOREX.r3.6HG0582650 | 30.108 | 1.00E-12 | AT3G42170 | DAY-SLEEPER | - | - |
| HORVU.MOREX.r3.6HG0592100 | 29.042 | 1.69E-45 | AT3G42170 | DAY-SLEEPER | - | - |
| HORVU.MOREX.r3.6HG0571480 | 28.319 | 1.11E-13 | AT3G42170 | DAY-SLEEPER | - | - |
**Candidates associated with early flowering:**
| gene name | MoresV3 gene ID |
| - | - |
| PSEUDO RESPONSE REGULATOR 1 (PRR1) | HORVU.MOREX.r3.6HG0595250 |
| CRYPTOCHROME 1a (CRY1a) | HORVU.MOREX.r3.6HG0587680) |
| GLYCIN-RICH RNA-BINDING-PROTEIN 1 (GR-RBP1) | HORVU.MOREX.r3.6HG0592840) |
| LIGHT-REGULATED WD 1 (LWD1) | HORVU.MOREX.r3.6HG0583670 |
**Candidates with consistent mutations across *eam7* / *EAM* genotypes:**
LIGHT-REGULATED WD 1 (LWD1) (HORVU.MOREX.r3.6HG0583670)
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| ------ | ------ | ------ | ------ | ------ | ------ | ------ |
| M-PPD-H1 | F | ACGTGAATGGTGGATCGGC | BstUI | 60°C | 506 | 433, 73 |
| | R | TATAGCTAGGTGCGTGGCG |
**Analysis:**
- based on observed recombinations in combination with flowering time data, the eam7 introgression area can be redefined
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## redefine eam7 introgression area
- based CAPS marker results
- combine observed recombinations with flowering time data to reduce the eam7 introgression area
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## calculate sample volumes
- from RNA concentrations: calculate the volume of RNA to use 1 µg RNA in cDNA synthesis reaction
- max amount: 11 µl
- fill the remaining amount with water
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**Crossing:**
- emasculate florets from mother plants before pollen are shed (about Waddington stage 8.5 - 9.0)
- pollinate florets with pollen from father plants, pollinate all florets from one spike with the same father: this is one cross
- cover the spike with a paper bag, count later how many of the pollinated florets develop into seeds
- cover the spike with a paper bag
- harvest seeds from each spike / each cross individually when they are completely dry
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## Score successful crosses
- all crossed spikes were covered with a paper bag
- number of pollinated florets per spike were documented
- when seeds were ripe: count how many of the polinated florets developed into seeds
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