sonia phillips phd supervisors: h van heerden ia dubery molecular characterization of...
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Sonia Phillips
PhD Supervisors: H van Heerden IA Dubery
Sonia Phillips
PhD Supervisors: H van Heerden IA Dubery
Molecular Characterization of Elicitor-responsive Genes in
Cotton
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Background Cotton is a multi-billion rand industry (> $ 500 billion) and Gossypium hirsutum accounts for > 95%
cotton fibre
Verticillium wilt = vascular plant disease caused by Verticillium dahliae, a destructive soil-borne, multi-host fungus (> 400 plant species afflicted)
Cotton production severely hampered by V. dahliae: in SA yield losses are around 30 % and can be as high as 70 %
Symptoms:
Wilting
Discolouration
Stunted growth
Defoliation
Smaller fruits
Poor fiber quality
Ultimately - death
Soil fumigation and fungicides are costly and have a negative ecological and health impact – alternative eco-friendly control measures are required: need to investigate the genetic components of plant immunity to V. dahliae
DDRT-PCR with V. dahliae elicitor in cotton (Michelle Zwiegellar, 2003, MSc dissertation) ID several ESTs differentially induced upon treatment of cell suspensions with V. dahliae elicitor
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Objectives
Obtain, analyze and characterize the full genomic sequences (+ promoter) of 3 genes represented by ESTs ID in DDRT-PCR study by genome walking, RACE, PCR, RT-PCR, bioinformatics analysis and Southern blots:
C4B5 (kinase?)
C4B4 (arm repeat?)
C1B10 (?)
Elicitation studies: Isolate elicitor from V. dahliae cell wall
Investigate if CWD elicitor induces PR protein production
Viability studies to determine appropriate elicitor concentrations for expression analysis
RT-qPCR to determine the time frame and kinetics of induction with CWD elicitor
Molecular characterization and genomic assignment of GhLIPN homoeologs (GhLIPN N and I)
GhLRK = Lectin receptor-like kinase (RLK) gene
GhARM = Armadillo-repeat gene
GhLIPN = Lipin gene
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Polyploidy in cotton
AADD tetraploid2n = 521 – 2 mya
G. hirsutum
G. darwinii
G. barbadense
G. mustelinum
G. tomentosum
AA diploid 2n = 26represented byG. herbaceum and G. arboreum
DD diploid 2n = 26represented byG. raimondii
Common ancestor5 – 10 mya
Hybridization and Endoreduplication
New allotetraploid species
B
Endoreduplication
New autotetraploid species
A
Autoployploidy Alloployploidy
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Genome Walking
Genomic DNA digested with four different restriction enzymes to obtain a genomic library from each
Genomic DNA isolated from the young leaves of Gossipium hirsutum cv. ‘OR-19’
Adapters ligated to digested fragments. Primary PCR with first gene-specific primers and adaptor primer 1
SP 1
AP 1
Secondary or ‘nested’ PCR with the second gene-specific primers and adaptor primer 2
SP 2
AP 2
Major bands cut from the gel, cloned, sequenced and characterized
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5` RACE
PCR product ready for agarose gel analysis and cloning
SP 1
(A)n 3`5`Synthesis of first strand cDNA with gene-specific primer SP 1 and reverse transcriptase
mRNA
(A)n 3`3`
cDNA
Degradation of the mRNA template by the RNase H activity of reverse transcriptase
3` (A)nAAA
Tailing of the purified cDNA with dATP by terminal transferase
Purify cDNA
Amplification of the tailed cDNA by primary PCR using the oligo (dT)-anchor primer and a nested gene-specific primer SP2
(A)nAAAA (T)nTTTTV
SP 2
Oligo dT-anchor primer
*V = A, C or G
SP 3
Inner PCR anchor primer
Secondary nested PCR with the PCR anchor primer and a SP 3 primer
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3` RACE
Amplification of the cDNA by PCR using gene-specific primers SP 4 and the outer PCR anchor primer
SP 4
Outer PCR anchor primer
*V = A, C or G
cDNA synthesis using the oligo (dT)- anchor primer by reverse transcriptase
5`mRNA AAAAAAAVTTTT
3`
Degradation of the mRNA by the RNase H activity of reverse transcriptase
cDNA
TTTT
PCR product ready for agarose gel analysis and cloning
SP 5
Inner PCR anchor primer
Nested secondary PCR, with the inner PCR anchor primer and gene-specific primer SP5
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Viability Study
0
10
20
30
40
50
60
70
80
90
100
110
-2.5 0 2.5 5 7.5 10 12.5 15 17.5 20
V. dahliae elicitor (g/ml)
% V
iabi
lity
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Genome Walking: GhLRK
2171 – 2501 bp
C4B5 EST
A
Stu
I
Dra
I
Eco
RI
PvuII
M
B
Stu
I
Dra
I
Eco
RI
PvuII
MUpstream from EST
Primary PCR Secondary/nested PCR
2240 bp
Stu
I
Dra
I
Eco
RI
PvuII
M
A B
Stu
I
Dra
I
Eco
RI
PvuII
M
Downstream from EST
Primary PCR Secondary/nested PCR
657 bp
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1080 bp
1 2
1259 - 2339
5` RACE: GhLRK
21
255 bp
312 - 567
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3` RACE: GhLRK
279 bp
132 bp2306 – 2583 bp
2306 – 2436 bp
2171 – 2501 bp
C4B5 EST
1 2
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Genomic analysis: GhLRK
1: SacI2: XbaI3: HindIII4: EcoRI
2277 bp
2007 bp30 bp 240 bp
309 bp2171 – 2501 bp
Full genomic sequence obtained from genome walking: 3011 bp
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Promoter analysis: GhLRK-309 + AAAGAAATTT TGAAATTTGT CGCAACATTC TAATCCCTCT TTACATGATA TTATTATGCT CTATTTTCAA CCCAAAATAT TTATTAGGGT GATTAAATAA AAAAAAGGAC - TTTCTTTAAA ACTTTAAACA GCGTTGTAAG ATTAGGGAGA AATGTACTAT AATAATACGA GATAAAAGTT GGGTTTTATA AATAATCCCA CTAATTTATT TTTTTTCCTG -199 + ATTCCGTGAT AAATTACTAA TGGGTCAATA TTTCAAGTTA GTACCTAAAA TTCAAACCCC ACTTCCCCAC GGCAACTGCC CAGATCATAT GTTTTGAGCC AATGCCATTT - TAAGGCACTA TTTAATGATT ACCCAGTTAT AAAGTTCAAT CATGGATTTT AAGTTTGGGG TGAAGGGGTG CCGTTGACGG GTCTAGTATA CAAAACTCGG TTACGGTAAA -89 + GATCAGTGTA TGACTTTGAA ATGTTCAAGT CATCCATTGG GGACTAATTG TTTCTATTCA CATATTTCTT ATGCTATATT ATTTTTTAG* - CTAGTCACAT ACTGAAACTT TACAAGTTCA GTAGGTAACC CCTGATTAAC AAAGATAAGT GTATAAAGAA TACGATATAA TAAAAAATC
Elicitor/pathogen-responsive
-309 + AAAGAAATTT TGAAATTTGT CGCAACATTC TAATCCCTCT TTACATGATA TTATTATGCT CTATTTTCAA CCCAAAATAT TTATTAGGGT GATTAAATAA AAAAAAGGAC - TTTCTTTAAA ACTTTAAACA GCGTTGTAAG ATTAGGGAGA AATGTACTAT AATAATACGA GATAAAAGTT GGGTTTTATA AATAATCCCA CTAATTTATT TTTTTTCCTG -199 + ATTCCGTGAT AAATTACTAA TGGGTCAATA TTTCAAGTTA GTACCTAAAA TTCAAACCCC ACTTCCCCAC GGCAACTGCC CAGATCATAT GTTTTGAGCC AATGCCATTT - TAAGGCACTA TTTAATGATT ACCCAGTTAT AAAGTTCAAT CATGGATTTT AAGTTTGGGG TGAAGGGGTG CCGTTGACGG GTCTAGTATA CAAAACTCGG TTACGGTAAA -89 + GATCAGTGTA TGACTTTGAA ATGTTCAAGT CATCCATTGG GGACTAATTG TTTCTATTCA CATATTTCTT ATGCTATATT ATTTTTTAG* - CTAGTCACAT ACTGAAACTT TACAAGTTCA GTAGGTAACC CCTGATTAAC AAAGATAAGT GTATAAAGAA TACGATATAA TAAAAAATC
Myb/Myc TF-binding
-309 + AAAGAAATTT TGAAATTTGT CGCAACATTC TAATCCCTCT TTACATGATA TTATTATGCT CTATTTTCAA CCCAAAATAT TTATTAGGGT GATTAAATAA AAAAAAGGAC - TTTCTTTAAA ACTTTAAACA GCGTTGTAAG ATTAGGGAGA AATGTACTAT AATAATACGA GATAAAAGTT GGGTTTTATA AATAATCCCA CTAATTTATT TTTTTTCCTG -199 + ATTCCGTGAT AAATTACTAA TGGGTCAATA TTTCAAGTTA GTACCTAAAA TTCAAACCCC ACTTCCCCAC GGCAACTGCC CAGATCATAT GTTTTGAGCC AATGCCATTT - TAAGGCACTA TTTAATGATT ACCCAGTTAT AAAGTTCAAT CATGGATTTT AAGTTTGGGG TGAAGGGGTG CCGTTGACGG GTCTAGTATA CAAAACTCGG TTACGGTAAA -89 + GATCAGTGTA TGACTTTGAA ATGTTCAAGT CATCCATTGG GGACTAATTG TTTCTATTCA CATATTTCTT ATGCTATATT ATTTTTTAG* - CTAGTCACAT ACTGAAACTT TACAAGTTCA GTAGGTAACC CCTGATTAAC AAAGATAAGT GTATAAAGAA TACGATATAA TAAAAAATC
Dof TF-binding
-309 + AAAGAAATTT TGAAATTTGT CGCAACATTC TAATCCCTCT TTACATGATA TTATTATGCT CTATTTTCAA CCCAAAATAT TTATTAGGGT GATTAAATAA AAAAAAGGAC - TTTCTTTAAA ACTTTAAACA GCGTTGTAAG ATTAGGGAGA AATGTACTAT AATAATACGA GATAAAAGTT GGGTTTTATA AATAATCCCA CTAATTTATT TTTTTTCCTG -199 + ATTCCGTGAT AAATTACTAA TGGGTCAATA TTTCAAGTTA GTACCTAAAA TTCAAACCCC ACTTCCCCAC GGCAACTGCC CAGATCATAT GTTTTGAGCC AATGCCATTT - TAAGGCACTA TTTAATGATT ACCCAGTTAT AAAGTTCAAT CATGGATTTT AAGTTTGGGG TGAAGGGGTG CCGTTGACGG GTCTAGTATA CAAAACTCGG TTACGGTAAA -89 + GATCAGTGTA TGACTTTGAA ATGTTCAAGT CATCCATTGG GGACTAATTG TTTCTATTCA CATATTTCTT ATGCTATATT ATTTTTTAG* - CTAGTCACAT ACTGAAACTT TACAAGTTCA GTAGGTAACC CCTGATTAAC AAAGATAAGT GTATAAAGAA TACGATATAA TAAAAAATC
Hormone-responsive
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Lectin domain Ser/Thr Protein Kinase domainTMS P
Protein analysis: GhLRK
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Predicted protein structure: GhLRK
Predicted carbohydratebinding site
Additional loopN-ter
N28
N52
C-ter
Activation segment
C-ter
N-ter
N577
Catalytic loop
Phosphate anchor
Lectin domain Protein kinase domainHomology to legume lectin from bark of Robinia pseudoacacia: 30% ID, E-score = 9.8e-25, EP = 100%
Homology to PK domain from AvrPto: 33% ID; E-score = 1e-35, EP = 100%
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RT-qPCR: GhLRK
0 2 4 6 8 100.000
1.000
2.000
3.000
4.000
5.000
6.000
7.000
8.000
9.000
10.000
Time (h)
Rela
tive
conc
entr
ation *
*
**
*
* p-value > 0.05
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Genome Walking: GhARMC4B4 EST
2759 - 3218
Stu
ID
raI
Eco
RI
PvuII
MB
Stu
I
Dra
I
Eco
RI
PvuII
MA
Secondary/nested PCRPrimary PCR
Downstream from EST
1078 bp
BStu
I
Dra
I
Eco
RI
PvuII
MA
Stu
I
Dra
I
Eco
RI
Pvu
II
M
Secondary/nested PCRPrimary PCR
Upstream from EST
2883 bp
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5` RACE: GhARM
2
2
1
1
832 bp
2202 - 3034
2126 - 2321
197 bp
1
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3` RACE: GhARM
1
2
2
1
2 1
2595 - 2969
2512 - 3213
3107 - 3213
367 bp
402 bp231 bp
1762 bp
C4B4 EST
2759 - 3218
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Genomic analysis: GhARM
Retrotransposon(777 bp)
1: SacI2: XbaI3: EcoRI4: HindIII
2759 – 3218 bp
1780 bp
1713 bp67 bp
986 bp
Full genomic sequence obtained from genome walking: 4131 bp
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-986 + GAATAA TAGCAGTTAA CAGTTGCAGT TAACAATTAA GGAAGTTAAG TTTTCTGTTA CAGTTGGTTA GCTAGACTCT TTATATACCT - CTTATT ATCGTCAATT GTCAACGTCA ATTGTTAATT CCTTCAATTC AAAAGACAAT GTCAACCAAT CGATCTGAGA AATATATGGA -900 + AAAGTTGTAT TCATTCGATA CAATGAAAAT ATAGAAGTAA TTCAGTTTCT ACAGCTTGTT TGCTGAGATT TAACAAGCAA GCACAGCCAA ATGCGGAGTG - TTTCAACATA AGTAAGCTAT GTTACTTTTA TATCTTCATT AAGTCAAAGA TGTCGAACAA ACGACTCTAA ATTGTTCGTT CGTGTCGGTT TACGCCTCAC -800 + AGTGAGGCTC GGCGCAGTTA TAGGAAACAA AGTCTAAATT TGATACAGTA AAAAGAGGCT AAAAAAGCGG GTCCCAAGAA GCAAGAGTAC AGCGACATCA - TCACTCCGAG CCGCGTCAAT ATCCTTTGTT TCAGATTTAA ACTATGTCAT TTTTCTCCGA TTTTTTCGCC CAGGGTTCTT CGTTCTCATG TCGCTGTAGT -700 + CATTCATCTA GGAAAAAGAA ACATTATAAA AATATACTAC TTGACTACTC CAACTCTAGA TTTATCCACC CCCAAGGATT GAGTGGTCCA TTTTTTCTTT - GTAAGTAGAT CCTTTTTCTT TGTAATATTT TTATATGATG AACTGATGAG GTTGAGATCT AAATAGGTGG GGGTTCCTAA CTCACCAGGT AAAAAAGAAA -600 + CATCCCCCCT CCCCAACACC TTCACATGCT AATGCTTTTG CTTCATGCTC ATTTCCTCCC TTTTCTTTAA TCTTTGATGT ACAGTTCAAT TAGTGGTCGT - GTAGGGGGGA GGGGTTGTGG AAGTGTACGA TTACGAAAAC GAAGTACGAG TAAAGGAGGG AAAAGAAATT AGAAACTACA TGTCAAGTTA ATCACCAGCA -500 + TATTTCCAAT ATATACAGTG AAAAACTGAA AATTACCCTA CCAATGCCTG CAGTCACTAT GTATCAATAT GCCTGCTAAG AGTTAAATGC TAAATGCTAA - ATAAAGGTTA TATATGTCAC TTTTTGACTT TTAATGGGAT GGTTACGGAC GTCAGTGATA CATAGTTATA CGGACGATTC TCAATTTACG ATTTACGATT -400 + GCAAACTGTT CCACTTTTCT TTTTTTATTT ATCGTTTAAT AGTGTTGGTA AAAGGTGATA AATAATGTGC CCCTTATACC CTTTTATTTA AAGCTGCTGT - CGTTTGACAA GGTGAAAAGA AAAAAATAAA TAGCAAATTA TCACAACCAT TTTCCACTAT TTATTACACG GGGAATATGG GAAAATAAAT TTCGACGACA -300 + GTGAACAAGC TGTAAAGCTT CATGCTGCCT CCTCTATTGC TTCTATCATT ACCTTTCACA ATTGTGTTCA GCCTTTTCCC TCTCTCATCT CTCGCGCCAG - CACTTGTTCG ACATTTCGAA GTACGACGGA GGAGATAACG AAGATAGTAA TGGAAAGTGT TAACACAAGT CGGAAAAGGG AGAGAGTAGA GAGCGCGGTC -200 + ATACAACCAA CATTACTAAT TTACTGTTAA TGTTATTGGT ATTTCTATTT TTTTCGAATC CAATGTTATT TGCCCCGTGA ACTCAGTCCA GTACTGGACT - TATGTTGGTT GTAATGATTA AATGACAATT ACAATAACCA TAAAGATAAA AAAAGCTTAG GTTACAATAA ACGGGGCACT TGAGTCAGGT CATGACCTGA -100 + TGTTTTTTCA TTATTGGAAC TGTCTTTCTC TCCATTTCCT TGGCTCCCCT TTTCCGTCCA ACCAAAGTGC CTTTTTTTTC CCAAGTCTTT TCATGTTTTT * - ACAAAAAAGT AATAACCTTG ACAGAAAGAG AGGTAAAGGA ACCGAGGGGA AAAGGCAGGT TGGTTTCACG GAAAAAAAAG GGTTCAGAAA AGTACAAAAA
Promoter analysis: GhARM
Elicitor/pathogen-responsive
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Promoter analysis: GhARM
Hormone-responsive
-986 + GAATAA TAGCAGTTAA CAGTTGCAGT TAACAATTAA GGAAGTTAAG TTTTCTGTTA CAGTTGGTTA GCTAGACTCT TTATATACCT - CTTATT ATCGTCAATT GTCAACGTCA ATTGTTAATT CCTTCAATTC AAAAGACAAT GTCAACCAAT CGATCTGAGA AATATATGGA -900 + AAAGTTGTAT TCATTCGATA CAATGAAAAT ATAGAAGTAA TTCAGTTTCT ACAGCTTGTT TGCTGAGATT TAACAAGCAA GCACAGCCAA ATGCGGAGTG - TTTCAACATA AGTAAGCTAT GTTACTTTTA TATCTTCATT AAGTCAAAGA TGTCGAACAA ACGACTCTAA ATTGTTCGTT CGTGTCGGTT TACGCCTCAC -800 + AGTGAGGCTC GGCGCAGTTA TAGGAAACAA AGTCTAAATT TGATACAGTA AAAAGAGGCT AAAAAAGCGG GTCCCAAGAA GCAAGAGTAC AGCGACATCA - TCACTCCGAG CCGCGTCAAT ATCCTTTGTT TCAGATTTAA ACTATGTCAT TTTTCTCCGA TTTTTTCGCC CAGGGTTCTT CGTTCTCATG TCGCTGTAGT -700 + CATTCATCTA GGAAAAAGAA ACATTATAAA AATATACTAC TTGACTACTC CAACTCTAGA TTTATCCACC CCCAAGGATT GAGTGGTCCA TTTTTTCTTT - GTAAGTAGAT CCTTTTTCTT TGTAATATTT TTATATGATG AACTGATGAG GTTGAGATCT AAATAGGTGG GGGTTCCTAA CTCACCAGGT AAAAAAGAAA -600 + CATCCCCCCT CCCCAACACC TTCACATGCT AATGCTTTTG CTTCATGCTC ATTTCCTCCC TTTTCTTTAA TCTTTGATGT ACAGTTCAAT TAGTGGTCGT - GTAGGGGGGA GGGGTTGTGG AAGTGTACGA TTACGAAAAC GAAGTACGAG TAAAGGAGGG AAAAGAAATT AGAAACTACA TGTCAAGTTA ATCACCAGCA -500 + TATTTCCAAT ATATACAGTG AAAAACTGAA AATTACCCTA CCAATGCCTG CAGTCACTAT GTATCAATAT GCCTGCTAAG AGTTAAATGC TAAATGCTAA - ATAAAGGTTA TATATGTCAC TTTTTGACTT TTAATGGGAT GGTTACGGAC GTCAGTGATA CATAGTTATA CGGACGATTC TCAATTTACG ATTTACGATT -400 + GCAAACTGTT CCACTTTTCT TTTTTTATTT ATCGTTTAAT AGTGTTGGTA AAAGGTGATA AATAATGTGC CCCTTATACC CTTTTATTTA AAGCTGCTGT - CGTTTGACAA GGTGAAAAGA AAAAAATAAA TAGCAAATTA TCACAACCAT TTTCCACTAT TTATTACACG GGGAATATGG GAAAATAAAT TTCGACGACA -300 + GTGAACAAGC TGTAAAGCTT CATGCTGCCT CCTCTATTGC TTCTATCATT ACCTTTCACA ATTGTGTTCA GCCTTTTCCC TCTCTCATCT CTCGCGCCAG - CACTTGTTCG ACATTTCGAA GTACGACGGA GGAGATAACG AAGATAGTAA TGGAAAGTGT TAACACAAGT CGGAAAAGGG AGAGAGTAGA GAGCGCGGTC -200 + ATACAACCAA CATTACTAAT TTACTGTTAA TGTTATTGGT ATTTCTATTT TTTTCGAATC CAATGTTATT TGCCCCGTGA ACTCAGTCCA GTACTGGACT - TATGTTGGTT GTAATGATTA AATGACAATT ACAATAACCA TAAAGATAAA AAAAGCTTAG GTTACAATAA ACGGGGCACT TGAGTCAGGT CATGACCTGA -100 + TGTTTTTTCA TTATTGGAAC TGTCTTTCTC TCCATTTCCT TGGCTCCCCT TTTCCGTCCA ACCAAAGTGC CTTTTTTTTC CCAAGTCTTT TCATGTTTTT * - ACAAAAAAGT AATAACCTTG ACAGAAAGAG AGGTAAAGGA ACCGAGGGGA AAAGGCAGGT TGGTTTCACG GAAAAAAAAG GGTTCAGAAA AGTACAAAAA
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Promoter analysis: GhARM
-986 + GAATAA TAGCAGTTAA CAGTTGCAGT TAACAATTAA GGAAGTTAAG TTTTCTGTTA CAGTTGGTTA GCTAGACTCT TTATATACCT - CTTATT ATCGTCAATT GTCAACGTCA ATTGTTAATT CCTTCAATTC AAAAGACAAT GTCAACCAAT CGATCTGAGA AATATATGGA -900 + AAAGTTGTAT TCATTCGATA CAATGAAAAT ATAGAAGTAA TTCAGTTTCT ACAGCTTGTT TGCTGAGATT TAACAAGCAA GCACAGCCAA ATGCGGAGTG - TTTCAACATA AGTAAGCTAT GTTACTTTTA TATCTTCATT AAGTCAAAGA TGTCGAACAA ACGACTCTAA ATTGTTCGTT CGTGTCGGTT TACGCCTCAC -800 + AGTGAGGCTC GGCGCAGTTA TAGGAAACAA AGTCTAAATT TGATACAGTA AAAAGAGGCT AAAAAAGCGG GTCCCAAGAA GCAAGAGTAC AGCGACATCA - TCACTCCGAG CCGCGTCAAT ATCCTTTGTT TCAGATTTAA ACTATGTCAT TTTTCTCCGA TTTTTTCGCC CAGGGTTCTT CGTTCTCATG TCGCTGTAGT -700 + CATTCATCTA GGAAAAAGAA ACATTATAAA AATATACTAC TTGACTACTC CAACTCTAGA TTTATCCACC CCCAAGGATT GAGTGGTCCA TTTTTTCTTT - GTAAGTAGAT CCTTTTTCTT TGTAATATTT TTATATGATG AACTGATGAG GTTGAGATCT AAATAGGTGG GGGTTCCTAA CTCACCAGGT AAAAAAGAAA -600 + CATCCCCCCT CCCCAACACC TTCACATGCT AATGCTTTTG CTTCATGCTC ATTTCCTCCC TTTTCTTTAA TCTTTGATGT ACAGTTCAAT TAGTGGTCGT - GTAGGGGGGA GGGGTTGTGG AAGTGTACGA TTACGAAAAC GAAGTACGAG TAAAGGAGGG AAAAGAAATT AGAAACTACA TGTCAAGTTA ATCACCAGCA -500 + TATTTCCAAT ATATACAGTG AAAAACTGAA AATTACCCTA CCAATGCCTG CAGTCACTAT GTATCAATAT GCCTGCTAAG AGTTAAATGC TAAATGCTAA - ATAAAGGTTA TATATGTCAC TTTTTGACTT TTAATGGGAT GGTTACGGAC GTCAGTGATA CATAGTTATA CGGACGATTC TCAATTTACG ATTTACGATT -400 + GCAAACTGTT CCACTTTTCT TTTTTTATTT ATCGTTTAAT AGTGTTGGTA AAAGGTGATA AATAATGTGC CCCTTATACC CTTTTATTTA AAGCTGCTGT - CGTTTGACAA GGTGAAAAGA AAAAAATAAA TAGCAAATTA TCACAACCAT TTTCCACTAT TTATTACACG GGGAATATGG GAAAATAAAT TTCGACGACA -300 + GTGAACAAGC TGTAAAGCTT CATGCTGCCT CCTCTATTGC TTCTATCATT ACCTTTCACA ATTGTGTTCA GCCTTTTCCC TCTCTCATCT CTCGCGCCAG - CACTTGTTCG ACATTTCGAA GTACGACGGA GGAGATAACG AAGATAGTAA TGGAAAGTGT TAACACAAGT CGGAAAAGGG AGAGAGTAGA GAGCGCGGTC -200 + ATACAACCAA CATTACTAAT TTACTGTTAA TGTTATTGGT ATTTCTATTT TTTTCGAATC CAATGTTATT TGCCCCGTGA ACTCAGTCCA GTACTGGACT - TATGTTGGTT GTAATGATTA AATGACAATT ACAATAACCA TAAAGATAAA AAAAGCTTAG GTTACAATAA ACGGGGCACT TGAGTCAGGT CATGACCTGA -100 + TGTTTTTTCA TTATTGGAAC TGTCTTTCTC TCCATTTCCT TGGCTCCCCT TTTCCGTCCA ACCAAAGTGC CTTTTTTTTC CCAAGTCTTT TCATGTTTTT * - ACAAAAAAGT AATAACCTTG ACAGAAAGAG AGGTAAAGGA ACCGAGGGGA AAAGGCAGGT TGGTTTCACG GAAAAAAAAG GGTTCAGAAA AGTACAAAAA
Dof TF-binding
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-986 + GAATAA TAGCAGTTAA CAGTTGCAGT TAACAATTAA GGAAGTTAAG TTTTCTGTTA CAGTTGGTTA GCTAGACTCT TTATATACCT - CTTATT ATCGTCAATT GTCAACGTCA ATTGTTAATT CCTTCAATTC AAAAGACAAT GTCAACCAAT CGATCTGAGA AATATATGGA -900 + AAAGTTGTAT TCATTCGATA CAATGAAAAT ATAGAAGTAA TTCAGTTTCT ACAGCTTGTT TGCTGAGATT TAACAAGCAA GCACAGCCAA ATGCGGAGTG - TTTCAACATA AGTAAGCTAT GTTACTTTTA TATCTTCATT AAGTCAAAGA TGTCGAACAA ACGACTCTAA ATTGTTCGTT CGTGTCGGTT TACGCCTCAC -800 + AGTGAGGCTC GGCGCAGTTA TAGGAAACAA AGTCTAAATT TGATACAGTA AAAAGAGGCT AAAAAAGCGG GTCCCAAGAA GCAAGAGTAC AGCGACATCA - TCACTCCGAG CCGCGTCAAT ATCCTTTGTT TCAGATTTAA ACTATGTCAT TTTTCTCCGA TTTTTTCGCC CAGGGTTCTT CGTTCTCATG TCGCTGTAGT -700 + CATTCATCTA GGAAAAAGAA ACATTATAAA AATATACTAC TTGACTACTC CAACTCTAGA TTTATCCACC CCCAAGGATT GAGTGGTCCA TTTTTTCTTT - GTAAGTAGAT CCTTTTTCTT TGTAATATTT TTATATGATG AACTGATGAG GTTGAGATCT AAATAGGTGG GGGTTCCTAA CTCACCAGGT AAAAAAGAAA -600 + CATCCCCCCT CCCCAACACC TTCACATGCT AATGCTTTTG CTTCATGCTC ATTTCCTCCC TTTTCTTTAA TCTTTGATGT ACAGTTCAAT TAGTGGTCGT - GTAGGGGGGA GGGGTTGTGG AAGTGTACGA TTACGAAAAC GAAGTACGAG TAAAGGAGGG AAAAGAAATT AGAAACTACA TGTCAAGTTA ATCACCAGCA -500 + TATTTCCAAT ATATACAGTG AAAAACTGAA AATTACCCTA CCAATGCCTG CAGTCACTAT GTATCAATAT GCCTGCTAAG AGTTAAATGC TAAATGCTAA - ATAAAGGTTA TATATGTCAC TTTTTGACTT TTAATGGGAT GGTTACGGAC GTCAGTGATA CATAGTTATA CGGACGATTC TCAATTTACG ATTTACGATT -400 + GCAAACTGTT CCACTTTTCT TTTTTTATTT ATCGTTTAAT AGTGTTGGTA AAAGGTGATA AATAATGTGC CCCTTATACC CTTTTATTTA AAGCTGCTGT - CGTTTGACAA GGTGAAAAGA AAAAAATAAA TAGCAAATTA TCACAACCAT TTTCCACTAT TTATTACACG GGGAATATGG GAAAATAAAT TTCGACGACA -300 + GTGAACAAGC TGTAAAGCTT CATGCTGCCT CCTCTATTGC TTCTATCATT ACCTTTCACA ATTGTGTTCA GCCTTTTCCC TCTCTCATCT CTCGCGCCAG - CACTTGTTCG ACATTTCGAA GTACGACGGA GGAGATAACG AAGATAGTAA TGGAAAGTGT TAACACAAGT CGGAAAAGGG AGAGAGTAGA GAGCGCGGTC -200 + ATACAACCAA CATTACTAAT TTACTGTTAA TGTTATTGGT ATTTCTATTT TTTTCGAATC CAATGTTATT TGCCCCGTGA ACTCAGTCCA GTACTGGACT - TATGTTGGTT GTAATGATTA AATGACAATT ACAATAACCA TAAAGATAAA AAAAGCTTAG GTTACAATAA ACGGGGCACT TGAGTCAGGT CATGACCTGA -100 + TGTTTTTTCA TTATTGGAAC TGTCTTTCTC TCCATTTCCT TGGCTCCCCT TTTCCGTCCA ACCAAAGTGC CTTTTTTTTC CCAAGTCTTT TCATGTTTTT * - ACAAAAAAGT AATAACCTTG ACAGAAAGAG AGGTAAAGGA ACCGAGGGGA AAAGGCAGGT TGGTTTCACG GAAAAAAAAG GGTTCAGAAA AGTACAAAAA
Promoter analysis: GhARM
Myb/Myc TF-binding
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Protein analysis: GhARM
SP ARM ARM ARM Armadillo-type fold AB C
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Predicted protein structure: GhARM
• Homology to human β-catenin protein from ARM repeat family• 14% ID; E-score = 3.6e-25,
estimated precision = 100%
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RT-qPCR: GhARM
0 0.5 1 2 4 6 8 100.000
0.200
0.400
0.600
0.800
1.000
1.200
Time (h)
Rela
tive
conc
entr
ation
* *
*
*
*
* p-value > 0.05
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Genome Walking: GhLIPNC1B10 EST
6488 - 6704
B a
Stu
ID
raI
Eco
RI
PvuII
M SnaB
IS
spI
Pd
iIEco
47
III
M SacI
Xb
aI
Hin
dIII
Eco
RV
M
b
Stu
I
SacI
Xb
aI
Hin
dIII
Eco
RV
MSsp
I
Pd
iI
MDra
I
Eco
RI
PvuII
M Eco
47
III
SnaB
Secondary/nested PCRPrimary PCR
1462 bp
B
Stu
I
Dra
I
Eco
RI
PvuII
M
Secondary/nested PCR
A
Stu
I
Dra
I
Eco
RI
PvuII
M
Primary PCR
Downstream from EST
1649 bp
Upstream from EST
Stu
I
Dra
I
Eco
RI
PvuII
M Stu
I
Dra
I
Eco
RI
PvuII
M
aA b
Primary PCR Secondary/nested PCR
676 bp
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5` RACE: GhLIPN
21
4
41290 – 3685 bp
1 2
33
3577 – 4073 bp
21
2
23877 – 5243 bp1 2
1
15805 – 6542 bp
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3` RACE: GhLIPN
1 2
C1B10 EST
6488 - 6704
6458 - 6837
6458 - 6704
246 bp
379 bp
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Genomic analysis: GhLIPN
75666-5738
9 6179-6307
11 6617-7021
106394-6492
86029-6072
65421-5507
44375-4448
54767-4851
33682-4280
22146-3602
11465-1633
1: XbaI 2: EcoRI3: HindIII
Genomic sequence GhLIPN N: 8 423 bpGhLIPN I: 6 704 bp
Transcribed sequenceGhLIPN N: 4 467 bpGhLIPN I: 4 479 bp
ORFBoth: 2 643 bp
PromoterGhLIPN N: 1 464 bpGhLIPN I: 1 289 bp
Several indels: mostly SNPs; 13 and 17bp
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GhLIPN_I ccaaaaaataaaaaaataaaaattcaaagctaccagagaatcctaacagcgtcggatcgttg 62 1 GhLIPN_N ccaaaaaataaaaaaataaaaattcaaagctaccagagaatcctaacagcgtcggatcgttg 62 GhLIPN_I tat-----------------agtaaagagaatcttacaattgtttcgatttcgagctgccag 107 GhLIPN_N tatcatcattcatcgatcatagtaaagagaatcttacaattgttttgatttcgagctgccag 124 GhLIPN_I attcgttcatcgcgggaattttcttctttgcgcaattcttaatctgtaagcttaccgacttt 169 1 GhLIPN_N gttcgttcatcccgggaattttcttctttgtgcagttcttaatctgtaagtttactgacttt 186 GhLIPN_I gactgtatagttatattttagttaaattttgaaattagaaagttcgattttttttcccacaa 231 GhLIPN_N ggctgtatagttatattttagttcaattttgaaattagaaagttcgattttttctcc-acaa 247 GhLIPN_I ttctcgtcgtttaatctgtatgttatcaatcttgtttgattgcttttactgtatactggaca 293 GhLIPN_N ttctcgtcgtttaatctgtatgttatcgatcttgtttgattgcttttaccgtatactggaca 309 GhLIPN_I tttattttatgtttggttgctgagaaaattggaggttttttttcc---cttgtgtttctaaa 352 GhLIPN_N tttattttatgtttggttgctgagaaaattggaggtttttttccctttcttgtgtttctaaa 371 GhLIPN_I gtttaagcttttcttcttttacgataaatatgttttagcattctatattgggatcatacttc 414 GhLIPN_N gtttaagcttttcttcttttatgagaaatatgttttagcattctatattgggatcatacttc 433 GhLIPN_I atataatattcgttaaacttggatttctatatttggttccaagtggcagcttatagattccc 476 GhLIPN_N atataatattcgttaaacttggatttctatatttggttccaagtgacagcttatagattccc 495 GhLIPN_I atacatgctttcttccttttttt--ccccattttattttcttgctgagctaaagggaacatg 536 GhLIPN_N atacatgctttcttccttttttttaccccattttattttctcgctgagcaaaagggaacatg 557 GhLIPN_I ttatggtttaagttggcttcgtttaattttgaactgaacgcagatacctgaaagtaaagaac 598 GhLIPN_N ttatggtttaagttggcttcgtttaattttgaactgaacgcagataactgaaagtaaagaac 619 GhLIPN_I aaagatccttgattagaacgattttggatgtttatattgatttgtggattcattttttgata 660 GhLIPN_N aaagatccttgattagaacgattttggatgtttatattgatttgtggattcattttt-gata 680 GhLIPN_I gaattaa-ggaaactgcagaaggatttttgttaatctaagaaaggtttaattttgacaaATG 721 2 GhLIPN_N gaattaaaggaaactgcagaaggatttttgttaatctaaggaaggtttaattttgacaaATG 742 GhLIPN_I AATGTGGTTGGCAAAGTTGGGAGTTTAATTTCACAAGGTGTATATTCTGTTGCTACTCCTTT 783 GhLIPN_N AATGTGGTTGGCAAAGTTGGGAGTTTAATTTCACAAGGTGTATATTCTGTTGCTACTCCTTT 804 GhLIPN_I CCATCCTTTCGGTGGAGCGGTTGATATAATTGTTGTTCGGCAACCAGATGGGACTTTTCGGA 845 GhLIPN_N CCATCCTTTCGGTGGAGCGGTTGATATAATTGTTGTTCGGCAAGCAGATGGGACTTTTCGGA 866 GhLIPN_I GCACACCTTGGTATGTTCGGTTTGGGAAGTTTCAGGGTGTCTTGAAAGGGGCTGAGAAGGTC 907 GhLIPN_N GCTCGCCTTGGTATGTTCGTTTTGGGAAGTTTCAGGGTGTCTTGAAAGGGGCTGAAAAGGTC 928 GhLIPN_I GTTCGTATAACTGTTAATGGCATTGAAGTAGATTTTCATATGTATCTTGATAACTCTGGGCA 969 GhLIPN_N GTTCGTATAACTGTTAATGGCATTGAAGCAGATTTTCATATGTATCTTGATAACTCTGGGCA 990 GhLIPN_I AGCATATTTTTTAAAGGAGGTTGAATCTGGTAAAGGATTTGAGACAAATGGAGATTTGAAGG 1031 GhLIPN_N AGCATATTTTTTAAAGGAGGTTGAATCTGGTAAAGGATTTCAGACAAATGGAGATTTGAAGG 1052 GhLIPN_I ATTCTGATGGTGAAGTTGTTTCAGATTCCTGGGTGGCTCAATTGAGGGATGAATGTGATGCC 1093 GhLIPN_N ATTCTGATGGTGAAGTTGTTTCAGATTCCTCGGTGGCTCAATTGAGGGATGAATGTGATGCC 1114 GhLIPN_I ACAAATCAAAAACAGCTTGAAAGGGCAGAATCTGATACTAGGTTCTATGATTTTCAAGATGA 1155 GhLIPN_N ACAAATCACAAACAGCTTGAAAGGGCAGAATCTGATACTAGGTTCTATGATTTTCAAGATGA 1176 GhLIPN_I TCAGTTTTCTCAAGAGGGTCTGGTTAATTTTTCAGAATATGGGTCCAACCGATATGAGGGTT 1217 GhLIPN_N TCAGTTTTATCAAGAGGGTCTGGTTAATTTTTCAGAATATGGGTCCGACCGATATGGGGGTT 1238 GhLIPN_I TAGATAGTGAGTGTTTTGGGGAAGCAAAAGGTTTAGATTCAGTTGTCTTCTTCAGTGAGGAT 1279 GhLIPN_N TAGATAGTGAGTGTTTTGGGGAAGCACAAGGTTTAGATTCAGTTGTCTTCTTCAGTAAGGAT 1300 GhLIPN_I GGCCATATTCTTACTGCCCCTGTTTTGGCATCAGACAGGAGTGCTGAAAATGTGCAACTAAG 1341 GhLIPN_N GGCCATATTCTCACAGCCCCTGTTTTGGCATCAGACAGGAGTGCTGAAAATGTGCAACTAAG 1362 GhLIPN_I CACACCTCTGTTCCATATAGGAGCAGGTGAAGGGCCTGACTCTTGTGACGGTAATGGGGAAT 1403 GhLIPN_N CACACCTCTGTTCCATATAGGCGCAGGTGAAGGACCTGACTTTTGTGACGGTAATGGGGAAT 1424 GhLIPN_I TTAGTCCAGGTGGCAATGAATCAGATGCTGACTATATAGGTAAGCTCAATGCTGCGGCACCT 1465 GhLIPN_N TTAGTCCAGGTGGCAATGAATCAGATGCTGACTATATAGGTAAGCTCAATGCTGCGGCACCT 1486 GhLIPN_I AAGAATTCCTCTGATATTGTTTGCAGCTTGGACAATGATTCTACTGCTTTGAGACACCATCT 1527 GhLIPN_N AAGAATTCCTCTGATATTGTTTGCAGCTTGGACAATGATTCTACTGCTTTGAGACACCATCT 1548
GhLIPN_I AGAAGTTTGTGAAAAAGGGGGGGAACATGCTTGTCAAACTGAAGAAACTCGAAACCTTTTCA 1589 GhLIPN_N AGAAGTTTGTGAAAGAGGGGGGGAACATGCCTGTCATACTGAAGAAACTCGAAACCTTTTCA 1610 GhLIPN_I AGCATGAAAATGAATTTATCAGGCAAAGTTACAGTGAAGATGCATCCGTGCATATTAAGGAT 1651 GhLIPN_N AGCATGAAAATGAATTTATCAGGCAAAGTGACATTGAAGATGCATCCATGCATATTAAGGAT 1672 GhLIPN_I GATGTTTTTAAGAGCTGTCTTGAACTATCTGAATTGGGCAGACACGATGAGAACACCAATTC 1713 GhLIPN_N GATGTTTTTAAGAGCAGTCTTGAACTATCTGAATTGGGCAGACACGAAGAGAACACCAATTC 1734 GhLIPN_I TGAAGAAATAGATAGCCCACTGCAAGCTCAGATTTCACAAGATAAGCCTTCTTGCAGCCCCC 1775 GhLIPN_N TGAAGGAATAGATAGCCCACTGCAAGCTCAGATTTCACAAGATAAGCGTTCTTGCAGCCCCC 1796 GhLIPN_I CAGAAGTTGGTGAAACTGAAAATGGAGCTATTGGTGGCTCCAGAAATAAAGATGTGTTGTCT 1837 GhLIPN_N CAGAAGTTGGTGAAACTGAAGATGGAGCTATTGGTGGCTCCAGAAATAAAGATGTGTTGTCT 1858 GhLIPN_I TCCTCTTGTAGTCCCAATTATTCCAATGAAAATGGGTCTCCTGATTTACCAGTTGAAAAGAC 1899 GhLIPN_N TCCTCTTGTATTCCCAATTATTCCAATGAAATTGGGTCTCCTGATTTACCAGTTGAAAAGAC 1920 GhLIPN_I AATGTTGGCTACAGATAATATGGGTTCTAACAATGCATCTGTTGATTTGGTTGTTAATGACC 1961 GhLIPN_N AATGTTGGCTACAGATAATAAGGGTTCTAACAATGCATCTGTTGATTTGGTTGTTAATGACC 1982 GhLIPN_I CTGAATTGAGAGATGAACAGTTCAATACTTCAGCAGCAACTGAAGGGATGAATAGCAGTCTG 2023 GhLIPN_N CTGAATTGAGAGATGAACAGTTCGATACATCAGCAGCAACTGAGGGGATGAATAGCAGTCTG 2044 GhLIPN_I CAAAGTCCTCCACCTGAGGACAAGAGTAGCATAAGTGAGACTGTGGAAACTGAAACAAGTTG 2085 GhLIPN_N CAAAGTCCTCCACCTGAGGACAAGAGTAGCATAAGTGAGACTGTGGAAACTGAAACAAGTTG 2106 GhLIPN_I TGCTAAAGAGATAGATGTTCGTGCTAGCCTGGgtaaggaactaagtgcttttcctcctttaa 2147 2 GhLIPN_N TGCTAAAGAGATAGATGTTAGTGCTAGCCTGGgtaaggaactaagtgcttttcctccttgaa 2168 GhLIPN_I ttacttaacctcccttattcttgctctgattctcgccttaaatttccagGATTTGAGATCTC 2209 3 GhLIPN_N ttacttaacttcccttattcttgctctgattctcgccttaaatttccagGGTTTGAGATCTC 2230 GhLIPN_I ACTCTGTGGCAATGAACTTTATGTGGGTATGGGTTCAGATGCTGCAGCAGAAGTCTTTGAAT 2271 GhLIPN_N ACTCTGTGGCAATGAACTTTATGTGGGTATGGGCTCAGATGCTGCAGCAGAAGTCTTTGAAG 2292 GhLIPN_I CACATCGGATATCAATGGAGGAATACAAAAATAATGCAATGTCAATTATTAAGAATACAAAC 2333 GhLIPN_N CACATCGGATATCAATGGAGGAATACAAAAATAATGCAATTTCAATTATTAAGAATACAAAC 2354 GhLIPN_I CTAATCATCCGATTTGGAGAGATGTACTTCACATGGGAAAAAGCTGCTCCTGTTGTTCTCGG 2395 GhLIPN_N CTAATCATCCGATTTGGAGAGATGTACTTCACATGGGAAAAAGCTGCTCCTGTTGTTCTCGG 2416 GhLIPN_I AATGGCTGCATTTGGTTTAGAGTTAGCTATTGAGCCCCAGGATGCAATCCCTGTTGAAAAGG 2457 GhLIPN_N AATGGCTGCATTTGGTTTAGAATTAGCTATTGAGCCCCAGGATGCAATCCCTGTTGAAAAGG 2478 GhLIPN_I ATGAGTCATCAAAGCCTAAGGGTGGTGCTTCTGGTGTCACTTCTGCACCTTCTGGCCGCAGA 2519 GhLIPN_N ATGAGTCACCAAAGCCTAAGGGTGCTGCTTCTGGTGTCACTTCTGCACCTTCTGGCTGCAGA 2540 GhLIPN_I TGGAGGCTTTGGTCTATTCCCTTAAAAAGGGTCAAAACACTTGAGAAGACCGGTAGCAATTT 2581 GhLIPN_N TGGAGGCTTCGGTCTATTCCCTTAAAAAGGGTCAAAACACTTGAGAAGACCGGTAGCAATTT 2602 GhLIPN_I ATCTAGTGAAGAGGTATTTCTTGATACTGAATCTTCTCTACAAAATTCACCAGAAGATTTAA 2643 GhLIPN_N ATCTAGTGAAGAGGTATTTCTTGATACTGAATCTTCTCTACAAAATTCACCAGCAGATTTAA 2664 GhLIPN_I TTCCAACATCCAGTGGAAGGATTGAATCTCCTGGCAAACAATTTGTGAGGACAAATATTCCC 2705 GhLIPN_N TTCCAACATCCAGTGGAAGGATTGAATCTCCTGGCAAACAATTTGTGAGGACAAATATTCCC 2726 GhLIPN_I ACCAACGAGCAGATTGCTTCCTTGAATCTGAAAAATGGTCAAAATATGATTACTTTCAGTTT 2767 GhLIPN_N ACCAACGAGCAGATTGCTTCCTTGAATCTGAAAAATGGTCAAAACATGATTACTTTCAGTTT 2788 GhLIPN_I CTCCTCCAGGGTTCTGGGAATACAACAGgtttcaattttctccttactcaattcttctggta 2829 3 GhLIPN_N CTTCTCCAGGGTTCTGGGAACACAACAGgtatcaattttttctttacacaattcttctggta 2850 GhLIPN_I ggttcgcaaggtttgtgttctgcttaaattttagcctaaaaatctgtgtatctttaacagGT 2891 4 GhLIPN_N ggttcgcaaggtttgtgttctgcttaaattttagcctaaaaatctgtgtatctttaacagGT 2912 GhLIPN_I TGAAGCTCATCTTTACTTGTGGAAATGGAACGCAAAGATTGTAATTTCAGATGTGGATGGAA 2953 GhLIPN_N TGAAGCTCATCTTTACTTGTGGAAATGGAACGCAAAGATTGTAATTTCAGATGTGGATGGAA 2974 GhLIPN_I CTATTACCAAgtagggatttatctcctgatattgttgctctgctgtatttaaaattttcccg 3015 4 GhLIPN_N CTATTACCAAgtaaggatttatctcctgatattgttgctctgcagtatttaaaattttcctg 3036 GhLIPN_I tttcacccatttatggacctgtctaaactagctctgaatcttattatgatattattatggaa 3077 GhLIPN_N tttcacccatttatggacctgtctaaactagctctgaatcttattatgatattattatggaa 3098
Homoeolog analysis: GhLIPN N & I
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GhLIPN_I acagagaaattaaaccatatattcgcatttgatgctgaaattataacttccattatgatctt 3139 GhLIPN_N acagagaaattaaaccatatattcgcatttgatgctgaaattataacttccattatggtctt 3160 GhLIPN_I tagtattagagcagcagtgagcattctagaagcagtcataccctaattgatttggtgtgaaa 3201 GhLIPN_N tagtattagagcagcagtgagcattctagaagcagtcataccctaattgatttggtgtgaaa 3222 GhLIPN_I tctttctttcgaatatgttgtagttatgtatagtgtaatacaagatgcatgtttcaataacc 3263 GhLIPN_N tctttctttcgaatatgttttagtcatgtatagtgtaatacaagatgcatgtttcaaaaacc 3284 GhLIPN_I ttaacatatttctttcagGTCTGATGTCTTAGGCCAGTTTATGCCTTTAGTTGGAAGGGATT 3325 5 GhLIPN_N ttaacatatttctttcagGTCTGATGTCTTAGGCCAGTTTATGCCTTTAGTTGGAAGGGATT 3346 GhLIPN_I GGACACAATCTGGTGTAGCTAATCTTTTCTCAGCTATTAAGgtatgcttttctcaattgttt 3387 5 GhLIPN_N GGACACAATCTGGTGTAGCTAATCTTTTCTCAGCTATTAAGgtatgcttttctcaattgttt 3408 GhLIPN_I ctttgttcagcttgacttgactgagaagttgttcttttctggactatcatgcaaaactagat 3449 GhLIPN_N ctttgttcaacttgacttgactgagaagtttttcttttctggactatcatgcaaaactagat 3470 GhLIPN_I ctaggaaattgctccaccgttttttagtttcaccacaactgaaatcaaaacttgtctcatgt 3511 GhLIPN_N ctagtaaattgctccaccattttttagtttcaccacaacagaaatcaaaacttgtctcatgt 3532 GhLIPN_I ttcccttgcgctttacttttactagttctttataaaattccccactgatgtaagtgatgata 3573 GhLIPN_N ttcccttgcgctttacttttactagttctttataaaattccccactgatgtaagtgatgata 3594 GhLIPN_I aattttttaatgcaagtttatagagtaacttttgataaccttagtatcaagattatttgagc 3635 GhLIPN_N aattttttaatgcaagtttatagagtaacttttgataaccttagtatcaagattatgtgagc 3656 GhLIPN_I attatcctgtcaccattatgccccttaacatttatttttctgttgcgaaatgtatttttgca 3697 GhLIPN_N attatcctgtcaccattatgcccctcaacatttatttttctgttgtgaaatgtatttttgca 3718 GhLIPN_I ctccttagtttatatatgggtagtgatgaaattcttctcataagttgcatttatgtcatctt 3759 GhLIPN_N cttcttagtttatatatgggtagtgatgaaattcttctcataagttgcatttgtgtcatctt 3780 GhLIPN_I tatccatccctcctgtaacatgtgtgcatgcacaaacacaatattgtttgtagggaacaata 3821 GhLIPN_N tattcatccctcctgtaacatgtgtgcatgcacaaacacaatattgtttgtagagaacaata 3842 GhLIPN_I tcaaactgtgtggctcttctatttattccattaaccaacatattttggcatgcatattaacc 3883 GhLIPN_N tcaaacagtgtggctcttctatttattccattaaccaacatattttggcatgcatattaacc 3904 GhLIPN_I aaatgatttccttgggtgtct-atgtccaacactaactgtatgttcacaacagGAGAATGGG 3944 6 GhLIPN_N aatt-attttcttgggtgtccgatgtccaacactaactgtatgttcacaacagGAGAACGGA 3965 GhLIPN_I TATCAGCTTCTATTTCTCAGTGCACGTGCAATTGTTCAGGCATATCTAACCAGAAGTTTCTT 4006 GhLIPN_N TATCAGCTTCTATTTCTCAGTGCACGTGCAATTGTTCAGGCATATCTAACCAGAAGTTTCTT 4027 GHLIPN_I ACTTAACCTGAAACAGgtaatggaatagcctgaatacaaatttttaaatttcccttatggaa 4068 6 GhLIPN_N ACTTAACCTGAAACAGgtaatggaatagcctgaatacacttttttacatttccctaatggaa 4089 GHLIPN_I gcaaaaaggcattagtgaaagtgttaaccaaagagaaaagctaacatttggaaaccatcttg 4130 GhLIPN_N gcaaaaaggt-------------ttaaccaaagagaaaagctaacatttggaaaccatcttg 4138 GhLIPN_I ttaaaagactttgaaatcacga-tttgtaaccgattttaactgatatagctttgtttatgta 4191 GhLIPN_N ttaaaagactttaaaatcacaaatttgtaaccgattttaactgatatagctttgtttatgta 4200 GhLIPN_I gGATGGAAAAGCTTTACCCTCTGGACCTGTTGTTATTTCTCCTGACGGTTTGTTTCCCTCAT 4253 7 GhLIPN_N gGATGGAAAAGCTTTACCCTCTGGACCTGTTGTGATTTCTCCTGACGGTTTGTTTCCCTCAT 4262 GhLIPN_I TGTACCGTGAGGgtgagatctttcattctttattttctcttacattgtttgtctattgaagt 4315 7 GhLIPN_N TGTACCGTGAGGgtgagatctttcattctttattttctctgacattgtttgtctattgaagt 4324 GhLIPN_I aatctagctgtttttttagtttgcttcacaattttgaggcttcagctacatgagaggaaaaa 4377 GhLIPN_N aatctagctgtttttttagtttgcttcacaattttgaggctccagctacatgagaggaaaaa 4386 GhLIPN_I gaattcttattgcattccatcacaaaacctttaaatgataaacgtagcaatatttgttgtct 4439 GhLIPN_N gaattcttattgtattccatcacaaaacctttaaatgataaacgtggcattatttgttgtct 4448 GhLIPN_I tggccttttctttatatcagaatcattaggctttattatcaagttacattgcttttcttctt 4501 GhLIPN_N tggccttttctttatatcagaatcattaggctttatcatcaagttacattgcttttcttctt 4510 GhLIPN_I ttacttcctttttaattatttataggttccattctaataaactatacttggcagTGATAAGA 4563 8 GhLIPN_N ttacttcctttttaattatttatgggttccattctaataaactatacttggcagTGATAAGA 4572 GhLIPN_I AGAACACCTCATGAATTCAAAATAGCTTGTTTAAAGgtgaattctgaaatattcaaccttgt 4625 8 GhLIPN_N AGAACACCTCATGAATTCAAAATAGCTTGTTTAAAGgtgaattctgaaatattcaaccttgt 4634
GhLIPN_I ttctaaatgtacaacggaactattactgcatccggatcagtcttttcacctacgatttcttc 4687 GhLIPN_N ttctaaatatacaacggaactattactgcatccagatcagtcttttcacctatgatttcttt 4696 GhLIPN_I cttatacgtgcgttgcagAATATCAGGAAGCTTTTCCCTTCGGACTACAATCCATTTTATGC 4749 9 GhLIPN_N cttatacatgaattgcagAATATCAGGAAACTTTTCCCTTCCGACTACAATCCATTTTATGC 4758 GhLIPN_I AGGCTTTGGAAACAGAGACACAGATGAACTCAGTTATAAAGAAATTGGCATCCCAAAGGGAA 4811 GhLIPN_N AGGCTTTGGAAACAGAGACACAGATGAACTCAGTTATAAAGAAATTGGCATCCCAAAGGGAA 4820 GhLIPN_I AAAGGTTTATTATTAATCCAAAGgtacatgtttttcatgtcctatttatgagaaatctatat 4873 9 GhLIPN_N AAATATTTATTATTAATCCAAAGgtacatgtttttcatgtcctacttatgagaaatctatat 4882 GhLIPN_I tgcaattaccatatcaaatttataacaatcggactcaaaactggcagGGTAAGGTGGCTGTA 4935 10 GhLIPN_N tgcaattaccatatcaaatttataacaatcgggctcaaaactggcagGGTGAGGTGGCTGTA 4944 GhLIPN_I AGTTATTGTATGAACTCGAGGTCATACACATCATTGCATACTCTTGTAGAAGAGATGTTTCC 4997 GhLIPN_N AGTTATTGTATGAACACGAGGTCATACACATCATTGCATACTCTTGTAGAAGAGATGTTTCC 5006 GhLIPN_I ACCAACCTCATCCATTGAACCGgtaaggcatccatttctcgcctctttatatatgatgcaaa 5059 10 GhLIPN_N ACCAACCTCATCCATTGAACTGgtaaggcatcaatttctcgcctgtttatatatgatgcaaa 5068 GhLIPN_I aaaacaattgaaaatttatctacaactgtctcttatttgttttggagccttcttgatatcct 5121 GhLIPN_N agaacaattgaaaatttatctacaactgtctcttatttgttttggagccttcttgatatcct 5130 GhLIPN_I tgatattttaatacgtttgcagGAAGATTATAACGAATGGAATTTTTGGAAAGTGCCATTGC 5183 11 GhLIPN_N tgatattttaatacgtttgcagGAAGATTATAACGAATGGAATTTTTGGAAAGTGCCATTGC 5192 GhLIPN_I AAGATATTGAGTAAcatagttgattgcctaaagaatacggtttagttataccaaggagatga 5245 GhLIPN_N AAGATATTGAGTAAcatagttgattgcctaaagaatacggtttagttataccaaggagatga 5254 GhLIPN_I tcagtagcttaatttgtcccataggtaagaaattgttctgagattcttcaagttcttaaact 5307 GhLIPN_N tcagtagcgtaatttgtcccataggtaagaaattgttatgatattcttcaagttcttaaact 5316 GhLIPN_I cgcttcctcactgacattcatattctttggtgaccttgaaatggtctgttggggttcatctt 5369 GhLIPN_N cgcttcctcactgacattcatattctttggtgaccttgaaatggtctgttggggttcatctt 5378 GhLIPN_I gtaaggaataaataattcaagtggataaaatcaaagccattgccta 5377 GhLIPN_N gtaaggaataaataattcaagtggataaaatcaaagccattgccta 5386
Sequence divergence: 2.5% (ungapped); between introns: 2.97%, exons: 2.27%
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GhLIPN_N MNVVGKVGSLISQGVYSVATPFHPFGGAVDIIVVRQADGTFRSSPWYVRFGKFQGVLKGA 60 GhLIPN_I MNVVGKVGSLISQGVYSVATPFHPFGGAVDIIVVRQPDGTFRSTPWYVRFGKFQGVLKGA 60 ************************************.******:**************** GhLIPN_N EKVVRITVNGIEADFHMYLDNSGQAYFLKEVESGKGFQTNGDLKDSDGEVVSDSSVAQLR 120 GhLIPN_I EKVVRITVNGIEVDFHMYLDNSGQAYFLKEVESGKGFETNGDLKDSDGEVVSDSWVAQLR 120 ************.************************:**************** ***** GhLIPN_N DECDATNHKQLERAESDTRFYDFQDDQFYQEGLVNFSEYGSDRYGGLDSECFGEAQGLDS 180 GhLIPN_I DECDATNQKQLERAESDTRFYDFQDDQFSQEGLVNFSEYGSNRYEGLDSECFGEAKGLDS 180 *******:******************** ************:** **********:**** GhLIPN_N VVFFSKDGHILTAPVLASDRSAENVQLSTPLFHIGAGEGPDFCDGNGEFSPGGNESDADY 240 GhLIPN_I VVFFSEDGHILTAPVLASDRSAENVQLSTPLFHIGAGEGPDSCDGNGEFSPGGNESDADY 240 *****:*********************************** ****************** GhLIPN_N IGKLNAAAPKNSSDIVCSLDNDSTALRHHLEVCERGGEHACHTEETRNLFKHENEFIRQS 300 GhLIPN_I IGKLNAAAPKNSSDIVCSLDNDSTALRHHLEVCEKGGEHACQTEETRNLFKHENEFIRQS 300 **********************************:******:****************** GhLIPN_N DIEDASMHIKDDVFKSSLELSELGRHEENTNSEGIDSPLQAQISQDKRSCSPPEVGETED 360 GhLIPN_I YSEDASVHIKDDVFKSCLELSELGRHDENTNSEEIDSPLQAQISQDKPSCSPPEVGETEN 360 ****:*********.*********:****** ************* ***********: GhLIPN_N GAIGGSRNKDVLSSSCIPNYSNEIGSPDLPVEKTMLATDNKGSNNASVDLVVNDPELRDE 420 GhLIPN_I GAIGGSRNKDVLSSSCSPNYSNENGSPDLPVEKTMLATDNMGSNNASVDLVVNDPELRDE 420 **************** ****** **************** ******************* GhLIPN_N QFDTSAATEGMNSSLQSPPPEDKSSISETVETETSCAKEIDVSASLGFEISLCGNELYVG 480 GhLIPN_I QFNTSAATEGMNSSLQSPPPEDKSSISETVETETSCAKEIDVRASLGFEISLCGNELYVG 480 **:*************************************** ***************** GhLIPN_N MGSDAAAEVFEAHRISMEEYKNNAISIIKNTNLIIRFGEMYFTWEKAAPVVLGMAAFGLE 540 GhLIPN_I MGSDAAAEVFESHRISMEEYKNNAMSIIKNTNLIIRFGEMYFTWEKAAPVVLGMAAFGLE 540 ***********:************:*********************************** GhLIPN_N LAIEPQDAIPVEKDESPKPKGAASGVTSAPSGCRWRLRSIPLKRVKTLEKTGSNLSSEEV 600 GhLIPN_I LAIEPQDAIPVEKDESSKPKGGASGVTSAPSGRRWRLWSIPLKRVKTLEKTGSNLSSEEV 600 ****************.****.********** **** ********************** GhLIPN_N FLDTESSLQNSPADLIPTSSGRIESPGKQFVRTNIPTNEQIASLNLKNGQNMITFSFFSR 660 GhLIPN_I FLDTESSLQNSPEDLIPTSSGRIESPGKQFVRTNIPTNEQIASLNLKNGQNMITFSFSSR 660 ************ ******************************************** ** GhLIPN_N VLGTQQVEAHLYLWKWNAKIVISDVDGTITKSDVLGQFMPLVGRDWTQSGVANLFSAIKE 720 GhLIPN_I VLGIQQVEAHLYLWKWNAKIVISDVDGTITKSDVLGQFMPLVGRDWTQSGVANLFSAIKE 720 *** ******************************************************** GhLIPN_N NGYQLLFLSARAIVQAYLTRSFLLNLKQDGKALPSGPVVISPDGLFPSLYREVIRRTPHE 780 GhLIPN_I NGYQLLFLSARAIVQAYLTRSFLLNLKQDGKALPSGPVVISPDGLFPSLYREVIRRTPHE 780 ************************************************************ GhLIPN_N FKIACLKNIRKLFPSDYNPFYAGFGNRDTDELSYKEIGIPKGKIFIINPKGEVAVSYCMN 840 GhLIPN_I FKIACLKNIRKLFPSDYNPFYAGFGNRDTDELSYKEIGIPKGKRFIINPKGKVAVSYCMN 840 ******************************************* *******:******** GhLIPN_N TRSYTSLHTLVEEMFPPTSSIELEDYNEWNFWKVPLQDIE 880 GhLIPN_I SRSYTSLHTLVEEMFPPTSSIEPEDYNEWNFWKVPLQDIE 880 :********************* *****************
Homoeolog analysis: GhLIPN N & I
* Identical aa: Conserved aa (16). Semi-conserved aa (5)
40 mismatches (4.5%)
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GhLIPN_I CGTGCAATTGTTCAGGCATATCTAACCAGAAGTTTCTTACTTAACCTGAAACAGgta 4025 GhLIPN_N CGTGCAATTGTTCAGGCATATCTAACCAGAAGTTTCTTACTTAACCTGAAACAGgta 4046 GR_Ea05119 ------------------------------------------------------... 209 GhLIPN_I atggaatagcctgaatacaaatttttaaatttcccttatggaagcaaaaaggcatta 4082 GhLIPN_N atggaatagcctgaatacacttttttacatttccctaatggaagcaaaaaggt.... 4099 GR_Ea05119 ......................................................... 209 GhLIPN_I gtgaaagtgttaaccaaagagaaaagctaacatttggaaaccatcttgttaaaagac 4139 GhLIPN_N .........ttaaccaaagagaaaagctaacatttggaaaccatcttgttaaaagac 4147 GR_Ea05119 ......................................................... 209 GhLIPN_I tttgaaatcacga.tttgtaaccgattttaactgatatagctttgtttatgtagGAT 4195 GhLIPN_N tttaaaatcacaaatttgtaaccgattttaactgatatagctttgtttatgtagGAT 4204 GR_Ea05119 ......................................................--- 212 GhLIPN_I GGAAAAGCTTTACCCTCTGGACCTGTTGTTATTTCTCCTGACGGTTTGTTTCCCTCA 4252 GhLIPN_N GGAAAAGCTTTACCCTCTGGACCTGTTGTGATTTCTCCTGACGGTTTGTTTCCCTCA 4261 GR_Ea05119 -----------------------------T--------------------------- 269 GhLIPN_I TTGTACCGTGAGGgtgagatctttcattctttattttctcttacattgtttgtctat 4309 GhLIPN_N TTGTACCGTGAGGgtgagatctttcattctttattttctctgacattgtttgtctat 4318 GR_Ea05119 -----------------------------------------t--------------- 326 GhLIPN_I tgaagtaatctagctgtttttttagtttgcttcacaattttgaggcttcagctacat 4366 GhLIPN_N tgaagtaatctagctgtttttttagtttgcttcacaattttgaggctccagctacat 4375 GR_Ea05119 -----------------------------------------------t--------- 383 GhLIPN_I gagaggaaaaagaattcttattgcattccatcacaaaacctttaaatgataaacgta 4423 GhLIPN_N gagaggaaaaagaattcttattgtattccatcacaaaacctttaaatgataaacgtg 4432 GR_Ea05119 ---------------------.-c--------------------------------a 439 GhLIPN_I gcaatatttgttgtcttggccttttctttatatcagaatcattaggctttattatca 4480 GhLIPN_N gcattatttgttgtcttggccttttctttatatcagaatcattaggctttatcatca 4489 GR_Ea05119 --gt------------------------------------------------t---- 496 GhLIPN_I agttacattgcttttcttcttttacttcctttttaattatttataggttccattcta 4537 GhLIPN_N agttacattgcttttcttcttttacttcctttttaattatttatgggttccattcta 4546 GR_Ea05119 --------------------------------------------a------------ 553 GhLIPN_I ataaactatacttggcagTGATAAGAAGAACACCTCATGAATTCAAAATAGCTTGTT 4594 GhLIPN_N ataaactatacttggcagTGATAAGAAGAACACCTCATGAATTCAAAATAGCTTGTT 4603 GR_Ea05119 --------------------------------------------------------- 610 GhLIPN_I TAAAGgtgaattctgaaatattcaaccttgtttctaaatgtacaacggaactattac 4651 GhLIPN_N TAAAGgtgaattctgaaatattcaaccttgtttctaaatatacaacggaactattac 4660 GR_Ea05119 ---------------------------------------g----------------- 667 GhLIPN_I tgcatccggatcagtcttttcacctacgatttcttccttatacgtgcgttgcagAAT 4708 GhLIPN_N tgcatccagatcagtcttttcacctatgatttctttcttatacatgaattgcagAAT 4717 GR_Ea05119 -------g------------------t--------t-------g--cg--------- 724 GhLIPN_I ATCAGGAAGCTTTTCCCTTCGGACTACAATCCATTTTATGCAGGCTTTGGAAACAGA 4765 GhLIPN_N ATCAGGAAACTTTTCCCTTCCGACTACAATCCATTTTATGCAGGCTTTGGAAACAGA 4774 GR_Ea05119 --------G-----------G------------------------------------ 781 GhLIPN_I GACACAGATGAACTCAGTTATAAAGAAATTGGCATCCCAAAGGGAAAAAGGTTTATT 4822 GhLIPN_N GACACAGATGAACTCAGTTATAAAGAAATTGGCATCCCAAAGGGAAAAATATTTATT 4831 GR_Ea05119 -------------------------------------------------GA------ 838 GhLIPN_I ATTAATCCAAAGgtacatgtttttcatgtcctatttatgagaaatctatattgcaat 4879 GhLIPN_N ATTAATCCAAAGgtacatgtttttcatgtcctacttatgagaaatctatattgcaat 4888 GR_Ea05119 ------------............................................. 850 GhLIPN_I taccatatcaaatttataacaatcggactcaaaactggcagGGTAAGGTGGCTGTAA 4936 GhLIPN_N taccatatcaaatttataacaatcgggctcaaaactggcagGGTGAGGTGGCTGTAA 4945 GR_Ea05119 .........................................---G------------ 866 GhLIPN_I GTTATTGTATGAACTCGAGGTCATACACATCATTGCATACTCTTGTAGAAGAGATGT 4993 GhLIPN_N GTTATTGTATGAACACGAGGTCATACACATCATTGCATACTCTTGTAGAAGAGATGT 5002 GR_Ea05119 --------------A------------------------------------------ 923 GhLIPN_I TTCCACCAACCTCATCCATTGAACCGgtaaggcatccatttctcgcctctttatata 5050 GhLIPN_N TTCCACCAACCTCATCCATTGAACTGgtaaggcatcaatttctcgcctgtttatata 5059 GR_Ea05119 ------------------------C-............................... 949
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GhLIPN_I tgatgcaaaaaaacaattgaaaatttatctacaactgtctcttatttgttttggagc 5107 GhLIPN_N tgatgcaaaagaacaattgaaaatttatctacaactgtctcttatttgttttggagc 5116 GR_Ea05119 ......................................................... 949 GhLIPN_I cttcttgatatccttgatattttaatacgtttgcagGAAGATTATAACGAATGGAAT 5164 GhLIPN_N cttcttgatatccttgatattttaatacgtttgcagGAAGATTATAACGAATGGAAT 5173 GR_Ea05119 ....................................--------------------- 970 GhLIPN_I_ TTTTGGAAAGTGCCATTGCAAGATATTGAGTAAcatagttgattgcctaaagaatac 5221 GhLIPN_N_ TTTTGGAAAGTGCCATTGCAAGATATTGAGTAAcatagttgattgcctaaagaatac 5230 GR_Ea05119 --------------------------G------------------------------ 1027 GhLIPN_I ggtttagttataccaaggagatgatcagtagcttaatttgtcccataggtaagaaat 5278 GhLIPN_N ggtttagttataccaaggagatgatcagtagcgtaatttgtcccataggtaagaaat 5287 GR_Ea05119 --------------------------------t------------------------ 1084 GhLIPN_I tgttctgagattcttcaagttcttaaactcgcttcctcactgacattcatattcttt 5335 GhLIPN_N_ tgttatgatattcttcaagttcttaaactcgcttcctcactgacattcatattcttt 5344 GR_Ea05119 ----c---t-—c--------------------------------------------- 1141 GhLIPN_I ggtgaccttgaaatggtctgttggggttcatcttgtaaggaataaataattcaagtg 5392 GhLIPN_N ggtgaccttgaaatggtctgttggggttcatcttgtaaggaataaataattcaagtg 5401 GR_Ea05119 ----c---------------------------------------------------- 1198 GhLIPN_I gataaaatcaaagccattgccta 5415 GhLIPN_N gataaaatcaaagccattgccta 5458 GR_Ea05119 ----------------------c 1222
11
GhLIPN_I acagGTTGAAGCTCATCTTTACTTGTGGAAATGGAACGCAAAGATTGTAATTTCAGA 2942 GhLIPN_N acagGTTGAAGCTCATCTTTACTTGTGGAAATGGAACGCAAAGATTGTAATTTCAGA 2963 GR_Ea05119 .........................................---------------- 16 GhLIPN_I TGTGGATGGAACTATTACCAAgtagggatttatctcctgatattgttgctctgctgt 2999 GhLIPN_N TGTGGATGGAACTATTACCAAgtaaggatttatctcctgatattgttgctctgcagt 3020 GR_Ea05119 ---------C-T---------.................................... 37 GhLIPN_I atttaaaattttcccgtttcacccatttatggacctgtctaaactagctctgaatct 3056 GhLIPN_N atttaaaattttcctgtttcacccatttatggacctgtctaaactagctctgaatct 3077 GR_Ea05119 ......................................................... 37 GhLIPN_I tattatgatattattatggaaacagagaaattaaaccatatattcgcatttgatgct 3113 GhLIPN_N tattatgatattattatggaaacagagaaattaaaccatatattcgcatttgatgct 3134 GR_Ea05119 ......................................................... 37 GhLIPN_I gaaattataacttccattatgatctttagtattagagcagcagtgagcattctagaa 3170 GhLIPN_N gaaattataacttccattatgatctttagtattagagcagcagtgagcattctagaa 3191 GR_Ea05119 ......................................................... 37 GhLIPN_I gcagtcataccctaattgatttggtgtgaaatctttctttcgaatatgttgtagtta 3227 GhLIPN_N gcagtcataccctaattgatttggtgtgaaatctttctttcgaatatgttttagtca 3248 GR_Ea05119 ......................................................... 37 GhLIPN_I tgtatagtgtaatacaagatgcatgtttcaataaccttaacatatttctttcagGTC 3284 GhLIPN_N tgtatagtgtaatacaagatgcatgtttcaaaaaccttaacatatttctttcagGTC 3305 GR_Ea05119 ......................................................--- 40 GhLIPN_I TGATGTCTTAGGCCAGTTTATGCCTTTAGTTGGAAGGGATTGGACACAATCTGGTGT 3341 GhLIPN_N TGATGTCTTAGGCCAGTTTATGCCTTTAGTTGGAAGGGATTGGACACAATCTGGTGT 3362 GR_Ea05119 ----------------------- C-------------------------------- 97 GhLIPN_I ------------------------.gtatgcttttctcaattgtttctttgttcagc 3398 GhLIPN_N AGCTAATCTTTTCTCAGCTATTAA.gtatgcttttctcaattgtttctttgttcaac 3419 GR_Ea05119 ------------------------G................................ 121 GhLIPN_I ttgacttgactgagaagttgttcttttctggactatcatgcaaaactagatctagga 3455 GhLIPN_N ttgacttgactgagaagtttttcttttctggactatcatgcaaaactagatctagta 3476 GR_Ea05119 ......................................................... 121 GhLIPN_I aattgctccaccgttttttagtttcaccacaactgaaatcaaaacttgtctcatgtt 3512 GhLIPN_N aattgctccaccattttttagtttcaccacaacagaaatcaaaacttgtctcatgtt 3533 GR_Ea05119 ......................................................... 121 GhLIPN_I tcccttgcgctttacttttactagttctttataaaattccccactgatgtaagtgat 3569 GhLIPN_N tcccttgcgctttacttttactagttctttataaaattccccactgatgtaagtgat 3590 GR_Ea05119 ........................................................ 121 GhLIPN_I gataaattttttaatgcaagtttatagagtaacttttgataaccttagtatcaagat 3626 GhLIPN_N gataaattttttaatgcaagtttatagagtaacttttgataaccttagtatcaagat 3647 GR_Ea05119 ......................................................... 121 GhLIPN_I tatttgagcattatcctgtcaccattatgccccttaacatttatttttctgttgcga 3683 GhLIPN_N tatgtgagcattatcctgtcaccattatgcccctcaacatttatttttctgttgtga 3704 GR_Ea05119 ......................................................... 121 GhLIPN_I aatgtatttttgcactccttagtttatatatgggtagtgatgaaattcttctcataa 3740 GhLIPN_N aatgtatttttgcacttcttagtttatatatgggtagtgatgaaattcttctcataa 3761 GR_Ea05119 ......................................................... 121 GhLIPN_I gttgcatttatgtcatctttatccatccctcctgtaacatgtgtgcatgcacaaaca 3797 GhLIPN_N gttgcatttgtgtcatctttattcatccctcctgtaacatgtgtgcatgcacaaaca 3818 GR_Ea05119 ......................................................... 121 GhLIPN_I caatattgtttgtagggaacaatatcaaactgtgtggctcttctatttattccatta 3854 GhLIPN_N caatattgtttgtagagaacaatatcaaacagtgtggctcttctatttattccatta 3875 GR_Ea05119 ......................................................... 121 GhLIPN_I accaacatattttggcatgcatattaaccaaatgatttccttgggtgtctatgtcca 3911 GhLIPN_N accaacatattttggcatgcatattaaccaattattttcttgggtgtccgatgtcca 3932 GR_Ea05119 ......................................................... 121 GhLIPN_I acactaactgtatgttcacaacagGAGAATGGGTATCAGCTTCTATTTCTCAGTGCA 3968 GhLIPN_N acactaactgtatgttcacaacagGAGAACGGATATCAGCTTCTATTTCTCAGTGCA 3989 GR_Ea05119 ........................-G---C--G------------------------ 155
5
4
5
4
6
Genomic assignment : A- or D-genome
Agreements between GhLIPN I and D-genome = 18
Agreements between GhLIPN N and D-genome = 8
Agreements between both homoeologs = 9
Therefore GhLIPN I likely derives from the ancestral D-genome and GhLIPN N from the ancestral A-genome
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GhLIPN_I_prom TTTCTTAACGCTGATTATGAGATTGTGAGAGAACGAGTATATATAGAGTGAAAAAAGA -943 GHLIPN_N_prom TTGGTTCACGTTGATTATGTGATTGTGAGAAAAAGAGAATATATAGAGTGAAAAAAGG -1383 GhLIPN_I_prom GAGATGATGATGTAACGTAATTAGGCAACGAGTGGATAGGCTAACGAGGTTTATTTTA -885 GHLIPN_N_pro1 GAGATGATGATGTAACGTAATTAGGCAAAGAGTGGATAGGCTAATGAGGTTTATTTTA -1325 GhLIPN_I_prom TTTTGACCAGAAGGGGCCGCCTTTAAAAGTAGATTGGAGTATGATATAAGTAGGAGTT -827 GHLIPN_N_prom TTTTGACCAGAATGGGCCGCCTTTAAAAGTAGAATGGAGTATGATATAAGTAGGAGTT -1267 GhLIPN_I_prom GCGATGTGAAGCAGGGGATGAATGTGTAATAAAAAAGAAAGTAAAATTCCTGCCTTCT -769 GHLIPN_N_prom GCGATATGAAGCAGGGGATGAATGTGTAATAAGAAAGAAAATAAAATTCATGGCTTCT -1209 GhLIPN_I_prom CCTCATCTCA-----ACCAGTCGGGTAAAACATAAAATC----TAAATTAT--TTAG- -723 GHLIPN_N_prom CCTCATCTCAACTCAACCCGTAAGGTAAAACATAAAAACAAACTAAAATATAGTTAAG -1151 GhLIPN_I_prom -TATTTATTTAAATGAGATTTGGTTGGTGATCATGGTA-TTATAGTTTTGGTAGAAGA -667 GHLIPN_N_prom TTAATAATCTAAATGA-ATTAG--TGGTGATCATGGTTGTTGTAGTTTTGGTAGAAGA -1096 GhLIPN_I_prom AGATGGGTC---TT--ACAAAAAACAAACATCCCAAACTATAATTTAGATTTTAATTT -614 GHLIPN_N_prom AGATGGGTGAGTTTCAACAAAAACAAAACATCCCAAACTATAATTTAGAATTTA---- -1042 GhLIPN_I_prom CTATTAATTACGTGTTAAATGCCATCTTAGCCACGACACAGAGCATGATATCTTAAAC -556 GHLIPN_N_prom ---------------TAAATG---TTTAATTCAATGCTCACCG-------TCTTAAAC -1009 GHLIPN_I_prom ACGGGAATTAAATC----------TTTACTTCTAA-----ATTCACTTATTTGGTCGC -513 GHLIPN_N_prom ACCGGAATTAAATCTAAATTCACTTTTGTTTCTTATTATTATTACAAAATTTTGTCGC -951 GHLIPN_I_prom ATTTAATTTTAATCATTTCTTTTCTTTTCCAAAACAAGCTCAATATTTGTTATGTTTG -455 GHLIPN_N_prom ATTTAATTTTAATCATTTCTTTTCTTTTCCAAAACAAGCTCAATATTTGGTATGTTTG -893 GHLIPN_I_prom AATTAAGCT------------------------------------------------- -446 GHLIPN_N_prom AATTAAGCTTTTAGTTGTTTCAAACACTTACAAAAATAACAGAATTTTATGTTGGTTC -835 GHLIPN_I_prom ---------------------------------------------------------- -445 GHLIPN_N_prom ACTGGAAAGTGTATCACTAACACTCATTTTTTATTTTTGTTATTTTGTCACATGTAAG -777 GHLIPN_I_prom ---------------------------------------------------------- -445 GHLIPN_N_prom ATTTGAAATATACAAGTTTCAAGGTTTTTATTATTCAATCAGAATAAATATGAATAAT -719 GHLIPN_I_prom ---------------------------------------------------------- -445 GHLIPN_N_prom TTTCACTAATATGATAGAAAATACTCCTGAATAATGTCTTTTATTTCGATCTCATTAT -661 GHLIPN_I_prom ---------------------------------------------------------- -445 GHLIPN_N_prom AGAATGATTAGATTTTTGAATTTCAATTTTAGATATGTAGGAAAATATAAAATGTAAG -603 GHLIPN_I_prom ---------------------------------------------------------- -445 GHLIPN_N_prom GGAGGAATTTTTGAGGAAAACTAACAAATTAGATGTTAGACTTTGATGTTTGAGATAG -545 GHLIPN_I_prom ---------------------------------------------------------- -445 GHLIPN_N_prom ATTATAATCTACATTTAAAAAAAAAAAAGAAAAAGAAAAAGAATTTCGACTTCTTTTT -487 GHLIPN_I_prom ----------------------------------------CAGGAGGTTTCTTTTC-A -429 GHLIPN_N_prom AAACAAAAGTTGATTAGTGTTAGTGATACACTGTCATTCTCAGGAGGTTACTTTTCTA -429 GHLIPN_I_prom CGTGTTTGAAACAACTAACAGCTTAATTCAAACCTAACAAATATTGAGCTTGTTTTGG -371 GHLIPN_N_prom AGTGTTTGAAACAACTGAAAGCTTAATTCAAACATAACAAATATTGAGCTTGTTTTGG -371 GHLIPN_I_prom AA---------------TTATATGCGACCAAATTTTCTAATAATAGACAAGAAACAAA -328 GHLIPN_N_prom AAAAGAAATGATTTAAATTATATGCGACAAAATTTTCTAATAATATATAAGAAACAAA -313 GHLIPN_I_prom TGTGAAATGAAGGGTAATTTAGTCAATTTAGATGTAAAGATT-------TTTTTTTAG -277 GHLIPN_N_prom AGTGAAATGAAGGGTAATTTAGTCAATTTAGAAGTTAATAATTTTTTTCTTTTTTTCG -255 GHLIPN_I_prom CATTACTGTAGAAGTTTCAATAATTATAATGTTTGTTCATTCCGGCGGCTCTACCATC -219 GHLIPN_N_prom GAATAAAACTCAAGTTTCAATAATTATAATGTTTCTTCATCCCGGCGGCTCTACCATC -197 GHLIPN_I_prom CTTAAG-CCTGAAAAAGCGGGACAATATTTCAAATATCCTAAATTACCCTTCCATTAA -162 GHLIPN_N_prom CTTAAGACCGGAAAAAGCGGGACAATATTTCAAATATCCAAAATTACCCTTCCATTAA -139 GHLIPN_I_prom TCACAAATTTACACCTCATATTTCTCGCTTCGTAGAGTCTTTCTAAGCAAAACTAAGG -104 GHLIPN_N_prom TCACAAATTTACACGTCATATTTCTCG----------TATTTTTAAGCAAAACTAAGG -91 GHLIPN_I_prom GACCTATTTATGTCTTTTCACAAGGTTACTTGTTTATTGCATGGCGGTTGCGGTTAG- -47 GHLIPN_N_prom GACCTATTTATGTCTTTTCGCA--------------TTGCATGGCGGTTGCGGTTAGG -47 GHLIPN_I_prom TCGCCGTTGCCAACTTCTGACTCGTTTTCAAAATTTCCATTTTTTT -1 GHLIPN_N_prom TCGCCGTTGCCAACTTCTGACTCGTTTTCTAAATTTCCATTTTTCC -1
Elicitor/pathogen-responsive
Promoter analysis: GhLIPN
Dof TFBS
Myb/Myc TFBS
Hormone-responsive
CAAT-box
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Protein analysis: GhLIPN 1 105 672
829
630 858 N-terminal lipin C-LIP HAD-like
domain
BNLS775-791
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Predicted protein structure: GhLIPN
• Homology to phosphatase domain of a polynucleotide kinase (HAD superfamily)• 18% ID; E-score = 3.7e-07,
estimated precision = 100%
Haloacid dehalogenase (HAD) domain
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RT-qPCR: GhLIPN
0 2 4 6 8 100.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
4.000
4.500
5.000
Time (h)
Rela
tive
conc
entr
ation
* p-value > 0.05
*
*
*
*
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Hypothetical Gene Interaction Model