sonia phillips phd supervisors: h van heerden ia dubery molecular characterization of...

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

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

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

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

Endoreduplication

New autotetraploid species

Autoployploidy Alloployploidy

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

Secondary or ‘nested’ PCR with the second gene-specific primers and adaptor primer 2

Major bands cut from the gel, cloned, sequenced and characterized

5` RACE

PCR product ready for agarose gel analysis and cloning

(A)n 3`5`Synthesis of first strand cDNA with gene-specific primer SP 1 and reverse transcriptase

(A)n 3`3`

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

Oligo dT-anchor primer

*V = A, C or G

Inner PCR anchor primer

Secondary nested PCR with the PCR anchor primer and a SP 3 primer

3` RACE

Amplification of the cDNA by PCR using gene-specific primers SP 4 and the outer PCR anchor primer

Outer PCR anchor primer

*V = A, C or G

cDNA synthesis using the oligo (dT)- anchor primer by reverse transcriptase

5`mRNA AAAAAAAVTTTT

Degradation of the mRNA by the RNase H activity of reverse transcriptase

PCR product ready for agarose gel analysis and cloning

Inner PCR anchor primer

Nested secondary PCR, with the inner PCR anchor primer and gene-specific primer SP5

Viability Study

-2.5 0 2.5 5 7.5 10 12.5 15 17.5 20

V. dahliae elicitor (g/ml)

Genome Walking: GhLRK

2171 – 2501 bp

C4B5 EST

MUpstream from EST

Primary PCR Secondary/nested PCR

2240 bp

Downstream from EST

657 bp

1080 bp

1259 - 2339

5` RACE: GhLRK

255 bp

312 - 567

3` RACE: GhLRK

279 bp

132 bp2306 – 2583 bp

2306 – 2436 bp

2171 – 2501 bp

C4B5 EST

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

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

Dof TF-binding

Hormone-responsive

Lectin domain Ser/Thr Protein Kinase domainTMS P

Protein analysis: GhLRK

Predicted protein structure: GhLRK

Predicted carbohydratebinding site

Additional loopN-ter

Activation segment

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%

RT-qPCR: GhLRK

0 2 4 6 8 100.000

10.000

Time (h)

ation *

* p-value > 0.05

Genome Walking: GhARMC4B4 EST

2759 - 3218

Secondary/nested PCRPrimary PCR

Downstream from EST

1078 bp

Upstream from EST

2883 bp

5` RACE: GhARM

832 bp

2202 - 3034

2126 - 2321

197 bp

3` RACE: GhARM

2595 - 2969

2512 - 3213

3107 - 3213

367 bp

402 bp231 bp

1762 bp

C4B4 EST

2759 - 3218

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

-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

Hormone-responsive

Dof TF-binding

Myb/Myc TF-binding

Protein analysis: GhARM

SP ARM ARM ARM Armadillo-type fold AB C

Predicted protein structure: GhARM

• Homology to human β-catenin protein from ARM repeat family• 14% ID; E-score = 3.6e-25,

estimated precision = 100%

RT-qPCR: GhARM

0 0.5 1 2 4 6 8 100.000

Time (h)

* p-value > 0.05

Genome Walking: GhLIPNC1B10 EST

6488 - 6704

M SnaB

M SacI

1462 bp

Secondary/nested PCR

Primary PCR

Downstream from EST

1649 bp

Upstream from EST

676 bp

5` RACE: GhLIPN

41290 – 3685 bp

3577 – 4073 bp

23877 – 5243 bp1 2

15805 – 6542 bp

3` RACE: GhLIPN

C1B10 EST

6488 - 6704

6458 - 6837

6458 - 6704

246 bp

379 bp

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

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

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%

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%)

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

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

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

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

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

Promoter analysis: GhLIPN

Dof TFBS

Myb/Myc TFBS

Hormone-responsive

CAAT-box

Protein analysis: GhLIPN 1 105 672

630 858 N-terminal lipin C-LIP HAD-like

domain

BNLS775-791

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

RT-qPCR: GhLIPN

0 2 4 6 8 100.000

Time (h)

* p-value > 0.05

Hypothetical Gene Interaction Model

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