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Multi-omics approach in plant characterization, domestication and breeding in a food safety context.
Dr. Gustavo H.F. Klabunde
[email protected] Agronomist - Post-Doc Research Fellow – UFSC, Brasil
RegionalSeminar“AdvancedresearchonpromissoryedibleplantsinLa6nAmerica:toolstoimproveFoodSecurityintheregion”
Dec4th,2017
Multi-omics approach in plant characterization, domestication and breeding in a food safety context. OutlineOmicsdefini6on–BriefhistoryandconceptOmicstoolsusedbyourresearchgrouponedibleplants.
Genomecharacteriza6on–MarkerdevelopmentandusePhylogeography–Araucariaangus+foliaChloroplastgenomics–Na6veMyrtaceaespeciesTranscriptomics–Acaurariaangus+foliaPhenomics–AccasellowianaMetagenomics–Bambusoideaespecies
Gene(Johannsen1909)–Evolvingconcept+Chromosome(Waldeyer-Hartz1888)“I propose the Genome expression for the set of haploidchromosomes, which together with their respec6veprotoplastspecifiesthefoundingmaterialofthespecies”Winkler,H.1920.VerbreitungundUrsachederParthenogenesisimPflanzen-undTierreiche.VerlagFischer,Jena.
Genome (Winkler 1920) Hybrid term - Fusion of affixes
ISSN:0888-7543
Genomics1:1-2(1987)
GenomicsTermcreatedin1987First“Omics”NewdisciplineNewpeer-reviewedjournal
Genomics Evolu3onStructure Func3on/Varia3on
Training–“capacitybuilding”
Omics
Bioinforma6cs Bioinforma6cs
Short Tandem Repeats – STR or Microsatellite - SSR
Molecular Marker Development
Acca sellowiana and Campomanesia xanthocarpa (Native Myrtaceae)
• DevelopmentofspecificnuclearandcpDNAmolecularmarkersforpoorlycharacterizedtreespecies
• RequiresclassicalLibraryconstruc6on(bacterialtransforma6on)ORasinglelowthroughputNGSrun(onlytoiden6fyrepe66vemofits)
• Requiressamplesfromatleast4distantpopula6onstomaximizetheprobabilityofvariant(alleles)finding
• RequiresaccesstoDNAgenotypingplagorms(C.E.orP.A.G.E.)• Timetodevelopandvalidatemarkers:from6to12months• Whenready,STRmarkersareabletoaccessgene6cdiversityfromundercharacterizedtreespecies
Short Tandem Repeats Development STR or SSR – Microsatellite
Accasellowiana–Feijoa-Na6veMyrtaceaeFruitswithuniqueflavourandtexture–HighMarketacceptanceIndomes6ca6onandbreedingprocessGene6cdiversity–Unknownformostpopula6ons(Lackofmoleculartools)
Samplescollectedformarkervalita6on
Newmarkers
Allelesrevealed
Short Tandem Repeats Development STR or SSR – Microsatellite
HighPolimorphysmInforma6onContent(PIC)
Short Tandem Repeats Development STR or SSR – Microsatellite
ABI3500XLDNASequencer
*Mul6plexgenotypingPanels(un6l24lociperrun)*96plantsgenotypedinlessthan120minutes
Laboratoryphase2AutomatedgenotypingCappilaryElectrophoresisE.C.
Laboratoryphase1PolymorphismscreeningPolyacrylamideGelElectrophoresisP.A.G.E.Costeffec6ve
Short Tandem Repeats Development STR or SSR – Microsatellite
Locus Primersequences(5'-3') Repeatmo3f
Allelesize(bp)
Ta(°C) 5´Dye GenBank
accessionno.
Cxant22 F:GCTTGGTGGTGCCTCTCTC (TCCA)3 209 55 VIC MG557627
R:GCTCTTCCCTTTGCCTCTCT Cxant26 F:ATGCAAAATCCCTACGTGCT (ATCG)3 162 57 NED MG557629
R:ATGACACATTTCGGCTGTGA Cxant36 F:TTCCCGCCTAACCCTAATG (AAG)4 323 57 PET MG557631
R:GTCAAATCTCGCTCCTCCAA Cxant50 F:CGCACAACCAGCACAAAAC (CTTT)3 477 66 6FAM MG557634
R:CTATCACCGAGGGAGGCAAG Cxant59 F:GAGGGACTTTCAGTTTGTGTGTC (GA)10 230 55 NED MG557635
R:GACCGTTTCCAACATTTCCA Cxant66 F:GCGAGACCATAAGCCACTAC (AGA)4 211 57 NED MG557636
R:TGAGAAGGAGACACACACAAAT Cxant69 F:CCCAACACTCTCCACAATCC (GA)7 294 55 6FAM MG557637
R:TCCTTCCCTCTTCTCTCCATC Cxant76 F:ATGTTTTTGTGCGTTCTGG (AAG)6 336 57 VIC MG557638
R:TTGACCTTTGTTCCTCTTCCT
Guabi05 F:TTCTCGTGATTTGTATCCAAGG (T)10 201 66 PETMG557633
R:TGCTTCAATCTTTCCTATCGAA Guabi11 F:TTGATTCAGGGAACAAATTCAA (ATTA)4 225 55 6FAM MG557632
R:TGGCTAGTGTGGTTCATTCAG
ChloroplastGenome
NucleargenomeIlluminarun
12Puta3vemo3fsdetected
77Puta3vemo3fsdetected
Polymorphismscreening4na3vepops.
2PolymorphicCpSSR
8PolymorphicCpSSR
Campomanesiaxanthocarpa(Myrtaceae)“guabiroba”Gene6cdiversity–Unknown(Lackofmoleculartools)Medicinalproper6es–FruitsCul6va6onop6on
Genetic characterization of wild populations of Araucaria angustifolia (Native conifer)
Short Tandem Repeats – STR
Molecular Marker Characterization
Short Tandem Repeats Characterization
• RequirespreviousmarkerDevelopmentandValida6on• STRsmarkersrevealpresentgene6cdiversityandgeneflowindexes• Insomecases,STRdatamaybeincorporedtoPhylogeographycanalysis
• STRAnalysisrevealsseveralPopula6onGene6csques6ons,suchasderivedfrom:
• Inbreeding/Ma6ngsystems• Migra6on/HabitatFragmenta6onEffects• RandomGene6cDrim• NaturalorAr6ficialSelec6on
Frequencedistribu6onofobserveddistancedispersaleventsof(a)pollenand(b)seed
dispersal,determinedbySTRparentageanalysis
Spa6algene6cStructureMul3plepurposestudy:Toaccessgene6cdiversityofFederalConserva6onAreasandevaluateits“efficiencyinconservategene6cresources”.Todetermineseedandpollendispersalranges.Increasingconcernaboutthehighlevelofforestfragmenta6onandassociatedlossofdiversity.
Chloroplast Genomics Plastome Campomanesiaxanthocarpa
Eugeniauniflora
Pliniaaureana
Wholeplastomesequenceprovides:
Dataforevolu3onarystudies Filogeny Ecology
Sourceofnewmolecularmarkers(SSR,SNPandintergenicspacers)
Gene3cdiversity Phylogeography
Tree Phylogeography Araucaria angustifolia
• Studyofprincipalsandprocessesgoverningthegeographicaldistribu6onofindividuals
• UseofChloroplastDNA(cpDNA)varia6ontoreconstructhistoricaleventsduetoitslowmuta6onrates
• Extensivefieldsamplingwork(wholedistribu6onrange)andDNAsequencing(SangerSequencing1stgen.)• Searchforbarriers,historicalgeneflowtendencies,glacialrefugiaandactualgenediversitystatusforconserva6onpurposes
JohnC.Avise
Sampling area x Range distribution 40 natural populations (n=594 trees)
SeveralChloroplastIntergenicSpacers–Sangersequencing
Chloroplast Variation
Laboratory workflow Extensive lab work
Forward / - Reverse AlignmentBioinformatics BidirecionalsequencingFinalConsensussequence
VariableSites29variablesitesin2.492bp
Araucaria angustifolia – Phylogeography Haplotypenetwork
Demographicscenarios–ABCanalysis
Mismatchdistribu6onfor3lineages
24dis3nctChloroplastforms
Central group
North group
South group
Admixture Zone (High gene flow)
BarrierNorthernhaplotypesarenotsharedwithCentralandSouthernVariantsHighGene6cdifferen6a6onbetweennaturalpopula6onsMul6pleGlacialRefugia
Speciesdistribu6onmodelingofA.angus+folia
Araucaria angustifolia – Phylogeography
Transcriptome Sequencing • AnalysisofallmRNAexpressedinagiven6ssueandcondi6on• Geneexpressioncompleteprofiles(includingmiRNAandotherRNAs)• Highlydinamicprofileanddependentonphysiologicalandenvironmentalcondi6ons,cells,6ssues,age,methodofsampling,stabiliza6onandstorage
• UseofNextGenera6onSequencing(NGS)Plagormsfordeepsequencing• Alterna6vewhenthegenomeofthespeciesofinterestisnotavailable(Crea6onofagenecatalogbasedontranscripts)
• Requiresextensivebioinforma6ctrainingandanalysis• Excellenttoolforthecharacteriza6onofdiversityandsearchforgenesofinterestforbreeding
RNA-Seq Workflow From field samples to complete transcriptomes
Field samples – Species Natural Environment
Leaf
Stem
Pollen
Leaf
Araucariaangus3folia
Podocarpuslamber3i
Transcriptomics *100Millionsofrawreadsper6ssue*ReadLength=80pbto(IonProton)*DenovoandRefferencebasedassembly(Piceaabies)*SeveralNGSplagormsavailable
Annotatedgenes
Annota3onEfficiency
GOlvl2 GOlvl3
P.lamber3iLeaf 4.649 28,43% CC:9 CC:17
MF:8 MF:12PB:15 PB:42
A.angus3foliaLeaf 4.354 29,25% CC:9 CC:17
MF:8 MF:12PB:15 PB:42
A.angus3foliaStem 3.643 28,43% CC:9 CC:17
MF:8 MF:12PB:15 PB:42
A.angus3foliaPollen 9.354 33,45% CC:12 CC:40
MF:13 MF:71PB:22 PB:196
Complete gene sequences from RNA-Seq data
Transcriptomics Functional annotation
Phenomics
• Largescalephenotypecharacteriza6on• Integra6onbetweenfactors
environmental,gene6c,epigene6cHowgene6cdiversityaffectsthephenotypes?
“P=G+E”
Borsuk,2015
Acca sellowiana - Phenomics Tree analysis - Field
Variables - Beggining of budding - Beggining of flowering (10% open) - End of flowering (90% fallen) - Beggining of harvest - End of harvest - Flower index - Fruit index
- Daily climate data
N= 235 genotypes
Evalua6onperiod-10years
Acca sellowiana - Phenomics Fruit analysis - Lab
Variables • Fruit diameter (cm), • Fruit length (cm), • Fruit weight (g), • Peel weight (g), • Pulp weight (g), • Pulp yield (%), • Peel thickness (cm), • Brix - Bxº • pH
N= 235 genotypes
Evalua6onperiod-10years
7 8 9 10 11 120.00
0.05
0.10
0.15
0.20 Inicio de Brotação - Inicio de Floração
Cv = 0.0033902 (Tb)^2 - 0.0526979 (Tb) + 0.3008925 R 2=0.9753 Tb=7,77°C
Temp. Base
CV
7 8 9 10 11 120.08
0.09
0.10
0.11
0.12 Inicio e Fim de Floração
Cv = 7.490e-04 (Tb)^2 - 1.143e-02 (Tb) + 1.261e-01 R 2=0.986 Tb=7,63°C
Temp. Base
CV
5 6 7 8 9 10 11 12 13 14 150.05
0.06
0.07
0.08
0.09
0.10Fim Floração - Inicio de Colheita
Tb=13,5°C
Temp. Base
CV
9 10 11 12 130.0
0.2
0.4
0.6
0.8 Inicio e Fim de Colheita
Cv = 0.05216 (Tb)^2 - 1.052549 (Tb) + 5.479667 R 2=0.9745 Tb=10,09°C
Temp. Base
CV
5 6 7 8 9 10 11 12 13 14 150.00
0.05
0.10
0.15
0.20Inicio de Brotação - Inicio de colheita
Tb=11,0°C
Temp. Base
CV
5 6 7 8 9 10 11 12 13 140.00
0.05
0.10
0.15Inicio de Brotação - Fim de colheita
Tb=11,0°C
Temp. Base
CV
Acca sellowiana - Phenomics
225
212
246
241
216
218
208
209
196
218
221
210
195
188
1069.61GD
1081.36GD
1058.41GD
1077.15GD
1083.93GD
1116.23GD
1058.43GD
1151.19GD
1105.52GD
1145.21GD
1159.58GD
1132.51GD
1091.73GD
1072.92GD
Dias
Acca sellowiana - Phenomics
L210
C10
20
L2
12C
L44.
38
L2
03C
212
L210
C10
17
L208
C34
1
Non
ante
L207
C29
1
L201
C53
B7
L202
C11
8
L210
C10
18
L214
C39
7
L204
C33
2
L214
C90
3
L210
C34
4
L309
C718
L201
C50-
2
L202
C103
L203
C242
L204
C301
L207
C152-
12
L205
C453
L215
C422
L204
C331
L213
C86
L305
CB24.27
L309
CB22.32
L206
C812
L310C719
L208C342
L206C511
L214C398
L203C240
L204C276
Helena
L207C358 L210C235 L207C138-2 L209C393 L208C339 L206C755 L210Cv1017 L210C535 L203C249 L212CB27.18 L301CB05.36 L304CB10.37
L207C712 L209C1010
L204C260
Mattos
L208C805
L214C382
L207CL33.11
L303CB25.01
L202C124
L208C716
L214C411
L214C381
L205C501
L213C21
L216C129
L206C522
L210CL44.29
L212CL54.02
L206C735
L216C80
L213C150
L302CB11.06
L202C135
L206C508
L202C138A
L210C259
L214C380
L207C392
L206C521B
L209C1004
L204C277
L215C389
L215C418
L209C1003
L310CB02.38
L218C831
L208C260
L215C350
L215C97
L210C1015
L215C15
L213C352
Alcantara L213C
128
L204C294 L210C1013 L210C533 L218C629 L209C629 L301CB08.30 L215C348 L213C722
L215C214 L209C1005
L215C720 L208C101
L302CB12.03
L204C373
L212CB10.04
L202C148
L217C432
L210C127
L213C14
L215C412
L213C20
L213C7
L205C504
L202C138
L203C159-27
L206C707
L206C509
L208C366
L210C333
L210C345
L212CB24.21
L306CB28.12
L209C902
L302CB15.16
L210C534
L213C806
L217C429
L306CB18.34
L207C520
L214C414
L306CB30.20
L305CB13.01
L214C415
L204C321
L212
CB2
5.29
L201
C50
2
L2
09C
502-
2
L2
02C
117
L208
C14
8
L2
05C
379
L204
C33
7
L2
07C
110
Alca
ntar
a (2
07)
L212
CB2
9.23
L203
C23
1
L2
12C
L49.
03
L302
CL5
1.02
L210
C53
6
L214
C458
L303
CL51
.02
L207
C228
L215
C347
L214
C519
L215
C320
L202
C119
L209
C53
L209
C721
L201
C79
L209
C1007
L201
C66
L205
C377
L205C401
L207C526
L216C427
L206C521A
L207C250
L208C91
L206C804
L303CB24.16
L307C336
L303CB22.32
L303CB27.16
L212CB12.03
L303CB10.37
L215C425
L217C522
L304CB23.16 L307C129 Distancia euclidiana pelo metodo UPGMA
Correlação cofenetica, r=0,6906
Primeiroranqueamento:L307C336,L301CB08.30,L209C629,L217C522,L303CB24.16,L303CB10.37,L310C719,L208C716,L206C812e,L215C425.Segundoranqueamento:L210C534,L217C522,L303CB10.37,L213C806,L215C425,L307C336,L303CB27.16,L212CB12.03,L203C249e,L304CB23.16.
Clones Group Alcantara 1 Helena 1 Mattos 1 Nonante 1 Outros 163 acessos 1 L204C294 2 L210C1013 2 L210C533 2 L218C629 2 L209C629 2 L301CB08.30 2 L215C348 3 L213C722 3 L215C214 3 L209C1005 3 L215C720 3
Com a ACP e CLUSTER Com o IS Método dos Ranks
Phenomics + Genomics AllPhenotypicallyevaluatedcul6vars/accessionsarebeingsequencedandgenotypedExtensivesetofinforma6onsforplantbreedersandresearchersLess6metoreleasenewandadaptedcul6vars
Metagenomics Associated genomes
• Applica6onofgenomictechniquesforthestudyofmicrobialcommuni6esdirectlyfromen6resamples;
• Dispensingtheneedofclassicmicrobiologictechniques;
Completegenomesor Metabarcode16S/ITS
Associated genomes metagenome
?
?
Associated genomes metabarcode workflow
-80°C
MS medium
30 days
Total DNA isolation and QC
In vivo: 10 nodal segments/tube
In vitro: 10 nodal segments
Type Region FragmentArquea 16S V4Bacteria 16S V3-V4Fungi ITS ITS2
PCR
NGS - Illumina MiSeq
NGS Data processing and OTU characterization
Associated genomes metagenome Bacterial preliminary results
44,3% 37,9% 17,9%
25,8% 72% 2,3%
36,3% 60,3% 3,4%
A
C
B
51,9 55,1
13,2 9,2
13,2 12,2
11,6 13,3
3,1 4,12,3 2,0
0102030405060708090100
SegmentoNodal Meiodecultura
Rela6veabun
dance(%
)
Spirochaetes
Planctomycetes
FBP
Cyanobacteria
Arma6monadetes
[Thermi]
OutrosemBactériaAcidobacteria
Laboratório de Fisiologia do Desenvolvimento e Genética Vegetal - LFDGV
Rgv.ufsc.brLfdgv.paginas.ufsc.br
Thankyouforyouraven6on
Multi-omics approach in plant characterization, domestication and breeding in a food safety context.
Dr. Gustavo H.F. Klabunde
[email protected] Agronomist - Post-Doc Research Fellow – UFSC, Brasil
RegionalSeminar“AdvancedresearchonpromissoryedibleplantsinLa6nAmerica:toolstoimproveFoodSecurityintheregion”
Dec4th,2017
