plant genomics and emerging techniques dr. j p khurana
TRANSCRIPT
Plant Genomics and Emerging TechniquesPlant Genomics and Emerging Techniques
Jitendra P. Khurana
Inter-disciplinary Centre for Plant Genomics
Department of Plant Molecular Biology,
University of Delhi South Campus,
New Delhi-110021, India
1
2
3
4 Jaillon eat al. 2007
Nature 449: 463-467Sanger475 MbVitis
Tuskan et al. 2006
Science 313:1596-1604Sanger550 MbPopulus
IRGSP 2005
Nature 436: 793-800Sanger389 MbOryza sativa
AGI 2000
Nature 408:796-815Sanger125 MbArabidopsis thaliana
ReferenceSequencing Technology
Genome Size
Plant S. No.
Sequenced Plant Genomes
5
7
6
Pre-publication draft releaseSanger+ 454 + SOLiD950 MbSolanum lycopersicum10
Pre-publication draft releaseSanger + Illumina GA840 MbSolanum tuberosum9
Schnable, et al. 2009
Science 326:1112-1115Sanger2.3 GbZea mays8
Paterson et al. 2009
Nature 457: 551-556Sanger730 MbSorghum
Huang et al. 2009
Nat. Genet. 41: 1275-1281Sanger + Illumina GA367 MbCucumber
Ming et al. 2008
Nature 452:991-997Sanger372 MbCarica papaya
International Tomato Genome Sequencing Project
0 µm
1 2 3 4 5 6 7 8 9 10 11 12
24 26 26 19 12 20 27 17 16 10 13 11Mb T=220
246 268 274 193 111 213 277 175 164 108 135 113BACs T=2276To sequence
Chromosome
Country USA Korea China UK India NL FranceJapan Spain USA China Italy
70 µm108.0 Mb
85.6 Mb
83.6 Mb
82.1 Mb 80.0 Mb
53.8 Mb
80.3 Mb
64.7 Mb
81.8 Mb
88.5 Mb
64.7 Mb
76.4 Mb
Euchromatin
Heterochromatin
International Tomato Genome Sequencing Project
New Initiative
Whole Genome Sequencing using NextGen Sequencing Technologies
Italy 10X 15XNetherlands 10X 15XFrance 10XIndia 05XSpain 30X
The technology
The countries and their
commitment
Resources Whole genome shotgun library (The Netherlands and Italy)
454 SOLiD
New physical map using HindIII, EcoRI and MboI libraries (Keygene)
New randomly-sheared insert BAC library (EU-SOL and US)
The new proposed resources
Resources available
Assembly
Data available
Whole genome shotgun library (The Netherlands and Italy)
3 kb/20 kb paired-end libraries (Roche)
15X SOLiD and 454 data produced
(Available at dedicated ftp site to the consortium)
NextGen Sequences + BAC sequences (Sanger’s) + Selected BAC Mixture (SBM) + BAC ends + fosmid ends
First draft of tomato genome by 2009 endTarget
deadline
ABI 3730xl 454 Flx system Solexa 1G SOLiD
Technology Automated Sanger’s di-deoxy
Emulsion PCR Bridge PCR Emulsion PCR
Polymerase; Fluorescent-di-
deoxy terminators
Polymerase; Pyrosequencing Polymerase; Reversible terminators
Ligase; Octamer with
two-base encoding
1 Instrument cost US$ 500,000 US$ 550,000 US$ 500,000 US$ 591,000
Sequencing Technologies: A Comparison
1 Instrument cost (approx.)
US$ 500,000 US$ 550,000 US$ 500,000 US$ 591,000
2 Running cost/ Mb data ~US$ 8,000 ~US$ 60 ~US$ 2 ~US$ 1
3 Data generated per run 50 Kb 500 Mb 1 Gb 6 Gb
4 Read length 500– 1000 bp 300 bp 36 bp 35 bp
5 Read pair information Available Available Available Available
Overview of Next Generation Sequencing applications in crop genetics and breeding
Parental lines of mapping populations
Reference genotypes
Different tissues of the same genotype/ same tissues of
different genotypes (e.g. NILs)
Wild relatives
Germplasm lines/natural population
Next Generation Sequencing
Pools of PCR products/
metagenomicscDNAsgDNA/cDNAgDNA/cDNABACsRRGcDNA
Varshney et al., 2009, Trends in Biotechnology
Genomic resources
SSRs
SNPs/Indels/haplotypes SNPs/Indels/haplotypesCandidate genes
Phenotype data
Genetic maps and QTLs
Expression QTLs (eQTLs)
Candidate markers/ genes
Alien introgression
Population biology
Crop breedingGenome structure
and dynamics
ESTs Gene space
Genome assembly
Sequence variations Gene expression data
Vol 436, August 11, 2005
Pseudomolecules of the 12 rice chromosomes along with the participating nations responsible for sequencing each chromosome
Examples of agriculturally important genes isolated from rice
Locus or gene Function Identification method
Xa21 Bacterial resistance Map-based cloning
Sub1 Submergence tolerance Map-based cloning
Moc1 Tillering number control Map-based cloning
Pi9 Fungal resistance Map-based cloning
Pi2 Fungal resistance Map-based cloning
Gid1, Gid2 or Slr1 Gibberellin signalling pathway Map-based cloning
Sd1 Gibberellin synthesis Map-based cloning
Lsi1 Silicon transport Map-based cloning
qSH1
Jung et al., 2008, Nature Reviews Genetics
qSH1 Grain abscission control Map-based cloning
Spl18 Fungal resistance Activation tagging
Fon1 Tillering number control and the number of seeds
T-DNA
Lhs Floral organ formation and seed setting
T-DNA
Udt1 Early anther development T-DNA
Xb3 Bacterial resistance Yeast two-hybrid
NH1 Bacterial resistance Yeast two-hybrid
NRR Bacterial susceptibility Yeast two-hybrid
Control of Tillering in Rice
Phenotype and complementation of the moc1 mutant
Activity of the MOC1
Li et al. (2003)
qSH1 is Required for Formation of the Abscission Layer at the Base of the Rice Grain
(Konishi et al. 2006)
A QTL for rice grain width and weight : RING-type E3 ubiquitin ligase
Song et al., 2007, Nat. Genet.
Control of rice grain-filling and yield by GRAIN INCOMPLETE FILLING 1 (GIF1)
gif1
WT
INCREASED grain size and weight in transgenic rice overexpressing GIF1
Grain-filling and sugar content of gif1 mutant and wild-type rice
Wang et al., 2008, Nature Genetics
Sub1A,Sub1A, an Ethylenean Ethylene--responseresponse--factorfactor--like Gene like Gene Confers Confers
Submergence Tolerance to RiceSubmergence Tolerance to RiceXu et al. (2006) NatureXu et al. (2006) Nature
Introgression of the FR13A Sub1 Haplotype into an Intolerant Variety by MAS Confers Submergence Tolerance
Sub1 Region Gene Composition and Submergenceinduced mRNA Accumulation in Rice
Developmental TimeDevelopmental Time--points (19) Under Investigationpoints (19) Under Investigation
3Leaf, Root, Seedlings
4SAM, P1-1, P1-2, P1-3
5
VegetativeVegetative Floral TransitionFloral Transition SeedSeed
7
PaniclePanicle
P1P100--3 cm3 cm
P2P233--5 cm5 cm
P3P355--10 cm10 cm
P4P41010--15 cm15 cm
P5P51515--22 cm22 cm
P6P62222--30 cm30 cm
MMAA
MMPP
Developmental Stages of Maximum
Differential Expression Activity(Gene Up-regulation in comparison to previous stage)
6000
7000
8000
Genes upregulated
Genes with signal value <15 in four controls
SpermSpermCellsCells
0
1000
2000
3000
4000
5000OrganogenesisOrganogenesis
MeiosisMeiosis
PollenPollenMitosisMitosis FertilizationFertilization
Embryo DevEmbryo Dev
Rice Genome AnalysisRice Genome Analysis
AnnotationAnnotation andand expressionexpression ofof RLCKRLCK genegene familyfamily MolMol PlantPlant 20082008
RiceRice genomegenome databasedatabase NARNAR 20082008
GlobalGlobal analysisanalysis ofof AA2020/AN/AN11 zinczinc fingerfinger genegene familyfamily FuncFunc.. && IntegInteg.. GenomicsGenomics 20082008
AnnotationAnnotation andand expressionexpression ofof HomeoboxHomeobox genegene familyfamily FEBSFEBS JJ 20082008
AnnotationAnnotation andand expressionexpression ofof bZIPbZIP genegene familyfamily PlantPlant PhysiolPhysiol.. 20082008
AnnotationAnnotation andand expressionexpression ofof TCSTCS genegene familyfamily FuncFunc.. && IntegInteg.. GenomicsGenomics 20072007
Rice/ArabidopsisRice/Arabidopsis GenomeGenome AnnotationAnnotation GenomeGenome ResearchResearch 20072007
IntronIntron--lessless genesgenes ofof Rice/ArabidopsisRice/Arabidopsis FuncFunc.. && IntegInteg.. GenomicsGenomics 20072007IntronIntron--lessless genesgenes ofof Rice/ArabidopsisRice/Arabidopsis FuncFunc.. && IntegInteg.. GenomicsGenomics 20072007
AnnotationAnnotation andand expressionexpression ofof FF--boxbox genegene familyfamily PlantPlant PhysiolPhysiol.. 20072007
AnnotationAnnotation andand expressionexpression ofof ZOSZOS genegene familyfamily PlantPlant MolMol.. BiolBiol.. 20072007
AnnotationAnnotation andand expressionexpression ofof MADSMADS boxbox genegene familyfamily BMCBMC GenomicsGenomics 20072007
AnnotationAnnotation andand expressionexpression ofof CDPKCDPK genegene familyfamily MGGMGG 20072007
AnnotationAnnotation andand expressionexpression ofof SAURSAUR genegene familyfamily GenomicsGenomics 20062006
AnnotationAnnotation andand expressionexpression ofof GHGH33 genegene familyfamily FuncFunc.. && IntegInteg.. GenomicsGenomics 20062006
AnnotationAnnotation andand expressionexpression ofof AUX/IAAAUX/IAA genegene familyfamily FuncFunc.. && IntegInteg.. GenomicsGenomics 20062006
AnnotationAnnotation andand expressionexpression ofof CRRCRR genegene familyfamily BMCBMC PlantPlant BiolBiol.. 20062006
GenomeGenome--widewide analysisanalysis ofof thethe SAPSAP genegene familyfamily MGGMGG 20062006
Expression Profiles of 69 Rice MADS Box Genes(BMC Genomics, 2007)
PanicleSeed Str
ess
•OsMADS1
Panicle & Seed
Panicle•OsMADS3
Panicle
Seed
Os01g52980FBX
Os01g41280FBD
Os01g64700FBT
Os01g22430FBDUF
Os02g30210FBK
Os02g06520FBLD
Os02g10600FBA
Os02g10700FBL
Os11g07970FBW
Os09g27660FBO
Os01g72310FBO
Family TIGR ID Protein structure
Subfamily Domain content No. in category
FBX Unknown/none 465
FBDUF DUF 66
FBL LRR 61
FBK Kelch 25
FBD FBD 17
Classification and domain organization of F-box proteins in rice(Plant Physiology, 2007)
F-boxFBD
TUB
DUF
Kelch LRR
FBA_1
WD40
KNOT1 PAC
PPR
H2B
RRM
DEXD
RING
HELICcHATC
Zn F
MYND
TPR
LYSMJmjC
SEL1
HGWP
UBQ
ANTH
VWA
Peptidase C48
HLH
PAS
Os01g05970FBO
Os02g29150FBO
Os12g04130FBO
Os10g04850FBO
Os09g39190FBO
Os06g02400FBO
Os04g40030FBO
Os03g12940FBO
Os03g27250FBO
Os03g49250FBO
Os07g36360FBO
Os08g20480FBO
Os01g72310FBO
Os01g69270FBO
Os02g41910FBO
Os02g05700FBO
Os11g36790FBO
Os11g34460FBO
FBT TUB 14
FBLD LRR and FBD 9
FBA FBA 4
FBW WD40 2
FBO Others 24
Protein Structure
bZIP6
1 bZIP
4 5bZIP2 3
bZIP8 9
bZIP7
bZIP6 7
bZIP131210
bZIP131211
bZIP13121110
bZIP1311
14
bZIP131210
14
14
14
OsbZIP proteins having similar protein structure
OsbZIP67, 73OsbZIP15, 20
OsbZIP13, 26, 86OsbZIP54, 55, 56
OsbZIP05, 45OsbZIP34, 59
OsbZIP09, 10, 23, 42, 46, 72
OsbZIP66OsbZIP12, 40
OsbZIP62OsbZIP29
Structural features in OsbZIP transcription factors
18bZIP
bZIP15
bZIP25
bZIP24
bZIP 21 22 23
2018 19bZIP
18 19bZIP
bZIP1311
bZIP16
bZIP16 17
bZIP 17
14
OsbZIP02, 06, 19
OsbZIP17OsbZIP64OsbZIP03, 08, 11, 28, 37, 41, 43, 47, 49, OsbZIP39, 60OsbZIP01, 18, 48OsbZIP25, 30, 35, 36, 61, 68, 75, 76, 78,
OsbZIP12, 40
OsbZIP53OsbZIP44OsbZIP07
6
7 13
1210
14
Motifs
24Part of Pro-rich domain
GCB
Potential
phosphorylation sites
COP1 interaction motif
Determination of gene function by a COMBINATION of whole-genome transcriptome and predicted-pathway analysis
Whole-genome expression profiling
to identify light responsive genes
Tos17 insertion in
gene 5-1
Jung et al., 2008, Nature Reviews Genetics
AV-M3 Plate2 (Column) 49-96
TILLING: Traditional mutagenesis meets functional genomics
LE ACS-2
SMALL
Development
Nutrient Deprivation
SMALL NON-CODING RNAs : KEY REGULATORS WITH DIVERSE ROLES
Leaf Polarity and
DevelopmentFlowering
SMALL RNAs
Abiotic Stress Biotic Stress
Bacterial Pathogensand
Effectors
Salt Stress
Cold StressOxidative Stress
Drought Stress
Nutrient Deprivation
Fungal Pathogens
Insect Attack
Outlook for Future
� Sequencing/Re-sequencing of Indian crops
� Discovery of SNPs in Indian core germplasm
� Functional analysis of genes/alleles for trait-specific genetic enhancementspecific genetic enhancement
� Uncover relationship of genotype to phenotype in systems mode
� Genomics-assisted breeding
� Academia/Industry partnership
THANK YOU !
Our work is financially supported by the Department of Biotechnology
and
Department of Science and Technology, Government of India