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