Pathways for Gene Discovery

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Pathways for Gene Discovery
- Single Gene Approach Reverse Genetic
Mapping - Global Approach Transposon
tagging EST Whole-genome sequencing
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Expressed genes in rice cDNA clones
RFLP map physical map
UNDERWAY
Characterization of cDNA
Future purpose
estimation of product function analysis of
tissue specificity
Sequencing of genomic region around these
genes Analyse switch mechanism causing their
specific expression
leaf root
cDNA library
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Genome Projects
Whole Genome Sequence
Genomic Structure All genes Genome evolution

Rice Gene Discovery, BIOTEC and Kasetsart Univ.
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Genome being Sequenced
Organisms
Genome Size (bp)
Number of Gene
Gene density (kb/gene)
Coding
H. influenzae 1.8 x106
1 1,800 85 E. coli 4.6 x106
2 2,300 84 Yeasts 12.1
x106 2 6,000 70 C.
elegans 100 x106 5
20,000 50 Arabidopsis 100 x106
5 20,000 50 Human 3,000 x106
200 150,000 10 Rice 400
x106 5 gt30,000 40 Maize 2
,500 x106 30
gt30,000 10 Wheat 16,000 x106
200
gt30,000 10
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Rice The Pivotal Genome for Gene Discovery

• Economically most important food
• Well-studied genome
• Reliable transformation systems
• Smallest nuclear genome among cereals
• Extensive synteny among cereal genomes

Rice Gene Discovery, BIOTEC and Kasetsart Univ.
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Rice Genome Projects

• IRGSP International Rice Genome
• Sequencing Project
• Monsanto
• Syngenta
• BGI Bejing Genomic Institute

Rice Gene Discovery, BIOTEC and Kasetsart Univ.
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How rice genomes were sequenced ?

• BAC-by-BAC shotgun sequencing
• - IRGSP
• - Monsanto
• Whole genome shotgun sequencing
• - Syngenta
• - BGI

Rice Gene Discovery, BIOTEC and Kasetsart Univ.
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Thailand s Rice Genome Project
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Thailand 's Rice Genomic Program
Rice Genome Sequencing
Genome Mapping
Gene Discovering
Integrated Physical Map
Wild rice Diversity
Molecular Breeding
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Aims
Combine efforts with multinationals to sequence
the rice genome
Chr.10
Chr.4
Chr.12
Chr.9
Chr.5
Chr.8
Chr.11
Chr.7
Chr.3
Chr.6
Chr.2
Chr.1
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Discovering agricultural useful genes from
local,landrace and wildrice
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Utilization of the sequence information for rice
breeding
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Capacity building
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End Part I
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Components of Rice Genome project
Genome Sequencing
Wild Rice Diversity
Gene Discovery
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Step in Shotgun Sequencing
- 150-200Kb genomic BAC library - Physical
mapping - Sequence-ready map - Sublibrary of
individual BAC seed - Shotgun sequencing -
Sequence assembling
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Flowchart of genome sequencing
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Selected region for sequencing

• Genetic map

• Physical map

• Sequence ready-map

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Chr1
Chr3
Chr4
Chr5
Chr6
Chr7
Chr8
Chr9
Chr10
Chr11
Chr12
Chr2
C161
RM205
6.60
RM251
0.90
OSR15
RM31
R2869
C213
1.30
R1963
2.20
RM228
C950
6.80
RM12
1.90
8.30
OSR14
5.70
RM84
12.80
OSR12
13.40
RM7
1.10
RM248
0.70
RM224
RM17
1.10
RM233F
1.80
4.00
RM26
17.30
19.40
RM1
RM232
RM245
1.70
5.50
5.80
C901
9.00
1.30
RM233
1.90
8.50
0.00
RM204
R1944
OSR13
RM215
R1877
2.90
3.50
RM235
2.80
RM254
RM233C
RM225
2.90
C1018
RM201
OSR33
10.90
OSR16
32.30
RM230
3.50
27.70
5.90
4.50
RM241
7.10
6.60
1.20
RM217
OSR28
1.10
20.60
OSR7
RM233B
1.70
13.80
RM53
RM252
RM258
RM253
OSR29
10.70
RM80
4.90
9.00
9.30
RM242
15.10
RM256
4.50
RM206
2.90
R1553
RM257
RM243
R2147
RM210
C449
RM234
3.50
5.50
RM246B
RM35
9.90
12.70
RM164
1.90
12.20
30.10
58.20
12.20
R2289
RM42
15.90
10.00
5.60
RM21
R372
R2171
40.50
9.00
R210
RM10
8.10
2.50
2.90
OSR32
OSR4
RM23
RM44
1.80
36.80
RM229
4.40
2.20
7.10
32.10
17.60
C451
R1629
17.60
RM223
RM209
1.20
RM24
RM11
10.00
7.90
9.30
4.30
18.20
2.90
R1928
OSR22
RM216
8.00
RM3
RM25
C3
RM249
6.30
6.90
RM214
6.80
RM5
2.39
RM247
0.50
1.59
RM29
RM222
RM16
RM261
RM2
R79
RM202
RM9
0.00
3.50
G24B
5.80
R288
19.50
RZ206
1.20
12.50
RM237
0.50
11.30
G103
5.90
C122
11.50
2.50
C1454
23.80
7.39
R642
R250
35.30
RM246A
1.59
25.60
4.60
OSR30
25.40
R2373
RM219
RM20A
0.60
OSR27
2.20
RM212
14.50
50.80
5.30
13.10
RZ698
C86
C1057
7.89
RM4A
RM55
RM250B
1.40
RM262
R2414
RM13
5.60
1.89
RM4B
1.30
OSR35
RM20B
11.60
10.60
OSR1
C499
SSC1
0.00
R1164
C358
S10709
0.00
13.90
RG757
1.00
G36
0.70
RM221
55.30
9.09
RM240
2.70
RM6
2.09
RM250A
2.39
C560
11.50
OSR34
RM208
3.30
RM48
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Shot-gun Sequencing
126G1R
EH0457
RB0783
74D12L
147C2L
118E2R
S10709
180D1R
44A3R
R1164
EST and STS marker
P0651-G5 (148)
P0663-H05 (156)
P0603-H10 (133)
P0567-C7 (148)
Physical map by Nipponbare PAC library
P0645-D04 (136)
P0645-D04
3,437 reads, 45 contigs, 170,783 bp
P0651-G05
2,127 reads, 26 contigs, 148,789 bp
P0603-H10
296 reads, 39 contigs, 91,939 bp
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Gene Identification and Genome Annotation System
Similarity Search
Similarity Search
with Rice
cDNAs
with Rice
cDNAs
Similarity

Search
Similarity

Search
with

non-Redundant
with

non-Redundant
Protein
DB
AGCCCGAGGTA
Protein
DB
AGCCCGAGGTA
Interpretation
GAACGTGCTGC
Interpretation
GAACGTGCTGC
GCCGTGGTGGG
Integration
by
GCCGTGGTGGG
Integration
by
AGGGGTTCTGC
Search of Long
AGGGGTTCTGC
Search of Long
Researcher
Researcher
GCGCGGCCCAC
Terminal Repeat of
GCGCGGCCCAC
Terminal Repeat of
CCAGGTATGAG
CCAGGTATGAG
Retrotransponson
Retrotransponson
GTACGGCGCCA
GTACGGCGCCA
GTAATTGCACG
GTAATTGCACG
Coding Region
Coding Region
Prediction
Prediction
Splice Site
Splice Site
Prediction
Prediction
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Searching for genes from genomic sequence
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Genome Annotation Around Submergence Tolerance
Region
Transcription factor
Putative retroelement pol polyrotein
Hypothetical Protein
C e n t r o m e r e
S10709
R1164
TRG1
AA
DX,AR
Zing-finger protein
Putative retroelement pol polyrotein
RAS-GTP-binding protein
Gramma response I Protein
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• Fast, reliable transformation system

Rice Plant
Stable Transformation
Nipponbare Black Rice
Plant Tissue Culture for Gene transfer
Selection, Manipulation of Transformed cells
4 - 6 months
Embryogenic suspension cultures
Agrobacterium-mediated Gene Transfer
Transient Expression
- BIBAC vector
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• High capacity vector

Tolerance QTL
intron
intron
intron
exon
exon
exon
exon
50-100 kb Genomic Sequence
Hind III Spe I Not I
RB
LB
PNo
NoT
iGUS
P35S
NoT
NoT
NPT
Lac
Z
HPT
P35S
intron
Ri ori
RK2(ori)
BIBAC vector