genomics assisted crop improvement- a new paradigm
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Genomics Assisted Crop Improvement
A New Paradigm
28 June 2010
Melaku Gedil
Monday 28 June, 2010
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 1
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PhenomicsBiochemical Physiological Agronomical
ProteomicsMetabolomics Interactomics
TranscriptomicssiRNA mRNA rRNA
GenomicsRegulatory Gene Other (IRE)
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0.00.51.01.52.02.53.03.54.0
cmd1 cmd2 cmd3 cmd4 cmd5 cmd6
Cassava mosaic disease
Ave
rage
sco
re
Ib07Ib08On07
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Frequency distribution of CMD score in two crosses (top) and two selfed (bottom) populations
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Clone Location Year N min max range1M-35 Onne 2007 2 1 3 21M-43 Onne 2007 5 1 3 21M-44 Onne 2007 5 1 3 21M-10 Onne 2007 3 1 4 31M-17 2007 2 1 4 31M-42 Onne 2007 3 1 4 31M-5 Onne 2007 3 1 4 3
23M-10 Onne 2007 5 1 3 223M-25 Onne 2007 4 1 3 223M-3 Onne 2007 4 1 3 2
23M-31 Ibadan 2007 5 1 3 223M-40 Onne 2007 3 1 3 223M-42 Ibadan 2007 5 1 3 223M-45 2007 4 1 3 223M-22 Onne 2007 5 1 4 323M-3 Ibadan 2008 2 1 4 3
23M-30 Ibadan 2007 3 1 4 323M-32 Ibadan 2007 4 1 4 323M-33 Ibadan 2007 5 1 4 323M-39 Onne 2007 4 1 4 323M-41 2007 3 1 4 323M-5 Onne 2007 4 1 4 3
23M-14 Ibadan 2007 3 1 5 423M-40 Ibadan 2007 5 1 5 423M-41 Ibadan 2008 5 1 5 423M-9 2007 3 1 5 4
Improved phenotyping – PCR & RT-PCR
Different virusesAfrican cassava mosaic virus (ACMV)
East African cassava mosaic virus (EACMV)
East African cassava mosaic Cameroon virus (EACMCV)
East African cassava mosaic Malawi virus (EACMMV)
East African cassava mosaic Zanzibar virus (EACMZV)
South African cassava mosaic virus (SACMV)
EACMV-UG: a recombinant virus,
EvaluationPCR diagnostics to determine the type of infectionReal-time PCR to determine virus loadResistance profiling
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• 12 clones
• Three replications
• Repeated sampling and
assay
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Replication-1
Rep# Score ACMV
EACMV
101 3 +++ +102 1 - -103 3 +++ ++104 1 - -
105a 3 +++ ++105b 2 +++ +++106 2 - +107 1 - +
108a 1 - -108b 1 - -109 3 +++ ++110 1 - -111 2 - -
112a 1 - +112b 1 - +
Replication-2
Rep# ScoreACMV
EACMV
201 3 +++ +202 1 - +203 - + -204 -205 1 - -206 1 + -
207a 2 +++ -207b 1 + +208a 1 - -208a 1 - +209 1 - +210 3 +++ +
211a 2 +++ +211b 2 +++ +++211c 2 +++ -212 4 +++ -
Replication-3
Rep# ScoreACMV
EACMV
301 4 +++ -302 - - -303 1 - -
304a 3 +++ +++304b 4 +++ -305a 1 - -305b 1 - +306a 1 - -306b 1 - -306c 1 - -306d 1 - -307 3 - -308 1 - -309 - - -
310a 1 - -310b 1 - -311 1 - -312 1 - -
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Sample cmd2 Cт Cт
Name Severity score Mean SD
1M-01 4 7.1 0.2
1M-02 5 17.6 0.8
1M-03 4 25.4 0.1
1M-04 1 21.3 1.5
1M-05 2 23.6 0.2
1M-06 2 19.5 0.3
1M-07 1 10.8 0.3
Example of real-time determination of ACMV quantity in cassava samples
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria
2. Marker Screening/genotyping
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SSR set Type No. Tested Valid Ref
New-SSR 1 EST 846 192 124 Lokko, 2007Raji, 2009
New SSR 2 EST 70 70 52 Ferguson in progress
Sub total 916 262 176
SSR set Type No. Tested Validated Ref
SSRY-C&-T Genomic 343 171 137 Mba, 2001
NS-A and OS-A Genomic 156 39 33 CIAT
NS-L Genomic 140 - CIATEST-SSR EST 178 - CIAT
Total 817 210 170
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R R R R R R R R S S S S S S S S
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Set-1 Set-2
NS158* SSRY238*
SSRY28* SSRY20*
SSRY235* SSRY51
SSRY44 SSRY76
NS136
SSRY169*
RME1 (SCAR)*
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Primers
Severity
Score
= R
Severity Score
= S
Genotype
= R
Genotype
= S
No. of false
positives
No. of false
negatives
NS158 20 18 19 18 2 1
SSRY28 20 18 17 20 1 1
SSRY235 20 18 21 17 7 4
NS169 20 18 18 20 1 1
RME-1 20 18 25 13 7 0
SSRY 20 17 19 13 23 7 10
SSRY 238 17 21 25 13 9 5
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Primers Severity Score
= R
Severity
Score
= S
Genotype
= R
Genotype
= S
No. of
false
positives
No. of false
negatives
NS158 24 21 27 17 6 3
SSRY28 24 21 26 19 6 4
SSRY235 24 21 26 19 15 13
NS169 24 21 24 21 13 14
RME-1 24 21 21 24 2 5
SSRY20 24 21 18 16 6 4
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Primers N
Severity
Score
= R
Severity
Score
= S
Genotype
= R
Genotype
= S
No. of
false
positives
No. of false
negatives
NS158 40 28 8 33 7 5 3
SSRY28 40 28 8 33 6 6 3
SSRY235 40 28 8 19 21 0 12
NS169 40 28 8 35 5 7 3
RME-1 40 28 8 29 11 4 7
SSRY20 37 28 8 20 17 0 9
SSRY76 37 28 8 23 14 2 9
SSRY238 21 28 8 16 6 4 3
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Primer 1M 2007 (%) 23M 2007
(%)
1M 2008 (%) 23M 2008
(%)
% Average
Association
NS158 92 80 90 91 88
SSRY28 95 78 94 88 89
SSRY235 71 38 71 28 52
NS169 95 40 94 35 66
RME-1 82 84 74 93 83
SSRY20 53 68 50 46 54
SSRY238 61 - 71 - 66
3. Resistance Gene Analogs (RGA)
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Cloning/Sequencing
Sequence Analysis
Markers
PCR-Primer(Degenerate)
NBSCC LRR
TIR NBS
NBSCC
TIR NBS
LRR
LRR
LRR
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Species # Acc
M. esculenta 7
M .epruinosa 1
M. glaziovii 1
M. brachyandra 1
M. tripartita 1
Other Manihots 3
Ricinus communis 1
DNA … PCR Cloning Colony PCR Purify Sequence
* Surveys genomic DNA
RNA cDNA PCR Cloning Colony PCR Purify Sequence
*Surveys expressed genes
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Template ClonesNumber of sequences
Genomic cDNA TotalCassavaTME 117 30 17 59 76TME 3 7 1 18 19TME 7 8 1 - 1TME 6 14 4 - 4
TME 279 32 21 - 21TME 52 24 16 - 16TME 56 43 30 - 30
Cassava subtotal 158 90 77 167Wild species
M.brachyandra 19 15 59 74M.epruinosa 147 111 1 112M.glaziovii 43 40 22 62M.tripartita 103 83 44 127
Wild subtotal 312 249 126 375Castor
Castor bean 51 31 - 31Castor-purple 84 - 53 53Castor-green 78 - 21 21
Castor subtotal 213 31 74 105Grand Total 683 370 277 647
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Number of matching scaffolds
RGA source species Number of Unique RGAs
On Manihotgenome
On Castor beangenome
Cassava 167 79 70M.brachyandra 74 37 37M.epruinosa 112 38 35M.glaziovii 62 14 14M.tripartita 127 35 27Castor bean 105 52 36Total 647 298 237
BLAST based sequence comparison of all RGA clones to draft:cassava genome (phytozome.net/cassava) and castor bean genome (phytozome.net/ricinus)
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A combination of analysis used to group the sequences
• 521 grouped in to 41 classes with 3-99 members
• 126 were not grouped
• About half contained open reading frame (ORF)
• Different NBS-LRR specific motifs found in the sequences
• Domain/motif characterization and search for species
specific features to be performed
• Phylogenetic analysis together with published R gene
sequences
• Sequences will be submitted to the GenBank
4. Tools for Hybrid Verification
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A B C
FED
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Search Genbank – core nucleotide
Download and cluster – BioEdit, ClustalW
Primer design -FastPCR BLAST against Cassava EST
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A. Primer 2F1-1R1, 22 castor genotypes, 1 cassava, and NTC
B. Primer 2F1-1R1 on cassava, hybrids, and castors
Amplification of different castor, cassava and progenies with castor- and cassava-specific primers.
C. Primer 2F1-1R1 in 24 ‘hybrids’ and - ctrl (Me) and + ctrl (Rc)
D. Primer NS33 (cassava specific)
Histogram representing the relative nuclear DNA content of G0/G1 cells isolated from the leaf tissue of cassava (A), castor bean (B), and hybrids (C, D).
Gedil et al. 2009, JFAE
Castor x Cassava
C
Cassava
A
1. Castor2. Cassava
B1 2
Castor x CassavaD
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TME117 x M. glazioviitested with SSRY20, SSRY76, SSRY238
M.glaziovii x TME117tested with SSRY20, SSRY76, SSRY238
Sample Name Mean CV
Glaz x TME117-M 106 2.12
Glaz x TME117-A 104 1.68
Glaz x TME117-B 105 2.62
TME117 X Glaz-A 100 2.26
TME117 X Glaz-B 99 1.77
TME117(Source)-A 98 2.29
TME117(Source)-B 100 2.75
Glaz(Source)-A 108 2.08
Glaz(Source)-B 108 2.09
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1089A x TME14, assayed with SSRY76
TME3 x Tme117, assayed amplified with SSRY20
5. TILLING in the Lab: A Novel High throughput Reverse
Genetics Technology for Detection of Mutation
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***
*
* *
**
-
+
--
Nuclease
Populations with chemically induced mutations “TILLING”
Populations with natural nucleotide variation “Ecotilling”
Pool DNAPooled or unpooled DNA
* *PCR Form heteroduplexes
gel
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T --> NAltered
ACC --> AACmissense
P --> STOPTruncated
CCA --> TGAnonsense
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Batch Number of seeds
Seedlings(no.)
Total numbers of seedlings recovered per Concentrations
0% 0.1% 0.2% 0.5% 1%1 917 106 36 0 26 30 142 920 31 8 0 10 9 43 960 245 65 03 68 59 504 960 130 38 0 53 22 175 960 136 0 15 52 25 44Total 4,717 648 147 18 219 145 129
Seed treated, 30572 HS
Clones: 96/1089A; TME3; TME7; TME117
Concentration: 0.1%; 0.2%; 0.5%; 1%
Time of treatment: 3h; 6h; 24h
Type of treatment: spot application or dipping
Cuttings treated
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Gene symbol Description Accession Targeted
regionNucleotide
typeAmplicon size (bp)
Length (bp)
No of SNPs detected
SUSY Sucrose synthase DQ443534 Gene mRNA 857 2775 18
SBE Sstarch branching enzyme X77012 Gene mRNA 884 2843 4
SOD Superoxide dismutase 3 DI139016 Promoter Genomic 875 1568 16
BglA beta glucosidase X94986 Promoter Genomic 833 1486 18
ACCO31-amino-cyclopropane-1-carboxylic oxidase
EF055260 Promoter Genomic 885 1536 15
GBSS Granule-bound starch synthase X74160 Gene mRNA 851 2168 0
CYP79D1
N-hydroxylating cytochrome p450-D1
AY834391 Gene mRNA 500 1629 In progress
CYP79D2
N-hydroxylating cytochrome p450-D1
AY834390 Gene mRNA 500 1626 In progress
Linam Linamarase (PLIN-Gene) U95298 Gene Genomic 700 2782 In progress
6. Marker for pro Vitamin A carotenoids - cassava
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Deep yellow Yellow Cream White
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ZDS
GGPP
Phytoene
Phytofluene
ζ- carotene
Neurosporene
PSY
PDS
Lycopene
α- carotene β- carotene
HYDHYD
β-crytptoxanthinHYD
ZeaxanthinLutein
β- LCYε- LCY
β- LCY
vp9
vp5
y1
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Lycopene epsilon cyclase
LYCE DB947186Library from several development stages and tissues
32 49
Lycopene beta cyclase LYCB DV445903
Normalized library from different tissues
32 62
Phytoene synthase PSY1 BH794911.1 Genomic survey
sequence.32 59
Beta-carotene Hydroxylase
HYD1 DB955222Library from several development stages and tissues
32 58
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Gene NCBI Accession DescriptionQuery
coverageE-value
Identity
HYDB1 XM_002513608.1Ricinus communis beta-carotene
hydroxylase, putative, mRNA.38% 1e-43 92%
LYCBXM_002531452.1
Ricinus communis Lycopene beta cyclase, chloroplast precursor, putative.
95% 6e-120 82%
PSY1XM_002527021.1
Ricinus communis Phytoene synthase, chloroplast precursor, putative
58% 3e-51 87%
LYCEAB238230.1
Citrus unshiu CitLCYe mRNA for lycopene epsilon-cyclase, complete.
36% 7e-39 86%
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Genesymbol
Homologous cassava database genes
E-value Identity
HYDB1cassava7257.valid.m1
cassava18978.valid.m11e-0662e-049
100%100%
LYCBcassava992.valid.m1cassava991.valid.m1 cassava993.valid.m1
e-176e-176e-176
100%100%100%
PSY1cassava32745.m1
cassava30274.valid.ml1e-088 100%
LYCEcassava43823.valid.m1
e-158 100%
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Gene Total positions
Putative positions
# of Heterozygous positions
Max # of bases
LycB 2 0 1 2
LycE 15 7 0 3 (T,C,G)
Hyd 1 10 7 7 2
PSY1 5 1 0 2
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High Total Carotenoid lines Low Total Carotenoid lines White Lines
S/N CloneTotal
Carotenoid (ug/g)
S/N CloneTotal
Carotenoid (ug/g)
S/N CloneTotal
Carotenoid (ug/g)
1
2
3
4
5
6
7
8
9
10
08/0054-1
08/0394-23
08/0073-1
08/0079-2
08/0081-2
08/0085-2
08/0389-15
08/0085-2
08/0380-3
08/0386-3
9.9
9.7
9.9
10.4
10.0
9.7
10.4
9.7
9.6
9.4
11
12
13
14
15
16
17
18
19
20
08/0284-4
08/0351-1
08/0380-2
08/0394-5
08/0283-1
08/0284-1
08/0284-4
08/0436-4
08/0436-5
08/0448-1
2.7
2.0
1.0
0.2
2.7
2.8
2.7
2.2
2.7
2.7
21
22
23
24
25
4(2) 1425
91/02324
TME117
TME1
30572
0.3
0.5
0.71
0.5
1.3
7. Marker for pro Vitamin A carotenoids - Maize
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Polymorphism site Types of polymorphism Expected Size of DNA fragment (bp) Amplified Size of DNA fragment (bp)
CrtRB15' INDEL/TE 800 (with 397 INS)-worst 600b
600 (with 206 INS)- best 800ab
400 (with no INS)-2nd best Nullab
600+800b
CrtRB13' INDEL/TE 543(without INS)-best 543b
296 + 875 (with 325 INS)-worst 296+1221+1800ab
296 + 1221+1800 (with 1250 INS)-2nd best 296+1221b
296+1800b
543+296b
296+875+1221b
543+296+1221b
543+1221b
296+1221b
296b
CrtRB1D4 DEL (bp) 129 (with 12 INS)-best 129b
117 (with 12 DEL)-worst 117ab
HYD3 Duplicated Seq 163-(worst) 163ab
transcript start site 473/476-(best); 608-(common) 163+608b
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Polymorphism site
Types of polymorphism
Expected Size of DNA fragment (bp)
Amplified Size of DNA fragment (bp)
LCYE5' INDEL/TE 993 (best) 150 + 280 +1700 (best + 2nd best)ab
150 + 280 (2nd best) Null ab
250 (3rd best) 250+380 (Worst )b
250+380 (worst) 280 + 250 + 1700+ 150b
280b
LCYE3' INDEL (DNA Seq) 144+502 (best) 144+502 (best)ab
399+502 (worst) 399+502 (best)a
null 502a
399+502+144ab
LCYE-Exn-216 SNP G (best) G (best)ab
A (worst) A (worst)a
null Nullb
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Gene/polymorphism
panel of 38#of genotypes
panel of 122#of
genotypes
Combined#of
genotypes
Frequencyfav allele inpanel of 38
Frequencyfav allele in
panel of 122
TotalAllele
frequency
LCYE5'INDEL/TEA1: 150 + 280
+1700 9 4 13 24 3.3 8.1LCYE3'INDEL
B1: 144+502 11 82 93 29 67 58LCYE-Exn-216
C1: G 37 120 157 97 98 98CrtRB15'INDEL/T
ED2: 600 0 17 17 0 14 11
CrtRB13'INDEL/TE
E1:543 0 14 14 0 11 9CrtRB1DEL4
F1: 129 0 1 1 0 0.8 0.6HYD3TF
G4:473/476+608 0 0 0 0 0 0
8. Soybean diversity and mapping
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Plant material 74 for SSR assay 68 for SNP assay (65 from above +3 new)
Released varieties Nigeria, Ghana, Benin, Togo, DRC, Uganda
Breeding lines Different maturity groups Promiscuously nodulating Specific nodulation Rust resistant from USA (SOY104) Rust resistant from Africa (UG5)
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Ibadan, Nigeria
Primer Screening Summary
Primers assayed 80
Genotypes 74Polymorphic (18 on PAGE; 16 on SFR)
34
Linkage groups9 x 1 locus8 x 2 loci3 x 3 loci
20
Number of alleles 2-7
Total alleles 125
#Alleles detected
#of loci
2 43 114 135 56 07 1
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Genomics in-house Capacity Building
in view of Emerging Technology
M OPAA1 OPAB5 OPT05
OPAB18 OPAA10
2k
16k
1k
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ABI's SOLiD Roche 454
400-600 million high-quality, filter-passed bases per run
1 billion bases per day
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Learning center•Nucleic acid isolation•Gel electrophoresis•PCR amplification
Bioinformatics•Sequence analysis/similarity search/databases•Linkage, QTL analysis•Association mapping
Genotyping service•Fragment analysis - ABI•Standard PCR- and elecrophoresis-based assays•Ploidy analysis
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Graduate Students (MS, PhD)•Competitive skills
Visiting Scientists/Training•Individual•Group training - workshops
Internship/educational visits•IT/NYSC•Group visitors from educational institutions
"Whoever could make two ears of corn or two blades of grass to grow upon a spot of ground where only one grew before, would deserve better of mankind, and do more essential service to his country, than the whole race of politicians put together".
Jonathan Swift (1667-1745)
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CassavaRoots
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