Genomics Assisted Crop Improvement- A New Paradigm

Genomics Assisted Crop
Improvement
A New Paradigm

Melaku Gedil
Monday 28 June, 2010

Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 1

Genomics
Regulatory Gene Other (IRE)

Transcriptomics
siRNA mRNA rRNA

Proteomics
Metabolomics Interactomics

Phenomics
Biochemical Physiological Agronomical

1. CMD gene tagging

Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010
5

4.0
3.5
3.0
Average score

2.5 Ib07
2.0 Ib08
1.5 On07
1.0
0.5
0.0
cmd1 cmd2 cmd3 cmd4 cmd5 cmd6
Cassava mosaic disease


Frequency distribution of CMD score in two crosses (top) and two selfed (bottom) populations


Clone Location Year N min max range
1M-35 Onne 2007 2 1 3 2
1M-43 Onne 2007 5 1 3 2
1M-44 Onne 2007 5 1 3 2
1M-10 Onne 2007 3 1 4 3
1M-17 2007 2 1 4 3
1M-42 Onne 2007 3 1 4 3
1M-5 Onne 2007 3 1 4 3
23M-10 Onne 2007 5 1 3 2
23M-25 Onne 2007 4 1 3 2
23M-3 Onne 2007 4 1 3 2
23M-31 Ibadan 2007 5 1 3 2
23M-40 Onne 2007 3 1 3 2
23M-42 Ibadan 2007 5 1 3 2
23M-45 2007 4 1 3 2
23M-22 Onne 2007 5 1 4 3
23M-3 Ibadan 2008 2 1 4 3
23M-30 Ibadan 2007 3 1 4 3
23M-32 Ibadan 2007 4 1 4 3
23M-33 Ibadan 2007 5 1 4 3
23M-39 Onne 2007 4 1 4 3
23M-41 2007 3 1 4 3
23M-5 Onne 2007 4 1 4 3
23M-14 Ibadan 2007 3 1 5 4
23M-40 Ibadan 2007 5 1 5 4
23M-41 Ibadan 2008 5 1 5 4
23M-9 2007 3 1 5 4


Improved phenotyping – PCR & RT-PCR
Different viruses
African 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,

Evaluation
PCR diagnostics to determine the type of infection
Real-time PCR to determine virus load
Resistance profiling
28 June 2010 Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 12

• 12 clones
• Three replications
• Repeated sampling and
assay


Replication-1 Replication-2 Replication-3
AC EAC AC EAC AC EAC
Rep# Score MV MV Rep# Score MV MV Rep# Score MV MV
101 3 +++ + 201 3 +++ + 301 4 +++ -
202 1 - + 302 - - -
102 1 - -
203 - + - 303 1 - -
103 3 +++ ++
204 - 304a 3 +++ +++
104 1 - - 304b 4 +++ -
205 1 - -
105a 3 +++ ++ 305a 1 - -
206 1 + -
105b 2 +++ +++ 305b 1 - +
207a 2 +++ -
106 2 - + 306a 1 - -
207b 1 + +
107 1 - + 306b 1 - -
208a 1 - -
108a 1 - - 306c 1 - -
208a 1 - + 306d 1 - -
108b 1 - - 209 1 - + 307 3 - -
109 3 +++ ++ 210 3 +++ + 308 1 - -
110 1 - - 211a 2 +++ + 309 - - -
111 2 - - 211b 2 +++ +++ 310a 1 - -
112a 1 - + 211c 2 +++ - 310b 1 - -
112b 1 - + 212 4 +++ - 311 1 - -
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria
312
28 June 2010
1 - - 14

Improved phenotyping – PCR & RT-PCR

28 June 2010 15

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


2. Marker Screening/genotyping

17

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 - CIAT
EST-SSR EST 178 - CIAT
Total 817 210 170

SSR set Type No. Tested Valid Ref
Lokko, 2007
New-SSR 1 EST 846 192 124 Raji, 2009

Ferguson
New SSR 2 EST 70 70 52 in progress

Sub total 916 262 176


R R R R R R R R S S S S S S S S


Set-1 Set-2

NS158* SSRY238*

SSRY28* SSRY20*

SSRY235* SSRY51

SSRY44 SSRY76

NS136

SSRY169*

RME1 (SCAR)*


Severity
Severity Score Genotype Genotype No. of false No. of false
Primers Score
=S =R =S positives negatives
=R

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


Severity No. of
Severity Score Genotype Genotype No. of false
Primers Score false
=R =R =S negatives
=S positives

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


Severity Severity No. of
Genotype Genotype No. of false
Primers N Score Score false
=R =S negatives
=R =S positives

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


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)

25

TIR NBS LRR

CC NBS LRR

TIR NBS LRR

CC NBS LRR

PCR-Primer
(Degenerate)

Cloning/Sequencing

Sequence Analysis
Markers
28 June 2010 26

Species # Acc
M. esculenta 7
M .epruinosa 1
M. glaziovii 1
M. brachyandra 1
M. tripartita 1

Other Manihots 3
Ricinus communis 1

 DNA  RNA
 …  cDNA
 PCR  PCR
 Cloning  Cloning
 Colony PCR  Colony PCR
 Purify  Purify
 Sequence  Sequence

* Surveys genomic *Surveys expressed
DNA genes

28 June 2010

TA-cloning, Qiagen

Purification before sequencing

28 June 2010

Number of sequences
Template Clones Genomic cDNA Total
Cassava
TME 117 30 17 59 76
TME 3 7 1 18 19
TME 7 8 1 - 1
TME 6 14 4 - 4
TME 279 32 21 - 21
TME 52 24 16 - 16
TME 56 43 30 - 30
Cassava subtotal 158 90 77 167
Wild species
M.brachyandra 19 15 59 74
M.epruinosa 147 111 1 112
M.glaziovii 43 40 22 62
M.tripartita 103 83 44 127
Wild subtotal 312 249 126 375
Castor
Castor bean 51 31 - 31
Castor-purple 84 - 53 53
Castor-green 78 - 21 21
Castor subtotal 213 31 74 105
Grand Total 683 370 277 647

BLAST based sequence comparison of all RGA
clones to draft:
cassava genome (phytozome.net/cassava) and
castor bean genome (phytozome.net/ricinus)
Number of matching scaffolds

Number of On Manihot On Castor bean
RGA source species
Unique RGAs genome genome

Cassava 167 79 70
M.brachyandra 74 37 37
M.epruinosa 112 38 35
M.glaziovii 62 14 14
M.tripartita 127 35 27
Castor bean 105 52 36
Total 647 298 237


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

A B C

4. Tools for Hybrid Verification
D E F

35

Search Genbank – core
nucleotide
Download and cluster – BioEdit,
ClustalW

Primer design -
FastPCR
BLAST against Cassava EST


A. Primer 2F1-1R1, 22 castor genotypes, 1 cassava, and
NTC

B. Primer 2F1-1R1 on cassava, hybrids, and castors

C. Primer 2F1-1R1 in 24 ‘hybrids’ and - ctrl (Me) and + ctrl
(Rc)

D. Primer NS33 (cassava specific)

Amplification of different castor, cassava and progenies with
castor- and cassava-specific primers.


A B
1 2

Cassava 1. Castor
2. Cassava

C D
Castor x Cassava
Castor x Cassava

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


TME117 x M. glaziovii M.glaziovii x TME117
tested with SSRY20, tested with SSRY20,
SSRY76, SSRY238 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

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

41

Populations with chemically Populations with natural
induced mutations nucleotide variation
“TILLING” “Ecotilling”
Pool DNA
Pooled or unpooled DNA

* *
PCR
Form heteroduplexes

*
*
Nuclease
* *
* *
*
*

gel
-
-
-
28 June 2010 + 44

missense nonsense
ACC --> AAC CCA --> TGA
T --> N P --> STOP
Altered Truncated


Seed Number Seedlings Total numbers of seedlings recovered per
Batch
treated, of seeds (no.) Concentrations
30572 HS
0% 0.1% 0.2% 0.5% 1%
1 917 106 36 0 26 30 14
2 920 31 8 0 10 9 4
3 960 245 65 03 68 59 50
4 960 130 38 0 53 22 17
5 960 136 0 15 52 25 44
Total 4,717 648 147 18 219 145 129

Cuttings Clones: 96/1089A; TME3; TME7; TME117
treated Concentration: 0.1%; 0.2%; 0.5%; 1%
Time of treatment: 3h; 6h; 24h
Type of treatment: spot application or dipping


Gene Targeted Nucleotide Amplicon Length No of SNPs
Description Accession
symbol region type size (bp) (bp) detected

SUSY Sucrose synthase DQ443534 Gene mRNA 857 2775 18
Sstarch branching
SBE X77012 Gene mRNA 884 2843 4
enzyme
Superoxide
SOD DI139016 Promoter Genomic 875 1568 16
dismutase 3
BglA beta glucosidase X94986 Promoter Genomic 833 1486 18

1-amino-
ACCO3 cyclopropane-1- EF055260 Promoter Genomic 885 1536 15
carboxylic oxidase
Granule-bound
GBSS X74160 Gene mRNA 851 2168 0
starch synthase
N-hydroxylating
CYP79
cytochrome p450- AY834391 Gene mRNA 500 1629 In progress
D1
D1
N-hydroxylating
CYP79
cytochrome p450- AY834390 Gene mRNA 500 1626 In progress
D2
D1
Linamarase (PLIN-
Linam U95298 Gene Genomic 700 2782 In progress
Gene)


6. Marker for pro Vitamin A
carotenoids - cassava

Deep yellow Yellow Cream White

50

GGPP Lycopene
PSY
y1
Phytoene β- LCY
β- LCY
PDS
Phytofluene ε- LCY
vp5
ζ- carotene α- carotene β- carotene
ZDS HYD
vp9 Neurosporene HYD β-crytptoxanthin
HYD
Lutein Zeaxanthin

28 June 2010

Lycopene Library from several
epsilon LYCE DB947186 development 32 49
cyclase stages and tissues

Normalized library
Lycopene
LYCB DV445903 from different 32 62
beta cyclase
tissues
Phytoene Genomic survey
PSY1 BH794911.1 32 59
synthase sequence.

Beta- Library from several
carotene HYD1 DB955222 development 32 58
Hydroxylase stages and tissues


Query Identi
Gene NCBI Accession Description E-value
coverage ty

Ricinus communis beta-carotene
HYDB1 XM_002513608.1 38% 1e-43 92%
hydroxylase, putative, mRNA.

Ricinus communis Lycopene beta cyclase,
XM_002531452.1 95% 6e-120 82%
LYCB chloroplast precursor, putative.

Ricinus communis Phytoene synthase,
XM_002527021.1 58% 3e-51 87%
PSY1 chloroplast precursor, putative

Citrus unshiu CitLCYe mRNA for lycopene
AB238230.1 36% 7e-39 86%
LYCE epsilon-cyclase, complete.


Gene Homologous cassava
E-value Identity
symbol database genes

cassava7257.valid.m1 1e-066 100%
HYDB1
cassava18978.valid.m1 2e-049 100%

cassava992.valid.m1 e-176 100%
LYCB cassava991.valid.m1 e-176 100%
cassava993.valid.m1 e-176 100%

cassava32745.m1
PSY1 1e-088 100%
cassava30274.valid.ml

LYCE e-158 100%
cassava43823.valid.m1


MSA of cassava Hydroxylase

28 June 2010 55

Gene Total Putative # of Max # of
positions positions Heterozygou bases
s positions
LycB 2 0 1 2

LycE 15 7 0 3 (T,C,G)

Hyd 1 10 7 7 2

PSY1 5 1 0 2


High Total Carotenoid lines Low Total Carotenoid lines White Lines

Total Total Total
S/N Clone Carotenoid S/N Clone Carotenoid S/N Clone Carotenoid
(ug/g) (ug/g) (ug/g)

1 08/0054-1 9.9 11 08/0284-4 2.7 21 4(2) 1425 0.3
2 08/0394-23 9.7 12 08/0351-1 2.0 22 91/02324 0.5
3 08/0073-1 9.9 13 08/0380-2 1.0 23 TME117 0.71
4 08/0079-2 10.4 14 08/0394-5 0.2 24 TME1 0.5
5 08/0081-2 10.0 15 08/0283-1 2.7 25 30572 1.3
6 08/0085-2 9.7 16 08/0284-1 2.8
7 08/0389-15 10.4 17 08/0284-4 2.7
8 08/0085-2 9.7 18 08/0436-4 2.2
9 08/0380-3 9.6 19 08/0436-5 2.7
10 08/0386-3 9.4 20 08/0448-1 2.7


7. Marker for pro Vitamin A
carotenoids - Maize

58

Types of
Polymorphism site Expected Size of DNA fragment (bp) Amplified Size of DNA fragment (bp)
polymorphism
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)-
296+1221b
2nd best
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
473/476-(best); 608-(common) 163+608b
site


Polymorphism Types of Expected Size of DNA fragment Amplified Size of DNA fragment
site polymorphism (bp) (bp)
150 + 280 +1700 (best + 2nd
LCYE5' INDEL/TE 993 (best)
best)ab
150 + 280 (2nd best) Null ab
250 (3rd best) 250+380 (Worst )b
250+380 (worst) 280 + 250 + 1700+ 150b
280b
INDEL (DNA
LCYE3' 144+502 (best) 144+502 (best)ab
Seq)
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


panel of 122 Combined Frequency Frequency Total
Gene/ panel of 38
#of #of fav allele in fav allele in Allele
polymorphism #of genotypes
genotypes genotypes panel of 38 panel of 122 frequency

LCYE5'INDEL/TE
A1: 150 + 280
+1700 9 4 13 24 3.3 8.1
LCYE3'INDEL
B1: 144+502 11 82 93 29 67 58
LCYE-Exn-216
C1: G 37 120 157 97 98 98
CrtRB15'INDEL/T
E
D2: 600 0 17 17 0 14 11
CrtRB13'INDEL/T
E
E1:543 0 14 14 0 11 9
CrtRB1DEL4
F1: 129 0 1 1 0 0.8 0.6
HYD3TF
G4:473/476+608 0 0 0 0 0 0

8. Soybean diversity and
mapping

66

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)

Melaku Gedil: Contract Review seminar,
28 June 2010 Ibadan, Nigeria 67

Primer Screening Summary

Primers assayed 80 #Alleles #of loci
Genotypes 74 detected
Polymorphic (18 on 34 2 4
PAGE; 16 on SFR) 3 11
Linkage groups 20 4 13
9 x 1 locus 5 5
8 x 2 loci 6 0
3 x 3 loci 7 1
Number of alleles 2-7
Total alleles 125

28 June 2010 68

Soy104, UG5
TGM 80(sp)
TGX- 1987 RR+
Released varieties
Other TGX lines
28 June 2010 69

Planned Activities

70

Genomics
in-house Capacity Building
in view of
Emerging Technology

74

M OPAA1 OPAB5 OPT05
OPAB18 OPAA10

2k

16k

1k


Affymetrix Illumina


ABI's SOLiD Roche 454

400-600 million high-
quality, filter-passed
bases per run

1 billion bases per day


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

Graduate Students (MS, PhD)
•Competitive skills

Visiting Scientists/Training
•Individual
•Group training - workshops

Internship/educational visits
•IT/NYSC
•Group visitors from educational institutions


Roots Cassava

"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)


Genomics Assisted Crop Improvement- A New Paradigm

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Genomics Assisted Crop Improvement- A New Paradigm