Genomics tools for discovery and tagging of genes can enhance the efficiency of breeding.CMD gene tagging,Marker Screening/genotyping,Resistance Gene Analogs (RGA),Tools for Hybrid Verification,TILLING in the Lab:
A Novel High throughput Reverse Genetics Technology for Detection of Mutation,Marker for pro Vitamin A carotenoids -cassava and Maize,Soybean diversity and mapping
10. Frequency distribution of CMD score in two crosses (top) and two selfed (bottom) populations
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 10
18. 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
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 18
19. R R R R R R R R S S S S S S S S
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 19
31. 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
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 31
32. 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
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 32
35. A B C
4. Tools for Hybrid Verification
D E F
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010
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36. Search Genbank – core
nucleotide
Download and cluster – BioEdit,
ClustalW
Primer design -
FastPCR
BLAST against Cassava EST
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 36
37. 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.
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 37
38. 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
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 38
39. 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
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 39
40. 1089A x TME14, assayed with SSRY76
TME3 x Tme117, assayed amplified with SSRY20
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 40
41. 5. TILLING in the Lab:
A Novel High throughput Reverse
Genetics Technology for Detection of
Mutation
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010
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44. 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
45. missense nonsense
ACC --> AAC CCA --> TGA
T --> N P --> STOP
Altered Truncated
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 45
46. 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
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 46
50. 6. Marker for pro Vitamin A
carotenoids - cassava
Deep yellow Yellow Cream White
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010
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51. 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
52. 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
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 52
64. 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
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 64
65. 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
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 65
66. 8. Soybean diversity and
mapping
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010
66
67. 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
68. 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
77. ABI's SOLiD Roche 454
400-600 million high-
quality, filter-passed
bases per run
1 billion bases per day
28 June 2010 Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 77
78. 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
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 78
79. Graduate Students (MS, PhD)
•Competitive skills
Visiting Scientists/Training
•Individual
•Group training - workshops
Internship/educational visits
•IT/NYSC
•Group visitors from educational institutions
Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 28 June 2010 79
81. 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)
28 June 2010 Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 81