genomics assisted crop improvement- a new paradigm

Genomics Assisted Crop Improvement

A New Paradigm

28 June 2010

Melaku Gedil

Monday 28 June, 2010

Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 1

28 June 2010Melaku Gedil: Contract Review seminar, Ibadan, Nigeria 2


PhenomicsBiochemical Physiological Agronomical

ProteomicsMetabolomics Interactomics

TranscriptomicssiRNA mRNA rRNA

GenomicsRegulatory Gene Other (IRE)

1. CMD gene tagging



0.00.51.01.52.02.53.03.54.0

cmd1 cmd2 cmd3 cmd4 cmd5 cmd6

Cassava mosaic disease

Ave

rage

sco

re

Ib07Ib08On07


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


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

28 June 2010 12Melaku Gedil: Contract Review seminar, Ibadan, Nigeria


• 12 clones

• Three replications

• Repeated sampling and

assay


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 - -

Improved phenotyping – PCR & RT-PCR

28 June 2010 15

28 June 2010 16

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



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


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)*


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


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


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


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)


Cloning/Sequencing

Sequence Analysis

Markers

PCR-Primer(Degenerate)

NBSCC LRR

TIR NBS

NBSCC

TIR NBS

LRR

LRR

LRR

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 … PCR Cloning Colony PCR Purify Sequence

* Surveys genomic DNA

RNA cDNA PCR Cloning Colony PCR Purify Sequence

*Surveys expressed genes

28 June 2010

28 June 2010

TA-cloning, Qiagen

Purification before sequencing


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


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)


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

28 June 2010

4. Tools for Hybrid Verification


A B C

FED


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

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



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


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


***

*

* *

**

-

+

--

Nuclease

Populations with chemically induced mutations “TILLING”

Populations with natural nucleotide variation “Ecotilling”

Pool DNAPooled or unpooled DNA

* *PCR Form heteroduplexes

gel

28 June 2010 44


T --> NAltered

ACC --> AACmissense

P --> STOPTruncated

CCA --> TGAnonsense


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


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


Deep yellow Yellow Cream White

28 June 2010

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


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


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%

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=255545185&dopt=GenBank&RID=SUM83VYP01N&log$=nucltop&blast_rank=1�

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=255581378&dopt=GenBank&RID=SUMGFE8K014&log$=nucltop&blast_rank=1�

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=255572255&dopt=GenBank&RID=SUKX731J01S&log$=nucltop&blast_rank=1�

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=92109235&dopt=GenBank&RID=SUMBFH4W016&log$=nucltop&blast_rank=2�


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%

MSA of cassava Hydroxylase

28 June 2010 55


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


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



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


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


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


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)

28 June 2010 67Melaku Gedil: Contract Review seminar,

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

28 June 2010 68

Soy104, UG5

TGM 80(sp)

TGX- 1987 RR+

Released varieties

Other TGX lines28 June 2010 69

Planned Activities



Genomics in-house Capacity Building

in view of Emerging Technology

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

"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)


CassavaRoots

genomics assisted crop improvement- a new paradigm

Technology

contract review seminar

assaymelaku gedil

populationsmelaku gedil

cassava samples melaku

cmd gene taggingmelaku

s positives ns158

recombinant virus

score mv mv rep