Download PPT - GCP21

I.Moreno, J. Tomkins, P. Hurtado, E. Okogbenin,
A. Dixon & M.Fregene
Global Cassava Partnership GCP-I
July 22, 2008
Outline
I.
II.
III.
IV.
V.
Introduction
Fine Mapping
Screening of the TME-3 BAC library
Chromosome walking
Conclusions
Cassava: A Tropical Crop of WorldWide Importance
Fifth staple after rice, wheat, maize, and potato
World-wide production exceeds 180million
tons/year, over half is produced in sub-Saharan
Africa (SSA)
Demand is expected to increase 2% per year for the
next 3 years
Constraints: Pests and Diseases
Cassava Brown
Streak Disease
Cassava Mosaic
Disease
Whiteflies
Cassava
mealybug
Cassava
bacterial
blight
Green spider
mite
Potential Yield Losses due to Cassava
Pests
P. herreni
P. manihoti
Cassava
Green Mite
Hornworm
E. ello
Mealybugs
21-53%
34 – 88%
18 – 64%
Brown Streak
Disease
Whiteflies
33 – 79%
30-100%
40-100%
Cassava Mosaic
Disease
The Cassava Mosaic
Disease (CMD)
A
viral disease
endemic in SS-Africa
and India
 Not recorded in the
Americas
 Vectored by the white
fly (Bemisia tabaci)
The Cassava Mosaic Disease (CMD)

A viral disease and number one production
constraint in SS Africa, yield loss estimated at
>US$1billion/year
A potential risk in South America and South East
Asia, and complicates germplasm transfer from the
center of diversity to to SS-Africa
Viruses can recombine to more virulent forms and
some have associated satellite molecules known to
breakdown resistance
The most efficient form of control is resistant
varieties which take 6-10 years to develop
Background
IITA (1930)
- Sources of resistance
I. 3rd Backcrossing : Cassava X Manihot glaziovii
(1947)
II. Local Nigerian varieties (1990)
CIAT (1997)
• Fregene 2000 (gen CMD1)Recessive gene
• Akano et al 2002 (gen CMD2) Dominant gene
• Linkage SSRY28 y NS158 (Akano 2002; Zárate 2002)
A single genome region explains >70% of phenotypic
variance for CMD resistance and dominance gene action.
SSRY NS158
TMS30555 XTME3
Resistant
Susceptible
R
Dist
cM
Marker
Name
rGY115
rGY9
7.9
15.6
RP/SP/RB/SB
rGY1
200bp
CMD2
16.1
rSSRY28
175bp
11.3
Ai19
150bp
A single genome region explains 48% of phenotypic variance for CMD
resistance and recessive gene action.
Resistant
Susceptible
D
Y66
18.5
rI18b
20.5
rJ1a
20.0
rGY57
21.0
rGY25
SSRY 40
TMS30572 X CM7857-4
21.2
SSRY9
23.9
SSRY3
16.2
CMD1
SSRY23
Fine mapping of the CMD2 region
1. Segregant population ( > 1000 individuals)
2.
Molecular markers of high resolution.
3. Bulk Segregant Analysis (BSA).
4.
Evaluation of molecular markers candidates in
recombinant individuals
1. Fine Mapping Population
SUSCEPTIBLE PARENTAL
RESISTANT PARENTAL
TMS 30572
TME-3
X
(1690 Individuals)
2. Phenotypical Evaluation
Seedling trial- low pressure - Mokwa (IITA)
Clonal trial- High pressure of disease (IITA)
3. Bulk Segregant Analysis (BSA)
PCR
Resistant
Bulk
Susceptible Recombinant
Bulk
Resistant
Bulk
Recombinant
Susceptible
Bulk
R
S
Monomorphic profile
R
S
Polymorphic profile
1st Level of selection: Parentals and Bulks
2nd Level of Selection: Open Bulks and recombinants individuals
Scheme of Fine Mapping
F1 POPULATION
(TME-3 X TMS 30572)
MAPPING OF
SSRY28-NS158
MARKERS.
PHENOTYPICAL
EVALUATION
DNA EXTRACTION
( Dellaporta.1993)
IDENTIFICATION OF
RESISTANCE,
SUSCEPTIBLES AND
RECOMBINANTS
INDIVIDUALS
SELECTION OF
MOLECULAR MARKERS
ASSOCIATED TO CMD2
BULK SEGREGANT
ANALYSIS (“BSA”)
MAPPING OF MOLECULAR
MARKERS
DATA ANALYSIS
Log-Likelihood : -515.93
Iterations : 3
Longest Seg cM : 27.895
Loop Tolerance : 0.010
Inner Tolerance: 0.010
Linkage map of molecular markers associated to
CMD2
Rec
Frac.
(10.2 %)
Dist
cM
(5)
RME-2
(4)
(1)
RME-1
CMD2
(2)
NS158
(3)
SSRY28
10.4
( 4.5 %)
4.5
( 7.1 %)
7.1
( 5.9 %)
Marker
Id
Name
5.9
MAPMAKER 2.0 ( θ= 0.3; LOD=5.0)
New linkage map ( SSRY28, NS158, RME-1 y RME-2)
27.9 cM
Screening of the TME-3 BAC
Library
1.
Library construction
2.
Characterization
3.
PCR screening of BAC-Pools
1.Cassava BAC Library Construction
HindIII partial
digestion
Purification and
cloning
388b
194kb
48.5kb
Mega base pair-sized
DNA in Agarose Plugs
Size selection
Cloning vector
BAC LIBRARY
(73,728 clones In
Micro-titre plates
and high density
filters)
Clone picking by
Robotics
97kb
48.5kb
6.9kb
Sizing of clones
Ligation and
Sizing of clones
2. Characterization
kb
194.0
145.5
97.0
48.5
pCUGI-1
Coverage 10X
N= Ln ( 1-P)/ Ln ( 1- L/G)
P= 99%
•73,728 clones
•4 high density filters
•Inserts average = 93 kb (25-250)
3. PCR Screening of the TME-3 library
2 NS158 positive clones
13 RME-1 positivos clones
1
MARKER
NS158
PP
MARKER
RP
CP
2
RME-1
LOCATION
#
90 N18
189 M19
LOCATION
#
12 M14
17 N21
34 L16
47 O3
51 I1
52 A23
54 I1
85 J23
130 D18
136 J8
139 G22
155 K24
173 F1
Chromosome Walking
1.
Restriction profile
2.
Contig construction
3.
BAC-ends Mapping
1. Restriction profile
2 3 4 5 6 7 8
9 10 11 12 13 14 15 16 17
Λ PstI
1
RME-1
NS158
Positive clones were digested with Hind III
2. Contig Construction
“Fingerprinting Contig” (“ FPC”) ( Marra et al 1997)
3. BAC-ends Mapping
Identification of BAC
ends and primer design
(RME=6,NS158=4)
Cloning and sequencing of
amplification products
Sequence analysis
Design of alleloespecific
primers
Identification of SSCP markers
Results
BS
BR
PS
PR
1. SSCP-SNP marker =Bac33b
BR
BS
BRecS
2. SSCP-SNP marker =SBac33c
BAC 35
BAC 36
BAC 33
RME--2
RME--1
SBAC 33c
BAC 33b
Fine mapping
BAC 18
BAC 23
BAC 9
Contig Construction
SBac33c
“Fingerprinting Contig” (“ FPC”) ( Marra et al 1997)
Ongoing work
Sequencing
Minimum tilling path
Annotation
Candidate genes
Genetic
Complementation
Conclusions
• Application of Positional cloning in Cassava for a dominant
gene (Fine mapping, Screening of BAC library and
Chromosome walking).
• Identification of new molecular markers associated to
CMD2: RME-1, RME-2, BAC33b and SBAC33c.
• Successive screening of the BAC library led to the
construction of a contig of BAC clones that stretches for
about 500Kb around CMD2.
• Five BAC clones that traverse a 500 Kb region around the
gene are currently being sequenced to search for candidate
R genes that can be tested by genetic complementation.
Acknowledgements
CIAT
Cassava Genetics (Jaime Marin, Edgar Barrera, Paola Alfonso)
Myriam Cristina Duque-Statistical support
Fernando Rojas- Bioinformatics support
CUGI (B. Blackmon, Michael Atkins, Mónica Muñoz, Maria
Delgado, Michael Saski)
University of Maryland (Pablo Rabinowicz- Comparative
Genomics)
Cornell University (Sharon Mitchel and Bunmi Olasanmi)
University of California-Davis (Ming Cheng Luo and Yanquin
Ma).
Thank You