Presentation at the November 2012 dialogue workshop of the Biosciences for Farming in Africa media fellowship programme in Arusha, Tanzania. Please see www.b4fa.org for more information

1. BIOTECHNOLOGY APPLICATION TO COMBAT
CASSAVA BROWN STREAK DISEASE (CBSD)
Presenter:
Kapinga, Fortunus Anton
Contact address:
Naliendele Agricultural Research Institute
10 Newala Road
P. O. Box 509
Mtwara
Tanzania
E-mail:
Cell phone:
fakapinga@yahoo.com
+255 784 327881

2. Presentation content
- Introduction
- Production constraints of cassava
- Conventional efforts taken to combat CBSD
- Use of biotechnology to combat CBSD
- Methodology
- Establishment of mapping population
- DNA extraction
- Estimation of DNA quantity and quality before genotyping
- Genotype screening based on allele size
- Heritable traits, DNA and molecular marker
- Principle underlying separation of DNA molecules in agarose gel during electrophoresis
- Identification of molecular marker associated with disease resistance
- Identification of progenies inherited disease resistance
- Advantages of molecular over conventional breeding
- Conclusion

3. Introduction
Cassava (Manihot esculenta Crantz) is one of the world’s
major food crops grown throughout the tropical and
subtropical regions (0 to 2000 masl)
It can potentially provide Africa with sufficient food despite
of the prevailing climatic changes (Jarvis et al., 2012)
Cassava is a staple food and provide food security for
over 800 million people worldwide (Ferguson et al., 2011)
In sub-Saharan Africa, more than 200 million people
derive over 50% of their carbohydrate intake from cassava
(IITA, 1992)

4. Various uses of cassava products
chips
vegetable
bread
beer
cake
ethanol
biofuel
beer
firewood

5. Production constraints of cassava
Despite the usefulness of cassava, actual yields are far
lower than potential yields
While cassava productivity in Africa is on average nearly
10 t/ha, productivity in some South Asian countries are
much higher, ranging from 16.3 - 31.4 t/ha (FAO, 2009)
Possible causes include:
-Cassava brown streak disease (CBSD), which can
cause up to 100% yield loss
-Susceptibility of cassava genotypes to the disease
-Biotic and abiotic stresses
-In the past CBSD was regarded as lowland or coastal

6. CBSD symptoms
and vector
foliar necrosis
leaf yellowing
root browning
dry necrotic
root rot
B. tabaci

7. Conventional efforts taken to combat CBSD
Breeding so far has been mainly based on mass phenotypic
recurrent selection (Ceballos, 2005)
Substantial progress on breeding for CBSD has been made
using conventional breeding methods
Nevertheless, the selection process is rather inefficient
The inefficiency increases when breeders try to select more
than one trait simultaneously
This problem can be solved using biotechnology techniques

8. Use of biotechnology to combat CBSD
Biotechnology techniques (molecular or maker assisted
breeding (MAB) or marker assisted selection (MAS)) is a
useful breeding tool for supplementing the inefficient
conventional breeding
Objective
Identify molecular markers associated with CBSD tolerance
in the cassava genotype NDL06/132
Expected output
Known molecular markers associated with tolerance to
CBSD in cassava variety NDL06/132

9. Methodology
-F1 seeds were generated from NDL06/132 (resistant) and
AR37-80 (susceptible)
-F1 plants were grown at disease-free location
-Leaf samples were collected for genotyping
-Initially, 26 SSR primers were screened for polymorphism
using two parental lines, from which 12 primers were
polymorphic
-These 12 primers were used to distinguish true crosses
from offtypes and selfs using SSR markers by scoring allele
sizes using GeneMapper software
-Phenotype the true crosses and their parents in two CBSD
hotspot locations for two years
-Identify molecular markers associated with CBSD tolerance

10. Establishment of mapping population
♀ X♂

11. DNA extraction
Pouring liquid nitrogen
in ceramic crucible
(mortar) ready for
grinding cassava
leaves
Grinding cassava leaf
sample in liquid
nitrogen to obtain fine
powder

12. Estimation of DNA quantity and quality before genotyping
Estimation by Nanodrop 1000
spectrophotometer
Estimation by agarose gel
electrophoresis

13. Theory underlying genotype screening
♂
Parents
♀
A
B
C
AC
BC
D
AD
BD
Note:
- Progenies processing AA, BB, CC and DD are selfs
- Progeny processing any allele out of A, B, C and D is offtype

14. Results and Discussion
Genotype screening based on allele size
Panel
(Marker)
Dye
color
NDL06/132
(♀)
AR37-80
(♂)
Allele Allele Allele Allele
1
2
1
2
Expected allele combinations
1
2
3
4
97
99
99
89
99
99
SSRY
Blue
169
97
99
89
99
97
89
SSRY
Green
148
110
112
112
118
110
112
110
118
112
112
112
118
SSRY
Red
52
257
267
267
267
257
267
257
267
267
267
267
267
123
123
113
123
123
113
123
123
123
113
123
123
NS
911
Green

15. Genotype screening cont ……..
113
123
Primer NS911 identified NDLAR10 as a true cross F1
(with alleles 113 from male and 123 from female parent)

16. Heritable traits, DNA and molecular marker
- Heritable traits are carried in a DNA molecules
- They are transferred from one generation to the next
- The DNA molecules are negatively charged and have
different sizes
- During agarose gel electrophoresis, the negatively
charged molecules are moved from -ve to positive pole
- Small DNA molecules are moved further forward
- This allow sorting of the DNA molecules based on weight
- It is possible to identify molecular markers associated
with a trait such as disease
- Identified molecular marker can used to identify resistant
genotype (marker = indicator of position or presence)

17. Principle underlying separation of DNA molecules
in agarose gel during electrophoresis
_
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
+
Electrophoresis = electrically induced movement of particles
P11

18. Identification of molecular marker associated
with disease resistance
_
Lane
Resistant
genotype
Susceptible
genotype
1
2
3
4
5
6
+
The third band is associated with disease resistance

19. Identification of progenies inherited disease resistance
_
RP
SP
P1
P2
P3
P4
P5
P6
P7
P8
P9
+
RP and SP stand for resistant and susceptible parents respectively
P1 …., P9 progeny 1, ….., progeny 9;
Resistant progenies are P1, P2, P5 and P8.
Susceptible progenies are P3, P4, P6, P7 and P9.

20. Advantages of molecular over conventional breeding
- Easy identification of disease tolerance gene(s)
- Easy screening for disease tolerant genotypes
(heterozygotes/homozygotes)
- Preliminary screening of genotypes at seedling stage
- Reduce population size for field evaluations
- MAB is not affected by environmental conditions e.g. soil
types and fertility, rainfall and temperature
- Late expressed traits (such as taste, flower and fruit
colour) can be identified at seedling stage
- Several traits can be studied simultaneously
- Breeding for disease resistance can be done even if
there is no disease in a particular

21. Conclusion
Biotechnology techniques (molecular breeding or marker
assisted selection (MAS) or marker assisted breeding
(MAB)) are not aiming at replacing conventional breeding
But, they are tools for simplifying breeding work
Thus, for efficient breeding work, where necessary, it is
important to apply both conventional and molecular
breeding techniques

22. End of Presentation
Thank You
For
Your Participation
22