BIOTECHNOLOGY APPLICATION TO COMBAT
CASSAVA BROWN STREAK DISEASE (CBSD)

Presenter:

Kapinga, Fortunus Anton

Contact addr...
Presentation content
- Introduction
- Production constraints of cassava
- Conventional efforts taken to combat CBSD
- Use ...
Introduction
Cassava (Manihot esculenta Crantz) is one of the world’s
major food crops grown throughout the tropical and
s...
Various uses of cassava products

chips
vegetable

bread

beer

cake

ethanol

biofuel

beer

firewood
Production constraints of cassava
Despite the usefulness of cassava, actual yields are far
lower than potential yields
Whi...
CBSD symptoms
and vector
foliar necrosis
leaf yellowing
root browning
dry necrotic
root rot

B. tabaci
Conventional efforts taken to combat CBSD
Breeding so far has been mainly based on mass phenotypic
recurrent selection (Ce...
Use of biotechnology to combat CBSD
Biotechnology techniques (molecular or maker assisted
breeding (MAB) or marker assiste...
Methodology
-F1 seeds were generated from NDL06/132 (resistant) and
AR37-80 (susceptible)
-F1 plants were grown at disease...
Establishment of mapping population

♀ X♂
DNA extraction
Pouring liquid nitrogen
in ceramic crucible
(mortar) ready for
grinding cassava
leaves

Grinding cassava le...
Estimation of DNA quantity and quality before genotyping

Estimation by Nanodrop 1000
spectrophotometer

Estimation by aga...
Theory underlying genotype screening

♂

Parents

♀
A
B

C
AC
BC

D
AD
BD

Note:
- Progenies processing AA, BB, CC and DD ...
Results and Discussion
Genotype screening based on allele size

Panel
(Marker)

Dye
color

NDL06/132
(♀)

AR37-80
(♂)

All...
Genotype screening cont ……..

113

123

Primer NS911 identified NDLAR10 as a true cross F1
(with alleles 113 from male and...
Heritable traits, DNA and molecular marker
- Heritable traits are carried in a DNA molecules
- They are transferred from o...
Principle underlying separation of DNA molecules
in agarose gel during electrophoresis

_

P1

P2

P3

P4

P5

P6

P7

P8
...
Identification of molecular marker associated
with disease resistance

_

Lane

Resistant
genotype

Susceptible
genotype

...
Identification of progenies inherited disease resistance

_

RP

SP

P1

P2

P3

P4

P5

P6

P7

P8

P9

+
RP and SP stand...
Advantages of molecular over conventional breeding
- Easy identification of disease tolerance gene(s)
- Easy screening for...
Conclusion
Biotechnology techniques (molecular breeding or marker
assisted selection (MAS) or marker assisted breeding
(MA...
End of Presentation
Thank You
For
Your Participation
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2.1 combating cassava brown streak disease kapinga

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2.1 combating cassava brown streak disease kapinga

  1. 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. 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. 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. 4. Various uses of cassava products chips vegetable bread beer cake ethanol biofuel beer firewood
  5. 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. 6. CBSD symptoms and vector foliar necrosis leaf yellowing root browning dry necrotic root rot B. tabaci
  7. 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. 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. 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. 10. Establishment of mapping population ♀ X♂
  11. 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. 12. Estimation of DNA quantity and quality before genotyping Estimation by Nanodrop 1000 spectrophotometer Estimation by agarose gel electrophoresis
  13. 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. 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. 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. 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. 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. 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. 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. 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. 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. 22. End of Presentation Thank You For Your Participation 22

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