2.1 combating cassava brown streak disease kapinga
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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: [email protected] Cell phone: +255 784 327881
Transcript of 2.1 combating cassava brown streak disease kapinga
BIOTECHNOLOGY APPLICATION TO COMBAT
CASSAVA BROWN STREAK DISEASE (CBSD)
Presenter: Kapinga, Fortunus Anton
Contact address: Naliendele Agricultural Research Institute10 Newala RoadP. O. Box 509MtwaraTanzania
E-mail: [email protected] phone: +255 784 327881
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
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)
Various uses of cassava products
vegetablechips
breadcake beer
beer ethanol biofuelfirewood
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
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 (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
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
ObjectiveIdentify molecular markers associated with CBSD tolerance in the cassava genotype NDL06/132
Expected outputKnown molecular markers associated with tolerance to CBSD in cassava variety NDL06/132
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
Establishment of mapping population
X♀ ♂
Pouring liquid nitrogen in ceramic crucible (mortar) ready for grinding cassava leaves
Grinding cassava leaf sample in liquid nitrogen to obtain fine powder
DNA extraction
Estimation of DNA quantity and quality before genot yping
Estimation by Nanodrop 1000 spectrophotometer
Estimation by agarose gel electrophoresis
Theory underlying genotype screening
Parents ♂
♀ C DA AC ADB BC BD
Note: - Progenies processing AA, BB, CC and DD are selfs- Progeny processing any allele out of A, B, C and D is offtype
Results and Discussion Genotype screening based on allele size
Panel (Marker)
Dye color
NDL06/132 (♀)
AR37-80 (♂)
Expected allele combinations
Allele 1
Allele 2
Allele 1
Allele 2
1 2 3 4
SSRY169
Blue 97 99 89 9997
89
97
99
99
89
99
99
SSRY148
Green 110 112 112 118110
112
110
118
112
112
112
118
SSRY52
Red 257 267 267 267257
267
257
267
267
267
267
267
NS911
Green 123 123 113 123123
113
123
123
123
113
123
123
113 123
Genotype screening cont …… ..
Primer NS911 identified NDLAR10 as a true cross F 1(with alleles 113 from male and 123 from female par ent)
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)
_ P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11
+
Electrophoresis = electrically induced movement of particles
Principle underlying separation of DNA molecules in agarose gel during electrophoresis
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
Identification of progenies inherited disease resis tance
_ 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.
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
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
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