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Research Project Proposal

Applicant’s full names: Josephine Sarah Kunguni

1. Title of your proposed research project

The title should be short and descriptive, and clearly describe the main objective of the proposed work. Avoid using

acronyms or abbreviations.

Characterization of Eleusine species for response to Striga

2. Research area

Choose one or more of the Research Areas that best describes your project. If none of the areas is relevant then please

select ‘Other’ and describe your research area(s).

a) Improved control of priority livestock and fish diseases including African swine fever (ASF); Contagious Bovine and Caprine Pleuropneumonia (CBPP and CCPP); Peste des Petits Ruminants (PPR); Rift Valley Fever (RVF); East Coast Fever (ECF); Capripox Virus diseases of ruminants;

b) Harnessing genetic diversity for conservation, resistance to disease and improving productivity of crops and livestock and fish (livestock focus: African indigenous breeds, particularly of goats, chickens, and cavies and

other micro-livestock);

c) Molecular breeding for important food security crops in Africa;

d) Plant transformation to address food insecurity in Africa;

e) Plant-microbe interactions;

f) Tissue culture and virus indexing for production of virus-free planting materials in Africa;

g) Orphan / underutilized species of crops and livestock

h) Crop pests, pathogens and weed management research, including biological control;

i) Food safety, including addressing Aflatoxin and other mycotoxin contamination in food and feeds;

j) Nutritional analysis of food and feeds;

k) Rapid diagnostics for crop, livestock and fish diseases;

l) Genomics and metagenomics, including for microbial discovery;

m) Studies on climate-smart forage grasses and mixed livestock-crop systems;

n) Microbial technology for improving adaptation of staple food crops and grasses to biotic and abiotic stresses;

o) Soil health in agricultural systems;

p) Special opportunities also exist to connect with leading international scientists linked with the BecA-ILRI Hub in the following areas: wheat rusts, insect pests, and nitrogen fixation.

q) Other special opportunities exist to connect with CGIAR Research Programs (CRPs): Livestock & Fish, Agriculture for Nutrition & Health, Humid tropics etc. Such collaboration would allow the candidate’s

research to contribute more directly to an impact-oriented research-for-development agenda, and offer additional opportunities for joint activities.

r) Other ……………………………………………………….

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3. Short summary (abstract) of your research project

Give the relevant background information, state the problem, identify the solution and why it is novel, specify the

objectives and the expected outputs.

Also describe the direct and longer-term impacts of your research.

(300 words maximum)

4. Statement of the problem, background & justification

Describe the food and nutritional security or food safety problem that your research will address.

What is the significance of the problem in Africa?

Give current state of knowledge, and describe how your research will build upon what has already been done

and why your work is novel.

Justify why the proposed research should be conducted, and the importance of the science.

(500 words maximum)

Finger millet is a staple food crop of in Africa due to; desirable attributes like being highly

nutritious, good grain storagability, drought tolerant and strong cultural value. In Kenya, finger

millet is on high demand for food, culture and trade. Low yield on farmer’s fields is due to poor

genetic potential varieties, poor management practices, unpredictable market prices, unimproved

varieties, biotic and abiotic factors. The most important biotic stresses in Kenya are blast disease

Pyricularia oryzae and Striga weed. Striga, also known as Witch weed is one of the most dangerous

parasitic weed that attacks several tropical African cereals leading to huge reduction in food production.

The life cycle of Striga ssp. is composed of five stages; germination, haustorium initiation,

penetration of host tissue, physiological compatibility and parasite growth and maturation. Striga

germinates and extends an infectious structure (haustorium) to the host and sticks to the root

invading the xylem and phloem tissues and initiates shoot development and sucks the essential

nutrients for plant growth from the hosts leading to stagnation of the host plants by altering

hormonal balance of the host, stimulating root growth and reducing shoot growth. Striga seed

production can be reduced by hand weeding, use of herbicides, biological control, use of suicidal

germination stimulants, toxic fumigants, and soil solarization. The purpose of this study will be to

evaluate variation among different Eleusine genotypes in their interaction with Striga upon

infestation: through identification of Eleusine spp. responding differently to the same Striga strain;

determine interactions of Eleusine spp. at the 3 critical stages of Striga infestation and determine

interactions between different Eleusine spp. and Striga for resistance, tolerance or susceptibility.

The proposed research will contribute to national food and nutrition security that will in turn reduce

poverty and generate income to low class people in the society. (300 words)

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Finger millet (Eleusine coracana (L.) Gaertn) is one of the most important crops to women

smallholder farmers in eastern and southern Africa because of its high nutritive value (Barbeu and

Hilu, 1993).It is a tetraploid crop (2n=4x=36), having a genome composition of (AABB) belonging

to the grass family Poaceae, subfamily Chloridoideae. ). Finger millet is the third most widely

cultivated millet after pearl millet (Pennisetum glaucum) and foxtail millet (Setaria italica) in the

semi-arid tropical and sub-tropical regions (Reddy et al., 2009). The grains contain 65–75%

carbohydrates, 5–8% protein, 15–20% dietary fiber and 2.5–3.5% minerals (Rai, 2000; Chetan and

Malleshi, 2007) and therefore a practical solution to malnutrition problems (Singh and Raghuvanshi,

2012) often experienced among women and children in sub-Saharan Africa. The color of finger millet

grains vary from white through orange-red, deep brown, purple to almost black (Crawford and Lee,

2003); colored ones having good levels of phenolics, tannins (Ramachandra et al., 1977) and do not

allow oxidation of other molecules in the body than white grains.

Although consumption demand for finger millet is increasing, yield on farmer’s field is low, at about

15-16% of its potential in Kenya (Oduori, 1998). The low yield is attributed to low research priority,

limited uses, difficulty in crop management, lack of improved varieties, poor crop husbandry,

competition from other crops like maize (Zea mays) and sorghum (Sorghum bicolor), lack of

commercial food products, poor technology in processing, pests and diseases including Striga,

lodging and moisture stress in dry areas ( Oduori, 1993).

Finger millet is highly resilient with incredible ability to grow under semi-arid conditions, especially

due to the leaf blade anatomy that uses C4 photosynthetic pathway (Brown, 1999). The crop is

tolerant to infertile and acidic soils and grows in marginal lands where other crops like rice (Oryza

sativa) and maize cannot survive (Padulosi et al., 2009; Bhag Mal et al., 2010). The nutritious grains

once harvested, can be stored for a long period of time without insect damage making it a desirable

subsistence crop (Dida et al., 2007). This makes it a most promising cereal crop for small holder

farmers in Africa.

Out of the thirty Striga species in sub-Saharan Africa known to originate from different regions

infesting different crops, S. hermonthica is the most important causing up to 100% yield losses on

farmers’ fields (Ejeta, 2007). Besides, due to the nutritive composition of finger millet, farmers in

some have continued to cultivate the crop. Striga is a major menace in Kenya among small holder

farmers (Midega, et al., 2010).

In as much as, studies have been carried out on resistance on host plants like in sorghum (Scholes and

Press 2008), transcriptome analysis in response to Striga has not been exploited in Eleusine spp. This

will be a great possibility of finding host resistance in east Africa because finger millet is native of

this place and has been growing side by side with sorghum for years. (491 words)

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5. Research question and hypothesis

State the research question(s), and give the hypothesis that you are testing with the research.

A research hypothesis is a statement of expectation or prediction that will be tested by the research.

This hypothesis should determine and drive the data collection for your study.

(200 words maximum)

6. Research objectives The objectives are what you want to achieve by your research activities.

Give no more than three objectives.

Each objective should flow logically to the next.

(150 words maximum)

Research Questions

1. Is there natural variation in Eleusine spp. for response to Striga infestation?

2. How is this variation manifested at the 3 critical stages of Striga infestation?

3. Will the difference observed result in susceptibility or resistance of the finger millet to

Striga under farmer conditions?

Hypotheses

1. Different Eleusine spp. will respond differently to the same Striga strain;

2. The different interactions can be observed at the 3 critical stages of Striga infestation both

at conventional and molecular level;

3. The different interactions between different Eleusine spp and Striga will result in

resistance, tolerance or susceptibility of the genotype to Striga. (105 words)

Main objective

The main objective will be to evaluate variation among different Eleusine genotypes in their

interaction with Striga upon infestation.

Specific objectives

The objectives of the experiment will be:

1. To identify Eleusine spp. that will respond differently to the same Striga strain;

2. To determine interactions of Eleusine spp. at the 3 critical stages of Striga infestation both at

conventional and molecular level;

3. To determine interactions between different Eleusine spp and Striga for resistance, tolerance

or susceptibility. (80 words)

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7. Research plan Describe how you will accomplish each objective.

Justify the research methods and explain why each step is important.

Give details of organisms to be studied.

It is very important that you describe the sampling design and data analysis.

(1000 words maximum)

Experimental Site and climate

The experiment will be conducted in the screen house at the Kenya Agricultural and Livestock Research Organization (KALRO) Kisii station in Kisii County and laboratory

at ILRI BecA in Nairobi County.

Genetic Materials

Striga hermonthica seed populations will be selected from two sites; KALRO Kakamega and Alupe fields will be used in this study. It will be important to collect same

species of Striga seeds from different locations in order to check on variations that may be introduced into the experiment as a result of ecotypes. The seeds will be stored in

ambient temperatures in polythene containers. Striga seeds will be conditioned by exposing them to water and incubating in an ambient temperature for a suitably long period

(Worshma, 1987).

Striga conditioning

Striga seed surfaces will be sterilized using 1% sodium hypochloride (NaOCl) solution for 30 minutes and rinsed three times in double distilled water to remove sand and any

debris before use (Worsham, 1987). Floating seeds will be discarded and the remaining seeds will be dried in an aseptic environment ready for pre-conditioning. Small disks

of glass fibre paper will be put in a wide Petri-dish and wetted with distilled water for conditioning. Two pieces of 11cm ordinary filter paper will be placed in a sterilized

11cm dish and moistened with distilled water. The moist disks will be picked with sterilized forceps and arranged in circles to cover the moistened filter paper in the Petri

dishes. Fifty sterilized Striga seeds will be dabbed onto each pre-arranged glass fibre disks in the Petri dish. The dishes will then be wrapped in aluminium foil to exclude

light and kept in the incubator at 28°C for conditioning for two weeks in the dark to respond to germination stimulants.

Eleusine genotypes

Four different Eleusine genotypes with different responses to Striga with one check will be germinated In vitro and then subjected to Striga 5 days after germination. This

include; E.coracana, E.kigeziensis, E.indica and KNE796 as a control.

Germinating Eleusine and Striga infestation (whole seedling assay)

The experiment will be conducted both in the screenhouse and laboratory. Eleusine seeds will be surface-sterilized using 1% solution of sodium hypochlorite which will be

made by adding 1ml of 10% NaOCl to 99 mls of sterile water. Each seed variety will undergo sterilization separately. Thirty millilitres of NaOCl solution will be poured onto

the seeds in separate beakers stirred using a glass rod to mix thoroughly and left to stand on the bench for 30 minutes. Floating seeds and any other debris together with the

solution will be drained. The remaining seeds in the beaker will be rinsed three times with double distilled water. The seeds will be dried under laminar flow hood and the

crop seeds will be ready for planting.

Screen house study

Plastic pots of 15- 20 cm diameter will be filled with 1 kg autoclaved sand in the screen house for whole-plant assay as described by (Ahonsi and Emechebe, 2005). Sterilized

seeds of Eleusine crop varieties will be planted at 1cm depth in well labelled plastic pots containing autoclaved sand, each pot will have four seedlings in the screen house at

KALRO Kisii. Watering will be done during this period when necessary. After planting, the seeds will be allowed to grow for two weeks, after which seedlings will be

harvested for the experiment. Plant tissues at Striga germination, attachment and penetration will be harvested and stored at -80°C freezer for RNA extraction. Finger millet

seeds planted in the absence of Striga seeds will be used as control.

Laboratory study

Sterilized petri-dishes lined with two moist 90 mm Whatmans no. 1 filter papers will be used to test for Striga growth on Eleusine genotypes. The paper will be moistened

with 300 ml distilled water. The dishes will be sealed with parafilm and incubated at 28°C for a day. Then, the dishes will be opened and a 15 pre-conditioned Striga seeds

and 15 Eleusine seedlings will be placed on the dish. The dishes will be moistened with 500 ml distilled water through the slit and sealed with parafilm. They will be left to

grow in the dark for three days at 28°C in an incubator. Then RNA will be extracted from the germination, attachment and penetration stages of Striga life cycle.

RNA Extraction

Eleusine plant tissues will be sampled and immediately stored in liquid nitrogen container. RNA will be extracted from genotype tissue at three stages of Striga infestation

using Zymo Research Plant MiniPrep RNeasy kit (ZR, Irvine, CA, USA) according to manufactures instructions.

Library preparation and Sequencing

The cDNA will undergo synthesis, and then RNA will be denatured, using Illumina’sTruSeq kits Stranded Total RNA Sample Preparation kit with Ribo-ZeroTM Plant

(Illumina, San Diego, California, USA) according to manufactures instructions, followed by PCR and transcription. The samples will be treated with RiboZero to remove

Ribosomal RNA before sequencing. Sequencing will be done using 300 PE v3 kit (Illumina, San Diego,. California, USA) according to manufactures instructions.

Transcript Assembly, annotation and SNP identification

The traces will be downloaded and transcript reads generated will be assembled using de novo TRINITY (Grabherr et al. 2011). The transcripts will be mapped to a reference

genome and identify single nucleotide polymorphism (SNP) variations will be detected from the resulting alignments using Samtools and Bcftools (Li et al. 2009).

Experimental design

The screenhouse and laboratory experimental design will be; 4 varieties × 1 Striga type x 2 screening technique in a completely randomized design (CRD).

Data Collection and Analysis

Striga seed germination will be determined by observing the germination of Striga seeds in response to germination stimulants present in host root exudates, attachment and

penetration of haustorium to the host root using a binocular light microscope. The data collected from observations in the laboratory will be subjected to analysis of variance

(ANOVA) using general linear models (GLM) procedure and means separated by Fischer’s protected least significant differences (p≤0.05) resident using SAS 8.2 ed

statistical package (SAS Institute, 2001). (994 words)

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8. Biological materials

Give details of the biological materials that you propose to bring to the BecA-ILRI Hub for your research project.

Are the materials infectious to plants or animals?

(100 words maximum)

9. Expected outputs of the research

Expected outputs of a research project are the deliverables from the research

(150 words maximum)

1. Eleusine genotypes will be screened for Striga infestation both in the screenhouse and

laboratory (E.coracana, E.kigeziensis, E.indica and KNE796).

2. Striga seeds.

The genotypes will be obtained from ICRISAT Nairobi and Striga seeds from KALRO Kakamega

and alupe. (39 words)

Finger millet being a highly nutritious crop and mostly cultivated by women in our society; I expect

to use my improved knowledge from the research to advice farmer’s to choose varieties wisely that

have better agronomic traits. This will in turn reduce poverty and generate income to low class

people in the society. I will also share the information with other researchers to provide way forward

to correct the increased negative attitude from farmer’s on orphan crops and enormous destruction

of Striga on finger millet. (85 words)

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10. Tentative project timetable and start date

List each activity in chronological order, and give start and finish weeks (week 1 is the first week of your

project at the BecA-ILRI Hub).

Be realistic about what can be completed in the time frame.

ABCF research fellowship projects are normally 3-12 months. Only in exceptional cases will projects for

longer than 12 months be considered.

Start Date: April, 2015

Activity Start Week End Week

a). Germplasm assembly-April 2016

b). Conditioning of Striga seeds-May 2016

c). Sterilization and planting of Eleusine

seeds-May-June 2016

d). In vitro Striga germination-June 2016

1

1

1

1

1

2

3

2

a). Data collection-July 2016

b). RNA extraction-July 2016

c). Transcriptome sequencing-August 2016

1

1

1

2

2

4

a). Transcript assembly-September-October

2016

1 8

Gene annotation-November-December 2016 1 8

Single nucleotide polymorphisms (SNPs)

identification-January 2017

1 4

Data analysis and Report writing-February-

April 2016

1 12

TOTAL WEEKS 48

*Please note that BecA-ILRI Hub has the final decision on when you should report/ commence your fellowship

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11.Skills for the proposed research project

List the research skills you currently possess that you will apply to the proposed research project

I. Field preparation procedures

II. DNA extraction, Polymerase chain reaction

(PCR), DNA purification and gel

electrophoresis

III. Trace assembly using Geneious software,

sequence editing

List the research skills that you would like to gain during the proposed research project at the BecA-ILRI Hub

1. Statistical analysis of phenotypic and genotypic

data independently using software’s such as

Genstat, SAS, SPSS, Bioedit etc.

2. RNA extraction

3.SNP identification using Next Generation

Sequencing (NGS)

2. Potential impact of your research outputs

What will you do with the outputs of your research project when you return to your home institute?

Describe how outputs of your project could lead to direct or longer-term impact to address food and nutritional

security, food safety, poverty reduction or livelihoods.

Describe your plan to ensure the outputs will ultimately lead to impact.

(500 words maximum)

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3. How does the proposed project link to your own research interests or a program of research at your institute?

(250 words maximum)

This project will be used to prepare a publication paper at BeCA. The knowledge and skills

acquired will be used at KALRO, ICRISAT and beyond for enlightening upcoming

researchers and farmers in planting cereals and legumes for food and nutritional security,

food safety, poverty reduction to improve livelihoods.

Finger millet SNPs that will be developed will help breed Striga resistant genotypes.

Screening of QTL regions contributing to traits of interest will improve on the breeding

activities at the stations that will result in increased yields on farmers’ fields from as low as

(<1 ton/ha) to (up to 7 tons/ha). The use of molecular markers in breeding programs being

more efficient than conventional methods will increase breeding output by more than 50%.

I believe my knowledge in analyzing molecular data will lead to more efficient finger

millet breeding and hence a faster release of improved varieties.

In vitro characterization of Eleusine spp in response to Striga weed will open a way for

more breeding and farming for varieties with valuable traits in order to bridge the gap of

food and nutritional security thus reducing malnutrition. Striga resistant finger millet

genotypes will be a great avenue in achieving the set goal for food and nutritional security

in Kenya for vision 2030 in both arid and semi-arid areas; finger millet is a drought tolerant

highly nutritious crop that is grown mostly by women in two seasons per year. (235 words)

Finger millet being a crop where transcription analysis and bioinformatics has not been

worked on, then it will be pleasure to exploit the opportunity: This project together with

breeders, geneticists and agronomists will exploit the use of Eleusine spp in

characterization for Striga response for germination, attachment and penetration through

in vitro screening. This will contribute towards capacity building especially on young

scientists in the agricultural sector KALRO) for future leaders in finger millet breeding.

Although Striga response variation is known across some species, no studies have been

performed to determine the extent of variation for response to Striga within specific

finger millet. Screening for Striga resistant genes has been done in other cereals like rice

(Oryza sativa) sorghum (Sorghum bicolor L.), so this study will impart positively on

agriculture. The results from this experiment will form a starting point to develop a

similar technique in finger millet at KALRO. (150 words)

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4. Describe how an ABCF Research Fellowship will benefit your career as a researcher, and how it will build capacity for your research Programme, department or institution

(250 words maximum)

5. References cited in the proposal

List references that you have cited in support of your proposal. Some of the references should be recent publications,

indicating that you are aware of the current research in your research area. List references in alphabetical order. All references listed must be cited in the text

Capacity development: I believe the research will strengthen my research skills and help

me better mentor and inspire other young upcoming scientists at KALRO thus come up

with solutions in this field of science. The knowledge gained will be advanced on the

already existing to contribute to agriculture in KALRO that will directly contribute to the

welfare of women and youth due to their high contribution to agriculture. Together with

other researchers at the institution, this study will improve on our skills especially on

transcription and Bioinformatics and will profitably contribute to solving existing hunger

problem in some regions. (99 words)

Ahonsi, M. O. and Emechebe, A. M. (2005). In-vitro whole-seedling assay for evaluating non-host crop plant induction of germination of witch weed seeds. African Crop

Science, 13(1), 61–69.

Barbeau, W.E., and Hilu, K.W. (1993). Protein, calcium, iron, and amino acid content of selected wild and domesticated cultivars of finger millet. Plant Foods for Human

Nutrition, 43(2), 97-104.

Bhag Mal, Padulosi S. and Bala Ravi, S. (eds) (2010) Minor Millets in South Asia: Learnings from IFAD-NUS Project in India and Nepal, Bioversity International, Maccarese,

Rome, Italy and the M.S. Swaminathan Research Foundation, Chennai, India, p.185.

Brown, R. H. (1999). Agronomic implications of C4 photosynthesis. C4 plant biology, 473-507.

Chetan, S. and Malleshi, N.G. (2007). Finger millet polyphenols: Characterization and their nutraceutical potential. American Journal of Food Technology, 2(7):582–592.

Crawford, G. and Lee, G. (2003). Agricultural origins in the Korean Peninsula. Antiquity, 295(77), 87–95.

Dida, M.M., Ramakrishnan, S., Bennetzen, J.L., Gale, M.D., and Devos, K.M. (2007). The genetic map of finger millet, Eleusine coracana. Theoretical and Applied

Genetics, 114(2), 321-332.

Ejeta, G. and Butler, L.G. (1993). Host-parasite interactions throughout the Striga Life cycle, and their contributions to Striga resistance. African Crop Science, 2(1), 75–80.

Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I. and Chen, Z. (2011). Full-length transcriptome assembly from RNA-Seq data without a

reference genome. Nature biotechnology, 29(7), 644-652.

Li, Z., and Yan, Z.W. (2009). Homogenized daily mean/maximum/minimum temperature series for China from 1960–2008. Atmos. Oceanic Sci. Lett, 2(4), 237-243.

Midega, C.A., Khan, Z.R., Amudavi, D.M., Pittchar, J. and Pickett, J.A. (2010).Integrated management of Striga hermonthica and cereal stemborers in finger millet

(Eleusinecoracana (L.)Gaertn.) through intercropping with Desmodium intortum. International journal of pest management, 56(2), 145-151.

Oduori, C.O.A. (1993). Small millets production and research in Kenya.Advances in smal. New Delhi, India: Oxford and IBH Publishing.

Oduori, C.O.A. (1998). Finger millet better varieties–Better crop care-More Food. DFID and GON produced by DEVCOM & AIC.

Padulosi, S., Bhag Mal, Bala Ravi, S., Godwa, J., Godwa, K.T.K., Shanthakumar, G., Yenagi, N. and Dutta, M. (2009) ‘Food security and climate change: role of plant genetic

resources of minor millets,’ Indian Journal of Plant Genetic Resources, 22(1), 1–16.

Ramachandra, G., Virupaksha, T. K. & Shadaksharaswamy, M. (1977). Relation between tannin levels and in vitro protein digestibility in finger millet (Eleusine coracana

Gaertn.). Journal of Agricultural and Food Chemistry, 5(25), 1101–1104.

Rai, M. (2000). Productivity grain focus. Survey of Indian Agriculture. The Hindu, pp 53-55.

SAS Institute. (2001). SAS/STAT user’s guide. NC, USA.: SAS Institute Inc.

Singh, P. and Raghuvanshi R.S. (2012). Finger millet for food and nutritional security. Afr J Food Sci 6(4):77–84.

Scholes, J.D. and Press, M.C. (2008). Striga infestation of cereal crops-an unsolved problem in resource limited agriculture. Curr. Opin Plant Biol, (11), 180–186.

Worsham, A.D. (1987). Germination of witchweed seeds. In: Musselman, L.J. (Ed.), Striga. Parasitic Weeds in Agriculture (1), 45–61). FL. CRC, USA: Boca Raton.

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If you have co-funding for the proposed research project, please give details (including the amount) and why you

need ABCF funding

Please give any other information that you think is relevant to your application but was not captured elsewhere.

Yes: I will get support from ICRISAT (Nairobi) and KALRO (Kisii).

The reason for request for ABCF funding is to work on the proposed research in order to

improve on crop productivity thus imparting positively on livelihood of small farmers of finger

millet and also improve on Transcriptome scientific research skills in developing Striga resistant

lines.

Commitment:

If given the funds and chance; am ready to abid to the rules set by the ABCF committee.

Thank you

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