Allanblackia In Agroforestry Systems Iv 9 Feb Caroline Kadu

ALLANBLACKIA IN AGROFORESTRY SYSTEMS: DEVELOPING THE TOOLS TO MANAGE A NEW TREE CROP FOR SMALL-SCALE FARMERS

9th FEBRUARY 2007

CAROLINE KADU – AFP ROTHAMSTED INTERNATIONAL JOANNE RUSSELL & MARY WOODHEAD – SCRI SUPERVISORSIAN DAWSON – CONSULTANT ON NOVELLA PROJECTRAMNI JAMNADASS – ICRAF SUPERVISOR

CONTENT

•Background

•Objectives of the molecular approach to managing Allanblackia

•Activities planned for the molecular diversity study

•Geographic focus

•RNA ISOLATION

•cDNA synthesis and cloning

•Genomic library construction

•SSR identification

•Future work on Allanblackia

•Outputs

Rothamsted International A UK non-profit organisation working for sustainable agricultural

development in under-developed countries around the World.Their main activities are managing Fellowship schemes and project

coordination; Fellowships are directed towards mid-career researchers in

agricultural sciences. Two types of schemes are offered, one open to scientists from all developing countries and another for African

scientists.

African Fellows Programme (AFP)

The Rothamsted International African Fellows Programme aims to provide problem-focused training in Europe for mid-career African scientists. The

Programme started in 2004.The purpose of the programme is to assist in capacity building, institutional strengthening and knowledge transfer in order to find relevant solutions to the problems of achieving sustainable agricultural production, as well as

improving rural development and conservation of biodiversity.The development of effective partnerships is fundamental to ensuring the success of the programme in order to build long-term strategic alliances.

SCRI is Scotland's leading institute for research on plants and their interactions with the environment, particularly in managed ecosystems. The research products are internationally recognised.

As such, the institute's mission is to conduct excellent research in plant and environment sciences. SCRI's objective is to deliver innovative products, knowledge and services that enrich the life of the community and address the public goods of sustainability and high quality and healthy food.

BACKGROUND INFORMATION I

•The World Agroforestry Centre (ICRAF) is one of the 15 International research Institutes within the Consultative Group of International Agricultural Research

• Our Vision is that of an 'agroforestry transformation' in the developing world resulting in a massive increase in the use of working trees on working landscapes by smallholder rural households that helps ensure security in food, nutrition, income, health, shelter and energy and a regenerated environment.

• 6 Regions: West and Central AfricaEast AfricaLatin AmericaSouthern AfricaSouth Asia South East Asia

BACKGROUND INFORMATION II

• 4 Cross cutting themes1. Land and people 2. Trees and Markets3. Environmental Services 4. Strengthening Institutions

• Trees and Markets theme has 3 focal areas and 8 outputs

• The focal area on Agroforestree Germplasm (TM1) is supported globally by the Germplasm Resources Unit (GRU). The other two are Tree Domestication (TM2) and Marketing of Agroforestry Tree Products (TM3)

• The Genetic Resources Unit (GRU) at ICRAF provides global support to ICRAF regional staff and partners for tree germplasm and tree information needs.

• It holds separately and/or in conjunction with national programmescollected and procured germplasm in both live and seed gene banks around the world.

BACKGROUND INFORMATION III

• At headquarters a centralized facility for storage, testing, characterization (including molecular) and dispatch for orthodox species exists.

• A nursery and field facility is also maintained at Meru (400 km north of Nairobi) for quarantine, testing and dispatch of introductions to all regions in Africa.

• Databases which compile information on tree taxonomy, uses, suitability and sources of seed are developed and maintained by the unit. Collectively these are part of the genetic resource activities of ICRAF.

BACKGROUND INFORMATION III ALLANBLACKIA

• Novella Africa: A public-private partnership project to domesticate and use the Africa oil tree (Allanblackia spp.) is one of the most recent projects undertaken by the GRU

Our partners are• Unilever• The World Conservation Union (IUCN), • Netherlands Development Organisation (SNV) • A number of African regional organisations (NGO’s, research

institutes, local- and national government)

The project promotes development, poverty alleviation and biodiversity in the African tropical forest belt.

BACKGROUND INFORMATION IVALLANBLACKIA

• The goal of this partnership is to domesticate, conserve and use the indigenous Allanblackia tree on a commercial scale through extraction of edible oil from the seeds. It is viewed as a superior substitute for Palm Oil because it requires less chemical processing and refraction thus would reduce Unilever’s “ecological foot print”

Seed kernels amount to 60-80% of the whole seed weight. The unusual hard white fat consists of 52-58% stearic acid and 39-45% oleic acid. Oleic and Stearicacids are reported to lower plasma cholesterol levels thus reducing the risks of heart attack.

Allanblackia floribunda

The Allanblackia tree is commonly found in parts of West, Central and East Africa.

The genus is thought to contain nine species (though some may be synonyms and distributions have not been fully delineated).

It grows primarily in tropical rainforests, but can also be found in farmland areas.

Allanblackia is a tall evergreen forest tree of up to 40 m tall, with a straight, occasionally buttressed bole and drooping branches which are often conspicuously whorled

It is Dioecious

BACKGROUND INFORMATION VALLANBLACKIA

• The partners will help and encourage local communities and smallbusinesses to cultivate the seeds for extraction of oil. The project will also help to achieve greater sustainability in the region by using Allanblackia trees where previously “slash and burn” methods have been practiced and thus diversify existing system.

• The guaranteed market will ensure long-term economic viability of the project whilst the planting of trees will positively affect the environment. This initiative is dubbed the Novella Africa.

• Work on Allanblackia, which began in earnest in 2002, consists of a diverse range of elements.

Economic evaluation of production optionsthe development of policies and guidelines to promote sustainable harvestingTree inventory and reproductive ecology studiesEnvironmental impact assessmentsDevelopment of market delivery structures and processing methods to bring product to the consumer.

BACKGROUND INFORMATION VIALLANBLACKIA • Early in the Allanblackia

initiative, a need for planting of the genus (on farm and in forest enrichments), rather than reliance on sourcing oil solely from natural stands, was identified as a crucial task. Recognising this need, project partners made a commitment to encourage domestication of the genus within smallholder agroforestry systems. In 2003, a domestication programme began in Cameroon, Ghana, Nigeria and Tanzania.

• From 2007 onwards, it is estimated that, if rural communities are to benefit fully from the initiative, several million trees annually will need to be planted across these countries.

Problem statement•Developing strategies for the sustainable cultivation and conservation of Allanblackia of this magnitude is limited because its biology is not known

•A reliance on limited sources of germplasm or germplasm whose genetic structure and variation is not known may result in a serious loss of biological diversity and species may suffer from geneticbottlenecks thus affecting their productivity

•At ICRAF, the two most commonly employed molecular approaches for determining genetic variation are

•RAPDs (randomly amplified polymorphic DNA analysis). • AFLPs (amplified fragment length polymorphisms analysis) The arbitrary fingerprinting techniques offer large numbers of polymorphic loci, scored as biallelic dominant markers, with little development time required & no prior knowledge of the species sequence•The drawbacks of these methods are

dominance non-specificity of the polymerase chain reaction (PCR) co-migrating fragments may be homoplastic. In case of RAPDS, non reproducibility between labs

Problem statement II

•Another commonly used approach is that of assessments of the frequency and distribution of length variants at simple sequencerepeat loci (SSRs, also called microsatellites or short tandem repeats). • Microsatellites are codominant, locus-specific markers showing high levels of allelic variability and thus have a robustness exceeding that of arbitrary fingerprinting approaches and this has led to theirpopularity for forensic, as well as ecological, evolutionary, and conservation applications. •Their limitations

the lengthy development phase required for each species or group of species

variability in priming sites leading to null alleles hyper-variability leading to the homoplastic origins of alleles a downward bias in estimators of population differentiation such as

FST These limitations of microsatellites are likely to be extenuated in range-wide studies of species where population divergence can be substantial and null alleles and homoplasy become more.

Problem statement III

The limitations of RAPDs, AFLPs and genomic microsatellites have led to continued attempts to refine and develop marker systems. One approach that has been advocated recently is the use of SSRs from expressed sequence tags (ESTs) (EST-SSRs) as genomics technologies have led to huge amounts of sequence information being available in public and private databases that can be 'mined' for potential SSR loci

Neither ICRAF nor the region has the capacity to develop these for plants. EST-SSRs based on cDNA libraries are definitely the way to go because of their better (when compared to standard SSRs) cross-taxaamplification, good clarity and higher transferability (both across laboratories and detection techniques).

These points more than offset the possibly somewhat lower allelic variation that EST-SSRs detect compared to regular SSRs.

Problem statement IV

EST-SSRs are particularly effective for identifying genetic bottlenecks

This is because the techinque reveals a high number of alleles and is known for the sensitivity of allelic richness to genetic bottlenecks

Ensuring bottlenecks do not enter cultivation during the early stages of Allanblackia cultivation is a crucial prerequisite for ensuring the success of Allanblackia domestication

Similarly, material entering cultivation ought to be of sufficient diversity to provide an adaptive capacity to potential changes in environment and user requirements.

OBJECTIVES OF THE MOLECULAR APPROACH TO MANAGING ALLANBLACKIA

1. Develop EST-SSR markers to investigate and understand the level, structure and origin of the genetic variation within and betweenpopulations of Allanblackia.

2. Use information derived from the diversity studies to contribute to development of optimum collection strategies for on-farm cultivation and conservation within national genebanks

3. Monitor and prevent potential bottlenecks in on-farm introductions during cultivation

4. Resolve taxonomic confusion existing among species within the genus

Activities Planned For The Molecular Diversity Study

•Survey and collection of plant material for nucleic acid isolation

•DNA extraction

•Total RNA extractions

•mRNA isolation, cDNA synthesis and cloning

•Sequencing of transformed clones, EST-SSR identification and testing

•EST-SSR application on range wide samples by ABI 3730 genotyping

GEOGRAPHIC FOCUS

Country Species Geographical Site

No. of individuals sampled

No. of individuals sampled for DNA

Cameroon A. gabonensis Bangangté 25 12 A. floribunda Edea 27 12 A. stanerana Edea 26 12 A. floribunda Sangmelima 25 15 A. gabonensis Sangmelima 16 3 Total 119 54 Ghana A. parviflora Wet evergreen 24 6 A. parviflora Moist evergreen 28 7 A. parviflora Moist/Wet evergreen 19 5 A. parviflora MSNW 5 2 A. parviflora MSSE 8 0 Total 84 20 Tanzania A. stuhlmannii Amani nature reserve 25 15 A. ulugurensis Uluguru 25 9 A. stuhlmannii Mazumbai Forest reserve 25 15 A. stuhlmannii Manyangu forest reserve 25 15 A. stuhlmannii/A. sacleuxii Mufindi Forest reserve 25 11 A. stuhlmannii Ndelema Forest Reserve 23 0 Total 148 65 7 species 3 countries 351 individuals 139 individuals

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Sudan

AlgeriaLibya

Mali

Chad

Niger

Congo DR

Egypt

Angola

EthiopiaNigeria

South Africa

Namib ia

Mauritania

Zambia

Tanzania

Kenya Somali

a

Mozambique

Botswana

Morocco

Congo

Mad

agas

car

Cam

ero o

n

Zimbabwe

Gabon

Ghana

Guinea

Uganda

Central African Repub lic

Tunisia

Senegal

Burkina Faso

Eritrea

W. S

ahara

Liberia

Sierra Leone

Djibouti

Swaziland

Country boundaryCountry with study sites

# Species location point

Key

Lakes

A. parvifloraA. stuhlmannii, A. ulugurensis, A. sacleuxii

A. floribunda, A. stanerana, A. gabonensis

A map showing the distribution of Allanblackia in 3 countries

Daniel Ofori and Theresa Peperah, colleagues from the Forest Research Institute in Ghana showing us the 200 A. parviflora seedlings that germinated from thousands planted in 2005

DNA EXTRACTION

•Optimization of the DNA extraction procedure was carried out. •A modified CTAB method was used which included the use of proteinase K and 1% Sodium sulphite, 1.5 M NaCl and 0.5 M EDTA•The Qiagen columns was then used to clean up the product after the Cloroform:IAA stage.•Number of individuals used for the whole run is 139

Total RNA isolation

Plant material was collected and stored in RNALater then frozen at -80oC until ready for extraction

Various methods were tried • Qiagen RNeasy kit• Tri Reagent Sigma product• CTAB method by Chang et al• SDS method by Mary Woodhead• Qiagen Rneasy kit by Gehrig et al 2000• Tri reagent by Gehrig et al 2000• Addition of HMW-Polyethylene Glycol

made a big difference to the extraction

• QIAGEN RNEASY protocol was selected because it gave good RNA for 3 out of the four tissues used

Barley

Qia

gen

Bar

ley

SD

S

AB r

oot

100 b

pm

arke

r

SE

SC

RO

OT

LEAF

SE

SC

LEAF

SE

SC

RO

OT

LEAF

SE

SC

LEAF

BARLE

Y 1

BARLE

Y 6

QIAGENRLT/ RLC

TRI GITC/GHCL

2.042.0779.33809.4415:48 22/03/2006 ABSE

2.012.0781.92835.9515:47 22/03/2006 ABSR 2

1.92.0867.57689.5115:46 22/03/2006 ABPE 1

1.792.0324.14246.315:44 22/03/2006 ABPC 2

1.9214.31145.9715:44 22/03/2006 ABPR

1.152.0618.98193.6715:43 22/03/2006 ABPE 2

1.981.8357.53587.0115:41 22/03/2006 ABSC 2

2.071.9151.17522.115:41 22/03/2006 ABSC 1

2.262.1363.15644.3815:39 22/03/2006 ABSR 1

1.761.9842.06429.1715:38 22/03/2006 ABPC 1

-1.63-0.12-0.1715:37 22/03/2006 H20

260/230 260/280 TOTAL AMOUNT OF µg RNA IN 98 µLng/ulTime Date Sample ID

NANODROP READINGS OF ISOLATED RNA

NANODROP READINGS OF ISOLATED RNA

Sample ID Date Time ng/ul 260/280 260/230

C 10/03/2006 17:39 -0.32 0.37 0.37

V 10/03/2006 17:42 0.4 2.81 -0.39

100306 T7 SE1 10/03/2006 17:43 92.79 3618.81 2.01 1.09

100306 T7 SE2 10/03/2006 17:45 108.45 4229.55 2.01 1.57

100306 T7 SE3 10/03/2006 17:46 85.35 3328.65 2.03 1.88

100306 T7 SE4 10/03/2006 17:47 97.14 3788.46 2.02 0.46

100306 T7 SE5 10/03/2006 17:48 69.03 2692.17 1.98 0.87

100306 T7 SE6 10/03/2006 17:49 102.36 3992.04 2.04 2.17

100306 T7 SE7 10/03/2006 17:50 2.63 102.57 1.3 0.07

21752.25

21.75225

21.75 µg

mRNA isolation, cDNA synthesis, cloning Sequencing and Primer design and testing

Poly (A)+ RNA isolations was carried out according to manufacturer's instruction using DynaBeads (Dynal)

1st Strand cDNA synthesis was synthesised using Ready-to-go You Prime First beads (AP Biotech) and the NotI primer-adapter from the Superscript Choice System (Invitrogen)

2nd Strand cDNA synthesis was synthesized according to standard protocols. cDNa fragments above 500 bp were excised from an agarose gel, ligated into the pSport 1 vector (Invitrogen) and used to transform electroMax DH10B cells (Invitrogen)

Transformed colonies were used to inoculate 96 – well plates containing 1 mL per well of 2x Luria-Bertani (LB) and 100 µg/mLAmpicillin and grown for 24 hrs at 37oC & 250 rpm

Bacteria was harvested by centrifugation at 3000 rpm for 5 min and plasmid DNA were prepared using the Multiscreen Plasmid Minipreparation system (Millipore)

Plasmid DNA 3 µL was sequenced using M13 Forward Primer and Big Dye Terminator version 3.1 chemistry (Applied Biosystems) and analyzed on the ABI 3730

Homologue searches were performed using BLAST against non redundant databases (blastn and blastx). Blastn searches were also made against dbEST, and SSRs were identified using the SPUTNIK program

Primers were designed to SSRs of ≥11 bps using PRIMER 3. SSRs≤20 bps have been found to be polymorphic in other plant species

For each of the primers designed, the left primer was end-labelled with γ[33P] and 16 individuals of Allanblackia representing the 7 species and 3 countries were amplified by touchdown PCR.

A typical 10 µL reaction contained 25 ng DNA, 1.0µM each primer, 200 µM dNTPs, 1x PCR buffer and 1 unit Taq Polymerase (Roche)

mRNA isolation, cDNA synthesis, cloning Sequencing and Primer design and testing II

EST-SSR identification and analysis of Allanblackia samplesPCR was performed as follows 5 min at 94oC; 7 cycles of 30 s at 94oC, 30 s at 65oC, and 30 s at 72oC decreasing to 58oC at 1oC/cycle, followed by 25 cycles of 30 s at 94oC, 30 s at 58oC and 30 s at 72oC, followed by 7 min at 72oC

Products were resolved on 6% Acrylamide gels that were dried and autoradiographed.

For primers that yielded single locus, polymorphic products, the left primer of each was fluorescently labelled with FAM and used to amplify the microsatellite loci from the populations. PCR products were analysed on 4% polyacrylamide gels using an ABI 3730 and GeneScan™ Rox 500 as an internal size standard.

Samples were analysed using GENEMAPPER v3.7 and tables with Alleles and their sizes were exported to Excel. Data was analysed for heterozygosity and genetic diversity using GENSTAT v9.0 and Microsatelite toolkit

RESULTS AND PROBLEMS ENCOUNTERED WITH THE EST-SSR DEVELOPEMENT•Results from Blast searches showed that 43.45% homology was to bacteria. However, one EST-SSR was obtained which was polymorphic across species. We did not pursue this one because it was not consistent but apparently may need annealing at 55oC

•Dilution of the ligation did not increase the transformation efficiency

•Using a new kit of Dynabeads and Invitrogen Superscript did not change this

•Increasing the amount of mRNA during the first strand cDNA synthesis didn’t change this ratio either

•When the procedure was repeated with Barley leaves we noticed that Barley maintained a 260/230 ratio of above 1.8 and 260/ 280 ratio of above 1.8 while for Allanblackia the 260/230 ratios varied and some were as low as below 0.5•Carrying out a Phenol:Chloroform:IAA extraction on the balance of the RNA however did not improve the result

•We thus resolved to develop a Genomic Library and search SSRs due to the recalcitrant nature of the genus

GENOMIC LIBRARY CONSTRUCTION ON ALLANBLACKIA I

•Genomic library was constructed from genomic DNA isolated from seedling leaves stored in RNAlater

•DNA (80 µg) was digested overnight at 65oC with Tsp509 (New England Biolabs, Inc) in a volume of 500 µL and purified using a Micron YM-50 column (Millipore)

•Purified DNA was size fractionated on a 2% agarose gel and DNA between 200 – 700 bp was excised and purified using MinElute Gel extraction Qiagen columns

•Tsp509 specific adaptors were ligated to 2 µL of the digested DNA in a 50 µL reaction

•10 PCR reactions were set up each 50 µL using 5 µL of DNA•PCR reactions were combined to 200 µL each purified on the MinElutecolumn and eluted in 25 µL

•Hybond N+ membrane carrying the oligonucleotides [CA]15, [GA]15, [AAG]8 and [ATG]8 were prepared and used to enrich the denatured PCR products by hybridisation

GENOMIC LIBRARY CONSTRUCTION ON ALLANBLACKIA II•Enriched DNA was eluted & Purified using the MinElute columns and subjected to a second round of PCR as before.

•Amplification was confirmed by gel electrophoresis and the five PCRS were combined, purified and elute in 25 µL sterile distilled water

•Enriched DNA (2 µL) was cloned into the pGEM-T Easy vector (Promega) and 1 µL was used to transform Electromax DH10B cells (Invitrogen). Transformed colonies were picked by the Genetics robot colony Picker into 16 384-well plates containing freezing medium with 100 µg/mL ampicillin.

•These plates were grown at 37oC for 24 hr, replicated and stored at -70oC. Aliquots (5µL were used to inoculate 96 – well plates containing 1 mL per well of 2 x Luria Bertani and the process was carried out as for the EST-SSR library.

Results from the genomic library construction

•1344 clones from the genomic library have been sequenced so far of which 1133 were good quality sequences. Sequences were organized into 92 contigs and 302 singletons

•59 SSRs have been identified

•45 primer pairs have been designed by PRIMER 3

•4 primers were found to be species or region specific

•7 primers were found to be polymorphic across species and individuals and these have been fluorescently labelled and will be used to assess the 139 individuals

Autoradiograph showing some of the alleles & CKSSR13 primer testing

45 primers designed from the 59 SSRs identified

Primer name Origin & motif PRODUCT SIZE P33 analysis

ckssr1 Contig 4_(TC)5 200 Polymorphic bands at 214, 215, 216, 218, 219, 220, 223, 222, 223, 224, 225

ckssr9 ABSGEN20_(AGT)6 202 Polymorphic band sizes range between 203 and 212

7 PRIMERS THAT CAN BE USED TO GENOTYPE THE WHOLE RANGE OF SAMPLES

ckssr19 ABSGEN16_(TTTTA2) 249 Didn't amplify very well with P33 but has a triplet band at 251 - 254 in the tanzanian region that also picks up Gabonensis. The Floribunda is at 255 -258

CKSSR27 ABSGEN609_(TCATC)2 189 Single product possibly monomorphicbut no amplification in Ghana & Bangangte

CKSSR38 ABSGen1005_(AG)20 201 Polymorphic across Tanzanian species

CKSSR39 ABSGen1020_(TC)10 191 Single product polymorphic across

all speciesCKSSR43 ABSGen1338_(TTTCC)2 178 Single product polymorphic across

all species

45 primers designed from the 59 SSRs identifiedPrimer name Origin & motif PRODUCT SIZE P33 analysisckssr11 ABSGEN252_(AG)6 181 6 bands Monomorpic acrss

species at 181. Cameroonian species have and extra triplet at 193

4 PRIMERS THAT MAY BE SPECIES SPECIFIC

ckssr13 ABSGEN324_(TGG)5 237 Triplet band across Tanzanian region that picks up Floribunda at 241

ckssr14 ABSGEN324_(GTG)5 191 Didn't amplify well and gel's a bit fuzzy but seems to a be a single band at 195 that picks the Tanzanian region

ckssr18 ABSGEN161_(CAG4) 247 Monomorphic double band at 251 in Tanzanian region that picks up Gabonensis

ckssr3 contig 7_(AC)5 234 Multiple bands 20 PRIMERS THAT HAVE MULTIPLE BANDS

ckssr4 contig 7_(CTA)4 248 Monomorphic multiple double bands for Tanzania. Amplifies in Parviflora & Floribunda but with a different pattern does not pick Gabonensis

ckssr5 Contig 12_(TG)6 250 Multiple bands

ckssr7 Contig 14_(TG)6 205 Has multiple bands at 210 and 260 which amplify across countries and show polymorphisms

45 primers designed from the 59 SSRs identifiedPrimer name Origin & motif PRODUCT SIZE P33 analysisckssr10 ABSGEN236_(GA)5 245 Monomorphic double band at 249.

ghan and Cameroon have extra doble bands at 259 & 267

20 PRIMERS THAT HAVE MULTIPLE BANDS

CKSSR23 CONTIG 2_(CA)6 209 Multiple products

CKSSR24 CONTIG 17_(AAC)5 217 Multiple products

CKSSR25 CONTIG 21_(GA)6 232 Multiple products

CKSSR26 CONTIG 49_(CT)6 227 Multiple products

CKSSR28 ABSGEN647_(AG)7153

Multiple products lower than expected product

CKSSR29 CONTIG 11_(TG)6 236 Multiple products

CKSSR30 CONTIG 13_(TC)6 213 Multiple products

CKSSR32 CONTIG 18_(CA)5 237 Multiple products

CKSSR33 CONTIG 21_(TG)7 248 Multiple products

CKSSR34 CONTIG 24_(TG)6 191 Multiple products

CKSSR35 CONTIG 24_(TG)6 205 Multiple products

CKSSR36 CONTIG 69_(CA)5 229 Multiple products

CKSSR37 CONTIG 70_(AAC)4 208 Multiple products

CKSSR40 ABSGen1035_(CT)8 173 Multiple products

CKSSR41 ABSGen1038_(TG)6 192 Multiple products

45 primers designed from the 59 SSRs identifiedPrimer name Origin & motif PRODUCT SIZE P33 analysisckssr2 Contig 5_(CA)5 195 didn't work at Annealing 58 on

Agarose10 PRIMERS THAT MAY NEEDMORE ADJUSTING OF ANNEALING

ckssr6 Contig 13_(TGT)4 221 No amplification at 58. Tanzania specific smeared bands at 55 on Agarose

TEMPERATURE

ckssr8 Contig 15_(CA)6 177 A smeared product at 55

ckssr12 ABSGEN308_(GA)19 232 Appeared as a smear though had amplifeid well on Agaros

ckssr15 ABSGEN408_(CA)6 166 No product

ckssr16 ABSGEN416_(AC)6 246 No product

ckssr17 ABSGEN416_(AC)12 175 No product

ckssr20 ABSGEN180_(GT5) 165 No Product on Agarose

ckssr21 ABSGEN180_(AG13) 250 No product on Agarose

CKSSR42 ABSGen1071_(TC)6 202 No product

CKSSR44 ABSGen1338_(TC)11 250 This primer includes CKSSR43 SSR site

3 PRIMERS WHOSE RESULT NOT KNOWN YET

CKSSR45 ABSGen1338_(CTTTT)3 221

CKSSR31 CONTIG 17_(GT)5 184

A scatter plot showing Allanblackia Populations and species relationships based on 2 Alleles ckssr1 and ckssr9

CKSSR1 & 9

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-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8

AMANIASBANGANGTEAGGHANAAPMANYANGUASMAZUMBAIASMUFINDIASSANGMELIMAAFSANGMELIMAAGULUGURUAUYALPENDAAFYALPENDAAS

Allele frequencies for all populations by locusLocus Populations..... Populations.....

CKSSR1A Amani Bangangte Ghana Manyangu Mazumbai Mufindi SangmelimaSGF SangmelimaSGG Uluguru YalpendaAF YalpendaAS Global213 100.00 15.38219 50.00 50.00 50.00 50.00 50.00 24.23221 50.00 50.00 50.00 50.00 50.00 24.23223 80.00 26.67 100.00 10.00225 20.00 73.33 100.00 100.00 26.15

CKSSR9A Amani Bangangte Ghana Manyangu Mazumbai Mufindi SangmelimaSGF SangmelimaSGG Uluguru YalpendaAF YalpendaAS Global202 41.67 10.00 38.89 100.00 55.00 16.67 50.00 26.36205 41.67 30.00 97.50 61.11 45.00 36.67 50.00 62.50 66.67 52.73208 16.67 55.00 2.50 36.67 100.00 33.33 27.78 18.18211 5.00 10.00 4.17 5.56 2.73

Population StatisticsPopulation Country and species Sample size Loci typed Unbiased Hz Obs Hz No AllelesAmani Tanzania AS 15 2 0.6009 0.5833 2.50Bangangte Cameroon AG 12 2 0.4816 0.2000 3.00Ghana Ghana AP 20 2 0.0250 0.0250 1.50Manyangu Tanzania AS 15 2 0.5103 0.5556 2.00Mazumbai Tanzania AS 15 2 0.2586 0.5000 1.50Mufindi Tanzania AS 11 2 0.5224 0.6500 2.00SangmelimaSGF Cameroon AF 15 2 0.5609 0.2667 3.00SangmelimaSGG Cameroon AG 3 2 0.0000 0.0000 1.00Uluguru Tanzania AU 9 2 0.5314 0.5000 2.00YalpendaAF Cameroon AF 12 2 0.2591 0.1667 2.00YalpendaAS Cameroon AS 12 2 0.2516 0.3333 2.00

Population Sample size Loci typed Unbiased Hz Obs Hz No AllelesGlobal 139 2 0.7025 0.3696 4.50

Future work and application of findings

SSRs will be used to assess the genetic structure and diversity of the seedlings currently being raised in the nurseries in Tanzania, Ghana and Cameroon.

SSRs will be shared with collaborators to establish mating system ofthe species in Tanzania

From publications of current work similar work could be initiated on other agroforestry species in collaboration with the BECA facility in Nairobi

A Principle coordinate analysis of 552 AFLP markers on 140 Allanblackia individuals showing the clustering relationship by country of origin

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First Principle coordinate (16% of variation)

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GhanaTanzania

A Principle coordinate analysis of 552 AFLP markers on 140 Allanblackia individuals showing the clustering relationship by species

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AFAGAPASASTAU

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A Principle coordinate analysis of 552 AFLP markers on 140 Allanblackia individuals showing the clustering relationship by Population

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AmaniBangangteGhanaManyanguMazumbaiMufindiSangmelimaAFSangmelimaAGUluguruYalpendaAFYalpendaASt

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138

Analysis of molecular variance (AMOVA) for 140 Allanblackia individuals sampled from three countries in Africa

---------------------------------------------------------------------- Source of MSD Variance % of P-Value variation d.f. components variation ---------------------------------------------------------------------- Among populations 10 209.22 13.55337 Va 26.11 Within populations 129 38.35 38.35391 Vb 73.89 b. Structured by country Among groups 2 569.24 9.88272 Va 17.94 Among populations within groups 8 119.22 6.86223 Vb 12.45 Within populations 129 38.35 38.35391 Vc 69.61 c. Structured by Species Among groups 5 323.15 10.56903 Va 19.73 Among populations within groups 5 95.3 4.65861 Vb 8.69 Within populations 129 38.35 38.35391 Vc 71.58

Allanblackia leaf samples from 3 countries and seven species in Africa for assessment of genetic variation

Reference Population Name N Country H 1 Amani 15 Tanzania 0.1438 2 Bangangte 12 Cameroon 0.1292 3 Ghana 20 Ghana 0.1226 4 Manyangu 15 Tanzania 0.1023 5 Mazumbai 15 Tanzania 0.1458 6 Mufindi 11 Tanzania 0.0998 7 Sangmelima AF 15 Cameroon 0.1372 8 Sangmelima AG 4 Cameroon 0.0883 9 Uluguru 9 Tanzania 0.1454 10 Yalpenda AF 12 Cameroon 0.1005 11 Yalpenda AS 12 Cameroon 0.1375 GST= 0.2432

+-------- Amani +--1 ! +--------Mazumbai +------4 ! ! +--------Manyangu +-------------6 +--3 ! ! +--------Uluguru ! ! +--8 +-----------------Ghana ! ! ! ! +--------Sangmelima AF ! ! +------2 +------------9 +---------------5 +--------Yalpenda AST ! ! ! ! ! +---------------Yalpenda AF ! ! -10 ! +------------------Bangangte ! +---------------7 ! +------------------Sangmelima AG ! +-----------------------------------------------Mufindi

A phenogram based on AFLP genetic distances between 12 populations of Allanblackia sampled from three countries in Africa

OUTPUTS I

The following outputs were achieved by this study:

• Training has been obtained in Isolation of RNA from tree material and in construction of cDNA libraries

• Exposure to capillary sequencing and bioinformatics, Designing of Primers, identification of appropriate EST-SSR markers for analysis of genetic diversity, Preliminary/pilot studies and associated dataanalysis.

From the preliminary/pilot studies the following outputs will be achieved:

(i) an indication of the relationship among species (ii) the likely broad impact of harvesting from natural stands on

cultivation and conservation strategies(iii) issues relating to sympatric distributions of species will be attained.(iv)This will also assist in resolving taxonomic confusion in the genus.

OUTPUTS IIv. from a preliminary study comparing A. parviflora sampled from

Ghana, (material currently in nurseries that will subsequently be distributed to small-holder producers for on-farm cultivation compared with germplasm originally obtained from natural stands), prospects for initial bottlenecks during on-farm cultivation in Ghana

The possible implications of harvesting from natural stands on their survival will be attained.

A methodology will be developed to enable the further testing and determination of populations suitable for on-farm introduction and conservation.

vi. from an initial assessment of Allanblackia material currently undergoing early propagation trials in Cameroon an indication ofthe potential dangers of vegetative propagation on effective population sizes entering cultivation will be obtained.

vii.This will allow development of strategies for the collection andmultiplication of an appropriate range of propagule source plants to minimise these risks.

ACKNOWLEDGEMENTS

I would like to express my gratitude to

•Rothamsted International for funding this project

•ICRAF for supporting me in taking this venture. In particurlar, Tony Simons for his Support. Ramni Jamnadass and Ian Dawson for the inception of the ideas and proposal development. Ramni for recommending me for the project. Samuel Lemurt for assistance with DNA extraction and logistics of sending material to SCRI. Lucy Mwaura and Moses Munjunga for assistance during sample collections. Hilary Kipruto for statistical analysis

•National partners and collaborators for assistance in sample collections

•Joanne Russell and Mary Woodhead from SCRI who supervised and guided me through the practical part of work

•Ingo Hein, Irene Tierney, Jodie Comodran, Linzi Jorgensen and Pete Hedley who were absolutely patient with all the questions I asked

•The entire genetics programme team who very kindly pointed me in the right direction

•Other members of SCRI staff eg. IT, sequencing unit, Bioinformatics and Ursula in the Library, stores, caretakers