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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 SUPERVISORS
IAN DAWSON – CONSULTANT ON NOVELLA PROJECT
RAMNI 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 Africa
East Africa
Latin America
Southern Africa
South Asia
South East Asia
BACKGROUND INFORMATION II
•    4 Cross cutting themes
1.   Land and people
2.   Trees and Markets
3.   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 programmes
  collected 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 IV
 ALLANBLACKIA
• 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 Stearic
                             acids 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 V
 ALLANBLACKIA
• The partners will help and encourage local communities and small
  businesses 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 options
   the development of policies and guidelines to promote sustainable
   harvesting
   Tree inventory and reproductive ecology studies
   Environmental impact assessments
   Development of market delivery structures and processing methods to
   bring product to the consumer.
BACKGROUND INFORMATION VI
ALLANBLACKIA      • 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
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 genetic
bottlenecks 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 sequence
repeat 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 their
popularity 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-taxa
amplification, 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 between
     populations 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              No. of individuals   No. of individuals
                                         Site                      sampled              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
A map showing the distribution of Allanblackia in 3 countries

                                                         Tun isia
                               Morocco

                                              Algeria
                                                                              Lib ya                  Egypt




                    a
                 ar
               ah
             .S
            W
                 Mauritania                  Mali
                                                           Niger
                                                                                                                           Eritrea
           Senegal                                                              Chad
                                                                                                      Sudan
                                   Bu rkina Faso                                                                                     Djibo uti
                     Gu inea
                                                        Nigeria                                                            Ethiopia




                                                                      oon
         Sierra Leone                  Gh ana                                 Cen tral African




                                                                  Cam er
                                      # ##
                                      #

                  Lib eria                                                    Repub lic
                                            ##
                                      ## # #
                                       ##
                                        ###
                                          #                   #

                                                                                                                                             a
                                                              #
                                                                                                                                          ali
                                                                                                                                      m
                                                                  #
                                                                  #



            A. parviflora                                                  Congo                           Ug an da
                                                                                                                      Ken ya       So
                                                              Gab on
A. floribunda, A. stanerana, A.                                                         Congo DR
                                                                                                                           #
                                                                                                                                          A. stuhlmannii, A.
                                                                                                           Tanzania
                                                                                                                       #   #


gabonensis                                                                                                                                ulugurensis, A.
                                                                                                                       #

                                                                                                                       #       #
                   Key
                                                                                                                  #

                               #   Species location point
                                   Country with study sites                   Angola
                                                                                                                                          sacleuxii
                                   Country boundary
                                                                                                 Zamb ia
                                   Lakes




                                                                                                                                                     ar
                                                                                                           Mozamb iqu e




                                                                                                                                                 asc
                                                                                                   Zim babwe




                                                                                                                                            dag
                                                                            Nam ib ia
                                                                                           Botswana




                                                                                                                                          Ma
                                                                                                           Swazilan d


                                                                                       South Africa
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
100 bp marker
                                                        Barley Qiagen
               Total RNA isolation




                                                                         Barley SDS

                                                                                      AB root
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
                                                                        TRI GITC/GHCL
• Qiagen Rneasy kit by Gehrig et al 2000     QIAGEN
                                             RLT/ RLC
• Tri reagent by Gehrig et al 2000




                                             BARLEY 1
                                             BARLEY 6
• Addition of HMW-Polyethylene Glycol




                                             ROOT




                                             ROOT
                                             LEAF


                                             LEAF



                                             LEAF


                                             LEAF
                                             SC



                                             SC


                                             SC



                                             SC
                                             SE



                                             SE


                                             SE



                                             SE
   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
NANODROP READINGS OF ISOLATED RNA
Sample ID   Date         Time    ng/ul      TOTAL AMOUNT OF µg RNA IN 98 µL   260/280       260/230



H20         22/03/2006   15:37      -0.17                                        -0.12         -1.63



ABPC 1      22/03/2006   15:38    429.17                 42.06                    1.98          1.76



ABSR 1      22/03/2006   15:39    644.38                 63.15                    2.13          2.26



ABSC 1      22/03/2006   15:41     522.1                 51.17                    1.91          2.07



ABSC 2      22/03/2006   15:41    587.01                 57.53                    1.83          1.98



ABPE 2      22/03/2006   15:43    193.67                 18.98                    2.06          1.15



ABPR        22/03/2006   15:44    145.97                 14.31                          2        1.9



ABPC 2      22/03/2006   15:44     246.3                 24.14                    2.03          1.79



ABPE 1      22/03/2006   15:46    689.51                 67.57                    2.08           1.9



ABSR 2      22/03/2006   15:47    835.95                 81.92                    2.07          2.01



ABSE        22/03/2006   15:48    809.44                 79.33                    2.07          2.04
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/mL
Ampicillin 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)
mRNA isolation, cDNA synthesis, cloning
Sequencing and Primer design and testing II
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)
EST-SSR identification and analysis of Allanblackia
samples
PCR 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 MinElute
column 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          7 PRIMERS THAT
                                                   between 203 and 212                   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
                                                   monomorphic
                                                   but no amplification in Ghana &
                                                   Bangangte
  CKSSR38      ABSGen1005_(AG)20          201      Polymorphic across Tanzanian
                                                   species

  CKSSR39      ABSGen1020_(TC)10          191      Single product polymorphic across

                                                   all species
  CKSSR43      ABSGen1338_(TTTCC)2        178      Single product polymorphic across

                                                   all species
45 primers designed from the 59 SSRs identified
     Primer name Origin & motif    PRODUCT SIZE P33 analysis
     ckssr11     ABSGEN252_(AG)6       181      6 bands Monomorpic acrss                 4 PRIMERS THAT
                                                species at 181. Cameroonian              MAY BE SPECIES
                                                species have and extra triplet at        SPECIFIC
                                                193

     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 identified
     Primer name Origin & motif    PRODUCT SIZE P33 analysis
     ckssr10     ABSGEN236_(GA)5       245      Monomorphic double band at 249. 20 PRIMERS THAT
                                                ghan and Cameroon have extra    HAVE MULTIPLE
                                                doble bands at 259 & 267        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)7                  Multiple products lower than
                                        153      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 identified
Primer name Origin & motif        PRODUCT SIZE P33 analysis
ckssr2      Contig 5_(CA)5            195      didn't work at Annealing 58 on       10 PRIMERS THAT
                                               Agarose                              MAY NEED
                                                                                    MORE
                                                                                    ADJUSTING OF
                                                                                    ANNEALING
ckssr6      Contig 13_(TGT)4           221       No amplification at 58. Tanzania   TEMPERATURE
                                                 specific smeared bands at 55 on
                                                 Agarose

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       3 PRIMERS
                                                 SSR site                           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

                               1.2

                                 1

                               0.8
                                                                         AMANIAS
                                                                         BANGANGTEAG
                               0.6
                                                                         GHANAAP
                               0.4                                       MANYANGUAS
                                                                         MAZUMBAIAS
                               0.2                                       MUFINDIAS
                                                                         SANGMELIMAAF
                                 0                                       SANGMELIMAAG
   -0.8   -0.6   -0.4   -0.2          0        0.2     0.4   0.6   0.8
                                                                         ULUGURUAU
                               -0.2
                                                                         YALPENDAAF
                                                                         YALPENDAAS
                               -0.4

                               -0.6

                               -0.8
Population Statistics
       Population         Country and species Sample size Loci typed Unbiased Hz    Obs Hz No Alleles
       Amani                 Tanzania AS         15            2       0.6009       0.5833   2.50
       Bangangte            Cameroon AG          12            2       0.4816       0.2000   3.00
       Ghana                  Ghana AP           20            2       0.0250       0.0250   1.50
       Manyangu              Tanzania AS         15            2       0.5103       0.5556   2.00
       Mazumbai              Tanzania AS         15            2       0.2586       0.5000   1.50
       Mufindi               Tanzania AS         11            2       0.5224       0.6500   2.00
       SangmelimaSGF        Cameroon AF          15            2       0.5609       0.2667   3.00
       SangmelimaSGG        Cameroon AG           3            2       0.0000       0.0000   1.00
       Uluguru               Tanzania AU          9            2       0.5314       0.5000   2.00
       YalpendaAF           Cameroon AF          12            2       0.2591       0.1667   2.00
       YalpendaAS           Cameroon AS          12            2       0.2516       0.3333   2.00

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




Allele frequencies for all populations by locus
 Locus  Populations.....                                                                                                       Populations.....
CKSSR1A    Amani         Bangangte Ghana Manyangu Mazumbai Mufindi SangmelimaSGF SangmelimaSGG Uluguru YalpendaAF YalpendaAS      Global
  213                              100.00                                                                                          15.38
  219       50.00                         50.00    50.00   50.00                                50.00                              24.23
  221       50.00                         50.00    50.00   50.00                                50.00                              24.23
  223                      80.00                                       26.67         100.00                                        10.00
  225                      20.00                                       73.33                             100.00     100.00         26.15

CKSSR9A      Amani     Bangangte Ghana Manyangu Mazumbai Mufindi SangmelimaSGF SangmelimaSGG Uluguru YalpendaAF YalpendaAS         Global
  202        41.67       10.00          38.89    100.00  55.00       16.67                    50.00                                26.36
  205        41.67       30.00   97.50  61.11            45.00       36.67                    50.00     62.50      66.67           52.73
  208        16.67       55.00    2.50                               36.67         100.00               33.33      27.78           18.18
  211                     5.00                                       10.00                               4.17       5.56            2.73
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 of
the 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

                                                                Cameroon
        Second Principle coordinate (14% of variation)          Ghana
                                                                Tanzania

                                                         0.2
                                                         0.0
                                                         -0.2
                                                         -0.4




                                                                 -0.4        -0.2         0.0          0.2          0.4

                                                                    First Principle coordinate (16% of variation)
A Principle coordinate analysis of 552 AFLP markers on 140 Allanblackia
individuals showing the clustering relationship by species

                                                            AF
    Second Principle coordinate (14% of variation)          AG
                                                            AP
                                                            AS
                                                     0.2    AST
                                                            AU
                                                     0.0




                                                                                              136

                                                                                                    137
                                                                                              135
                                                     -0.2




                                                                                                    138
                                                     -0.4




                                                             -0.4          -0.2         0.0               0.2     0.4

                                                                  First Principle coordinate (16% of variation)
A Principle coordinate analysis of 552 AFLP markers on 140 Allanblackia
individuals showing the clustering relationship by Population


     Second Principle coordinate (14% of variation)
                                                      0.2
                                                      0.0




                                                                                        136
                                                             Amani                             137
                                                                                              135
                                                             Bangangte                           135
                                                                                               137
                                                             Ghana
                                                      -0.2




                                                             Manyangu
                                                             Mazumbai
                                                             Mufindi
                                                             SangmelimaAF
                                                             SangmelimaAG                     138      138
                                                             Uluguru
                                                      -0.4




                                                             YalpendaAF
                                                             YalpendaASt




                                                              -0.4          -0.2       0.0              0.2     0.4
                                                                First Principle coordinate (16% of variation)
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
A phenogram based on AFLP genetic distances between 12 populations
of Allanblackia sampled from three countries in Africa


                                            +-------- 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
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 data
    analysis.

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 II
v. 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 of
    the potential dangers of vegetative propagation on effective
    population sizes entering cultivation will be obtained.

vii. This will allow development of strategies for the collection and
     multiplication 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

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Allanblackia In Agroforestry Systems Iv 9 Feb Caroline Kadu

  • 1. 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 SUPERVISORS IAN DAWSON – CONSULTANT ON NOVELLA PROJECT RAMNI JAMNADASS – ICRAF SUPERVISOR
  • 2. 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
  • 3. 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.
  • 4. 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.
  • 5. 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 Africa East Africa Latin America Southern Africa South Asia South East Asia
  • 6. BACKGROUND INFORMATION II • 4 Cross cutting themes 1. Land and people 2. Trees and Markets 3. 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 programmes collected and procured germplasm in both live and seed gene banks around the world.
  • 7. 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.
  • 8. 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.
  • 9. BACKGROUND INFORMATION IV ALLANBLACKIA • 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 Stearic acids are reported to lower plasma cholesterol levels thus reducing the risks of heart attack.
  • 11. 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
  • 12. BACKGROUND INFORMATION V ALLANBLACKIA • The partners will help and encourage local communities and small businesses 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 options the development of policies and guidelines to promote sustainable harvesting Tree inventory and reproductive ecology studies Environmental impact assessments Development of market delivery structures and processing methods to bring product to the consumer.
  • 13. BACKGROUND INFORMATION VI ALLANBLACKIA • 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
  • 14. 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 genetic bottlenecks 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
  • 15. Problem statement II •Another commonly used approach is that of assessments of the frequency and distribution of length variants at simple sequence repeat 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 their popularity 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.
  • 16. 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-taxa amplification, 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.
  • 17. 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.
  • 18. 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 between populations 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
  • 19. 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
  • 20. GEOGRAPHIC FOCUS Country Species Geographical No. of individuals No. of individuals Site sampled 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
  • 21. A map showing the distribution of Allanblackia in 3 countries Tun isia Morocco Algeria Lib ya Egypt a ar ah .S W Mauritania Mali Niger Eritrea Senegal Chad Sudan Bu rkina Faso Djibo uti Gu inea Nigeria Ethiopia oon Sierra Leone Gh ana Cen tral African Cam er # ## # Lib eria Repub lic ## ## # # ## ### # # a # ali m # # A. parviflora Congo Ug an da Ken ya So Gab on A. floribunda, A. stanerana, A. Congo DR # A. stuhlmannii, A. Tanzania # # gabonensis ulugurensis, A. # # # Key # # Species location point Country with study sites Angola sacleuxii Country boundary Zamb ia Lakes ar Mozamb iqu e asc Zim babwe dag Nam ib ia Botswana Ma Swazilan d South Africa
  • 22. 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
  • 23. 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
  • 24. 100 bp marker Barley Qiagen Total RNA isolation Barley SDS AB root 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 TRI GITC/GHCL • Qiagen Rneasy kit by Gehrig et al 2000 QIAGEN RLT/ RLC • Tri reagent by Gehrig et al 2000 BARLEY 1 BARLEY 6 • Addition of HMW-Polyethylene Glycol ROOT ROOT LEAF LEAF LEAF LEAF SC SC SC SC SE SE SE SE 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
  • 25. NANODROP READINGS OF ISOLATED RNA Sample ID Date Time ng/ul TOTAL AMOUNT OF µg RNA IN 98 µL 260/280 260/230 H20 22/03/2006 15:37 -0.17 -0.12 -1.63 ABPC 1 22/03/2006 15:38 429.17 42.06 1.98 1.76 ABSR 1 22/03/2006 15:39 644.38 63.15 2.13 2.26 ABSC 1 22/03/2006 15:41 522.1 51.17 1.91 2.07 ABSC 2 22/03/2006 15:41 587.01 57.53 1.83 1.98 ABPE 2 22/03/2006 15:43 193.67 18.98 2.06 1.15 ABPR 22/03/2006 15:44 145.97 14.31 2 1.9 ABPC 2 22/03/2006 15:44 246.3 24.14 2.03 1.79 ABPE 1 22/03/2006 15:46 689.51 67.57 2.08 1.9 ABSR 2 22/03/2006 15:47 835.95 81.92 2.07 2.01 ABSE 22/03/2006 15:48 809.44 79.33 2.07 2.04
  • 26. 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
  • 27. 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/mL Ampicillin 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)
  • 28. mRNA isolation, cDNA synthesis, cloning Sequencing and Primer design and testing II 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)
  • 29. EST-SSR identification and analysis of Allanblackia samples PCR 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
  • 30. 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
  • 31. 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 MinElute column 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
  • 32. 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.
  • 33. 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
  • 34. Autoradiograph showing some of the alleles & CKSSR13 primer testing
  • 35. 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 7 PRIMERS THAT between 203 and 212 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 monomorphic but no amplification in Ghana & Bangangte CKSSR38 ABSGen1005_(AG)20 201 Polymorphic across Tanzanian species CKSSR39 ABSGen1020_(TC)10 191 Single product polymorphic across all species CKSSR43 ABSGen1338_(TTTCC)2 178 Single product polymorphic across all species
  • 36. 45 primers designed from the 59 SSRs identified Primer name Origin & motif PRODUCT SIZE P33 analysis ckssr11 ABSGEN252_(AG)6 181 6 bands Monomorpic acrss 4 PRIMERS THAT species at 181. Cameroonian MAY BE SPECIES species have and extra triplet at SPECIFIC 193 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
  • 37. 45 primers designed from the 59 SSRs identified Primer name Origin & motif PRODUCT SIZE P33 analysis ckssr10 ABSGEN236_(GA)5 245 Monomorphic double band at 249. 20 PRIMERS THAT ghan and Cameroon have extra HAVE MULTIPLE doble bands at 259 & 267 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)7 Multiple products lower than 153 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
  • 38. 45 primers designed from the 59 SSRs identified Primer name Origin & motif PRODUCT SIZE P33 analysis ckssr2 Contig 5_(CA)5 195 didn't work at Annealing 58 on 10 PRIMERS THAT Agarose MAY NEED MORE ADJUSTING OF ANNEALING ckssr6 Contig 13_(TGT)4 221 No amplification at 58. Tanzania TEMPERATURE specific smeared bands at 55 on Agarose 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 3 PRIMERS SSR site WHOSE RESULT NOT KNOWN YET CKSSR45 ABSGen1338_(CTTTT)3 221 CKSSR31 CONTIG 17_(GT)5 184
  • 39. A scatter plot showing Allanblackia Populations and species relationships based on 2 Alleles ckssr1 and ckssr9 CKSSR1 & 9 1.2 1 0.8 AMANIAS BANGANGTEAG 0.6 GHANAAP 0.4 MANYANGUAS MAZUMBAIAS 0.2 MUFINDIAS SANGMELIMAAF 0 SANGMELIMAAG -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 ULUGURUAU -0.2 YALPENDAAF YALPENDAAS -0.4 -0.6 -0.8
  • 40. Population Statistics Population Country and species Sample size Loci typed Unbiased Hz Obs Hz No Alleles Amani Tanzania AS 15 2 0.6009 0.5833 2.50 Bangangte Cameroon AG 12 2 0.4816 0.2000 3.00 Ghana Ghana AP 20 2 0.0250 0.0250 1.50 Manyangu Tanzania AS 15 2 0.5103 0.5556 2.00 Mazumbai Tanzania AS 15 2 0.2586 0.5000 1.50 Mufindi Tanzania AS 11 2 0.5224 0.6500 2.00 SangmelimaSGF Cameroon AF 15 2 0.5609 0.2667 3.00 SangmelimaSGG Cameroon AG 3 2 0.0000 0.0000 1.00 Uluguru Tanzania AU 9 2 0.5314 0.5000 2.00 YalpendaAF Cameroon AF 12 2 0.2591 0.1667 2.00 YalpendaAS Cameroon AS 12 2 0.2516 0.3333 2.00 Population Sample size Loci typed Unbiased Hz Obs Hz No Alleles Global 139 2 0.7025 0.3696 4.50 Allele frequencies for all populations by locus Locus Populations..... Populations..... CKSSR1A Amani Bangangte Ghana Manyangu Mazumbai Mufindi SangmelimaSGF SangmelimaSGG Uluguru YalpendaAF YalpendaAS Global 213 100.00 15.38 219 50.00 50.00 50.00 50.00 50.00 24.23 221 50.00 50.00 50.00 50.00 50.00 24.23 223 80.00 26.67 100.00 10.00 225 20.00 73.33 100.00 100.00 26.15 CKSSR9A Amani Bangangte Ghana Manyangu Mazumbai Mufindi SangmelimaSGF SangmelimaSGG Uluguru YalpendaAF YalpendaAS Global 202 41.67 10.00 38.89 100.00 55.00 16.67 50.00 26.36 205 41.67 30.00 97.50 61.11 45.00 36.67 50.00 62.50 66.67 52.73 208 16.67 55.00 2.50 36.67 100.00 33.33 27.78 18.18 211 5.00 10.00 4.17 5.56 2.73
  • 41. 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 of the 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
  • 42. A Principle coordinate analysis of 552 AFLP markers on 140 Allanblackia individuals showing the clustering relationship by country of origin Cameroon Second Principle coordinate (14% of variation) Ghana Tanzania 0.2 0.0 -0.2 -0.4 -0.4 -0.2 0.0 0.2 0.4 First Principle coordinate (16% of variation)
  • 43. A Principle coordinate analysis of 552 AFLP markers on 140 Allanblackia individuals showing the clustering relationship by species AF Second Principle coordinate (14% of variation) AG AP AS 0.2 AST AU 0.0 136 137 135 -0.2 138 -0.4 -0.4 -0.2 0.0 0.2 0.4 First Principle coordinate (16% of variation)
  • 44. A Principle coordinate analysis of 552 AFLP markers on 140 Allanblackia individuals showing the clustering relationship by Population Second Principle coordinate (14% of variation) 0.2 0.0 136 Amani 137 135 Bangangte 135 137 Ghana -0.2 Manyangu Mazumbai Mufindi SangmelimaAF SangmelimaAG 138 138 Uluguru -0.4 YalpendaAF YalpendaASt -0.4 -0.2 0.0 0.2 0.4 First Principle coordinate (16% of variation)
  • 45. 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
  • 46. 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
  • 47. A phenogram based on AFLP genetic distances between 12 populations of Allanblackia sampled from three countries in Africa +-------- 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
  • 48. 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 data analysis. 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.
  • 49. OUTPUTS II v. 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 of the potential dangers of vegetative propagation on effective population sizes entering cultivation will be obtained. vii. This will allow development of strategies for the collection and multiplication of an appropriate range of propagule source plants to minimise these risks.
  • 50. 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