The document discusses developing molecular tools to manage Allanblackia, an African tree crop. [1] Objectives are to develop EST-SSR markers to study genetic variation within and between Allanblackia populations. [2] Activities include collecting samples, extracting DNA/RNA, cloning cDNA, identifying EST-SSRs, and genotyping samples. [3] The study focuses on populations in Cameroon, Ghana, and Tanzania to monitor genetic bottlenecks and resolve taxonomic confusion in the genus.
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Exploiting untapped potential of roots,tubers and bananas to improve nutrition and food security,increase income generation,foster improved livelihoods for women, youth, children &
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The Global Plan of Action for the conservation, sustainable use and development of forest genetic resources provides a framework for countries, regional bodies and international organizations to highlight and respond to the serious challenges as well as the opportunities facing forest genetic resources.
This plan is based on the first ever State of the World Forest Genetic Resources Report, which over 80 countries have contributed to. Learn more: http://www.bioversityinternational.org/research-portfolio/forest-tree-genetic-diversity/capacity-and-awareness/
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B4FA 2012 Ghana: Plant Genetic Resources in Ghana - Lawrence Aboagyeb4fa
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www.b4fa.org
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This plan is based on the first ever State of the World Forest Genetic Resources Report, which over 80 countries have contributed to. Learn more: http://www.bioversityinternational.org/research-portfolio/forest-tree-genetic-diversity/capacity-and-awareness/
The climate-smart village : a model developed by CCAFS program to improve the adaptive capacity of communities
Presented by Dr Jules Bayala, World Agroforestry Centre at Africa Agriculture Science Week 6, 15 July 2013, Accra, Ghana. http://ccafs.cgiar.org/events/15/jul/2013/africa-agriculture-science-week-2013
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Each “growing degree day” spent at a temperature above 30°C decreases yields by 1 percent under optimal (drought-free) rainfed conditions.Southern Africa faces the risk of more severe and protracted droughts and periods of extremely low and extremely high rainfall could become more common as temperatures increase from 2-4°C
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Sustainable management actions are needed for several indigenous agro forestry plant species like the black plum (Vitex doniana Sweet) because they are facing increasing pressures due to the rapid human growth and threats such as climate change. By combining species distribution modelling using the Maximum Entropy Algorithm (Max Ent) and representation gap analysis, this study accessed the impacts of current and future (2050) climates on the potential distribution of Vitex doniana in Benin with insight on the protected areas network (PAN). The model showed a high goodness-of-fit (AUC = 0.92 ± 0.02) and a very good predictive power (TSS = 0.72 ± 0.01). Our findings indicated annual mean rainfall, annual mean diurnal range of temperature and mean temperature of the driest quarter as the most important predictors driving the distribution of V. doniana. Under current climate, about 85 % of Benin area is potentially suitable for its cultivation. This potential suitable area is projected to increase by 3 to 12 % under future climatic conditions. A large proportion (76.28 %) of the national PAN was reported as potentially suitable for the conservation of the species under current climate with increase projections of 14 to 23 % under future climate. The study showed that V. doniana can be cultivated in several areas of Benin and that the PAN is potentially suitable for its conservation. These findings highlighted some of the opportunities of integrating V. doniana in the formal production systems of Benin and also its potentialities in ecosystems restoration under the changing climate. Get the full articles at: http://www.innspub.net/ijaar/climate-and-potential-habitat-suitability-for-cultivation-and-in-situ-conservation-of-the-black-plum-vitex-doniana-sweet-in-benin-west-africa/
GENOMIC AND TRANSCRIPTOMIC APPROACHES TOWARDS THE GENETIC IMPROVEMENT OF AN U...Faraz Khan
With the world population estimated to be nine billion by 2050, the need to exploit plant genetic diversity in order to increase and diversify global food supply, and minimise the over-reliance for food on a few staple crops is of the utmost importance. Bambara groundnut (Vigna subterranea (L) Verdc.), is underutilised legume indigenous to Africa, rich in carbohydrates, with reasonable amounts of protein. It is known to be drought tolerant, able to
grow on marginal lands where other major crops cannot with minimal rainfall (<700 mm) and chemical inputs. Crop improvement for abiotic stress tolerance and increasing/stabilising yield have been difficult to achieve due to the complex nature of these stresses, and the genotype x environment interaction (GxE). This review paper highlights how a number of recent technologies and approaches used for major crop research, can be translated
into use in research of minor crops, using bambara groundnut as an exemplar species. Using drought tolerance as a trait of interest in this crop, we will demonstrate how limitations can affect genomic approaches for understanding traits in bambara groundnut, and, how genomic and transcriptomic methodologies developed for major crops can be applied to underutilised crops for better understanding of the genetics governing important agronomic traits. Furthermore, such approaches will allow for cross species comparison between major and minor crops, exemplified by bambara groundnut leading to improved research in such crops. This will lead to a better understanding of the
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Analysis of genetic diversity of the African Locust Bean (Parkia biglobosa) to improve its strategy of conservation and breeding in the face of climate change
’Mainstreaming agrobiodiversity conservation and use in Sri Lankan agro-ecosy...Janathakshan Gte Ltd
Presentation by Ministry of Mahaweli Development and Environment
Technical Session 02: Climate Change Adaptation
Experience Sharing Forum on Climate Smart Initiatives of Sri Lanka
Sri Lanka Next – A blue Green Era – Conference and Exhibition 2017
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Each “growing degree day” spent at a temperature above 30°C decreases yields by 1 percent under optimal (drought-free) rainfed conditions.Southern Africa faces the risk of more severe and protracted droughts and periods of extremely low and extremely high rainfall could become more common as temperatures increase from 2-4°C
Diversity, Sustainability and Resilience in Natural Resource Management in Af...SIANI
This study was presented during the conference “Production and Carbon Dynamics in Sustainable Agricultural and Forest Systems in Africa” held in September, 2010.
ICRISAT newsletter - Happenings, featured 2 stories from TL III workshops held in Nairobi, Kenya. 1. TL III Monitoring Learning and Evaluation workshop and TL III Genetic Gains - Program Improvement Plan. Read the happenings document for detailed deliberations and way forward from both the workshops.
Presentation on the state of the art for the ASSETS project for the inception workshop in Southampton, May 21st 2012. Project is supported by the ESPA program, with funding coming from DFID and NERC.
A presentation of the head of the National Centre for Plant Genetic Resources: Polish Genebank concerning plant genetic resources conservation in Poland.
Global Information Systems for Plant Genetic Resources (2009)Dag Endresen
Global information systems for plant genetic resources. For the Caucasus germplasm network training course at the Nordic Genetic Resource Center (NordGen), Alnarp Sweden 29th January 2009.
Climate and potential habitat suitability for cultivation and in situ conserv...Innspub Net
Sustainable management actions are needed for several indigenous agro forestry plant species like the black plum (Vitex doniana Sweet) because they are facing increasing pressures due to the rapid human growth and threats such as climate change. By combining species distribution modelling using the Maximum Entropy Algorithm (Max Ent) and representation gap analysis, this study accessed the impacts of current and future (2050) climates on the potential distribution of Vitex doniana in Benin with insight on the protected areas network (PAN). The model showed a high goodness-of-fit (AUC = 0.92 ± 0.02) and a very good predictive power (TSS = 0.72 ± 0.01). Our findings indicated annual mean rainfall, annual mean diurnal range of temperature and mean temperature of the driest quarter as the most important predictors driving the distribution of V. doniana. Under current climate, about 85 % of Benin area is potentially suitable for its cultivation. This potential suitable area is projected to increase by 3 to 12 % under future climatic conditions. A large proportion (76.28 %) of the national PAN was reported as potentially suitable for the conservation of the species under current climate with increase projections of 14 to 23 % under future climate. The study showed that V. doniana can be cultivated in several areas of Benin and that the PAN is potentially suitable for its conservation. These findings highlighted some of the opportunities of integrating V. doniana in the formal production systems of Benin and also its potentialities in ecosystems restoration under the changing climate. Get the full articles at: http://www.innspub.net/ijaar/climate-and-potential-habitat-suitability-for-cultivation-and-in-situ-conservation-of-the-black-plum-vitex-doniana-sweet-in-benin-west-africa/
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With the world population estimated to be nine billion by 2050, the need to exploit plant genetic diversity in order to increase and diversify global food supply, and minimise the over-reliance for food on a few staple crops is of the utmost importance. Bambara groundnut (Vigna subterranea (L) Verdc.), is underutilised legume indigenous to Africa, rich in carbohydrates, with reasonable amounts of protein. It is known to be drought tolerant, able to
grow on marginal lands where other major crops cannot with minimal rainfall (<700 mm) and chemical inputs. Crop improvement for abiotic stress tolerance and increasing/stabilising yield have been difficult to achieve due to the complex nature of these stresses, and the genotype x environment interaction (GxE). This review paper highlights how a number of recent technologies and approaches used for major crop research, can be translated
into use in research of minor crops, using bambara groundnut as an exemplar species. Using drought tolerance as a trait of interest in this crop, we will demonstrate how limitations can affect genomic approaches for understanding traits in bambara groundnut, and, how genomic and transcriptomic methodologies developed for major crops can be applied to underutilised crops for better understanding of the genetics governing important agronomic traits. Furthermore, such approaches will allow for cross species comparison between major and minor crops, exemplified by bambara groundnut leading to improved research in such crops. This will lead to a better understanding of the
role of stress-responsive genes and drought adaptation in this underutilised legume.
In situ Conservation of Crop Wild Relatives in SADC Region – towards a region...ExternalEvents
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Presentation given by Bioversity International scientist Ehsan Dulloo at the side event 'Strengthening the in situ conservation of plant genetic resources for food and agriculture including Crop Wild Relatives in Protected Area Networks' during the 6th Meeting of the International Treaty on Plant Genetic Resources for Food and Agriculture Governing Body, Rome, Italy, 5-9 October 2015.
Bioversity International is leading an initiative on the in situ conservation and use of crop wild relatives in South Africa, Mauritius and Zambia. This three-year project is supported by the Secretariat of the African, Caribbean and Pacific (ACP) Group of States through its ACP-EU Co-operation Programme in Science and Technology (S&T II) Its overall objective is to enhance link between conservation and use of crop wild relatives as a means of underpinning regional food security and mitigating the predicted adverse impact of climate change.
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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
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
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