Why to assess genetic diversity? How IITA does it: Morphological characterization, Molecular characterization,Cytogenetic characterization.

1. Characterization of Genetic Diversity of Conserved Germplasm
A major component of IITA’s Research Portfolio
Outline
1. Why to assess genetic diversity?
2. How IITA does it?
3. Case studies
Crops:
- Yam
- Cowpea
- Bananas (Banana & Plantain)
- Bambara groundnut
- Maize
- African yam bean
By Irie Vroh Bi

2. 1. Why to assess genetic diversity?
-From domestication to selection to improvement, farmers have relied on
genetic variation for thousands of years.
-Genetic diversity gives species the ability to adapt to changing environments,
including new pests, new diseases and new climatic conditions.
-Efficient conservation of germplasm depends on the understanding
of the available diversity.
-Efficient utilization of germplasm by breeders depends on the understanding
of the extent, nature and structure of the available genetic diversity.
Conservation of a diversity in crop species, varieties and wild relatives
and efficient utilization are key to our survival
Need to assess and understand crop genetic diversity

3. 2. How IITA does it?
1. Morphological characterization: morphological and agronomical descriptors
2. Molecular characterization: Molecular markers (Biotech Ibadan & BECA, Nairobi)
-Simple Sequence Repeat (SSR)
-Amplified Fragment Length Polymorphism (AFLP)
-Single Nucleotide Polymorphism (SNP)
-Diversity Array Technology (DArT)
-IITA uses available DNA markers
-IITA in collaboration with partners participates in
developing new molecular markers
-Molecular analyses are performed in house or outsourced
3. Cytogenetic characterization : ploidy analysis

4. Morphological characterization
Bambara groundnut: 391 accessions
characterized using 39 descriptors
Bananas: ~200 accessions
characterized with 50 descriptors
African yam bean: 152 accessions
Characterized using 62 descriptors

5. Germplasm characterization
Crops Number Characterization
descriptors
Cowpea 46 Completed
Core/Minicore
Bambara 39 Completed
Core
Yam 125 Completed
Core
Cassava 42 Partial
In progress
Soybean 6 Partial
Wild vigna 70 Partial
In progress
Bananas 50 Completed
core
Maize 0 Started

6. Assessment of genetic diversity of yam germplasm with DNA markers
-Yam (Dioscorea sp): multi-species, polyploid and vegetatively propagated
-Over 90% of world yam production comes from West and Central Africa
-Farmers continue to grow local landraces that are low in productivity
-IITA holds >3000 landrace accessions from several African countries
-Identification of suitable diverse parents is key to breeding
-A core subset of 391 accessions characterized by IITA based on
morphological characteristics
-The core collection comprising six economically important species.
-Analysis of the core for inter- and intra-specific variability using 23 SSR markers

7. D. alata (red)
D. bulbifera (purple)
D. cayenensis (yellow)
D. dumentorum (ash)
D. esculenta (blue)
D. rotundata (green)
-Results generally consistent with
established taxonomical relationships
-High level of genetic variation in core
germplasm
-Greater resolution of genetic relationships
among and within species
Valuable information for trait mapping
and breeding in yam
Figure 1. Genetic diversity tree of 342 yam accessions base
on SSR data using unweighted neighbour- joining analysis

8. Assessment of genetic diversity of cowpea germplasm with DNA markers
-Cowpea (Vigna unguiculata L. ) plays an important nutritional role in tropical
and subtropical regions (e.g. Sub-Saharan Africa)
-High protein content “~20%” (cowpea is recognized as the poor man’s meat)
-Cowpea forage is also used to feed livestock
-IITA maintains 15,000 accessions of cowpea
-A core collection of 2000 accessions was selected based
on morphological characterization
-Subsets of the core collection were analyzed further using molecular markers
to make a mini core of more than 300 accessions
-Subsets of the mini core are often drawn for further analyses

9. Diverse geographical origins covered in Sub-Saharan Africa
Nigeria, Niger, Cameroon, Ghana,Senegal, Zambia, Malawi,
Swaziland, Chad, Tanzania, Keyna, Mozambique, Zimbabwe,
Congo, Botswana
48 wild cowpeas assessed using 12 SSR markers
Gel electrophoregram of SSR primer VM27 showing amplifications with forty eight wild cowpea
From the analysis of number of alleles, marker polymorphic information content (PIC)
and clustering:
-Three subspecies, dekindtiana, pubescens and rhomboidea were recognized
-Others were recognized as varieties of dekindtiana except mensensis
-Southern Africa region is a likely center of diversity of wild cowpeas
-Nine SNP markers were also used to assess the diversity of the subset

10. Genetic diversity in yard-long bean
(Vigna unguiculata subspecies unguiculata cv-gr sesquipedalis)
-50 accessions analyzed with 16 SSR primers
-68 polymorphic bands produced
-Accessions of yard-long-bean from India showed the highest level of genetic diversity
-The results support the suggestion that India is a likely center of diversity
Development of genomic resources for diversity analysis,
mapping and marker assisted breeding in cowpea
IITA in collaboration with UCR, GCP and others

11. Molecular analysis of genetic diversity of Maize
Sample study in a collaborative GCP funded project
(IITA, France, Kenya, China, India, Indonesia, Thailand, Vietnam, and Philippines)
Objective 1: Assess the global maize diversity (previous studies targeted
Latin and North American and European germplasm only)
Objective 2: Compare genotyping procedures from different laboratories
-237 landraces (Asia, Africa and Latin America). 10 teosinte accessions
(Northern and Central America) and six CIMMYT maize lines were used
- 80 African landraces provided by IITA and analyzed with 51 SSR markers
-The diversity within maize landraces was extensive
- Up to 36% of alleles were unique to teosintes

12. Caribbean - Cuba
2 Central America - Guatemala
Central America - Mexico
Eastern Africa - Ethiopia
Eastern Africa - Kenya
Eastern Africa - Malawi
Eastern Africa - Somalia
1.5 Eastern Africa - Uganda
Eastern Africa - Zambia
Eastern Africa - Zimbabwe
Eastern Asia - China
Eastern Asia - Japan
Middle Africa - Angola
Middle Africa - Chad
1
Middle Africa - Congo
North America - Canada
North America - USA
Northern Africa - Algeria
Northern Africa - Egypt
Northern Africa - Morocco
0.5 Northern Africa - Sudan
Oceania - Australia
South America - Argentina
South America - Bolivia
South America - Brazil
South America - Chilie
0 South America - Colombia
-2 -1.5 -1 -0.5 0 0.5 1 1.5 2 South America - Ecuador
South America - Paraguay
South America - Peru
South America - Peru
South America - Venezuela
-0.5 South-Eastern Asia - Indonesia
South-Eastern Asia - Philippines
South-Eastern Asia - Thailand
South-Eastern Asia - Vietnam
Southern Africa - South Africa
Southern Asia - Afghanistan
Southern Asia - India
-1
Southern Asia - Nepal
Southern Asia - Pakistan
Teosinte
Western Africa - Benin
Western Africa - Burkina Faso
Western Africa - Ghana
-1.5 Western Africa - Guinea
Western Africa - Mali
Western Africa - Niger
Comparison to data gathered from collaborators indicated
that data from IITA was of good quality

13. Molecular analysis of genetic diversity in bananas (banana & plantain)
-Bananas (Musa spp.) are major food crops in tropical and subtropical regions
-Major cultivars are triploid (AAA, AAB and ABB genomes) with high sterility levels
-Wild species are highly diverse genetically and fertile
-IITA maintains cultivated and wild accessions (in vitro and field germplasm)
-Diversity analysis in the wild diploids to group and use the most diverse
-Done with SSR analysis
-Diversity analysis in the triploid landraces for association mapping
-Done with SSR and AFLP markers
-Diversity in mapping populations (also conserved in vitro)
-Done with SSR, AFLP, DArT and SNP markers
Markers specific to Musa genomes
A genome
B genome
Reproducible PCR markers differentiating the A from the B genome in a diversity panel

14. EMS treatment to induced mutations in vitro
The most preferred cultivars
are evolutionary dead end
Plantain Agbagba (AAB) Cooking banana Bluggoe
(ABB)
-Meristems treated with various concentrations of EMS
-Duration of treatment from 2 hours to 72 hours
-In vitro culture of meristems and acclimatation
EMS treated plants transferred to the field for
evaluation of phenotypes and agronomic traits
Collaboration with IAEA?

15. Analysis of somaclonal variations in IITA germplasm
-Genetic integrity is of primary importance in germplasm conservation, plant breeding,
and in the adoption of new varieties
-IITA conserves vegetatively propagated crops in its in vitro Genbank
-The germplasm is maintained over years and distributed to national and international
requesters
-Unexpected variations can occur during in vitro propagation (somaclonal)
to generate off-types with or without agronomic value
-It is critical to track those variations and to possibly link them to traits

16. Characterization of somaclonal variations in banana germplasm
2nd round amplification of variant band by PCR
-In vitro culture
&
-Appropriate molecular techniques
Sequencing
-Potential functions of the variation assessed
-Sequences registered in public genomic databases
Examples:
GenBank Acc# ET165586 to ET165601 (US_NCBI)
-Plants tagged & transferred to the field to link
genomic variations to phenotypes
-Applying the procedure to other IITA Sequence of a variant fragment in Bluggoe (ABB genome)
mandate crops (e.g. Yam)

17. IITA participates to international efforts to generate genomic resources
useful for diversity analysis, mapping and functional genomics
Cowpea
• Over 40,000 ESTs sequenced from cowpea drought
differential cDNA libraries
• 17,000 unique contiguous and single sequences yielded
• Over 5000 potential new markers identified
– 3226 single nucleotide polymorphisms
– 1806 microsatelite markers
• Microarray chip under manufacture
Bananas
• 5494 ESTs sequenced from musa drought differential
cDNA libraries, combined with existing consortia data
• 32,000 unique contiguous and single sequences yielded
• Over 30000 potential new markers identified
– 28800 single nucleotide polymorphisms
– 1937 microsatelite markers
Participation to generating DArT arrays in IITA
mandate crops
-Bananas
-Yam

18. Characterization of germplasm by Cytogenetics
-Two flow cytometers (Nigeria and Uganda)
-Ploidy analysis in yam, cassava, bananas, Cowpea and other orphan crops
-Throughtput of 100 samples a day
CV-Accessions File # Ploidy CV-Accessions File # Ploidy
D. rotundata 907 4x Balbisiana-selfed2 310 2x
D. rotundata 927 4x Balbisiana-selfed3 317 2x
D. rotundata 929 4x Balbisiana-selfed4 301 2x
D. cayenensis 908 8x Balbisiana-selfed5 320 2x
D. cayenensis 909 8x Balbisiana-selfed6 389 2x
D. cayenensis 910 8x Balbisiana-selfed7 302 2x
D. cayenensis 911 8x Balbisiana-selfed8 330 2x
D. cayenensis 912 8x Balbisiana-selfed9 303 2x
D. cayenensis 916 8x Balbisiana-selfed10 340 2x

19. Acknowledgments:
-Morphological description of Germplasm: Dr. Dumet D.
-Molecular analysis of Yam : Dr. Asiedu R.
-Analysis of Bananas: Dr. Vroh B. I.
-Cowpea analysis by SSR markers: Dr Fatokun C.
-Maize analysis by SSR markers: Dr. Hearnes S.
-Cytogenetics-ploidy analysis: Dr. Vroh B. I.
-Generating genomic resources (Cowpea, bananas): Dr. Hearnes S.
-Participating to DArT markers activities: Dr. Vroh B. I.
-Several Donors
-Several collaborators (NARS, ARIs)
-Several students and national partners trained
(List available with scientists mentioned above)
THANK YOU