Presentation at RTB Annual Review and Planning Meeting (Entebbe, Uganda, 29 Sep-3 Oct 2014)

1. Complex genetic structure of African cassava
and genetic architecture of key traits
Ismail Y. Rabbi
IITA
Ibadan – Nigeria
2014 Annual RTB Meeting

2. Team members and funding
acknowledgements
IITA
Cornell University
Peter Kulakow,
Jean-Luc Jannink,
Melaku Gedil,
Martha Hamblin,
Elizabeth Parkes,
Charlotte Acharya,
Lava Kumar,
Delphine Ly,
Nzola Mahungu,
Puna Ramu,
Rachid Hanna,
Pheneas
Ntewaruhunga,
Edward Kanju,
Morag Ferguson,
US DoE-JGI and UC
Berkeley
Dan Rokhsar,
Simon Prochnik,
Jessen Bredesen,
Cindy Ha
CIAT
Luis-Augusto Becerra
National Programs
NRCRI – Chiedozie
Egesi
NaCCRI – Robert
Kawuki, Yona
Baguma,
Funding: CRP-RTB/HarvestPlus/BMGF and DFID

3. Objective: increase genetic gain through use
of molecular markers in cassava breeding
• Limited use of
markers in cassava
research.
• Advances in next-generation
sequencing should
change (and is
changing) this
situation.

4. PstI sites ApekI sites
GBS 96-plex Protocol (cont.)
Primers
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1. Plate DNA &
adapter pair
4. Pool
DNAs
Primers
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5. PCR
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Sample digestion, barcode and
2. Digest DNA with RE
3. Ligate adapters
Illumina F and R adapters
(may be done simultaneously)
ApeKI (5 base-cutter)
Clean-up
Clean-up
6. Evaluate
fragment sizes
GBS 96-plex Protocol (cont.)
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1. Plate DNA &
adapter pair
4. Pool
DNAs
5. PCR
2. Digest DNA with RE
3. Ligate adapters
(may be done simultaneously)
A
ApeKI (5 base-cutter)
6. Evaluate
fragment sizes
GBS 96-plex Protocol (cont.)
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1. Plate DNA &
adapter pair
4. Pool
DNAs
5. PCR
2. Digest DNA with RE
3. Ligate adapters
(may be done simultaneously)
ApeKI (5 base-cutter)
6. Evaluate
fragment sizes
Sample DNA
A/G
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Step 1
Sample digestion using enzyme
Step 2
Barcode adapter ligation
Step 3
Pooling of samples, PCR
amplification and
sequencing
Reference sequence
Step 4
DNA sequences aligned to reference
genome, sorted by barcodes and
SNPs called.
DNA
barcode
Universal
forward
primer
Digestion
site
Digestion
site
Pooling PCR Illumina sequencing
Sequence sorting (bioinformatics)
Technical overview

5. Application of GBS in cassava
improvement research areas
1. Understanding genetic diversity and population
structure for targeted breeding strategy (e.g.
heterotic grouping and hybrid breeding)
2. Development of genomic resources (e.g. snp
markers, annotated reference genomes, genetic
linkage maps).
3. Determining genetic architecture of target traits
(e.g. disease resistance, nutrition).
4. Genomic selection for accelerated breeding.

6. Population structure and
genetic diversity of African cassava
• Very little is known about the population structure, levels
of diversity, and ancestry of African cassava.
• Such information is required for targeted breeding
strategy (e.g. heterotic grouping and hybrid breeding).
• Most historical studies relied on at most few dozen
markers and limited germplasm set.

7. Populations genotyped so far
Population N Description
IITA improved varieties 1055 Improved genotypes cloned since 1970s
IITA-GRC 278 IITA genebank’s core collection
NRCRI 383 National Root Crops Research Institute (Nigeria)
IITA regional breeding 543
Breeding germplasm/landraces from DRC, Tanzania,
Ghana, Malawi, Zambia, Cameroon
IITA landrace collection 809
Landraces assembled from sub-Saharan Africa since
early 1980’s
CIAT* 285
Total 3353
*** Sequencing just completed, analysis in progress
60326 SNP loci from ApekI GBS

8. Population structure/ancestry of
African cassava
100%
50%
0%
West Africa, East Africa, Central Africa
• Admixture analysis detected about nine subpopulation
(divergent founders).
• Most clones derive their ancestry from more than two
subpopulations
• Improved varieties typically show more ancestries
(expected?)

9. Identification of genetic duplicates:
Reduce cost of germplasm maintenance
Zambia_M86_0016
Zambia_00_0093_ZAMBIA
Tanzania_NDL_2003_111
MexMglaziovii_MExMGlaziovii_003
Tanzania_NDL_2005_1471
Zambia_TME_594_ZAMBIA
Cameroon_Le_blanc_Bakou
Cameroon_Doumbouno(Dongali)
Ghana_BANKYE_(6MONTHS)
Cameroon_Tibedop(Betare)
Zambia_MWAKA_UMO_3
Cameroon_Obala
Ghana_DAGYEAEBANKYE
Zambia_NULUMINO
Zambia_192_034_ZAMBIA
Ghana_AFS_2000_053
Ghana_SW_2000_210
Ghana_OGhFaFn_a2_0B0D0__0986_141
Ghana_UCC_2001_138
Ghana_AKS_2000_052
Ghana_NKS_2000_024
Tanzania_Lwakitangaza
Zambia_MUKUNTA
Zambia_KABALA
Zambia_UNKNOWN_4B5
Ghana_DNA_2000_008 Ghana_KW_2000_161
GGCORE_mm990477
Cameroon_MI_alor
Cameroon_Mi_alot
Cameroon_Be_ysrgp
Ghana_BD_96_121
Cameroon_Libongo(s alando)
Cameroon_Mbouyombo(salando)
Cameroon_Ba_Mabongo
Cameroon_Var_Atin_odzoe
Ghana_BD_96_186
DRC_VUABIKA_VUABIKA
Cameroon_MI_kolo_2
Cameroon_Babank
Cameroon_Banya
Cameroon_EP_chou
Cameroon_Etoko_abomo(cook)
Ghana_UCC_2001_015
Cameroon_NM_RC
Cameroon_Ni_Panya
Ghana_BD_96087
Cameroon_Pendere(Betare)
Cameroon_Dg_Agric
Cameroon_Ni_locale_musate
Cameroon_Be_arougnchong
Cameroon_BPA_mukonde
Cameroon_Bg_re d
Cameroon_Mboumpe
Cameroon_AO_YSRP
Cameroon_Bbe_rc
Cameroon_Libongo(Betare)
Cameroon_Bititi(Betare)
Cameroon_Peau_rouge (petit_Koundji)
Cameroon_Ni_mogheuti
Cameroon_TL_WC(bs_v1)
Cameroon_Yara(moun_bourou)
Cameroon_Feofouo
Cameroon_NO_WC_RSS
Cameroon_Monembong
Ghana_AFS_2000_131
Cameroon_Etuku
Cameroon_Ajimbi(petit_Koundji)
Cameroon_Be_white_root
Cameroon_Napol_odo(ka ya_II)
Cameroon_Yokadouma_foufoue ng
Cameroon_Pk_peau_rouge
Cameroon_Mi_mambo
Cameroon_BI_mwende YM_White_GSRP
Cameroon_Be_peppep
Cameroon_Cathalan(nkonagena)
Cameroon_Yahoroup(Bi djoro)
Cameroon_SO_Akossi
Cameroon_Yahoroup(ngoura gadi)
Cameroon_Campou_Monatele
Cameroon_BSGP_Kotbotie_Essere
Cameroon_NI_marigo
Cameroon_LMR
Cameroon_Mambo(Widikum)
Cameroon_Dg_nkolo_bitcho
GGCORE_caricass_1
DRC_KIZELANA
DRC_LEMBE
DRC_NSOKOMBU
DRC_DINKONDO
DRC_NSUMAKANI
DRC_KIMBILA_1
DRC_KIMBODILA
DRC_KIMUISI_KIMUISI
DRC_94_0330
DRC_DIYILU
DRC_MANDOMBE
DRC_KIYIZILA_2
DRC_MULUTU
DRC_SADISA
DRC_MVUAMA(83_138)
DRC_KINSAKI_NSAKI
DRC_BOMA
DRC_NGUNGILA
DRC_KIMONEKENE
DRC_ZIZILA
DRC_PAPAYI
DRC_DISANKA
DRC_MAMBIKA_2
DRC_NGAYI_NGAYI
DRC_AKOKOLIA
DRC_KIVIEKI
DRC_KIVANGA
DRC_MOYINDO
DRC_NDEKE_LUKA
DRC_NTITI
DRC_RAV
DRC_MBABA
DRC_BOLILO
DRC_MUKULU_DADA
DRC_KIYIZILA_1
DRC_BUTAMU
DRC_KIMBUNGU_2
DRC_MANDUDI
DRC_KULA_NSATU
DRC_NGONGA_NABUTU_2
DRC_NGABIDU
DRC_KINTUENGE
DRC_BATENDE
DRC_SALONGO_2
DRC_KAYEBU
DRC_NAMBIYO_MBIYO
DRC_LIYAYI
DRC_NKENI
DRC_ETIENNE
DRC_MAMBIKA_1
DRC_DOLODUA
Ghana_UCC_2001_011
Ghana_KS12000196
Ghana_1235
Ghana_AFS_2000_048
Ghana_AGA_97208
Ghana_AFS_2000_043
Ghana_WCH_2000_004(6M ONTHS)
Ghana_BD_96010
Ghana_BD_96184_SUS_30572
Ghana_KW_2000_148
Ghana_BD_96_136
Ghana_BD_96_087
Ghana_BD_96_178
Ghana_I011412_GHANA
Ghana_NKABOM
Ghana_TA_97_123
Ghana_TA_97_034
Ghana_AFS_2000_027
Ghana_AFS_2000_081
Ghana_UCC_2001_157
Ghana_NKZ_2000_109
Ghana_SW_2000_146
Ghana_NKZ_2000_011
Ghana_TA_97_137
Ghana_AFS_2000_050
Ghana_SW_2000_095
Ghana_AFS_2000_071
Ghana_OFF_2000_022
Ghana_NKZ_2000_034
Ghana_UCC_2001_062
Ghana_UCC_2001_325
Ghana_DMA_2000_070_A
Ghana_H_008
Ghana_SW_2000_220
Ghana_KSI_2000_036
Ghana_AFS_2000_011
Ghana_DMA_2000_070_B
Ghana_UCC_2001_432
Ghana_DMA_2000_008
Ghana_KSI_2000_151
Ghana_OFF_2000_025
Ghana_SW_2000_187
Ghana_BD_96_154
AFS_2000_020
Ghana_UCC_2001_153
Ghana_WENCHI
Ghana_I011371_GHANA
Ghana_BD_96_021
Ghana_KSI_2000_191
Ghana_SW_2000_077
Ghana_UCC_2001_417
GGCORE_i011371
GGCORE_i011551
GGCORE_i011797
GGCORE_i30572-2
GGCORE_i940330
GGCORE_i960249
GGCORE_i960590
GGCORE_i990313
Malawi_Chisalanzo
Malawi_Chiwombola
Malawi_Chikwela
Malawi_Phoso
Malawi_Mbundumali
MexMglaziovii_MExMGlaziovii_001
MexMglaziovii_MExMGlaziovii_002
MexMglaziovii_MExMGlaziovii_004
GGCORE_mm990276
Tanzania_Maktub
Tanzania_46106_27
Tanzania_Sepide
Tanzania_Kipusa
Tanzania_Mwari
Tanzania_Namikonga
Tanzania_NDL_2003_031
Tanzania_Lwabahimba
Tanzania_Rubona
Tanzania_Liongo_Kigoma
Tanzania_Aipin_Valenca
Tanzania_Kalingisi
Tanzania_Ging'hi_Magu
Tanzania_KBH_2006_29
Tanzania_KBH_2006_74
Tanzania_KBH_2006_22
Tanzania_MIGYERA
Tanzania_KIROBA
Tanzania_KBH2006_94
Tanzania_KBH2002_505
GGCORE_z970474
Zambia_KASAI
Zambia_CHILA
Zambia_MANYOPOLA
Zambia_KATOBA_MPUTA
Zambia_KARIBA
Zambia_MWAKA_UMO_1
Zambia_MWAKA_UMO_2
Zambia_BANGWEULU_1
Zambia_CHIMAMBA
Zambia_NAMUNYONGO
Zambia_BUNGABUTU
Zambia_KALABA
Zambia_KASWESHI
Zambia_NAKAMBUMA
Zambia_NDELE_MUKAKA
Zambia_UNKNOWN_1
Zambia_MULOSHA
Zambia_NDOLA
Zambia_MANGABWA
Zambia_NAJOYCE
Zambia_LUSAKA
Zambia_MABUMBA_1
Zambia_NAKAPAYI
Zambia_UNKNOWN_1B
Zambia_UNKNOWN_3
Zambia_UNKNOWN_1BG
Zambia_NAKASHI_MBI
Zambia_TANKENA
Zambia_BANGWEULU_2
Zambia_KASONTA
Zambia_TOMO
Zambia_MABUMBA_2
Zambia_L9_304_36
Zambia_MH95_0414_ZAMBIA
Zambia_99_0359_ZAMBIA
Zambia_14(2)1425_Z AMBIA
Zambia_00_1093_ZAMBIA
Zambia_96_1432_ZAMBIA
Zambia_L9_304_73
Zambia_L9_92_032
Zambia_MM96_1757_ZAMBIA
Zambia_01_1551_ZAMBIA
Zambia_01_1649_ZAMBIA
Zambia_MM96_1759_ZAMBIA
Zambia_L9_304_26
Zambia_180_142_ZAMBIA
Zambia_L9_304_175
Zambia_96_1565_ZAMBIA
Zambia_L9_304_101
Zambia_99_3575_ZAMBIA
Zambia_L9_304_5
Zambia_I92_0342_Z AMBIA
IITA GRC
Zambia
DRC
Cameroon
Ghana
Tanzania
Histogram of RTB.IBD
Distance (1−PSA)
Frequency
0.00 0.05 0.10 0.15 0.20 0.25
0 50000 100000 150000
Duplicate
distance
threshold

10. Application of GBS in cassava
improvement
1. Understanding genetic diversity and population
structure for targeted breeding strategy (e.g.
heterotic grouping and hybrid breeding)
2. Development of genomic resources (e.g. snp
markers, annotated reference genomes, genetic
linkage maps).
3. Determining genetic architecture of target traits
(e.g. disease resistance, nutrition).
4. Genomic selection for accelerated breeding.
10/30/2014

11. Mapping populations genotyped
through RTB/other projects
Population Cross N Genotyping
IITA_MP1 TMSI961089AxTMEB117 205 IGD
IITA_MP2 TMEB117 x TMSI961089A 207 IGD
IITA_MP3* TMS I011412 x
TMS4(2)1425
177 IGD, Berkeley
IITA_MP4* TMS30001 x I961089A 271 Berkeley
IITA_MP5* TMSI961089A x
TMS30001
243 Berkeley
IITA_MP6 I011371xTMEB117 300 Berkeley
* Contributed to the cassava consensus map consortium

12. High-density GBS SNP maps
Genetic map PstI (GACGTC); 770 SNPs ApekI (GCWGC); 6756 SNPs
0
50
100
150
Genetic map
Chromosome
Location (cM)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
0
20
40
60
80
100
120
Chromosome
Location (cM)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
IITA_MP1 IITA_MP3
Crop Science Vol. 54 (2014) Virus Research (2014)

13. Improvement of reference genome
using high-density linkage maps
• High-quality genome assembly needed for QTL, GS,
GWAS, genetic transformation.
• Current cassava genome is assembled into 12977
pieces (scaffolds).
• 10 high-density GBS SNP maps used to anchor the
cassava reference genome.
• 71.9% of the assembled genome has been placed on
the 18 cassava chromosomes.
• Work done as part of the cassava genetic map
consortium

14. Application of GBS in cassava
improvement
1. Understanding genetic diversity and population
structure for targeted breeding strategy (e.g.
heterotic grouping and hybrid breeding)
2. Development of genomic resources (e.g. snp
markers, annotated reference genomes, genetic
linkage maps).
3. Determining genetic architecture of target traits
(e.g. disease resistance, nutrition).
a. QTL mapping using bi-parental populations
4. Genomic selection for accelerated breeding.
10/30/2014

15. QTL mapping in bi-parental
populations
• Mapping population:
– Full-sib F1 populations derived from pairs of non-inbred parents
• Phenotyping (>2 years):
– Susceptibility to Cassava Mosaic Disease
– Carotenoid accumulation (b-carotene) in storage roots
– Anthocyanin pigmentations
– Number of harvested roots
– root weight per plot.
• Genotyping:
– GBS using PstI Restriction enzyme (n = 770 SNPs)
• Data analysis:
– Genetic map – Joinmap®
– Phenotype data analysis - R/lme4
– QTL mapping using R/qtl

16. Log of odds ratio
QTL mapping in bi-parental
populations
Rabbi et al. Crop Science vol. 54:

17. Major locus underlying carotenoid
accumulation in cassava roots
80
60
40
20
0
S7520_912057 PULPCOL Phytoene synthase II
linkage group
lod
2011
2012
combined
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Rabbi et al. Crop Science vol. 54:

18. High-resolution mapping of the
CMD2 locus
40
30
20
10
0
CMD1S S5214_30876
linkage group
lod
CMD2 locus
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Resistant parent Susceptible parent

19. Narrow genetic base for major gene
resistance to CMD?
a 50
S5214_780931
40
30
20
10
0
-Log10(P)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
50
40
30
20
10
0
0 30 60 90 120 150
IITA TMS 011412
IITA TMS 4(2)1425
scaffold 5214
Position (cM)
b
-Log10(P)
a. Genome-wide scan
for linkage between
CMD resistance and
6756 SNPs across 18
cassava linkage groups.
b. A detailed view of the
of linkage group 16
Rabbi et al. Virus Research 2014 DOI: 10.1016/j.virusres.2013.12.028

20. Anchoring previously-mapped
resistance loci
S4175_365840
S4175_207572
S4175_58407
S4175_116369
S4175_207488
S4175_241208
S4175_208108
S4175_64514
S4175_62342
S4175_181543
S4175_255324
S7933_3549
S7933_41036
S5214_1427030
S5214_1273806
S5214_1274892
S5214_1073280
S5214_1084049
S5214_831886
S5214_780931
S5214_776689
S5214_514589
S5214_472282
S5214_566592
S6906_46617
S6906_46672
S6906_119465
S6906_119507
S6906_359407
S6906_359424
S6906_359496
20
40
60
80
100
120
CMD Resistance
LG16P1F2
r^2
0 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 1
NS198
PVE: 11%
SSRY106
PVE: 30%
SSRY28
PVE: 60-70%
NS158 and
NS169
PVE: N/A
All markers linked to
dominant, major gene
resistance to CMD from
different genetic
backgrounds occur in
same chromosome!
Linkage group 16

21. Application of GBS in cassava
improvement
1. Understanding genetic diversity and population
structure for targeted breeding strategy (e.g.
heterotic grouping and hybrid breeding)
2. Development of genomic resources (e.g. snp
markers, annotated reference genomes, genetic
linkage maps).
3. Determining genetic architecture of target traits
(e.g. disease/pest resistance, plant morphology,
nutrition)
b. Genome-wide association
4. Genomic selection for accelerated breeding.
10/30/2014

22. What next?
• So far, most of the applications of GBS SNPs relate
to germplasm characterization and QTL discovery.
• Next step is to change gears to ‘applications’ by
using the discovered information in the crops’
improvement.
– Heterotic pattern/grouping (on-going)
– Genomic selection and MAS
• www.nextgencassava.org
• www.cassavabase.org

23. Team members and funding
acknowledgements
IITA
Cornell University
Peter Kulakow,
Jean-Luc Jannink,
Melaku Gedil,
Martha Hamblin,
Elizabeth Parkes,
Charlotte Acharya,
Lava Kumar,
Delphine Ly,
Nzola Mahungu,
Puna Ramu,
Rachid Hanna,
Pheneas
Ntewaruhunga,
Edward Kanju,
Morag Ferguson,
US DoE-JGI and UC
Berkeley
Dan Rokhsar,
Simon Prochnik,
Jessen Bredesen,
Cindy Ha
CIAT
Luis-Augusto Becerra
National Programs
NRCRI – Chiedozie
Egesi
NaCCRI – Robert
Kawuki, Yona
Baguma,
Funding: CRP-RTB/HarvestPlus/BMGF and DFID

24. Also, check out our poster on tracking of
cassava varieties in farmers fields
•
•
•
Thank you !!!