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Recent progress on CBSD research at NRI
Maruthi M N Gowda
Natural Resources Institute
University of Greenwich, UK
Virus-vector relationships
0,0
10,0
20,0
30,0
40,0
50,0
60,0
5 min 30 min 1 h 4 h 24 h 48 h Life-long
16,0
32,0
40,0 40,045,0
40,045,0
% C
BS
V t
ran
smis
sio
n
AAP
AAP for CBSV transmission by Bemisia tabaci
0,0
10,0
20,0
30,0
40,0
50,0
60,0
5 min 30 min 1 h 4 h 24 h 48 h Life-long
0,0
6,7
20,0
33,3
40,0 40,045,0
% C
BS
V t
ran
smis
iso
n
IAP
IAP for CBSV transmisison by Bemisia tabaci
0,0
10,0
20,0
30,0
40,0
50,0
60,0
CBSUV CBSV
34,540,0
% C
BS
V t
ran
smis
sio
n
Virus type
Transmisison of CBSV v/s
CBSUV
Semi-persistent transmission
Assays developed to screen
Sources of resistance
Developing low-cost diagnostics
CMD + CBSD
Both mosaic and brown streak viruses can be detected in a single PCR
Cost per sample
Consumables costs only UK£ US$*
CTAB method + two-step RT-PCR for CBSV + Electrophoresis 2.01 3.02
CTAB method + one-step RT-PCR for CBSV + Electrophoresis 5.15 7.72
RNeasy method + two-step RT-PCR for CBSV + Electrophoresis 7.39 11.08
RNeasy method + one-step RT-PCR for CBSV + Electrophoresis 10.53 15.79
How much per sample?
*Based on an exchange rate of £1 = $1.5
Our protocols are five times cheaper than the currently used methods
Day 1Planting cassava
at NRI quarantineglasshouse
Months 1-3Visual observations for
CMD & CBSDsymptoms Plants with CBSD
& CMD symptoms – Discard or keep for treating
Month 4PCR testing for CMGs & CBSVsby multiplexing
Months 5-7Tissue culture, test a sub-set by PCR & ready for shipping
Symptom-free plants – Keep &
continue with further analysis
Month 4Virus indexing –
Grow stem cuttings or Tissue culture
Months 5-7Chemo & thermo therapies on tissue
culture plants
Repeat the process from the beginning on virus-infected plants
Summary protocol for generating
virus-free cassava plants
Phase I
SYMPTOMS
Phase III
PCR - II
Phase II
PCR - I
CYCLE 1 (8 months)
Cassava lines cleanedCountry of origin Variety No. of stems planted
Tanzania KBH 2002/ 066 21
Pwani 21
Mkumba 21
Kizimbani 21
KBH 2006/ 26 21
Kenya LM1/2008/363 22
F19 23
Tajirika 20
Shibe 21
F10-30-R2 20
Kibandameno 20
Mkumbozi 17
Malawi Yizaso 20
Mbundumali 21
Sauti 22
CHO 5/203 24
Sagonja 20
Kalawe 20
Mozambique Oekhumelela 21
Eyope 22
Nziva 22
Colicanana 21
Orera 22
Uganda Nase 3 12
TME 204 13
Tz 130 14
Nase 18 (109-TME 14) 10
72-TME 14 11
Nase 14 (MM 96/ 4271) 13
Nase 1 14
33 varieties
Received:
Aug-Sept 2012
Cleaned: 28 var. (85%)
July 2013 (cycle 1)
Cycle 2: 3 varieties
Cycle 3: 2 varieties
Identifying CBSD resistance
Three cassava varieties; Kaleso, Kiroba and Albert tested against bothCBSV and CBSUV
Kaleso resistant to root symptoms but not for virus infection
Relative virus concentrations
0
50
100
150
200
250
1 2 4 8 12 16 20 24 28 32 36
Rel
ativ
e vi
rus
titr
e
Kaleso
Kiroba
Albert
0
50
100
150
200
250
300
1 2 4 8 12 16 20 24 28 32 36
Rel
ativ
e vi
rus
titr
e
Kaleso
Kiroba
Albert
UCBSV
CBSV
Measuring virus concentration by qPCR
Negligible amounts of virus in
resistant variety Kaleso
0,1
1,0
10,0
100,0
2 3 4 8 12 16 20 24Re
lati
ve
CB
SV
qu
an
titi
es
Time (weeks)
Albert
Kalawe
F10-30-R2
Kaleso
C
0,2
1,0
5,0
25,0
125,0
2 3 4 8 12 16 20 24
Re
lati
ve
CB
SV
qu
an
titi
es
Time (weeks)
Mbundumali
Mkumbozi
Albert
Kaleso
D
0,0
0,1
1,0
10,0
100,0
2 3 4 8 12 16 20 24
Re
lati
ve
CB
SV
qu
an
tity
Time (weeks)
Mkumba
Kaleso
Albert
Oekhumelela
Pwani
A
0,1
1
10
100
2 3 4 8 12 16 20 24Re
lati
ve
CB
SV
qu
an
tity
Time (weeks)
Tme 204
Kaleso
Albert
Kizimbani
B
Virus behaviour in cassava varieties
time
logc
bsv
-3
-2
-1
0
1
2
5 10 15 20 25
Albert F10-30-R2
5 10 15 20 25
Kalawe Kaleso
5 10 15 20 25
Kizimbani
Mbundumali Mkumba Mkumbozi Nase3
-3
-2
-1
0
1
2
oekhumelela-3
-2
-1
0
1
2
Orera
5 10 15 20 25
Pwani TME204
Relative virus quantities & CBSD resistance
0 20 40 60 80
kaleso
Pwani
Mkumba
Nase 3
Oekhumelela
TME 204
Kizimbani
Kalawe
Orera
F10-30-R2
Albert
Mkumbozi
Mbundumali
Cluster 1
Cluster 2
Cluster 3
Outliers
Resistant
Tolerant
Susceptible
Susceptible
(Highly)
• RNA-Seq analysis of Kaleso and Albert varieties
• ~50 million reads per sample, 50-60% of reads mapped per sample
• 34,151 genes in total, about 28,667 genes expressed in at least one of 4 treatments
• High number of Kaleso-specific genes, compared to other treatments
Mechanism of resistance
Illumina HiSeq 2000
Albert Control Albert CBSV Kaleso Control Kaleso CBSV
Number of
Reads Percentage
Number of
Reads Percentage
Number of
Reads Percentage
Number of
Reads Percentage
All 54,045,667 - 60,070,579 - 38,949,010 - 49,681,907 -
Mapping to whole
genome 31,632,660 59 35,964,664 60 20,946,755 54 29,534,087 60
Non uniquely mapped 8.674,373 27 10,282,664 29 5,526,455 26 7,563,418 26
Uniquely mapped 23,261,749 74 26,036,303 72 15,618,148 75 22,243,065 75
Resulting Reads 23,261,749 74 26,036,303 72 15,618,148 75 22,243,065 75
• 28,667 genes expressed in at least one of 4 treatments• High number of Kaleso-specific genes, compared to other treatments
Sequence analysis
745 235
Kaleso Albert
101 373 239
Kaleso Albert
87
Kaleso Albert
Over expressed Under expressed
Assigning metabolic pathways to genes
(KEGG) KEGG
pathway
number Pathway details
Number
of genes
% of total genes
present in the
pathway P-Value
ath01061 Biosynthesis of
phenylpropanoids
20 3 7.8E-3
ath01070 Biosynthesis of plant
hormones
20 3 5.7E-2
ath01062 Biosynthesis of terpenoids and
steroids
16 2.4 1.6E-2
ath00520 Amino sugar and nucleotide
sugar metabolism
10 1.5 3.5E-3
ath00051 Fructose and mannose
metabolism
8 1.2 2.7E-3
ath00680 Methane metabolism 8 1.2 5.7E-2
ath03050 Proteasome 7 1 3.7E-2
ath00941 Flavonoid biosynthesis 6 0.9 9.7E-4
ath00906 Carotenoid biosynthesis 4 0.6 6.6E-2
Are there Resistant Gene Analogues (RGAs)?
• Dominant resistant genes• Searching using common elements
– Toll interleukin-1 receptor (TIR), coiled domain (CC), nucleotide binding site (NBS) or a leucine-reach repeat domain (LRR)
• 235 genes were expressed with NBS or LRR domains
• Also compared to other RGAs; RTM-1, RTM-2, Tm-1
• RTM-2 and Tm-1 were not differentially expressed
• None were significantly over expressed because of virus infection
• Recessive resistant genes• Common elements – translation initiation factors eIF4E or eIF4G
• Four eIF4E or one eIF4G were expressed in our data
• Amongst these, eIF4E transcript cassava4.1_016601m was increased two-fold in infected Kaleso compared to healthy and Albert controls
In Silico analysis of QTLs
• Kulembeka (2012) identified
– 12 CBSD resistance-associated markers
– spanning 10 linkage groups – 3 on an integrated map, 1 on Namikonga F1 map, and 8 on three Namikonga S1 maps.
• Candidate QTLs were identified using cassava genome assemblies and their chromosomal locations of were determined by BLASTN
• Linked markers of 12 CBSD R QTLs were mapped to the cassava chromosomes – chr. V, chr. IX, chr. XI and chr. XVII
Marker Trait Map category Scaffold Chromosome
Linkage
group
SSRY54 CBSD-RN_NDI2008 Namikonga S1 scaffold05938 IX S17
SSRY4 CBSD-RN_CHZ2007 Integrated scaffold05921 ND C18
SSRY4 CBSD-RN_CHZ2007 Namikonga S1 scaffold05921 ND S12a
SSRY295 CBSD-RN_CHZ2008 Namikonga S1 scaffold07523 V S1
NS945 CBSD-RN_CHZ2008 Namikonga scaffold02973 V N4
SSRY195 CBSD-RN_CHZ2008 Namikonga scaffold11821 V N4
NS667 CBSD-RN_CHZ2008 Namikonga scaffold02994 V N4
NS78 CBSD-RN_CHZ2007 Namikonga S1 scaffold05958 V N4
ESTsSSRY66 CBSD-RN_CHZ2007 Namikonga S1 scaffold11821 V N4
SSRY160 CBSD-RN_CHZ2007 Namikonga S1 scaffold02967 V N4
NS235 CBSD-RN_CHZ2007 Namikonga S1 scaffold02967 V N4
NS235 CBSD-RN_NDI2008 Namikonga S1 scaffold02967 V N4
NS945 CBSD-RN_CHZ2008 Integrated scaffold02973 V C4
ESSRY96 CBSD-RN_CHZ2007 Namikonga S1 scaffold08359 XI S12a
ESSRY96 CBSD-RN_CHZ2007 Integrated scaffold08359 XI C18
NS347 CBSD-RN_CHZ2007 Namikonga S1 scaffold10401 XVII S8
SSRY19 CBSD-RN_CHZ2008 Namikonga S1 scaffold10401 XVII S8
Scaffold and chromosome locations of QTLS
Conclusions
• RNA-Seq data on two varieties
• We’re planning to get additional sequences
for six more varieties
• Using information from known RGAs
• Using QTLs and generate more QTLs
• Identify CBSD resistance genes/ markers
Holistic approach to research
• Epidemiology & control
• Diagnostics
• Virus diversity and distribution
• Vector transmission
• Tissue culture & virus indexing
• Resistance – natural
• Resistance mechanism – RNA-Seq
• Socioeconomic impact
• Impact on value chain
• Training, capacity building, transfer of technology
NRI is a specialized institute
focused on agriculture, food
security, poverty alleviation
in developing countries.
It’s a school of the
University of Greenwich in
the UK
www.nri.org
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