Recent progress on CBSD research at NRI

Maruthi M N Gowda

Natural Resources Institute

University of Greenwich, UK

Main crops & viral diseases

Disease triangle – holistic researchHost

PathogenEnvironment/ Insects

Identifying CBSV vector - whiteflies

Whitefly adults CBSD leaf symptoms on a old cassava leaf

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

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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

Robust diagnostics

Our protocols are twice as efficient than the currently used methods

Virus distribution, biodiversity

Both CBSV and UCBSV are distributed throughout eastern Africa

Protocols for virus elimination in cassava

30 oC 35 oC

40 oC 45 oC

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

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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

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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

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es

Time (weeks)

Mbundumali

Mkumbozi

Albert

Kaleso

D

0,0

0,1

1,0

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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

Virus-vector team

Epidemiology

Diagnostics

DiversityVector

Control/ Resistance

Mike ThreshRory Hillocks

Sue SealMaruthi M N Gowda

John Colvin Richard Gibson