Striga hermonthica infesting a Sorghum field in Sudan

Striga gesnerioides

Striga hermonthica

Striga spp. (Witchweed)

•Obligate root parasite belongs to Orobanchaceae.

•Wide spread mainly in sub-Saharan Africa and Asia.

•Infect important crops, such as sorghum, maize, rice and cowpea

•S. hermonthica and S. asiaticaparasitize Poaceae species, whereas S. gesnerioidesparasitize legumes

•Spread over 50 million ha area

•Estimated yield losses are 7 billion US$ annually

Striga hermonthica infects Sorghum bicolor

Life cycle of Striga hermonthica

Modified from IITA Striga manual (1997)

Germination stimulant Haustorium inducer

Yoshida and Shirasu (2009) New

Phytologist

Striga hermonthica infects rice

Cross section

ShZm

Zm

Sh

Striga parasitizing Zea mays

Our aim:

Understand how parasitic plants

parasitize host plants

--Molecular mechanisms?

How to find important genes?

Sequence

information

Functional analysis

Reverse genetics

Forward genetics

Evolutional aspects

Genome comparison

Sequence

information

Functional analysis

Genome

TranscriptomeReverse genetics

Forward genetics

Evolutional aspects

Genome comparison

Expressed genes

Complete genes

How to find important genes?

How to find important genes?

Sequence

information

Functional analysis

Genome

TranscriptomeReverse genetics

Forward genetics

Evolutional aspects

Genome comparison

Expressed genes

Complete genes

EST :Expressed sequence tag

A catalog of the expressed

sequences ≈ functional genes

How to find important genes?

Material: S. hermonthicaseedling, flower, stem, root, leaf

Construction of full-length enriched cDNA library and EST analysis

Full length enriched cDNA library

Sequence (Sanger method)

37,710 clones

75,330 reads

17,137 unigenes (6,818 singletons, 10,319 contigs)

Now the web-page is available!! ���� http://striga.riken.psc.jp

EST summary

Independent clone 37,710

Raw sequence 75,330

High quality sequence 67,814

Unigenes 17,137

Putative SSRs 1,445

Average unigene length 810.3 bp

Putative SNPs 9,299

Average number of EST per contig 5.9

Yoshida et al 2010, BMC Plant Biol.

S triga only2624

Grape

Arabidopsis

Triphysaria

P oplar

G +S36

G +A+S36

T+S662

A+S19

P +S58

P +T+S87

G +T+S81

A+T+S34

P +G+T+S220

A+G +T+S72

P +G +S114

A+P +G +T+S11711

A+P +T+S246

A+P +G+S1075

A+P +S62

���� Possible parasitic plant specific genes

Blast results of S. hermonthica unigenes

Application of ESTs: SSR markers

Independent clone 37,710

Raw sequence 75,330

High quality sequence 67,814

Unigenes 17,137

Putative SSRs 1,445

Average unigene length 810.3 bp

Putative SNPs 9,299

Average number of EST per contig 5.9

*SSR: Simple Sequence Repeat

Striga hermonthica biotypes

SorghumMillet

Striga hermonthica biotypes

Dirweesh SorghumCentral Sudan

Tandalti Pearl milletWestern Sudan

Elkaraiba SorghumCentral Sudan

El Obeid SorghumWestern Sudan

Agadi MaizeEastern Sudan

Kenya MaizeKenya

Host speciesLocation RegionNo.

1

6

5

4

3

2

Extracted DNAs from seeds collected at different

locations and hosts.

High genetic diversity of S. hermonthica in the

same field.

Identify SSR markers

(Pooled

DNA)

64 SSR primer sets were tested. 10 could be used for analysis.

PIC value = Polymorphic information content

= probability that the parent is heterozygous x probability that the offspring is informative

Identify SSR markers

Agadi (Maize)66

73

70

0.1

Tandalti (Pearl millet)

Kenya (Maize)

El Obeid (Sorghum)

Dirweesh (Sorghum)

Elkaraiba (Sorghum)

Correlation between polymorphisms and host specificity

We could not detect correlation between polymorphisms

and host specificities

� Relatively recent diversification

Yoshida et al BMC Plant Biol. 2010

Horizontal gene transfer from a host to a parasite

Yoshida et al Science2010

Summary of blast results

Eudicots: Arabidopsis, Poplar, Grape, Soybean;

Monocots: Rice, Sorghum Threshold e-value =10e-10

87% Sorghum bicolor cDNA

0.1

9898

9999

7794

Striga hermonthica ShContig9483

Sorghum bicolor Sb0015s004030

Sorghum bicolor Sb01g013250

Brachypodium distachyon Bradi1g00410

Brachypodium distachyon Bradi2g36840

Oryza sativa LOC_Os06g50670

Oryza sativa LOC_Os07g15430

Triticum aestivum TA50621_4565

Oryza sativa LOC_Os01g56900

Oryza sativa LOC_Os10g32020

Sh: Striga hermonthica, Om: Orobanche minor, Pj: Phtheirospermum japonicum

Nb: Nicotiana benthamiana, Am: Antirrhinum majus, At: Arabidopsis thaliana, Sb: Sorghum bicolor, OS: Oryza sativa

ShContig9483 clusters with sorghum homologues

and does not exist in dicots genome

Yoshida et al 2010, Science

An adjacent gene is clustered with eudicot genes

The sequences outside ORF are conserved between S. hermonthica and sorghum

The sequences outside ORF are conserved between S. hermonthica and sorghum

Does polyA sequence suggest the gene was

captured as mRNA?

The gene transfer occurred after differentiation of genera Orobanche

and Striga, but before speciation in the Striga genus

Phtheirospermum

Orobanche minor

Clade II

Clade I

Clade V

Antirrhinum majus

Striga gesnerioides

Solanum tuberosum

Orobanchaceae (48 Mya)

Scrophulariaceae (68 Mya)

Solanaceae

Striga hermonthica

ShContig9483 homologs exist in Striga gesnerioides

More sequences

More new findings

Sequence

information

Functional analysis

Genome

TranscriptomeReverse genetics

Forward genetics

Evolutional aspects

Genome comparison

Striga asiatica Striga hermonthica

~600 Mb 1.6- 1.8 Gb

Self-crossing Out-crossing

Photo by Lytton Musselman

Towards complete genome sequences of Striga spp.

Methods

Illumina HiSeq 2000

Next generation sequencer

90 bp/read

10 Gbp/lane

Construction of jump libraries

500 bp, 3 kbp, 10 kbp, 20 kbp,

and a fosmid library

We will sequence more than

60 x genome coverage

Materials

Striga asiatica

in vitro culture

from a single plant

USA strain from Prof M. Timko

Sequence

information

Functional analysis

Transcriptome

Genome

Reverse genetics

Forward genetics

Evolutional aspects

Genome comparison

PPGP project: Striga, Orobanche, Triphysaria

Our group: Phtheirospermum � Talk at 11th

June

Summary

•Large-scale EST database of S. hermonthica is

available at http://striga.psc.riken.jp

•SSR markers can be useful for genetic diversity

analysis

•Horizontal gene transfer of a nuclear gene from

host to parasite was found.

•Striga genome sequence project is on-going.

Acknowledgments

Ken ShirasuJuliane Ishida

RIKEN, Bioinfo

Kenji Akiyama

Tetsuya Sakurai

Univ. Tokyo, Evolution

Shin-ichiro Maruyama

Hisayoshi Nozaki

Penn State, USA

Claude dePamphilis

Eric Walufa

S. hermonthica seeds and DNAAbdelbagi M. Ali (ARC Sudan)Nasrein Kamal (ARC Sudan) A.G. Babiker (Sudan Univ.)

Strigol synthesis

Kenji Mori

S. asiatica seeds

Mike Timko