Development of COS markers in grasses

Isabelle Bertin, Pauline Stephenson and Michelle Leverington-Waite John Innes Centre

Purposes of developing SSCP-SNP as COS (Conserved Ortholog Set) markers

• Develop markers that allow easier alignment not only of maps from different crosses but also different species

• Define synteny with model (rice and Brachypodium) and crops at a closer level

• Markers can be targeted to specific region

SSCP-SNP marker system background

• Exploit pearl millet EST available from NCBI

• Synteny

• Intron polymorphism > Exon polymorphism

• Single Strand Conformation Polymorphism (SSCP) gel

SSCP-SNP mining

NCBI 1900 Pearl Millet

EST sequences

650 Pearl millet EST show good homology

with rice (34%)

blastn

299 Pearl millet EST homologous to single copy rice gene (16%)

Select single

copy genes

Define intron/exon boundaries in pearl

millet EST

RiceGAAS

Test polymorphism on SSCP gel 102 markers polymorphic (34%)

design primer across intron

Bmc genetics 3: art-19; Ching et al., 2002 In maize the frequency of nucleotide change among varieties is high, at around one polymorphism per 31 bp in non-coding regions and 1 polymorphism per 124 bp in coding regions. Insertions and deletions (indels) are also frequent in non-coding regions (1 per 85 bp), but rare in coding regions SNP frequencies in more conserved crop species may be much lower.

Done in silico

Marker assay design

Pearl Millet EST CD726515

Rice genomic DNA BAC AP005071

EXON EXON

70844bp 71002 bp 71085 bp 71282

70859 bp 71002 bp 71089 bp 71269 bp

164 bp 308 bp 314 bp 497 bpF primer R primer

INTRON

PREDICTED INTRON

R primer

F primer

Intron

SSCP gel profiles and panel variety sequence data at Xpms30CD726044

1 2 3 4 5 76 81 3 61 3 61 3 6

1: ICMP 4512: 81B3: 841B4: 863B5: PT 732B6: P1449-27: ICMP 854108: LGD 1-B-10

-2

- -

1: ICMP 4512: 81B3: 841B4: 863B5: PT 732B6: P14497: ICMP 854108: LGD 1 B 10

Exon

Intron

50 bp

65 bp 255 bp

22 bp 45 bp

37 bp

Exon

Exon

Intron

50 bp

65 bp 255 bp

22 bp 45 bp

37 bp

Exon

• PCR products were denatured and separated on SSCP gels using MDE™ (Mutation Detection Enhancement)

• MDE™ gel solution reported to cause DNA separation on the basis of both size and conformation (Soto and Sukumar, 1992)

Polymorphism identification and frequency in pearl millet

• Type of polymorphism:

– 2/3 of variation are SNPs– 1/3 of variation are indels

• Polymorphism frequency:

– 1 SNP/Indel per 59 bp in intron– 1 SNP/Indel per 714 in exon

Transfer SSCP-SNP marker system to wheat - why?

• Over 603,492 EST sequences are publicly available

• International effort to develop SNPs failed

Transfer SSCP-SNP marker system to wheat - how

• Wheat is a polyploid (AABBDD)

• Physical map of ~6500 wheat ESTs available from GrainGenes http://wheat.pw.usda.gov/wEST/binmaps/

• Marker can be directly targeted to specific region of the genome

• Rice sequence and gene annotation databases http://ricegaas.dna.affrc.go.jp/rgadb/

• Sequence analysis program SNPF1.2 identifies

SNPs/HSVs and sorts ESTs into homoeologous groups

http://wheat.pw.usda.gov/ITMI/WheatSNP/ • Target primer design in region conserved between the 3 genomes

Wheat marker screening

BE496976

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B

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Op

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Syn

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Avalo

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Cad

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contig 1430.5 int6

Ch

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Nu

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A

Nu

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B

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Syn

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BE488921

• 32 wheat markers were screened on Chinese Spring aneuploid lines– 20 markers resolved product from 3 genomes– 10 markers resolved product from only 2 genomes

• Tested on parents of 5 different crosses– Approx 25% of the markers were polymorphic over the 5 crosses

Intron size comparison between rice and wheat

196

100

130

80

72 83

67

405

230

150

79

131

417

80

121

96 466 89472

679

942

87

~800

? 60-120

90-115

7-900

100-1406-900

135-240

4-900

Rice

intron size

Wheat intron size

BE500570

Wheat mapping data

BE444894 cysteine proteinase precursor

gwm210(2)

wmc4070

stm8acag19gwm63622

27

psp315344

gwm35956

gwm275gwm9581

gwm294102gwm312107gwm349117wPt-6894122 gwm356123BE444894124wmc181125 gwm382129gwm311131wPt-5887134barc122135

gwm515

Spark x Rialto – 2A

Wheat mapping data

BE444071 : DNAJ protein homolog ANJ1

barc1370

gwm113 gwm4136 gwm187BE4430718HMW7+8/17+1811wPt-070512

gwm153 gwm27417

wPt-903221gwm26822

wPt-094438

wmc4447

psp310053

gwm25960

Spark x Rialto – 1B

Could SSCP-SNP be transferred to other crop?

• Sequence homology in exon between pearl millet and rice is well conserved

• Sequence homology breaks down in introns

Develop those markers to COS marker

R primer

F primer

Intron

R millet primer

F millet primer

R primer

F primer

INTRON

Pile up EST from different species in order to develop primer in conserved region of genes

Use of SSCP-SNP as COS marker

Rice Maize

Rye Barley Brachypodium

58% 39% 94% 94% 74%

Wheat primer tested across species (32)

Wh

ea

t

Ric

e

Ma

ize

Ry

e

Ba

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Bra

ch

yp

od

ium

Polyacrylamide gel

SSCP-SNP compared to SSR

• How polymorphic are SSCP-SNPs compared to SSR?

• Advantages:– Target genes or chromosome regions– SSCP-SNP entirely developed in silico– Several introns in each gene– Transferable between species– Low-tech using gels or high-tech using capillary

electrophoresis

Opata/synthetic

Spark/Rialto

Avalon/Cadenza

CS/SQ1

Trintilla/Piko

SSCP-SNP 19% 34% 32% 28%

9%

SSR 42% 46% 53% - 50%

Summary

• Easy to develop and easy to use

• Highly transferable

• Target genic regions

• Much more informative for comparative genetics – synteny definition

Acknowledgements

• Mike Gale

• John Snape

• WheatPauline StephensonMichelle Leverington-WaiteYingkun Wang James Simmonds