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 CD Rice genomic DNA BAC AP EXON 70844bp71002 bp71085 bp bp71002 bp71089 bp71269 bp 164 bp308 bp314 bp497 bp F primer R primer INTRON PREDICTED INTRON R primer F primer Intron

SSCP gel profiles and panel variety sequence data at Xpms30CD : ICMP 451 2: 81B 3: 841B 4: 863B 5: PT 732B 6: P : ICMP : LGD 1-B : ICMP 451 2: 81B 3: 841B 4: 863B 5: PT 732B 6: P1449 7: ICMP : LGD 1B10 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 Marker can be directly targeted to specific region of the genome Rice sequence and gene annotation databases Sequence analysis program SNPF1.2 identifies SNPs/HSVs and sorts ESTs into homoeologous groups Target primer design in region conserved between the 3 genomes

Wheat marker screening BE Chinese Spring Nulli -5A Nulli -5B Nulli -5D Opata Synthetic SparkRialto Avalon Cadenza A A B D contig int6 Chinese Spring Nulli -3ANulli -3BNulli -3D OpataSynthetic SparkRialtoAvalon Cadenza A A B B D D BE 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 ~800? Rice intron size Wheat intron size BE500570

Wheat mapping data BE cysteine proteinase precursor gwm210(2) wmc4070 stm8acag19 gwm psp gwm35956 gwm275 gwm95 81 gwm gwm gwm wPt gwm BE wmc gwm gwm wPt barc gwm515 Spark x Rialto – 2A

Wheat mapping data BE : DNAJ protein homolog ANJ1 barc1370 gwm113 gwm4136 gwm187 BE HMW7+8/ wPt gwm153gwm27417 wPt gwm26822 wPt wmc4447 psp 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 RiceMaizeRyeBarleyBrachypodium 58%39%94% 74% Wheat primer tested across species (32) Wheat RiceMaizeRyeBarleyBrachypodium 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-SNP19%34%32%28%9% SSR42%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 Wheat Pauline Stephenson Michelle Leverington-Waite Yingkun Wang James Simmonds