1. Development of COS markers in grasses Isabelle Bertin, Pauline Stephenson and Michelle Leverington-Waite John Innes Centre

2. 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

3. SSCP-SNP marker system background Exploit pearl millet EST available from NCBI Synteny Intron polymorphism > Exon polymorphism Single Strand Conformation Polymorphism (SSCP) gel

4. 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

5. Marker assay design Pearl Millet EST CD726515 Rice genomic DNA BAC AP005071 EXON 70844bp71002 bp71085 bp71282 70859 bp71002 bp71089 bp71269 bp 164 bp308 bp314 bp497 bp F primer R primer INTRON PREDICTED INTRON R primer F primer Intron

6. SSCP gel profiles and panel variety sequence data at Xpms30CD726044 12345768136136136 1: ICMP 451 2: 81B 3: 841B 4: 863B 5: PT 732B 6: P1449-2 7: ICMP 85410 8: LGD 1-B-10 -2 -- 1: ICMP 451 2: 81B 3: 841B 4: 863B 5: PT 732B 6: P1449 7: ICMP 85410 8: 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)

7. 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

8. Transfer SSCP-SNP marker system to wheat - why? Over 603,492 EST sequences are publicly available International effort to develop SNPs failed

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

10. Wheat marker screening BE496976 Chinese Spring Nulli -5A Nulli -5B Nulli -5D Opata Synthetic SparkRialto Avalon Cadenza A A B D contig 1430.5 int6 Chinese Spring Nulli -3ANulli -3BNulli -3D OpataSynthetic SparkRialtoAvalon Cadenza A A B B D D 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

11. Intron size comparison between rice and wheat 19610013080728367405 230 150 79 131417801219646689472679942 87~800?60-12090-1157-900100-1406-900135-2404-900 Rice intron size Wheat intron size BE500570

12. Wheat mapping data BE444894 cysteine proteinase precursor gwm210(2) wmc4070 stm8acag19 gwm63622 27 psp315344 gwm35956 gwm275 gwm95 81 gwm294102 gwm312107 gwm349117 wPt-6894122 gwm356 123 BE444894 124 wmc181125 gwm382129 gwm311131 wPt-5887134 barc122 135 gwm515 Spark x Rialto – 2A

13. Wheat mapping data BE444071 : DNAJ protein homolog ANJ1 barc1370 gwm113 gwm4136 gwm187 BE4430718 HMW7+8/17+1811 wPt-070512 gwm153gwm27417 wPt-903221 gwm26822 wPt-094438 wmc4447 psp310053 gwm25960 Spark x Rialto – 1B

14. 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

15. 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

16. Use of SSCP-SNP as COS marker RiceMaizeRyeBarleyBrachypodium 58%39%94% 74% Wheat primer tested across species (32) Wheat RiceMaizeRyeBarleyBrachypodium Polyacrylamide gel

17. 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%

18. Summary Easy to develop and easy to use Highly transferable Target genic regions Much more informative for comparative genetics – synteny definition

19. Acknowledgements Mike Gale John Snape Wheat Pauline Stephenson Michelle Leverington-Waite Yingkun Wang James Simmonds