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Development of COS markers in grasses

This study aims to develop SSCP-SNP markers for easier alignment of maps across different crosses and species, define synteny, and target specific regions for marker design. The markers were tested on pearl millet and showed good homology with rice. The marker system was also successfully transferred to wheat, a polyploid species. The study highlights the potential of SSCP-SNP markers as a tool for marker-assisted breeding in various crops.

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Development of COS markers in grasses

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  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 blastn 650 Pearl millet EST show good homology with rice (34%) 299 Pearl millet EST homologous to single copy rice gene (16%) Select single copy genes Done in silico RiceGAAS Define intron/exon boundaries in pearl millet EST 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. design primer across intron Test polymorphism on SSCP gel 102 markers polymorphic (34%)

  5. Marker assay design 164 bp 308 bp 314 bp 497 bp R primer F primer PREDICTED INTRON Pearl Millet EST CD726515 70859 bp 71002 bp 71089 bp 71269 bp Rice genomic DNA BAC AP005071 EXON EXON INTRON 70844bp 71002 bp 71085 bp 71282 F primer Intron R primer

  6. 1 1 1 1 2 3 3 3 3 4 5 6 6 6 6 7 8 1: ICMP 451 2: 81B 3: 841B 4: 863B 5: PT 732B 6: P1449 7: ICMP 85410 8: LGD 1 B 10 Exon Exon 50 50 bp bp Intron Intron 65 65 bp bp 255 255 bp bp 22 22 bp bp 45 45 bp bp 1: ICMP 451 2: 81B 3: 841B Exon Exon 4: 863B 37 37 bp bp 5: PT 732B 6: P1449 - - 2 2 7: ICMP 85410 8: LGD 1 - - B - - 10 SSCP gel profiles and panel variety sequence data at Xpms30CD726044 • 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/ • Marker can be directly targeted to specific region of the genome • Rice sequence and gene annotation databaseshttp://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

  10. Chinese Spring Chinese Spring Cadenza Avalon Opata Spark Rialto Cadenza Opata Synthetic Spark Rialto Nulli -5B Avalon Synthetic Nulli -5A Nulli -3A Nulli -3B Nulli -3D Nulli -5D A B D D A B A B D contig 1430.5 int6 A BE488921 BE496976 Wheat marker screening • 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. 131 80 96 466 121 417 942 472 679 89 196 100 130 80 72 83 67 405 230 79 150 87 ? 90-115 7-900 60-120 ~800 4-900 6-900 135-240 100-140 Intron size comparison between rice and wheat BE500570 Rice intron size Wheat intron size

  12. Wheat mapping data Spark x Rialto – 2A 0 wmc407 19 stm8acag 22 gwm636 27 gwm210(2) 44 psp3153 56 gwm359 gwm275 gwm95 81 gwm515 102 gwm294 107 gwm312 117 gwm349 122 wPt-6894 123 gwm356 124 BE444894 125 wmc181 BE444894 cysteine proteinase precursor 129 gwm382 131 gwm311 134 wPt-5887 barc122 135

  13. Wheat mapping data Spark x Rialto – 1B 0 barc137 3 gwm11 6 gwm413 7 gwm18 8 BE443071 11 HMW7+8/17+18 12 wPt-0705 BE444071 : DNAJ protein homolog ANJ1 17 gwm153 gwm274 21 wPt-9032 22 gwm268 38 wPt-0944 47 wmc44 53 psp3100 60 gwm259

  14. Sequence homology in exon between pearl millet and rice is well conserved Sequence homology breaks down in introns Develop those markers to COS marker F primer Intron R primer Could SSCP-SNP be transferred to other crop?

  15. Pile up EST from different species in order to develop primer in conserved region of genes F primer R primer F millet primer INTRON R millet primer

  16. Use of SSCP-SNP as COS marker Brachypodium Rye Rice Barley Maize Wheat Wheat primer tested across species (32) 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

  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

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