Transcript Slide 1
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 blastn 650 Pearl millet EST show good homology with rice (34%) Select single copy genes 299 Pearl millet EST homologous to single copy rice gene (16%) RiceGAAS 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. Define intron/exon boundaries in pearl millet EST design primer across intron Test polymorphism on SSCP gel 102 markers polymorphic (34%) Done in silico Marker assay design 164 bp F primer 308 bp PREDICTED INTRON Pearl Millet EST CD726515 Rice genomic DNA BAC AP005071 314 bp R primer 497 bp 70859 bp 71002 bp EXON 70844bp 71089 bp INTRON 71002 bp 71085 bp F primer Intron R primer 71269 bp EXON 71282 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) 1 2 3 4 5 6 Exon 50 bp Intron 65 22 bp 45 bp 255 bp Exon 37 bp bp 1: ICMP 451 2: 81B 3: 841B 4: 863B 5: PT 732B 6: P1449 -2 7: ICMP 85410 8: LGD 1 --B-10 B-10 7 8 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 Chinese Spring Cadenza Avalon Rialto Spark Synthetic Opata Nulli -3D Nulli -3B Nulli -3A Chinese Spring Cadenza Avalon Rialto Spark Synthetic Opata Nulli -5D Nulli -5B Nulli -5A Wheat marker screening A B D D A B A B D contig 1430.5 int6 A BE496976 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 BE500570 Rice 131 80 96 466 121 417 942 472 679 89 intron size 196 Wheat intron size 100 87 230 ? 79 150 90-115 7-900 130 60-120 80 ~800 72 4-900 83 6-900 67 135-240 100-140 405 Wheat mapping data Spark x Rialto – 2A 0 wmc407 19 22 27 stm8acag gwm636 gwm210(2) 44 psp3153 56 gwm359 81 gwm275 gwm95 gwm515 102 107 117 122 123 124 125 129 131 134 135 gwm294 gwm312 gwm349 wPt-6894 gwm356 BE444894 wmc181 gwm382 gwm311 wPt-5887 barc122 BE444894 cysteine proteinase precursor Wheat mapping data Spark x Rialto – 1B 0 barc137 3 6 7 8 11 12 gwm11 gwm413 gwm18 BE443071 HMW7+8/17+18 wPt-0705 17 gwm153 gwm274 21 22 wPt-9032 gwm268 38 wPt-0944 47 wmc44 53 psp3100 60 gwm259 BE444071 : DNAJ protein homolog ANJ1 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 F primer Intron R primer Pile up EST from different species in order to develop primer in conserved region of genes F primer F millet primer INTRON R primer R millet primer Brachypodium Barley Rye Maize Rice Wheat Use of SSCP-SNP as COS marker Wheat primer tested across species (32) Polyacrylamide gel Rice Maize Rye Barley Brachypodium 58% 39% 94% 94% 74% SSCP-SNP compared to SSR • How polymorphic are SSCP-SNPs compared to SSR? Opata/ synthetic Spark/ Rialto Avalon/ Cadenza CS/ SQ1 Trintilla/ Piko SSCP-SNP 19% 34% 32% 28% 9% SSR 42% 46% 53% - 50% • 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 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