Development and Application of Axiom Wheat660K: A High-Density SNP Array

1. Development and application of Axiom Wheat660K, a high density SNP array Jizeng Jia Institute of Crop Sciences, CAAS  IWC 2015, Sydney

2. Background • Wheat was a leading crop on research in the world before rice genome sequenced • Wheat research lags behind rice after rice sequenced in 2001 Rice wheat wheat Rice 1985-2000 2001-2014 2001-2014

3. Background • Two major reasons resulted in this situation • Difficult to get gene: Shortage of genome sequence and fine mapping information • Difficult to confirm the gene function: Very low ratio of transformation

4. JT has made the breakthrough for wheat transformation ,and we introduced their techniques and the transformation efficiency reaches to 40% • We report here the development of Wheat660, a high density SNP array, which will accelerate the breakthrough of gene discovery and genomic breeding in wheat

5. SNP-the third generation molecular marker • First generation molecular marker:RFLP, its application lasted for about 5 years only • Second generation molecular marker:SSR, lasted for more than 20 years • SNP is the third generation marker, with advantage of huge number of loci, high throughput, it should be lasted much more longer

6. Outlines • Axiom Wheat660K development • Axiom Wheat660K application

7. Affy techniques can produce SNP array with a density SNP more than 600K/slides • However, the high density can be generated in diploid species only • Wheat is hexaploid, in order to develop the high density array, we developed a new strategy: select SNPs with diploid characters

8. Procedure of Weat660 development Screening 4x660K by Affymetrix Wheat accessions 2nd generation data Producing 4x660K mapping Test 4x660k array vs192 wheat accessions Genome-specific SNP Wheat660 Screening 10x660K

9. De novo, resequence, transcriptome and GBS Common wheat varieties come from world wide

10. SNPanalysis • Total51,380,485 SNPs discovered • SNPs outside of genes:50,375,584 • SNPs in genes: 1,004,901 • #SNP in exon: 589,236 • #SNP in inron: 415,665 • From all 99,386 wheatgenes • SNPs in Tandem repeat：1,516,247 • SNPs in TE： 37,945,716 • 23 million candidateSNPs to Affymetrix for further screening

11. Chromosome distribution of 51 millions SNPs

12. Procedure of Weat660 development 4x660K by Affymetrix Wheat accessions 2nd generation data Producing 4x660K mapping Screening 4x660k array vs192 wheat accessions Genome-specific SNP Wheat660K Screening 10x660K

13. X X

14. Wheat660 has good performance • The highest density array in polyploid • Genome-specific SNPs • “Diploidization” • A single arraywith 630,517 SNPs

15. Auto redding

16. Distribution on genome evenly Genetic density : 13 SNP/cM, Physical density: 3 SNP/100K

17. Cover almost genes of wheat • Containing630,517 SNPs • #SNPs out of genes: 503,173 • #SNPs in genes: 106,587 in 99,386 genes • One gene One SNP , every gene has a SNP in wheat, almost every gene has a functional marker • SNPs in exon: 63,604 • SNPs in inron: 43,059 Stop codon : 1247 Phase 1 14689 23.2% 2 12035 19.0% 3 36726 57.8%

18. Functional SNPsin Wheat660

19. 820SNPs on pathway of Oxidative phosphorylation

20. 702 SNPs on pathway of ribosome（translation）

21. 339 SNPs in pathway annotation : carbon metabolism

22. Wheat660 has good performance • High density • Call rate: 97% • Auto reading • Genotypin • PolyHighResolution: 19.3%, 14.5%, 21.8%and 24% respectively

23. Outlines • AxiomWheat660K development • Axiom Wheat660K application • Construction of high density of wheat genetic maps • Construction of Wheat Hapmap • Gene detection

24. Construction of fine genetic maps by use of Wheat660

25. Comparison genetic map between the Wheat660 maps and the international wheat genetic maps Internat I Na l Wheat660

33. SNP distribution on genetic maps of common wheat • 550 common wheat varieties were genotyped with Wheat660 • The SNP distribution evenly in general, however, super cluster were discovered in each chromosome log 625 125 25 5 1

34. log 625 125 25 5 1

35. log 625 125 25 5 1

37. Outlines • AxiomWheat660K development • Axiom Wheat660K application • Construction of high density of wheat genetic maps • Fine mapping of interesting genes

38. Gene detection • Natural population: 800 accessions • GMAIL (genome wide multiple allele introgression lines) population

39. Fine mapping of the heading date genes Gene fine mapping by using pooling strategy Heading date gene Segregation population: Am3/L953*6F4

40. 11 mapped on 2A, 6 on 58-59 cM region • 33 mapped on 2D, 9 mapped on 64-65 cM region Pool2 917 Pool1 916 Pool4 422 Pool3 478 44 SNP

41. Fine mapping of the plant height genes • 12 common SNP among 4 pools • 8 mapped on 2D, 6 4 located on 64-66 cM region Pool2 917 Pool1 916 Pool4 422 Pool3 478 12 SNP

42. Summary • We developed a highest density and efficient wheat SNP array, Wheat660k • Wheat660k cover almost wheat genes • Highest density genetic maps were constructed • Chromosomal inversions of some synthetic wheat were revealed • There are very low efficient recombination regions in wheat • Wheat 660K is an efficient tool for gene fine mapping

43. Works on the way • Construction of wheat HapMap • Fine mapping and cloning of genes conferring agronomic important traits • Development of Wheat50k for wheat genomic breeding

44. Acknowledge Aimin Zhang Ruilian Jing Zhonghu He Jirui Wang Guangyao Zhao Xiuxing Kong Lifeng Gao Dangqun Cui Zhengang Ru Affymetrix CapitalBio 973 Project