1. Institute of Crop Sciences, CAAS
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

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
凝胶时代：
限制性酶切片段长度多态性分析(RFLP)
寡核苷酸连接分析(OLA)
等位基因特异聚合酶链反应分析(ASPCR)
单链构象多态性分析(SSCP)
变性梯度凝胶电泳分析(DGGE)
高通量时代：
特异位点杂交(ASH)
特异位点引物延伸(ASPE)
特异位点切割(ASC)
特异位点连接(ASL)
单碱基延伸(SBCE)

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
Wheat accessions
Screening 4x660K
by Affymetrix
2nd generation data
Producing 4x660K
mapping
Test 4x660k array vs 192
wheat accessions
Genome-specific SNP
Wheat660
Screening 10x660K

9. Total data: 6+Tb De novo, resequence, transcriptome and GBS
Species
accessions
Genome
Data type
Data 1
T. urartu
G1812
UR203
UR206 AA
resequencing
170x 2
Ae. tauschii
AL8/78(Y2282)
Y2280 DD
92x 3
T. turgidum
LDN
PS4
AABB 5x 4
T. aestivum
AK58
32 accessions
78 accessions
AABBDD
De novo, transcriptome
and GBS sequencing
272x
500G
20x
Total data: 6+Tb
Common wheat varieties come from world wide

10. SNP analysis Total 51,380,485 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 wheat genes
SNPs in Tandem repeat：1,516,247
SNPs in TE： 37,945,716
23 million candidate SNPs to Affymetrix for further screening

11. Chromosome distribution of 51 millions SNPs1A
3,037,061 1B
2,527,422 1D
1,295,018 2A
4,213,482 2B
4,125,640 2D
998,762 3A
2,532,902 3B
5,055,792 3D
743,180 4A
3,443,133 4B
1,953,790 4D
1,071,141 5A
3,029,073 5B
3,298,191 5D
1,296,462 6A
2,559,818 6B
2,856,559 6D
1,194,569 7A
2,724,036 7B
2,083,129 7D
1,341,325
subtotal
21,539,505
21,900,523
7,940,457
total
51,380,485 %
41.9%
42.6%
15.5%

12. Procedure of Weat660 development
Wheat accessions
4x660K
by Affymetrix
2nd generation data
Producing 4x660K
mapping
Screening 4x660k array vs 192
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 array with 630,517 SNPs

15. Auto redding

16. Distribution on genome evenly
homoeologous group　
A genome
B genome
D genome
wheat660
90Kchip 1
30,978
2,072
24,199
3,471
23,966
1,024 2
31,371
2,090
39,062
3,250
28,228
1,491 3
20,176
1,800
50,067
2,491
19,779
883 4
28,068
1,928
15,159
1,444
17,036
296 5
18,209
2,454
38,075
2,964
27,007
1,004 6
20,147
2,133
26,178
2,528
20,870
571 7
30,226
2,662
18,750
2,461
31,570
1,250
subtotal
179,175
15,139
211,490
18,609
168,456
6,519 %
32.0%
37.6%
37.8%
46.2
30.1%
16.2
我将4个群体能够做上的标记汇总后总共为359811个，没有显示度。但和上述数字整合有难度，主要是去除冗余有难度。
Genetic density : 13 SNP/cM, Physical density: 3 SNP/100K

17. Cover almost genes of wheat
Containing 630,517 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 % % %
密码子相位（Phase ）：指SNP发生在三联体密码子的第几位

18. Functional SNPs in Wheat660
Gene
SNP of W660K
LR1 AX
Lr10 AX AX AX
Lr21 AX
Lr34 AX AX AX
Pm3-B AX AX AX
Pm6 AX AX AX
Sr33 AX AX AX
Sr35 AX AX AX
TaPHS AX AX AX
Ppd-D1 AX AX AX
VRN1 AX AX AX
VRN2 AX AX AX
VRN3 AX AX Q AX AX AX
密码子相位（Phase ）：指SNP发生在三联体密码子的第几位

19. 820 SNPs 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 Axiom Wheat660K 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
Jing_DH
Xia_DH
Zhang_RIL
Wang_RIL
Population type DH
RIL13
RIL10
#of individuals
141
181
182
170
#SNP
121,681
91,260
137,512
95,552
%HighPolymorphic SNP
19.3%
14.5%
21.8%
15.2%
Genetic density(SNP/cM) 29 22 33 23
Physical density (SNP/100k)
0.7
0.5
0.8
0.6
各图图距汇总
合计 名称 Jing zhang Wang xia 1a b d a b d a b d a b d a b d a b d a b d

25. Comparison genetic map between the Wheat660 maps and the international wheat genetic maps

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 1
log 5 25
125
625

34. 1
log 5 25
125
625

35. 1
log 5 25
125
625

36. BIN
#SNP
#in Gene
#Out Gene
% In Gene
Bin77
437
108
329
24.7%
Bin90
541
111
430
20.5%
Bin119
401 51
350
12.7%
allBin
27107
3438
23669

37. Outlines Axiom Wheat660K 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. 44 SNP 11 mapped on 2A, 6 on 58-59 cM region
Pool1
916
Pool4
422
Pool3
478
Pool2
917
11 mapped on 2A, on cM region
33 mapped on 2D, mapped on cM region

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

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