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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