Experimental validation, integration to the linkage map and
gene flow pilot study in common bean using
Single Nucleotide Polymorphisms
e
(SN )
NPs)
NP
Constanz Quintero
za
MSc. Pl t Genetic R
MS Plant Ge ti Resources
Universidad Nac
cional de Colombia

Con
ntents
Introduction
Objectives
Subjects
1. Experimental valid
dation of SNPs
2. Genetic map saturation
3. Gene flow pilot study
p y
Conclusions

Introduction (cont…
SNP
AGGCGGCGTAGCTTTGCAGTACA
AGGCGGCGTAG
GCTTTGCAGTACA
AGGCGGCGTAGCTTTGTAGTACA
AGGCGGCGTAG
GCTTTGTAGTACA
SNP discovery in Phaseolus vulgaris L. Glycine max L. Merrill.
CIAT-Gaitán-Solís et.al. 2008. Soybean Genomics Lab-USDA
Perry Cregan,
y g ,
Direct sequencing of coding and non-
-
coding regions (300-1000bp) Resequencing soybean unigenes in
10 genotypes Leguminosae species.
223 SNPs found in 20964pb 174 SNPs primer trios
trios.
25 primer trios

Objectives
General: To assess the potential of SNP markers for diversity, gene flow
and phenotype-genotype association studies in P. vulgaris.
Specific:
To validate available SNP markers identified in common bean and
s
soybean.
To integrate validated SNPs to th existing linkage map (DOR364/
he
G19883).
G19883)
To study the relationship between s some phenotypic characteristics and
individual SNP alleles or haplotypes.
To characterize the genetic diversity of a wild-weedy-crop complex from
y
Colombian and determine whether S SNPs could provide evidence of gene
dynamics i P vulgaris.
flow d
fl i in P. l i

1. SNP Validation
Plant material:
GNumber or ID Biological status
s Origin Genepool
DOR364 Cultivated-Bred Lin
ne M
G19833 Cultivated-Landrac
ce Perú A
G23421 Wild Perú A
G5773 Cultivated-Bred Lin
ne M
G4494 Cultivated-Bred Lin
ne A
G2771 Wild México M
G4342 Cultivated-Landrac
ce México M
G16110 Cultivated Perú A
G23432 Wild México M
G23422 Wild Perú A
130 soybean-derived SNP mar
y rkers

SNP GENOTYPING: Single Base Extension (SBE) in flow cytometer Luminex100
Two to three fragments
DNA template amplified per reaction
PCR mutiplex PCR number decreased
in 30%, due to multiplex
130 SNP-containing fragments
adjustment
dj t t
Pool of PCR-multiplex,
same SNP type
Up to 15 SNP loci screened in one SBE reaction
d
A
Allele A
Mean Fluorescenc Intensity
A A A A
2,000 A A
1,500 A A
A
1,000 A
500 92 SNPs
ce
SBE multiplex 0
464 938 526 691 976 709 791 79
93 799 858 893 497 523 527
G
578 validated as true
1,200 Allele G G polymorphisms
1,000
800 G
600
G
n
400
200
0
464 938 526 691 976 709 791 793
3 799 858 893 497 523 527 578
SN
NP

2. Integration of SN to the linkage map
NPs
2 0cM
B ng0 4 5 G 1 0 01 D GC10 E 0 71 G F 08 2 G M9DB1D
B ng0 8 3 S NP 3 4 1 B ng 0 2 3 S NP 7 9 1 L 04 5 G B ng0 9 1
S NP 9 3 8 S NP 1 7 4 S NP 6 0 7
A A1 9 8 G L 04 86 G
S NP 9 3 3 , S NP 5 1 0 S NP 1 9 C A S NP 2 6 T C
P S NP 8 0 5
S NP 1 8 9 B ng1 6 1 S NP 5 C A
S NP 2 8 A G S NP 6 6 2 B ng 0 7 1 b M130D
S NP 6 2 1 S NP 2 1 9 S NP 3 3 9
P S NP 2 6 5 S NP 4 5 5
B ng1 2 6 B ng0 3 2 S NP 1 7 TC A N0 3 4 D
S NP 7 5 8 S NP 6 3 7 S NP 6 0 9
S NP 2 4 C T AK 0 6 3 6 G GC09 S NP 4 7 8
S NP 7 9 3 D A3 9
3
S NP 3 0 1 K 1 21 D V1 0 3 G S NP 9 0 2
K 1 22 G A A1 9 3 D A N0 3 3 D S NP 8 G C
P
E 0 73 D U0 1 1 D
S NP 5 2 6
B ng 0 9 4 B ng0 5 2
S NP 2 8 2 S C AR 2 c D U1 0 0 2 D
S NP 5 2 7 S NP 5 9 1
P
K 1 23 D D A8 8 S NP 4 4 1 H2 0 3 D
S NP 9 9 2 L 04 97 D S NP 3 4 5 V1 0 4 D
G A2 6
P 1 60 1 D DC96a H1 9 1 G S NP 3 6 1
A0 1 6 D K 1 26 G
P9DB1D
Y 5 01 D S NP 2 7 G A
B ng0 0 3 b B ng0 2 4 S NP 2 0 G T
P1DB3G S NP 0 8 3
S NP 7 7 1 S NP 6 6 7 F 08 4 G H1 8 5 G
O 2 0 105 G
B ng1 0 8 S NP 2 9 8 S NP 4 6 2
Y 5 02 G S NP 9 5 7 S NP 8 3 1 G185D AS 8 .9 0 0 S NP 1 4 9
S NP 8 4 8 B ng0 2 5
B ng 0 2 7 B ng0 0 1
S NP 7 0 9 AG 1 3 0 1 D P1DB2D B ng0 0 5 B ng0 4 2 S NP 1 8 0
S NP 9 3 5 S NP 0 6 6
P B ng0 6 0 S C AR 1 a G S NP 2 6 7 H1 9 3 G
A1 4 1 D V1 0 8 D S NP 7 3 7 S NP 5 7 4 S NP 6 2 6 S NP 8 5 8
S NP 7 3 0 AK 0 6 4 4 D
S NP 5 8 8 S NP 5 9 3 A1 4 4 G S NP 2 7 6
B ng 0 4 6 S NP 1 6 2 S NP 0 3 8
M124D S NP 1 1 G T
S NP 2 0 1 K 1 28 1 G
G122G S NP 8 1 9
B ng2 0 4
D 1 80 1 D B ng0 4 9 S NP 8 4 7
P
S NP 9 0 8
P S NP 2 3 TC
S NP 8 5 6
AG 1 3 0 2 D O204G B ng0 7 1 S NP 8 5 0
P S NP 5 7 6
S NP 5 7 8 , S NP 7 8 5 S NP 3 3 7 , S NP 6 6 8
S NP 1 8 2 S NP 1 1 7 A A1 9 1 0 D
B ng0 4 8 S NP 6 9 1 D A1 1
S NP 0 1 5 AK 0 6 1 D
G A2 6 a S NP 5 8 3
B ng0 9 6 B ng1 2 9
S NP 5 0 9 D A6 2
S C AR 2 bD S W 1 2 .70 0 X 1 12 D S NP 0 8 4 Hin d4 3 4
D 0 54 G S NP 0 7 8
P H1 8 2 D
S NP 1 5 9
O 2 0 112 D W 2 03 D
H1 9 8 G S NP 3 0 5 , S NP 7 1 5 S NP 2 6 1
S NP 5 8 2
S NP 3 0 C T
S NP 2 0 4 S NP 0 5 5 L 04 7 D S NP 7 1 6
AD 1 8 0 1 G
S NP 5 0 1 , S NP 5 2 9 F 70 1 D A H1 7 4 G S NP 7 8 8
S NP 1 4 4 B ng1 6 0 S C AR 2 dD G A TS 1 1
GC07c B ng1 1 8 S NP 7 1 8
S NP 5 4 1 S NP 9 0 1 , S NP 5 0 0 S NP 0 9 1 , S NP 4 9 2
S NP 2 5 0 S NP 0 6 8
S NP 1 0 G T S NP 2 0 3 S NP 6 6 9 , S NP 4 9 4
S C AR 1 bD S NP 7 7 8 S NP 4 G C G A2 3
B ng1 1 7 S NP 8 2 2 B ng 0 8 0 c
S NP 7 9 9
G A TS 5 4 B 0 4 -b S NP 8 0 8 B ng 0 8 0 D 1 83 1
B ng0 4 7
D A5 9
H1 8 1 D B 1 1 -j
j
S NP 6 8 9 H1 9 6 2 D
G A TS 9 1
S NP 6 4 5 S NP 2 9 A T B ng 0 9 5 B ng0 2 6 B 0 9 -k
M 1 1 017 C
S NP 5 0 2 , S NP 5 2 1
S NP 2 9 G A B 0 8 -f DC87
P 1 01 D
S NP 6 1 6
B ng0 7 5 B 0 5 -e
S NP 0 4 5
S NP 8 2 4 AG 1 B 0 6 -g B 0 7 -a B 1 0 -i
S NP 0 4 4
S NP 1 5 G T
S NP 3 8 3 B ng0 1 1
B 0 1 -h
S NP 5 1 2
S NP 9 8 4
S NP 6 9 9
S NP 9 7 4
O207G
B ng0 1 2
B ng0 1 6
S NP 2 1 C T
135 added to the linkage map-Mapmaker
d
S NP 2 1 6
B 0 2 -d
G A2 1
S NP 5 3 9
S NP 1 5 3
S NP 5 2 3
Final map with 470 markers.
DC35
pr5 9 9 D
Several LG regions covered by SNPs
Gs
B 0 3 -c
SNP ord needed for haplotype inference
der

3.Gene flow pilot study. C Wee
W “G50879”
C
Colombia,
C Dept. Caldas
C
MEX
Wee
C
GTM C
C
CRI Wee Wee
COL
ECU Chloroplast haplotypes and SSR most
p p yp
frequent alleles were shared between wild
and cultivated types
PER
BLV
No clear discrimination between biological
forms at the DNA level could be observed
Wild-weedy-crop complexes
No gene flow direction could be determined
Source:URG-CIAT

Cont.. Gene flow pilot study.
1. Genotyping: 2. SNP haplotype inference (HAP)
20 SNPs (LGs B01 y B07), 3 blocks/LG
All polymorphic 2-12 haplotypes/block
(
(Total=43).
)
3. Gene flow estimation - Admixture coe
efficient (MY) (ADMIX1_0)
( ( )
SNP haplotype
MW1 S.D.2 distribution within and
0.58 0.12 between biological forms
Too high Parental Wild a Cultivated
and
MC3 S.D. S.D. values to be re-
-defined
0.42 0.16 Blocks with PIC≥0.7:
1 y 2 in B01;
1M
1 in B07
W: relative contribution from the wild parental population
2S.D: Standard deviation based on 1000 resampling events.
n
3M
C: relative contribution from the c
cultivated parental population

SNP haplotypes frequency in the complex G50879
100
W ild W eedy Cultivated
80 C Block 1 B01
1,
60
PIC = 0.70
40
W W
20
W
C W C
0
A B C D E F G H
Frequency (%)
100
80
Block 2, B01
PIC = 0.76
60
C W
40
W C
F
20
W
C C W C C C
0
A B C D E F G H I J K L
100
80
Block 1, B07
C PIC = 0.73
60
W
40
20
W C W
W C C
0
A B C D E F G H I J K
Haplotype v
variant

Re-definition of genetic diversity structu inside the wild-weedy-crop complex
ure
G500879
Parental “Truly” Wild
“Tr l ” Parental “Tr l ”c lti ated
“Truly”cultivated
Wild [Phenotype +haplotype] Cultivated [Phenotype +haplotype]
Admixture events
Wild SNP Haplotype
Cultivated SNP Haplotype
Hybrid
H b id populations
l ti
Mixed haplotypes
Wild-hybrid Weeedy Cultivated-hybrid
Ms 0,4669 4458
0,4 0,3237
DS 0,055 0436
0,0 0,0501
Mc 0,5281 5518
0,5 0,6766
DS 0,0588 043
0,0 0,0495
Mc/Ms 1,1 1,2 2,1
Probable
Geneflow No-preferential
bi-directional No-prefferential
bi-dire
ectional From cultivated
direction

Conclusions
A reproducible methodology fo simultaneous SNP genotyping
p gy or g yp g
could be adjusted using SBE met
thod in a Luminex100 platform.
Lessons learned on such multiplex SNP genotyping method were
p g yp g
useful to perform rapid mapping of validated SNPs, and were also
g
applied to other crops.
Description of LGs was increas
sed after the integration of SNPs
markers to linkage map of DOR3
364/G19833.

Conclusions
No association was found bet tween SNPs and the response to
isolates of ANT and ALS evalua
ated in the mapping population.
SNP haplotypes were inform mative enough to describe the
structure of the genetic diversit of Colombian germplasm.
ty
SNP markers provided evidence of gene flow dynamics in the
wild-weedy-crop complex G508 879, and allowed a more accurate
estimation of admixture coeficie
ents.

Acknowledgments
Ginés-Mera Fel
llowship Fund
FDRH-
-CIAT
Universidad Nacional de Col
lombia - Rigoberto Hidalgo
Soybean Genomics Lab USDA - Perry Cregan, Charles Quigley
CIAT-BRU CIAT-UURG CIAT-BEAN PROGRAM
Joe Tohme Rosa Goonzález Héctor F. Buendía
Eliana Gaitán Daniel De
ebouck Henry Terán
Myriam C. Duque Orlando Toro
o Carlos Jara
Janneth Gutiérrez

2. Integration of SN to the linkage map
NPs
8
87 RILs ( 9) ( eebe et a , 1998)
s (F9) (B e al, 998)
DOR 364 G19833
135 added to the linkage m (LOD ≥ 4)-Mapmaker
map
Final map with 470 markers.
s.
s

Introduction: Ph
haseolus vulgaris L.
Wild P. vulgaris
Diploid (2 22)
Di l id (2n=22)
Mainly autogamous
y g
Cultivated worldwide
High nutritional value
45% produced in Latin America
d d i L ti A

SNP GENOTYPING: Single Base Extension (SBE) in flow cytometer Luminex100
n

Results SNP validation
P
I. PCR validation II. SBE validation
Single PCR in DOR364 (130 SNPs) 20 PCR products pooled=20SNPs
Fragment size + optimal
Temp determined
PCR-multiplex groups defined
SNP 01 SNP 11 SNP 01
a a a
Reproducibility of PCR-multiplex SNP 10 SNP 20 SNP 20
DOR364 and G19833 confirmed
Reproducibility tested
Low vs high number pooled SBEs
PCR number products
decreased in
30%, due to
multiplex Luminex:
adjustment Mean Fluorescence Intensity
Masterplex GT
Threshold values defined,
Allele calling performed
10 genotypes
PCR 1 PCR 2 PCR 3 ... PCR n 92 SNPs validated as true polymorphisms
Same SNP
n SNPs n SNPs n SNPs n SNPs

Simultaneous SNP genotyping in DOR364
P
A
sity
Allele A A
A A
scence Intens
2,000
1,500 A
1,000
500
0
028 144 182 219
2 250 265 301 305 5CA
C
C
Mean Fluores
2,000 Allele C C
1,500
1,000
C
500
0
028 144 182 219
2 250 265 301 305 5CA
M
SNP
A
Allele A
cence Intensity
A A A A
A
2,000
2 000 A
1,500 A A
A
1,000
A
500
0
464 938 526 691 976 709 791 793 799 858 893 497 523 527 578
Mean Fluoresc
G
1,200 Allele G G
1,000
800
G
600
G
M
400
200
0
464 938 526 691 976 709 791 793 799 858 893 497 523 527 578
SNP