http://cbsu.tc.cornell.edu/lab/doc/GBS_Method_Overview1.pdf
RADseq: Restriction-site Associated DNA markers
• Uses Illumina sequencing technology
• Based on digestion with restriction enzymes. An adapter binds to the restriction site
and up to 5kb fragments are sequenced around the target size.
• Bioinformatics work used to find SNPs on the amplified regions
72.4
73.6
74.9
76.1
78.6
82.4
83.6
84.9
86.1
88.6
91.1
92.3
94.8
97.3
98.6
102.4
103.6
104.8
108.6
109.9
112.4
113.6
114.8
117.3
118.6
119.8
122.3
126.1
132.6
133.8
137.6
140.1
141.4
143.9
146.4
147.6
148.8
151.3
153.8
155.1
156.3
158.8
160.1
168.0
169.2
170.4
171.7
scssr00334 3_0245
FGX_OWB00313 FGX_OWB00072 1_0436 1_0640 1_0632
FGX_OWB00365
2_0374 1_1430 3_1252 2_0442 2_1476
1_1316 3_1256 ABG356
bPb-4837 GBM1023 1_1347 3_1307 2_1094
1_1072 bPb-6437 bPb-0885 bPb-7256
FGX_OWB00227 bPb-4040 bPb-5440 [222771] 1_1096
bPb-9754 bPb-6088
3_0205 3_1021 1_0407 bPb-8100 bPb-7243
2_0833
2_0528 2_0667
1_1402 FGX_OWB00003
3_1394 FGX_OWB00079
3_0178
scsnp03343 [222968]
1_1388 3_1445
1_0786 1_0859
1_1100
FGX_OWB00337 3_0900 1_1533 3_0897 2_0340
3_0901 vrs1
1_0936
1_0969 2_1351
2_0793 FGX_OWB00508 FGX_OWB00026 Bmag0125
DsT-41
FGX_OWB00273
MWG503
bPb-3563 3_0216 bPb-7991 1_1307 bPb-6194
bPb-4835 2_1007 1_1285 bPb-1926 GBM1062
FGX_OWB00074 FGX_OWB00022
KFP203
2_0923 MWG882A 1_0398 3_0200 FGX_OWB00526
2_1527 1_1466 1_0900
3_0095 3_0480
3_0049 bPb-8737 bPb-3653 3_0555 1_1323
bPb-4577 bPb-1772
1_1094 bPb-6822 FGX_OWB00464
FGX_OWB00183 FGX_OWB00047 FGX_OWB00209 2_1238 1_0263
bPb-3870 3_1402 bPb-8274 1_1043 bPb-2481
2_0064 1_0990 1_0989 1_0128 FGX_OWB00299
2_0955 FGX_OWB00060 ABG072 [223776]
1_1236 1_0707 3_0459 1_0429 2_0989
FGX_OWB00055
1_0739 FGX_OWB00173
1_0780 3_1406
bPb-1266 bPb-8949
1_1365 bPb-7992 3_0097 bPb-0541 3_0152
2_0141 [221435] 2_1315 bPb-6688
3_1100 1_0109 2_1459 [222960] 1_1486
3_0695 3_0041 1_0446 3_0310 2_1406
FGX_OWB00343 Ebmc0415 FGX_OWB00238 FGX_OWB00123 FGX_OWB00181
cnx1 bPb-2971 bPb-5412 FGX_OWB00011
[222464]
2_0215 1_0065 2_1125 2_1336 1_0383
1_0472 bPb-3858 3_0678
1_1227 FGX_OWB00198
bPb-1505 [221983] 3_0106 3_0396
Zeo1
3_0248
2_0590 2_1181
[223553]
FGX_OWB00152 bPb-0303 1_0315 bPb-4092 1_0566
bPb-1986 1_1023 bPb-5619 bPb-1184 bPb-5460
[221407] bPb-1815 bPb-7208 bPb-8530 bPb-1566
GBM1019 FGX_OWB00229
FGX_OWB00377 2_0943
3_0823 FGX_OWB00099 Aglu4 FGX_OWB00322 FGX_OWB00132
Aglu5 FGX_OWB00312
MORPHOLOGICAL
RFLP
SSR
STS
DArT
SNP
RAD
Marker Usefulness
• Informativeness - measured by the number of
alleles and allele frequencies
– heterozygosity (H)
– polymorphic information content (PIC)
• Throughput - multiplex ratio (number of
simultaneously assayed loci)
• Utility
– reproducibility of the marker assay
– clarity of the marker genotypes
Heterozygosity or
Gene/Marker Diversity
k
H  1  p
i 1
2
i
pi is the frequency of the ith allele
k is the No. of alleles
(Nei 1973)
Outbred population
• H estimates the probability that a randomly sampled
individual is heterozygous
Inbred population
• H estimates the probability that a randomly sampled
pair of lines are homozygous for different alleles
The Hardy-Weinberg Principle
AA
Aa
Aa
aa
p1 (freq. of A) = 0.5
p2 (freq of a) = 0.5
p12 (freq. of AA) = 0.25
p22 (freq. of aa) = 0.25
2 p1 p2 (freq. of Aa) = 0.50
p12 + 2 p1 p2 + p22 = 1.00
freq. of homozygotes = p12 + p22
freq. of heterozygotes = 1 - (p12 + p22)
k
H  1  p
i 1
2
i
Examples
ABO alleles in humans
If p1 = 0.28 , p2 = 0.06 , and p3 = 0.66
H = 1 – (0.282 + 0.062 + 0.662) = 0.48
SSR markers in sunflower
If p1 = 0.13 , p2 = 0.19 , p3 = 0.62 , and p4 = 0.06
H = 1 – (0.132 + 0.192 + 0.622 + 0.062) = 0.56
Heterozygosity
No. Alleles
Alleles
1
2
3
4
5
2
3
4
5
p1
1.
0
0.95
0.90
0.85
0.92
0.50
0.33
0.25
0.20
0.05
0.05
0.05
0.02
0.50
0.33
0.25
0.20
0.05
0.05
0.02
0.34
0.25
0.20
0.05
0.02
0.25
0.20
p2
p3
p4
p5
Heterozygosity for a genetic
marker with k equally frequent
alleles
H
0.02
0.
0
0.10
0.19
0.27
0.15
0.20
0.50
0.67
0.75
0.80
Polymorphism Information Content (PIC)
k 1
k
k 1
k
k
PIC  1   p    2 p p  H    2 p p
i 1
2
i
i 1 j i 1
2
i
2
j
i 1 j i 1
(Botstein et al. 1980)
2
i
2
j
pi is the frequency of the ith
allele
pj is the frequency of the jth
allele
k is the No. of alleles
• A measure of the informativeness of a genetic
marker for linkage analysis
• Probability that a marker will be polymorphic
in a set of untested genotypes
H vs. PIC
Heterozygocity and PIC for a
genetic marker with k equally
frequent alleles
Mean Heterozygosity
n
H  Hj /n
j 1
Hj is the heterozygocity of the jth markers
n is the No. of genetic markers
Mean Heterozygosity
• HT mean heterozygocity for polymorphic and
monomorphic markers
• Hp mean heterozygocity for polymorphic markers
HT 
np
n p  nm
np
H
j 1
np
j
/ n p  p H j / n p
j 1
Hj is the heterozygocity of
the jth markers
np is the No. of
polymorphic markers
nm is the No. of monomorphic
markers
HT  H
When averaging over polymorphic and nonpolymorphic markers
Mean number of polymorphic genetic
markers per assay
b
P  HT ( ma / b)  m H T
a 1
ma is the No. of bands produced by ath assay
b is the No. of assays
m is the mean No. of bands per assay
HT is the mean heterozygosity
• Product of the mean number of bands per
assay and mean heterozygocity