p 2 - Barley World

Report
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
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97.3
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102.4
103.6
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108.6
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112.4
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117.3
118.6
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122.3
126.1
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133.8
137.6
140.1
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143.9
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168.0
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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

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