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

[223556]0.0FGX_OWB00455 bPb-0611 FGX_OWB00196 bPb-0516 1_1407[222367] 3_1446 bPb-7151 1_0970 FGX_OWB00098bPb-3598 bPb-0485 bPb-3608 [220862] bPb-7892bPb-8959 FGX_OWB00453 FGX_OWB00441 bPb-5191

1.3

FGX_OWB004631.9FGX_OWB004612.52_1466 ABG0583.7scind02622 1_0718 [222211]5.02_14156.2FGX_OWB00147 bPb-0205 1_1030 FGX_OWB00005 [223937]bPb-7969 bPb-5033 bPb-5900 FGX_OWB00496 bPb-04717.4bPb-7557 bPb-5519 bPb-5688 bPb-7024 [223539]bPb-0615 [221144]9.91_0230 1_105912.4[223312] FGX_OWB0013813.7bPb-5444 bPb-9220 ABG008 3_0775 3_0155[221686] bPb-4295 bPb-4481 3_0631 bPb-1708[220974] bPb-6897 bPb-0715 FGX_OWB00061 3_0402bPb-8399 bPb-6128 FGX_OWB00263 FGX_OWB00403

14.9

[104949] [222714] 2_1423 bPb-2782 bPb-6494bPb-8986 bPb-2355 [222296] [221518] bPb-2108bPb-4715 bPb-8374 bPb-6879

16.1

FGX_OWB00012 bPb-8292 2_0724 FGX_OWB0050717.41_104019.9

scssr10226 bPb-4261 2_1261 bPb-4875 bPb-968242.72_059343.9scssr07759 1_0307 1_0891 bPb-3190 bPb-79062_0368 bPb-250145.12_1049 FGX_OWB0048046.41_1304 GBM106647.6FGX_OWB00289 FGX_OWB00412 1_0847 FGX_OWB0007348.8FGX_OWB00129 FGX_OWB00436 2_1461 3_0420 1_0342bPb-0722 1_0919 1_0837 3_0657 bPb-22301_0525 2_0326 1_0178 1_0296 Pox1_1057 FGX_OWB00522 FGX_OWB00483 FGX_OWB00363

51.3

1_0084 1_149352.6FGX_OWB00118 1_0715 3_0379 3_0029 FGX_OWB00324FGX_OWB0025553.83_0491 1_048655.01_1340 2_0281 1_020456.3FGX_OWB00338 bPb-5057 bPb-9925 1_0498 1_1505FGX_OWB0028758.8FGX_OWB0020159.52_0674 3_0691 3_0604 2_0688 2_10051_0156 bPb-0220 1_1428 [223952]60.0scssr03381 [221628] 1_1302 2_0929 1_06381_1131 [222438] 1_042261.21_1522 1_1400 3_147463.7FGX_OWB00505 2_0917 2_0748 bPb-3056 2_02341_0147 1_0927 2_0387 3_0195 bPb-6438bPb-6881 2_0235 3_0251 2_0602 FGX_OWB00419

66.2

3_0209 Hot1 1_0558 3_0487 2_06432_0004 3_055767.5FGX_OWB0018968.1scssr02236 [220842] Ebmac0684 3_0853 1_09482_0032 1_0474 2_0878 1_0154 1_0330bPb-1836 2_0500 2_0378 3_0206 1_12883_1218 2_1434 2_1337 3_0634 [220925]1_1155 2_0039 2_1286 1_0947 1_04853_0068 2_0458 2_0417 2_0196 1_12111_1369 3_0772 2_0181 3_1189 1_00991_0883 [222433] FGX_OWB00346 FGX_OWB00107 FGX_OWB00439FGX_OWB00220 FGX_OWB00175 FGX_OWB00121

68.7

1_138469.9FGX_OWB00401 FGX_OWB00280 2_033571.2FGX_OWB0012271.6FGX_OWB0010072.0scssr00334 3_024572.4FGX_OWB00313 FGX_OWB00072 1_0436 1_0640 1_0632FGX_OWB0036573.62_0374 1_1430 3_1252 2_0442 2_14761_1316 3_1256 ABG35674.9bPb-4837 GBM1023 1_1347 3_1307 2_10941_1072 bPb-6437 bPb-0885 bPb-725676.1FGX_OWB00227 bPb-4040 bPb-5440 [222771] 1_1096bPb-9754 bPb-608878.63_0205 3_1021 1_0407 bPb-8100 bPb-72432_083382.42_0528 2_066783.61_1402 FGX_OWB0000384.93_1394 FGX_OWB0007986.13_017888.6scsnp03343 [222968]91.11_1388 3_144592.31_0786 1_085994.81_110097.3FGX_OWB00337 3_0900 1_1533 3_0897 2_03403_0901 vrs198.61_0936102.41_0969 2_1351103.62_0793 FGX_OWB00508 FGX_OWB00026 Bmag0125104.8DsT-41108.6FGX_OWB00273109.9MWG503112.4bPb-3563 3_0216 bPb-7991 1_1307 bPb-6194bPb-4835 2_1007 1_1285 bPb-1926 GBM1062FGX_OWB00074 FGX_OWB00022

113.6

KFP203114.82_0923 MWG882A 1_0398 3_0200 FGX_OWB00526117.32_1527 1_1466 1_0900118.63_0095 3_0480119.83_0049 bPb-8737 bPb-3653 3_0555 1_1323bPb-4577 bPb-1772122.31_1094 bPb-6822 FGX_OWB00464126.1FGX_OWB00183 FGX_OWB00047 FGX_OWB00209 2_1238 1_0263bPb-3870 3_1402 bPb-8274 1_1043 bPb-24812_0064 1_0990 1_0989 1_0128 FGX_OWB002992_0955 FGX_OWB00060 ABG072 [223776]

132.6

1_1236 1_0707 3_0459 1_0429 2_0989FGX_OWB00055133.81_0739 FGX_OWB00173137.61_0780 3_1406140.1bPb-1266 bPb-8949141.41_1365 bPb-7992 3_0097 bPb-0541 3_01522_0141 [221435] 2_1315 bPb-6688143.93_1100 1_0109 2_1459 [222960] 1_14863_0695 3_0041 1_0446 3_0310 2_1406FGX_OWB00343 Ebmc0415 FGX_OWB00238 FGX_OWB00123 FGX_OWB00181

146.4

cnx1 bPb-2971 bPb-5412 FGX_OWB00011147.6[222464]148.82_0215 1_0065 2_1125 2_1336 1_03831_0472 bPb-3858 3_0678151.31_1227 FGX_OWB00198153.8bPb-1505 [221983] 3_0106 3_0396155.1Zeo1156.33_0248158.82_0590 2_1181160.1[223553]168.0FGX_OWB00152 bPb-0303 1_0315 bPb-4092 1_0566bPb-1986 1_1023 bPb-5619 bPb-1184 bPb-5460[221407] bPb-1815 bPb-7208 bPb-8530 bPb-1566GBM1019 FGX_OWB00229

169.2

FGX_OWB00377 2_0943170.43_0823 FGX_OWB00099 Aglu4 FGX_OWB00322 FGX_OWB00132Aglu5 FGX_OWB00312171.71_0937 3_1300 MWG720174.22_0957175.4bPb-1181 bPb-1611 bPb-4232 bPb-4601 bPb-7212FGX_OWB00004 FGX_OWB00294177.9GBM1012 1_0329180.4FGX_OWB00207 1_0085 bPb-6048 bPb-1085 bPb-62961_1050 bPb-3102 bPb-8698 [223337] bPb-0305bPb-9419

182.9

2_1414 FGX_OWB00308184.12_1496185.4FGX_OWB00293186.6FGX_OWB00471 scssr08447 3_0102 MWG949A FGX_OWB00408187.9

2H

MORPHOLOGICAL

RFLP

SSR

STSDArT

SNPRAD

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 orGene/Marker Diversity

Outbred population• H estimates the probability that a randomly sampled

individual is heterozygousInbred population• H estimates the probability that a randomly sampled

pair of lines are homozygous for different alleles

pi is the frequency of the ith allelek is the No. of alleles

(Nei 1973)

k

iipH

1

21

The Hardy-Weinberg Principle

AA Aa Aa aa

p1 (freq. of A) = 0.5p2 (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 + p2

2 = 1.00

freq. of homozygotes = p12 + p2

2

freq. of heterozygotes = 1 - (p12 + p2

2)

k

iipH

1

21

Examples

ABO alleles in humansIf p1 = 0.28 , p2 = 0.06 , and p3 = 0.66

H = 1 – (0.282 + 0.062 + 0.662) = 0.48

SSR markers in sunflowerIf 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

Heterozygosity for a genetic marker with k equally frequent

alleles

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

p2 0.05 0.05 0.05 0.02 0.50 0.33 0.25 0.20

p3 0.05 0.05 0.02 0.34 0.25 0.20

p4 0.05 0.02 0.25 0.20

p5 0.02 0.20

H0.0 0.10 0.19 0.27 0.15 0.50 0.67 0.75 0.80

Polymorphism Information Content (PIC)

• 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

1

1 1

221

1 1

22

1

2 221k

i

k

ijji

k

i

k

ijji

k

ii ppHpppPIC

pi is the frequency of the ith allelepj is the frequency of the jth allelek is the No. of alleles

(Botstein et al. 1980)

H vs. PIC

Heterozygocity and PIC for a genetic marker with k equally

frequent alleles

Mean Heterozygosity

n

jj nHH

1

/ Hj is the heterozygocity of the jth markersn is the No. of genetic markers

Mean Heterozygosity• HT mean heterozygocity for polymorphic and

monomorphic markers • Hp mean heterozygocity for polymorphic markers

HHT

pp n

jpj

n

jpj

mp

pT nHpnH

nnn

H11

//Hj is the heterozygocity of

the jth markersnp is the No. of

polymorphic markersnm is the No. of monomorphic

markers

When averaging over polymorphic and non-polymorphic markers

Mean number of polymorphic genetic markers per assay

• Product of the mean number of bands per assay and mean heterozygocity

T

b

aaT HmbmHP

)/(1

ma is the No. of bands produced by ath assayb is the No. of assays

m is the mean No. of bands per assayHT is the mean heterozygosity