Using&NGS*enabled&genetics&to&improve&marker ...4D | 171 5A | 149 5B | 220 5D | 224 6A | 184 6B |...

Using&NGS*enabled&genetics&to&improve&marker&selection&and&design&in&hexaploid&wheat&(Yr15)

Ricardo&H.&Ramírez*González

The&Genome&Analysis&Centre

Wheat&Yellow&Rust

• Puccinia striiformis.• Fungus.• Traditionally&controlled&by&

resistance&genes&(for&example,&Yr15).

• Yr15 locus is&an&introgressionfrom T.2dicoccoides

Segovia&VCereal&disease&http://www.hgca.com/minisite_manager.output/3625/3625/Cereal%20Disease%20Encyclopedia/Diseases/Yellow%20(Stripe)%20Rust.mspx?minisiteId=26


F 2 p

opul

atio

n Se

quen

cing

Bioi

nfor

mat

ics

Gen

otyp

ing

Prog

enito

rs

Avs Yr15a)

4 lanes Illumina HiSeq 2000RNA-Seq100 bp, read pair

Yr15R1 R2 R3 S1 S2S3 Avs

1x1082x1083x1084x108

Library

No. o

f rea

ds

31 2Lane 4

c) -SNPs between progenitors-Ratio of alleles between bulks

Sequence Alignments

Bulk Frequency Ratios

Primer design

-NCBI Unigenes v60 56,954 genes-Krasileva’s gene models 94,177 genes

BWA-0.5.9

bioruby-1.4.3bioruby-samtools 0.6.1

blat-0.3.4exonerate-2.2.0

MAFFT-7.055primer3-2.3.4

-Homoeologous in IWGSC scaffolds-Genome specific primer

d)

KASP Assays Genetic mapValidation by breeders

SNP Markerse)

ResistantR1: 70 ind.R2: 67 ind.R3: 50 ind.

SusceptibleS1: 15 ind.S2: 17 ind.S3: 13 ind.

F1

b)

⊗


Parental&PlantsAvocetAvocet+Yr1522*

*Isogenic&line&developed&by&the&University&of&Sydney

Segovia&VSegovia&V

Segovia&V


F 2 p

opul

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n Se

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cing

Bioi

nfor

mat

ics

Gen

otyp

ing

Prog

enito

rs

Avs Yr15a)



1x1082x1083x1084x108

Library

No. o

f rea

ds

31 2Lane 4


Sequence Alignments


Primer design


BWA-0.5.9





d)


SNP Markerse)



F1

b)

⊗


F2 population

Avocet ’S’Avocet ’S’ + Yr15



F1

Expected&segregation:&&3&resistant&:&1&susceptible!2 P&=&0.049;&187&resistant&and&45&susceptible&F2 plants

⊗


F 2 p

opul

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cing

Bioi

nfor

mat

ics

Gen

otyp

ing

Prog

enito

rs

Avs Yr15a)



1x1082x1083x1084x108

Library

No. o

f rea

ds

31 2Lane 4


Sequence Alignments


Primer design


BWA-0.5.9





d)


SNP Markerse)



F1

b)

⊗


Transcriptome&size

Wheat&genome:• ~17&Gbp• Hexaploid&AABBDD

• Coverage:&~2X

Coverage&per&Ilumina HiSeq 2000&lane&per&manufacturer&specification

Wheat&transcriptome:• ~76&Mnt• Prone&to&gene&expression&bias

• Coverage:&~440x


RNA*Seq

Genomic DNA

mRNA

Exon Intron Exon Intron Exon Intron


RNA*Seq

Yr15R1 R2 R3 S1 S2S3 AVS

1x1082x1083x1084x108

Library

No. o

f rea

ds

31 2Lane 4

4 lanes Illumina HiSeq 2000100 bp, read pair


F 2 p

opul

atio

n Se

quen

cing

Bioi

nfor

mat

ics

Gen

otyp

ing

Prog

enito

rs

Avs Yr15a)



1x1082x1083x1084x108

Library

No. o

f rea

ds

31 2Lane 4


Sequence Alignments


Primer design


BWA-0.5.9





d)


SNP Markerse)



F1

b)

⊗


Gene coverage per sample

0x

50x

100x

150x

200x

20x

Gen

e co

vera

ge

UCW NCBI UniGene

Yr15AVS

Sequence&alignment


F 2 p

opul

atio

n Se

quen

cing

Bioi

nfor

mat

ics

Gen

otyp

ing

Prog

enito

rs

Avs Yr15a)



1x1082x1083x1084x108

Library

No. o

f rea

ds

31 2Lane 4


Sequence Alignments


Primer design


BWA-0.5.9





d)


SNP Markerse)



F1

b)

⊗


Bulk&Frequency&Ratios

1AS c t G t g G g a1BS c t T t g G g a 1DS c t G t g G g a

Avocet S + Yr15

Avocet S

Genomic Sequence

1AS c t G t g G g a1BS c t T t g A g a 1DS c t G t g G g a

✓✗



1AS c t G t g G g a1BS c t T t g G g a c t K t g G g a1DS c t G t g G g a

Avocet S + Yr15

Avocet S

Genomic Sequence Consensus from parental

Homoeologous Allelic

1AS c t G t g G g a1BS c t T t g A g a c t K t g R g a1DS c t G t g G g a

✓✗


1AS c t G t g G g a1BS c t T t g G g a c t K t g G g a1DS c t G t g G g a

Avocet S + Yr15

Avocet S

Genomic Sequence Consensus from parental

Homoeologous Allelic

Susceptible bulk Resistant Bulk

1AS c t G t g G g a1BS c t T t g A g a c t K t g R g a1DS c t G t g G g a

c t G t g G g a. . . . . . . .. . T . . . . .. . T . . . . .. . . . . . . .. . . . . A . .. . T . . . . .. . . . . . . .. . T . . . . .

181 184c t G t g G g a. . . . . . . .. . T . . A . .. . T . . A . .. . . . . . . .. . . . . A . .. . T . . A . .. . . . . A . .. . T . . A . .

181 184PositionReference


SNP&Index&184A:1

8= 0.125

6

8= 0.75

Bulk&Frequency&Ratio:&&0.75

0.125= 6

✓✗



ALL 1 2 3 4 5 6 7 8 9 10

1,000

0

100

200

300

400

500

600

700

800

900

Minimum Bulk Frequency Ratio

Num

ber

of

SNPs

100

0

10

20

30

40

50

60

70

80

90

Perc

enta

ge o

f SN

Ps

SNPs on the short arm of chromosome group 1# SNPs AVS# SNP Yr15% SNPs Yr15


ALL

Scale 0.58

| 1621A

| 1751B

| 2101D

| 1862A

| 1892B

| 1532D

| 2083A

| 1573B

| 1673D

| 1674A

| 1234B

| 1714D

| 1495A

| 2205B

| 2245D

| 1846A

| 1286B

| 1626D

| 2457A

| 1897B

| 2427D

161#cM1A

174#cM1B

209#cM1D

185#cM2A

188#cM2B

152#cM2D

207#cM3A

156#cM3B

166#cM3D

166#cM4A

122#cM4B

170#cM4D

148#cM5A

219#cM5B

223#cM5D

183#cM6A

127#cM6B

161#cM6D

244#cM7A

188#cM7B

241#cM7D

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

|

Scale

17%0%

All&&SNPs SNPs&BFR>6

S. Wang, et al (2014). Characterization of polyploid wheat genomic diversity using a high*density 90,000 SNP array. Plant Biotechnology Journal.


BFRs&near&the&1B&centromere


Selection&criteria• Origin&(Yr15)

• Short&arm&Chromosome&group&1

• BFR&>&6

AVS Yr15

161#cM1A

174#cM1B

209#cM1D

|

|

|

0 20 40 60 80 100

0

50

100

150

200

Infinity

BFR

R11


UCW gene models

From Yr15(11,230)

11,230

Candidate&selection

Putative&genes&with&SNP:&16,022&(17.01%)

AVS Yr15


UCW gene modelsGroup 1S (554)

From Yr15(11,230)

385

169

10,845

Candidate&selection


AVS Yr15

161#cM1A

174#cM1B

209#cM1D

|

|

|


Candidate&selection


UCW gene modelsGroup 1S (554)

From Yr15(11,230)

BFR > 6(643)

98287 22

481 42

147

10,364

AVS Yr15

161#cM1A

174#cM1B

209#cM1D

|

|

|

0 20 40 60 80 100

0

50

100

150

200

Infinity

BFR

R11


F 2 p

opul

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Bioi

nfor

mat

ics

Gen

otyp

ing

Prog

enito

rs

Avs Yr15a)



1x1082x1083x1084x108

Library

No. o

f rea

ds

31 2Lane 4


Sequence Alignments


Primer design


BWA-0.5.9





d)


SNP Markerse)



F1

b)

⊗


Target&SNP&in&1B

1DS

1BS

1AS


PolyMarker:&Input


PolyMarker:&Search&sequence


PolyMarker:&Local&alignment

cgcatttGcgcgYgcgataccggcgcctKtgGgaatatttgcagcgaaggcgtg

cgcatttAcgcgYgcgataccggcgcctKtgAgaatatttgcagcgaaggcgtg

cgcatttGcgcgcgcgataccggcgcctGtgGgaatatttgcagcgaaggcgtg

cgcatttAcgcgcgcgataccggcgcctTtgGgaatatttgc---gaaggcgtg

c--atttGcgcgTgcgataccggcgcctGtgGgaatatttgcagcgaaggcgtg

SNP-1 A

SNP-1 B

IWGSC-1A

IWGSC-1B

IWGSC-1D







-------------------------------&----------------------

SNP-1 A

SNP-1 B

IWGSC-1A

IWGSC-1B

IWGSC-1D

SNPnon&homoeologous.

PolyMarker:&Candidate&SNP

✓


PolyMarker:&Candidate&SNP

✓✗cgcatttGcgcgYgcgataccggcgcctKtgGgaatatttgcagcgaaggcgtg





-------:-----------------------&----------------------

SNP-1 A

SNP-1 B

IWGSC-1A

IWGSC-1B

IWGSC-1D

SNPhomoeologous,


PolyMarker:&Genome&Semi*Specific

✓✗

✓






-------:----c------------------&----------------------

SNP-1 A

SNP-1 B

IWGSC-1A

IWGSC-1B

IWGSC-1D

semi%specific


PolyMarker:&Genome&specific






-------:----c---------------T--&----------------------

SNP-1 A

SNP-1 B

IWGSC-1A

IWGSC-1B

IWGSC-1D

specific

✓✗

✓ ✓✓


SNP-1 A

SNP-1 B

IWGSC-1A

IWGSC-1B

IWGSC-1D




cgcatttAcgcgcgcgataccggcgcctTtgGgaatatttgcagcgaaggcgtg

cgcatttGcgcgTgcgataccggcgcctGtgGgaatatttgcagcgaaggcgtg

-------:----c---------------T--&----------------------

PolyMarker:&Selected&primerTested&with&Primer3

✓✗

✓ ✓✓

Ramirez*Gonzalez, R, et al. (2014). RNA*Seq bulked segregant analysis enables theidentification of high*resolution genetic markers for breeding in hexaploid wheat. PlantBiotech. J, 12(9), 1–12.


Primer&Validation

R2 varieties0

1

Yr15Oc

Co

AVSRoCa

0.5

0.5R8 varieties

0

1

0.5

10.5

Yr15

Oc Bo

Co

AVSSh

Ro Ca

R8 F₂ population

0

1

0.5

10.5


F 2 p

opul

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n Se

quen

cing

Bioi

nfor

mat

ics

Gen

otyp

ing

Prog

enito

rs

Avs Yr15a)



1x1082x1083x1084x108

Library

No. o

f rea

ds

31 2Lane 4


Sequence Alignments


Primer design


BWA-0.5.9





d)


SNP Markerse)



F1

b)

⊗


Genetic&Map

R19R21R43wms0413

R8Yr15R11R5R16R22 Xbarc8

0.5 cM

1.791.020.511.280.26 0.26

IndividualsXbarc8 wms0413Yr Score R19R21R43R8R11R5R16R22

0.26

distal

B B B B B R B B B B B 51B B H H H R H H H H H 1B B B B B R B B B B H 6H H B B B R B B B B H 1H H B B B R B B B B B 1H H H B B R B B B B B 1H H H H H R H H B B - 1H H H H H R H H H H H 88A A A A A S A A A A A 37H H H H H S A A - A A 1H H H H H R H H A A A 2A H H H H R H H H H H 1A A H H H R H H H H H 2A A A A A R H H H H H 1A A A A A S H H H H H 1A A A A A S A A H H H 1


Validation&on&breeding&germplasm

R5 R8R11

C A T

T A T

T G C

SNP haplotype

Intermediate SusceptibleResistant

- 6 16

- 11 -

79 1 -

Reaction to P. striiformis

Validation&on&113&UK&varieties


F 2 p

opul

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n Se

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cing

Bioi

nfor

mat

ics

Gen

otyp

ing

Prog

enito

rs

Avs Yr15a)



1x1082x1083x1084x108

Library

No.

of r

eads

31 2Lane 4


Sequence Alignments


Primer design


BWA-0.5.9





d)


SNP Markerse)



F1

b)

⊗


Acknowledgments

• TGAC– Mario&Caccamo– Sarah&Ayling– Paul&Bailey– Jon&Wright

• JIC– Cristobal&Uauy– Nick&Bird– Vanesa Segovia– Martin&Trick&

• Limagrain– Paul&Fenwick– Simon&Berry

• RAGT&Seeds– Sarah&Holdgate– Peter&Jack

• University&of&Sidney– Robert&McIntosh


Building(Excellence(in(Genomics(and(Computational(Bioscience

Thank&you&for&listening.

Ramirez*Gonzalez, R, et al. (2014). RNA*Seq bulked segregant analysis enables theidentification of high*resolution genetic markers for breeding in hexaploid wheat. PlantBiotech. J, 12(9), 1–12.

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Using&NGS*enabled&genetics&to&improve&marker ...4D | 171 5A | 149 5B | 220 5D | 224 6A | 184 6B |...

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