1

18871887

TodayToday

RM 190

RM 481

RM 433

MarkerMarker--assisted Breeding forassisted Breeding for

Disease Resistance TraitsDisease Resistance Traits

Virginia BoyettVirginia BoyettUniversity of Arkansas Rice Research & Extension CenterUniversity of Arkansas Rice Research & Extension Center

Breeding and Genetics Biotechnical Support ProgramBreeding and Genetics Biotechnical Support Program

2

ObjectivesObjectives

1. Brief overview of marker development for 1. Brief overview of marker development for rice diseases rice diseases

2. General information on rice blast resistance 2. General information on rice blast resistance gene markers: background and application.gene markers: background and application.

3. Discussion of our work here at the UA RREC3. Discussion of our work here at the UA RREC

The BeginningThe Beginning

Bacterial blightGene Source Markers Chr Reference

Xa-1 Kogyoku XNpb 235 4 Yoshimura et al. 1996

Xa-3 Chugoku45 XNpb 181 11 Yoshimura et al. 1995

Xa-4 IR20 XNpb 181 11 Yoshimura et al. 1995

xa-5 IR1545-339 RG 556 5 Yoshimura et al. 1995Xa-7 IRBB7 M5 6 Porter et al. 2003Xa-10 IRBB10 CDO365 11 Yoshimura et al. 1995xa-13 IR66699-5-5-4-2 RG 136 8 Zhang et al. 1996Xa-21 O. longistaminata RG 103 11 Ronald et al. 1992Xa-22(t) Zhachanglong R1506 11 Lin et al. 1996Xa-26(t) Minghui 63 R1506, M224 11 Yang et al. 2003Xa-27(t) O. minuta M631/M1230 6 Gu et al. 2004

Subhudhi, Sasaki, and Khush. Genome Mapping and Molecular Cloning Vol. 1 (Kole ed.)

3

Major Resistance Genes identified:Major Resistance Genes identified:Bacterial leaf blightBacterial leaf blight——2020++

BlastBlast——4040++

Sheath blightSheath blight——00

••Deployment of major R genes in breeding resistant cultivars is Deployment of major R genes in breeding resistant cultivars is

simple, but simple, but major limitation is lack of durabilitymajor limitation is lack of durability..

••Can improve durability by pyramiding multiple resistance genesCan improve durability by pyramiding multiple resistance genes

••Both QTL and major genes are required for durable resistance Both QTL and major genes are required for durable resistance

Subhudhi, Sasaki, and Khush. Genome Mapping and Molecular Cloning Vol. 1 (Kole ed.)

Stages of GeneStages of Gene--Tag Development:Tag Development:1.1. QTL putatively mapped to a chromosomal regionQTL putatively mapped to a chromosomal region

2.2. QTL verified to reside in a chromosomal regionQTL verified to reside in a chromosomal region

3.3. MAS with flanking markersMAS with flanking markers——selected progeny proved to selected progeny proved to have phenotypehave phenotype

4.4. MAS with molecular tags within the gene sequence itselfMAS with molecular tags within the gene sequence itself

5.5. MAS with molecular tags within THE portion of the gene MAS with molecular tags within THE portion of the gene that causes the R (or S) reaction.that causes the R (or S) reaction.

Previously, major genes made it past Stage 3Previously, major genes made it past Stage 3——not not QTLQTL’’ss!!

RiceCAPRiceCAP effort to accomplish this for Sheath blight effort to accomplish this for Sheath blight QTLQTL’’ss!!Shannon Pinson

4

Candidate gene approach

Genes potentially involved in biochemical pathways responsible for expression of resistance are used as markers for genetic analysis

e.g., QTL for corn earworm resistance in maize include genes involved in flavone synthesis (Byrne et al., 1996; McMullen et al., 1998)

Conventional QTL mapping identifies chromosomal intervals carrying candidate genes, whereas the candidate gene approach begins with a target sequence.

Slide: Jan Leach

What are candidate DR genes for disease or pest resistance QTL?

Genes involved in initial recognitionResistance genes (R genes) Resistance gene analogs: NBS-LRRs (Bai et al., 2003)

Genes encoding structural proteins, enzymes of secondary metabolism, enzymes directly involved in defense, or regulatory genes obtained from:

Literature/database searchMicroarray experimentsSuppression subtraction hbyridization cDNA libraries

Slide: Jan Leach

5

“Consensus” candidate genes associated with QTL:

Oxalate Oxidase

Aldosereductase

NBS-LRR(maize, barley)

Dihydrofolatereductase-thymidylatesynthase

PeroxidaseThaumatinCatalase

SODPal

Chitinase

NBS-LRR (rice, barley)Dehydrin

Rice IR64 x Azucena: blast Ramalingam et al., 2003

Irrigated rice SHZ-2 X LTH: blast (Liu et al., 2004)

Wheat: tan spot and/or leaf rust (Faris et al. 1999)

NBS-LRR(rice) 14-3-3

PR1

Slide: Jan Leach

Locatio

n in

China

or Phil

ippine

s

0 1 2 3 4 5

20

60

100

Amou

nt o

f Disea

se

No. Candidate genes in 101 lines

Accumulation of candidate defense genes into rice to enhances resistance to blast

Liu et al., 2004, MPMI

More DR genes → Less disease

Slide: Jan Leach

6

Lines with five candidate genes exhibit less sheath blight (Rhizoctonia solani)

0

20

40

60

80

100

SHZ BC10-46 BC10-10 BC116 D6-256 GR978

Rice Line

Disea

sed

Plan

tAre

a (%

)

F. Correa et al., unpubl.Slide: Jan Leach

Practical Applications?

A small number of genes with quantitative effects can be accumulated into a line to achieve disease resistance

The candidate gene approach can be an effective means to address complex problems such as rice blast disease

Slide: Jan Leach

7

IB33 IB1 IB49 IB54 IB45 IH1 IG1 IC17 IE1 IE1KIB33 IB1 IB49 IB54 IB45 IH1 IG1 IC17 IE1 IE1K

Races

Races

Resistance G

enesR

esistance Genes

Pi-ta2

Pi-z

Pi-kh

Leah

Pi-ks

Pi-d *

Pi-i

Pi-b

Chr 12

Chr 11

Chr 11

Chr 11

Chr 11

Chr 6

Chr 9

Chr 2

**

* No markers available, ** Partial resistance to IB49

Major Resistance Genes to Predominant Major Resistance Genes to Predominant Races of Blast in the Southern U.S.Races of Blast in the Southern U.S.

Fjellstrom et al.

Chr 2 Chr 11 Chr 12

Genetic maps of marker loci on rice chromosomes.Genetic maps of marker loci on rice chromosomes.

Fjellstrom et al., 2004 Crop Sci.

8

Fjellstrom et al., 2005

Genetic map of Genetic map of PiPi--zz gene and marker loci.gene and marker loci.

PiPi--bb on on ChrChr 22‘‘SaberSaber’’

Markers: Markers: RMRM’’ss 138, 166, 208, 266, 138, 166, 208, 266, PibdomPibdomName Type Primer Sequence Anneal Dilution Size Range Resistant

f-ggtcctgggtcaataattgggttaccr-ttgctgcatgatcctaaaccggf-tctgcaagccttgtctgatgr-taagtcgatcattgtgtggaccf-gaacaatgcccaaacttgagar-gggtccacatgtcagtgagc

RM 166 SSR

RM 208 SSR 55°C 1:200

61°C

365 +

177

1:100 315-421 315

163-177

Pibdom Fragment 65°C

IB33 IB1 IB49 IB54 IB45 IH1 IG1 IC17 IE1 IE1KIB33 IB1 IB49 IB54 IB45 IH1 IG1 IC17 IE1 IE1K

Pi-b

**

Gramene, Fjellstrom et al., 2004 Crop Sci.

9

RM 166

RM 208

GeneScanGeneScan ElectropherogramElectropherogram of Saber with of Saber with PiPi--bb markers.markers.

PiPi--bb

Sónia NegrãoPiPi--bb Dominant MarkerDominant Marker

PortuguesePortuguese

Nipponbare

Nipponbare

Reiho

Reiho

IR36

IR36

TeTe -- quingquing

10

Name Type Primer Sequence Anneal Dilution Size RangePi-k h Pi-k s

f-tgccctggcgcaaatttgatccr-gctagaggagatcagatggtagtgcatgf-atcgatcgatcttcacgaggr-tgctataaaaggcattcggg

Resistant

255

120

243-255 255

120-156 139

RM 144 SSR 55°C 1:200

RM 224 SSR 55°C 1:1000

PiPi--kk on on ChrChr 1111‘‘CocodrieCocodrie’’ ——PiPi--kkhh ‘‘BengalBengal’’——PiPi--kkss

IB33 IB1 IB49 IB54 IB45 IH1 IG1 IC17 IE1 IE1KIB33 IB1 IB49 IB54 IB45 IH1 IG1 IC17 IE1 IE1K

Pi-kh

Markers: Markers: RMRM’’ss 144, 224144, 224

Gramene

GeneScanGeneScan ElectropherogramElectropherogram of of PiPi--kk marker.marker.

RM 224

11

PiPi--tata on on ChrChr 1212‘‘KatyKaty’’

IB33 IB1 IB49 IB54 IB45 IH1 IG1 IC17 IE1 IE1KIB33 IB1 IB49 IB54 IB45 IH1 IG1 IC17 IE1 IE1K

Pi-ta2

Markers: Markers: RMRM’’ss 155, 7102, OSM 89, YL100/YL102, 155, 7102, OSM 89, YL100/YL102, YL153/YL154,YL153/YL154, YL155/YL87, YL183/YL87, YL155/YL87, YL183/YL87, YL155/YL183/YL200 (SNLP). YL155/YL183/YL200 (SNLP).

Name Type Primer Sequence Anneal DilutionSize Range Resistant Susceptible

f-YL155-agcaggttataagctaggccf-YL183-agcaggttataagctagctatr-YL200-agagccaaatagccaattcaf-YL155-agcaggttataagctaggccr-YL87-ctaccaacaagttcatcaaaf-YL183-agcaggttataagctagctatr-YL87-ctaccaacaagttcatcaaaf-YL153-caacaatttaatcatacacgr-YL154-atgacaccctgcgatgcaaf-ttggtcaaagttagcatgggagggr-tttgaaccgggtggcccacatgf-aacgtcgctcccttccagggcr-ctccacggtgagcggtggcagf-ttgagagcgtttttaggatgr-tcggtttacttggttactcg

OSM91

SSR

SSR

55°C

67°C

61°C

YL153/YL154 Fragment 440

YL183/YL87 Fragment 55°C +

+1042

1042

YL155/YL87 Fragment 55°C

SNLP green 183blue 181

OSM89

+

61°CFragment 1:1000 181-183

1:200

1:100

290-300

269-292

296

269

167-187 187RM 7102 SSR 55°C

PiPi--tata

Jia et al., 2004 Phytopath, Jia et al., 2004 Euphyt., Fjellstrom et al., 2004 Crop Sci.

12

YL155Resistant

YL183Susceptible

GeneScanGeneScan ElectropherogramElectropherogramof Coof Co--dominant dominant PiPi--tata TriprimerTriprimermarker showing heterozygote.marker showing heterozygote.

1Kb DNA Ladder

1 10 20 30 40 48

1 10 20 30 40 48

Resistant Allele

Susceptible Allele

PiPi--tata Dominant Markers YL155 and YL183.Dominant Markers YL155 and YL183.

YL155/YL87

YL183/YL87

1Kb DNA Ladder

1.6Kb—

1.6Kb—

—1Kb

—1Kb

13

PiPi--zz on on ChrChr 66‘‘JeffersonJefferson’’

IB33 IB1 IB49 IB54 IB45 IH1 IG1 IC17 IE1 IE1KIB33 IB1 IB49 IB54 IB45 IH1 IG1 IC17 IE1 IE1K

Pi-z

Markers: AP5659Markers: AP5659--1, AP56591, AP5659--3, AP56593, AP5659--5, 5, MRG2431a, MRG4963, MRG5836MRG2431a, MRG4963, MRG5836

Name Type Primer Sequence Anneal DilutionSize Range Resistantf-atccaaatccaatggtgcr-gtggcgaaagggaacattctf-cgaaaagtgggaagcaaatgr-gcgtacccctagtggctgtaf-tgttgcatatggtgctatttgar-gatacggcttctaggccaaaf-gctgagatagccgagaaatcr-actagctgcccacctaagc

1:200 225-268 249

1:500 199-219 219

1:1000 148-165 148

1:200 166-175 172

55°C

55°C

55°C

61°C

MRG5836 SSR

AP5659-1 SSR

MRG2431a SSR

MRG4963 SSR

Gramene, Fjellstrom et al., 2005

GeneScanGeneScan ElectropherogramElectropherogram of Bengal with of Bengal with PiPi--zz markers.markers.

MRG2431a

MRG4963MRG5836

14

MarkerMarker--assisted Breeding: assisted Breeding: A DoA Do--ItIt--Yourself GuideYourself Guide

ClearClear--cut goalscut goalsGood DNA extraction methodGood DNA extraction methodGood markersGood markersData reasonable to interpretData reasonable to interpretDonDon’’t forget controls!t forget controls!

Successful MarkerSuccessful Marker--assisted selection programs include:assisted selection programs include:

15

ClearClear--cut goalscut goals

FF33, F, F55, and TC, and TC11 populationspopulationsYellow Yellow

Blue Green RM 190 RM 190 CommentsSample No. Pi-ta R Pi-ta S Peak 1 Peak 220309-1 181.03 182.76 105.40 120.18 Pedigree: 010110/Jing 185-720309-2 181.01 182.72 105.41 122.22 S120/S122//R10520309-3 180.96 182.63 105.35 122.1720309-4 180.96 182.67 105.36 122.21

Yellow YellowBlue Green RM 190 RM 190 Comments

Sample No. Pi-ta R Pi-ta S Peak 1 Peak 2 CT CT417-86 181.16 182.93 113.95 126.41 14 20 RU9901133/JING 185-7//RU0001124417-87 181.06 105.23 113.70 10 14 S (CT20) / R (CT10) // R (CT14)417-88 181.12 182.80 105.35 113.72 10 14417-89 181.05 105.27 113.68 10 14417-90 180.92 182.61 113.65 126.16 14 20

Not as useful.Not as useful.

UsefulUseful

Good DNA extraction methodGood DNA extraction method

How to extract 4,053 DNA samples in one week:How to extract 4,053 DNA samples in one week:

Steps:Steps:1.1. Leave the country!Leave the country!

2.2. Send email as a joke to dedicated volunteer assistants.Send email as a joke to dedicated volunteer assistants.

3.3. Have no further contact with volunteer assistants.Have no further contact with volunteer assistants.

4.4. Have a reliable highHave a reliable high--throughput extraction method.throughput extraction method.

16

HighHigh--throughput DNA Extraction Methodthroughput DNA Extraction Method

Advantages:Advantages:CheapCheapFast and easyFast and easyVersatileVersatile——fresh, frozen, freezefresh, frozen, freeze--drieddriedNo liquid nitrogenNo liquid nitrogenNo chloroformNo chloroformWorks well with Works well with SSRSSR’’ss and automated detectionand automated detection

Method:Method:9696--well PCR platewell PCR plateReRe--usable Silicone Sealing Matusable Silicone Sealing MatSingle Hole PunchSingle Hole PunchBuffer A, 100 Buffer A, 100 mMmM Sodium hydroxide, 2% Sodium hydroxide, 2% TweenTween 2020Buffer B, 100 Buffer B, 100 mMmM TrisTris--HCl, 2 HCl, 2 mMmM EDTAEDTAHeating Block or Heating Block or ThermalcyclerThermalcycler

1.1. Harvest leaf tissue into manila coin envelopes and sample using Harvest leaf tissue into manila coin envelopes and sample using a a single holesingle hole--punch, one leaf disk per well.punch, one leaf disk per well.

2.2. Prepare Buffer A.Prepare Buffer A.3.3. Add 50 Add 50 µµl Buffer A to each well.l Buffer A to each well.4.4. Seal plate and incubate at 95Seal plate and incubate at 95°°C for 10 minutes.C for 10 minutes.5.5. Add 50 Add 50 µµl Buffer B to each well. Mix.l Buffer B to each well. Mix.

Xin et al., 2003 BioTech.

17

FF33 seedlings ready seedlings ready for sampling.for sampling.

Leaf tissue sampled and ready Leaf tissue sampled and ready for extraction.for extraction.

Samples extracted and ready for PCR!Samples extracted and ready for PCR!

Good markersGood markers

Data reasonable to interpretData reasonable to interpret

DonDon’’t forget controls!t forget controls!

Segregate with geneSegregate with geneAmplify wellAmplify wellConsistentConsistent

MultiplexingMultiplexingMultigeneMultigene familiesfamiliesExotic Exotic GermplasmGermplasmLabeling dyesLabeling dyes

Waxy CT Repeat 6FAM HEX NED TAMRARM 190 CT 20 124 126 126 128

18

873 F873 F33 lines of 20 Crosseslines of 20 Crosses

Target: 7 samples of each line, or 6,111 samplesTarget: 7 samples of each line, or 6,111 samples

15 seeds each line (September & December 2004.)15 seeds each line (September & December 2004.)

Total planted:13,095 seedsTotal planted:13,095 seeds

1,595 samples harvested last week October1,595 samples harvested last week October——1818

4,053 samples harvested January/February 20054,053 samples harvested January/February 2005——4747

Total: 5,648 samples harvested; processed 65 templatesTotal: 5,648 samples harvested; processed 65 templates

Analyzed for Analyzed for PiPi--tata and RM 190 first, then and RM 190 first, then PiPi--bb and and PiPi--zz

140 PCR Plates, 70 ABI 3700 runs, 21120 data points140 PCR Plates, 70 ABI 3700 runs, 21120 data points

Analysis ProcedureAnalysis Procedure

A Case Study: Space Plants 2004A Case Study: Space Plants 2004

PiPi--tata SNLP PCRSNLP PCR25 25 µµl Reactionl Reaction

6.0 6.0 µµl Molecular Grade Hl Molecular Grade H22OO1.3 1.3 µµl PCR Grade BSA (2%)l PCR Grade BSA (2%)1.0 1.0 µµl PVP40 (25%)l PVP40 (25%)1.0 1.0 µµl MgCll MgCl22 (25mM)(25mM)

12.5 12.5 µµl l TaqTaq Master Mix (Master Mix (QiagenQiagen))0.4 0.4 µµl Primer F YL155 (10l Primer F YL155 (10µµM)M)0.4 0.4 µµl Primer F YL183 (10l Primer F YL183 (10µµM)M)0.4 0.4 µµl Primer R YL200 (20l Primer R YL200 (20µµM)M)2.0 2.0 µµl DNA Templatel DNA Template

Final reaction concentrations: 0.6 U Final reaction concentrations: 0.6 U TaqTaq, 0.1% BSA, , 0.1% BSA, 1% PVP40, 2001% PVP40, 200µµM M dNTPdNTP’’ss, 2.5mM MgCl, 2.5mM MgCl22, 0.16 , 0.16 µµM M each Primer F, and 0.32 each Primer F, and 0.32 µµM Primer R.M Primer R.

19

Cycling Conditions:Cycling Conditions:

1.1. Initial Initial denaturationdenaturation:: 9494°°CC⎯⎯55’’

2.2. DenaturationDenaturation:: 9494°°CC⎯⎯30s30s3.3. Annealing:Annealing: 6161°°CC⎯⎯30s30s4. Extension:4. Extension: 7272°°CC⎯⎯11’’

5. 35 cycles5. 35 cycles

6. Final extension:6. Final extension: 7272°°CC⎯⎯55’’

7. Hold7. Hold 1010°°CC⎯∞⎯∞

65%561304865Totals

5815112630491

3835830485

38581330484

4445930483

81121330479

4023530478

3710172730435

752793630434

206243030429

10030330420

671261830379

10050520320

43526812020296

6763920267

762583320266

10080820265

791694621520242

266172320241

2014520238

782015825920231

% DiscardDiscardKeepLinesCross No.

865 865 lineslinesReduced toReduced to

304304

Final set of data Final set of data presented on presented on March 24, 2005March 24, 2005..

20

March 28, 2005March 28, 2005PiPi--tata Selection is Made in Puerto RicoSelection is Made in Puerto Rico

AcknowledgementsAcknowledgements

Dr. Karen MoldenhauerDr. Karen MoldenhauerDr. James GibbonsDr. James GibbonsDr. Steve BrooksDr. Steve Brooks

Rice Research and Promotion BoardRice Research and Promotion Board

Dr. J. Neil Dr. J. Neil RutgerRutgerDr. David Dr. David GealyGealyMelissa Melissa JiaJia

Cindy LedbetterCindy LedbetterCuWandaCuWanda FlowersFlowersJill BullochJill BullochMaurice BlockerMaurice BlockerAmanda GriffithAmanda GriffithAaron JacksonAaron JacksonJason HillJason HillLorie BernhardtLorie Bernhardt

Dr. Fleet LeeDr. Fleet LeeDr. Dr. YulinYulin JiaJiaDr. Georgia Dr. Georgia EizengaEizengaDr. Dr. JundaJunda JiangJiangDr. Eugenia WinstonDr. Eugenia WinstonDr. Dr. PratibhaPratibha SinghSinghDr. Helen MillerDr. Helen MillerDr. Dr. HeshamHesham AgramaAgramaDr. Jim Dr. Jim CorrellCorrellDr. Chris Dr. Chris DerenDeren

USDAUSDA--Agricultural Research ServiceAgricultural Research Service

Dr. Anna McClungDr. Anna McClungDr. Bob Dr. Bob FjellstromFjellstromDr. Shannon PinsonDr. Shannon PinsonDr. Dr. RodanteRodante TabienTabien

Dr. Jan LeachDr. Jan LeachSSóóniania NegrNegrããoo

Marc Marc RedusRedus

Lisa HoffmanLisa HoffmanLora Lora BuekerBuekerAlishaAlisha NelmsNelmsGordon MillerGordon MillerYing ChenYing ChenKayla Kayla EarvineEarvineBrenda Brenda BusickBusickLola JacobsLola Jacobs

21