Marker-assisted Breeding for Disease Resistance Traits€¦ · Marker-assisted Breeding for Disease...
Transcript of Marker-assisted Breeding for Disease Resistance Traits€¦ · Marker-assisted Breeding for Disease...
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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
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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.)
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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
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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
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“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
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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
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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.
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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.
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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
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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
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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.
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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
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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
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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:
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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.
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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.
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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
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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.
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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..
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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
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