ABSTRACT
Breedingworkutilizingbothgenotypicandphenotypicmarkersisthemosteffectivewayofachievingtarget. Molecular markers eg restriction fragment length polymorphism (RFLP), single nucleotidepolymorphism (SNP) and simple sequence repeat (SSR) tightly linked to target gene have beenidentified in different chromosomes to impose the genetic selection iemarker assisted selection(MAS). This paper summarize the progress and achievementmade in breeding for blast resistancebasedonDNAmarkerswhichhelpinplanningblastresistancegenetagginginNepalesericegenotypesanddevelopingblast resistant inbred lineornear isogenic line (NIL).Blast fungus (PyriculariaoryzaeCav.)can infectplantsatanygrowthstage fromseedling tomaturityandatanyparteg leaf,node,internode, neck and seed. Qualitative resistance gene may occasionally be broken down due tonumerous races of blast fungus both physiological and geographical races available in Nepal.Quantitative gene resistance and gene pyramiding are the best alternative for creating durableresistancesystem.At least40genesconferringresistance toblast isolateswithmultiplealleleshavebeen described. Both dominant and recessive resistance alleles have been found in many ricelandraces.Morphologicaland isozymicmarkersare limited innumber.Therefore,highlypolymorphicandeasilydetectableSSRmarkersarebeingused inbreeding forblast resistance.Bulked segregantanalysis(BSA)isthesimplemethodfortaggingresistancegenebySSRmarkers.Quantitativetraitloci(QTLs)havealsobeenmappedandmostofthemarelinkedtoqualitativegenes.SSRmarkerslinkedtothe gene are being used to select plants possessing the desired trait andmarkers throughout thegenome are being used to select plants that are genetically similar to recurrent parent.Using SSRmarkersitmaybepossibletoselectblastresistancegenotypesatanystageofcropdevelopmentfromany small part of crop, to conductmany round of selection, to selectwithout inoculums,withoutscoring, and without testing in hot spot or artificial inoculation.Molecular based blast resistancebreeding work is initiated focusing on resistance gene tagging in Nepalese rice landraces andtransferringresistancegenesincvsJumliMarshi,Khumal4andMansuli.
Keywords:Blastresistance,DNAmarker,genetagging,rice
MolecularTagging,AlleleMiningandMarkerAidedBreedingforBlastResistanceinRice
BalK.Joshi1,HariP.Bimb1,GopalParajuli2andBedanandChaudhary3
1BiotechnologyUnitNARC,POBox1135Kathmandu,Nepal.Email:[email protected],Khumaltar,Kathmandu3NRRPNARC,Hardinath,Janakpur
Correspondingauthor:BiotechnologyUnit,NARC,Khumaltar,POBox1135Kathmandu
Email:[email protected]:5539658Fax:5545485
RunningTitle:Markeraidedblastresistancebreeding
MolecularTagging,AlleleMiningandMarkerAidedBreedingforBlastResistanceinRice
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INTRODUCTIONRice (Oryza sativa L.) is themostpreferred cropofNepalese farmersand they are growing about2000differentricelandracesand67improvedricecultivarsfrom60mto3050maltitude(UpadhyayandJoshi2003). In all rice growing areasblastdisease causedby (Magnaporthe grisea (Herbert)Borr. (anamorphPyriculariaoryzaCav.=P.griseaCav.)isthemostseriousfungaldiseasecausingheavyyieldlossesfrom10to 80% (Manandhar et al 1992). Blast occurs at different growth stages. The fungus produces spots orlesionson leaves,nodesanddifferentpartsofpaniclesandgrains.Theneckblastmakesmoresignificantyield and quality losses than leaf blast (Katsube et al 1970).While highly positive correlation betweenresistancetoleafandneckblasthasbeengenerallyobserved,inconsistencybetweenresistancetoleafandneckblastwasalsoevident(Zhuangetal2002).
Blast disease is themost destructive diseaseworldwide.Growing resistant varieties hasbeen themosteffectiveandeconomicwaytocontrolthisdiseasebutresistanceisoftenlostinafewyearsaftercultivarsreleasedbecauseofthehighvariabilityofthericeblastfungus.Tobreedricevarietieswithmoredurableblast resistance, multiple resistance genes utilizing both qualitative and quantitative genes must beincorporated into individual varieties. This necessitates the exploration ofmore efficient selection andbreeding strategies than those currently exist. Recent advancements in DNA marker technology mayprovidenewsolutionsforselectingandmaintainingmoredurableresistancegenotypesinrice.Incontrastto the traditional selection based on phenotypic screening molecular markers are refractory toenvironmental variation. Upon identification of molecular markers closely linked to desirable trait/s,markerassistedselection(MAS)canbeperformedformultipleresistanceinearlysegregatinggenerationsandatearly stagesofplantdevelopment.Thispaper summarize theprogressandachievementmade inbreedingforblastresistancebasedonDNAmarkerswhichhelpinplanningblastresistancegenetagginginNepalesericegenotypesanddevelopingblastresistantinbredlineornearisogenicline(NIL).
GENETICSThegeneticsofriceblasthavebeenextensivelystudiedandtwotypesofresistancehavebeendescribed,complete(trueorvertical)andfiledresistance(horizontal)(FukuokaandOkuno2001).Riceblastisoneofthe best characterizedmodels for understandingmolecularmechanisms of natural defense response.Resistanceto infectionbythisfungusfollowsaclassicgeneforgenetheory(Silueetal1992).Qualitativegeneandindividualquantitativetraitloci(QTL)inheritinMendelianprinciple.ThePibandPitagenesaretwomajorbalstresistance(R)genesthathavebeencharacterizedmolecularly(Wangetal1999).Geneticanalysis of blast resistance studied by several researchers indicated either monogenic dominant,monogenic recessive, two dominant independent genes, two dominant complementary genes and tworecessiveduplicate genesor resistance controlledbyminor genes.Mostly the genes resistance toblastfungus is monogenic dominant. Multiple alleles upto 5 are identified. Mackill and Bonman (1992)developed 22 near isogenic lines (NILs) each having a single complete R gene from CO39 to study thegeneticsofblast resistance.At least four independent loci,Pi1 toPi4were identifiedafter inheritancestudyusing theseNILs. Inukaietal (1994) showed thatPi1wasallelic toPiz.Structuraland functionalanalyses ofmanymajor R genes have shown that they encode proteinswith similar structuralmotifs
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nucleotidebinding site,kinasedomains, leucinerich repeats thatare responsible for ligand recognitionandsignaltransduction(Wuetal2004).Moststudieshavesuggestedthatfieldresistancetoblastisundercomplexgeneticcontrolandmultiplegenesareresponsiblefortheexpressionoffieldresistance(HigashiandSaito1985).
QUALITATIVEANDQUANTITATIVEGENESTrueresistanceisgovernedbyqualitativegenealsocalledmajorgeneandfieldresistancebyquantitativegenes also calledminor genes.More than 40 qualitative resistance genes for blast fungus have beendescribed (Table1).Mostof themaredominantand located inchromosome6,11and12. In some locimultiple resistanceallelesare identified.Thewidegeneticvariationavailable inblast fungusmaybe themain factor to evolvemany resistance genes in rice. These two resistance types, complete and fieldresistanceareundermonogenicandpolygenicsystemsrespectively.Pikhas5alleles,Pi2locushas2andPitahas2alleles.FourRgeneshavebeen identified inTetepand3Rgenes inPaiKanTai.Wangetal(1994)hasdescribed10QTLs.TwoQTLsare located inchromosome4(Table2).MajorityoftheQTLsareassociatedwithqualitativegenes.
Table1.Genesfortrueresistancetoblastfungusandmarkerslinkedtoresistancegenes
SN Gene Allele ChrN Markerlinkedto Variety/remarks
Type Marker
1. Pi1 11 RZ536,Npb181,RZ424 LAC23(japonica)
2. Pi2(t)(=Piz5)
Piz,Piz5,Pizt,PI9(t)
6 RFLP RG64,RZ612,RG16,RG456
A5173(Piz5),CO251(Pizt),O.minuta(Pi9(t),TKM11(Pizt),Toride1(Pizt),Toride21(Pizt),Zenith(Piz)
SSR RM527,RM6836,RM3431,RM5963,AP56591,AP56593,AP56595
SNP Z4792/Z605,Z5765.Z3943,Z4794,Z6050,Z5966,Z3542
3. Pi3 6 PaiKanTao
4. Pi4 Pi4a(t),Pi4b(t),Pita
12 RFLP RG869,RZ397
5. Pi5(t) Pi3(t),Pi 9 RFLP RG788,JJ80T3,JJ81T3, Fujisaka5(Pii),Moroberekan,Tetep(Pi
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SN Gene Allele ChrN Markerlinkedto Variety/remarks
Type Marker
i(t) JJ113T3 5(t)),Zenith(Pii)
6. Pi6 12 RFLP RG869
Isozyme
SHD1
7. Pi7 11 RFLP RG103A/RG16
8. Pi8, 11
9. Pi10(t) 5 RAPD RRF6,RRH18,RG13
10. Pi11(t)(=Pizh)
8 BP127,RG181B
11. Pi12 12 RG869,RG81,RZ397
12. Pi13
13. Pi14(t) Pi16(t),Pid(t)
2 RM262,G1314,XNpb223
Isozyme
Amp1
14. Pi15 9
15. Pi17
16. Pi18 11 RZ536 Suweon(japonica)
17. Pi19 AichiAsahi
18. Pi20 12 XNpb88 IR24
19. pi21 2 RFLP G271/G317 Recessive
20. Pi22
21. Pi24(t) 12 RGA3620,RG241A,N7520600
Zhong156
22. Pi25 2or SSR RM21 Gumei2,Leafandneck
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SN Gene Allele ChrN Markerlinkedto Variety/remarks
Type Marker
6? RFLP RG520,RGA7470/A7550 blastresistance
23. Pi29(t) 8 RZ617
24. Pi33(t) 8 RFLP G1010
25. Pi34
26. Pi44 11 CDO520
27. Pi? 12 RFLP RG869
28. Pik Pikm,Piksor5,Pikk,Pikp,Pikh
11 L198,R1506 Caloro(Piks),Dawn(Pikh),Fuji102(Pikh),Fujisaka5(Piks),Hokuriku12(Piks),HR22(Pikh),IshikariShiroke(Piks),K2(Pikp),K3(Pikh),
K60(Pikh),Kanto51(Pik),Norin18(Piks),Norin6(Piks),Shin2(Piks),Shin3(Piks),Taichung65(Piks),Tetep(Pikh)
SSR RM144,RM224
29. Pik? Dular,Tadukan
30. Pita(=sl=Pi4(t))
Pita2 12or9?
RZ397,OSM89,XNpb088,RG241,RZ869
IR36,IR64,K1(Pita),PaikanTao,Reiho(Pita2),Tadukan,Tetep
SSR RM155/RM7102
31
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