KITASHIBA  Hiroyasu

NISHIO  Takeshi

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NISHIO, Takeshi

KITASHIBA, Hiroyasu

Tohoku University Repository (TOUR) / 東北大学機関リポジトリ

Recent Research Topics in the Laboratory ofPlant Breeding and Genetics著者 NISHIO  Takeshi, KITASHIBA  Hiroyasujournal orpublication titleTohoku journal of agricultural researchvolume 60number 1-2page range 39-44year 2009-12URL http://hdl.handle.net/10097/41177TohoknJoumalof AgriculturalResearchVol. 60 No. 1-2, December 2009Pnlnted in JapanRecent Research Topics in the Laboratory ofPlant Breedin色and GeneticsTakeshi NISH{o and Hiroyasu KITASHIBALabwatory of Plant Breeding and鮎tics, Graduate School of AgriczLltural Sbienoe,Tohoku University(Received, Septx!mber 6, 2009)Summaryln the Laboratory of Plant Breeding and Genetics, we have studie'd geneticsand molecular biology of Brassica andrice for 60years. Based on the greatachievements of the past, We are studying molecular genetics of plant reproductionsystems, genetics of tolerances to environmental stresses inrice, and the Brass血and Raphanus genomeS. These studies are apparently independent, but advanceinone oontributes tothe other studies and leads tDfurt･her development of plant･breeding teclmiques.Molecular Genetics of Self-incompatibility and Male-sterilityThe molecular mechanism of self-incompatibility haぷbeen most intensivelystudied uslng Brassica･ Self-pollen rejection bythe stigma toinhibit self-fertilization is controlled by a single locus complex, S, having nultiplealleles･One set ofal1eles of the genes in the S locus complex is termed "S haplotype･"We have developed many lines homozygous for S haplotypeS, i･e･, S-tester lines,in Brassica rapa) Brassica olerMa, and Raphanus satimLS, Which are useful forgenetic study of self-incompatibility and Fl hybrid breeding uslng a Self-incompatibility system (Nishio et al., 2006). Each S haplotype h鮎auniqueS-locus structure (Fukni et al., 2003),and interhaplotype recombination seldomoocurs (Taknno et al., 2007). Althoughthe number of S haplotypes in a specieshas been estimated to be morethan 50, such haplotypes tend to be lost incultivated plants, eveningene banks (Takuno et all, 2009)･ In determination ofthe nucleotide sequences of a large number ofalleles of the pollen ligand gene(SPll/SCR) andthose of the stigma reoeptor gene (SRK), we found very similarS haplotypes between different species (Sato et al., 2002 ; Fujimoto et all, 2006a)･Pollination tests using int冶rSpeCiAc hybrids or transgenic plants revealedthatthese S haplotypes havethe same recognition specificity between different species3940 T. Nishio and H. Kitashiba(Kimura et al･, 2002 ; Sato et all, 2003). These results suggest that these similarS haplotypes were derived from the same ancestralS haplotypes and havemaintained the sane ancestralrecognition specificity since divergence of B和冶Sicaspecies･ Studying pollen-stigma recognition specificities of similar S haplotypesuslng reCOmbinant proteins and transgenic plants, we proposed an evolutionarymodel of the generation of new S hapIQtyPeS (Sate et al., 2004). We also revealedthe cause of self-Compatibility of self-compatible cultivars in B･ rapaand Blolerw and a self-Compatible amphidiploid species, Brassica napus (Fujimoto etall, 2006b ; Okamoto et al., 2007).Since there are many S haplotypes in a species, confusion and contaminationof S haplotypes sometimes occur･ For identification of S haplotypes, PCR-RFLP analysis of SLG,anS-loons gene homologous to SRK, has been used(Nishio et all, 1996)･ However, there are severalS haplotypes not identifiable bythis method･ We found dot-blotanalysis of SPll/SCRal1eles to be highlyusefdl for S haplotype identification (Fujimoto and Nishio, 2003). This methodwas improved usingallele-specific oligonucleotides and applied to S haplotypeidentification of Rosaceae fruit trees (Kitashiba et al., 2008).Male-sterility isalso an important trait used for Fl hybrid seed production･A novel cytoplasmio male-sterile line was developed by intergeneric hybridizationbetween Di'plotam's muralis and B･ Papa followed by repeated back-orosslngS OfBrassica in our laboratory (Hinata and Konno, 1979)･ Amitochondrialgeneresponsiblefor cytoplasmic male-sterility was identified using a newly developedrestorer line of this male-sterility (Shinada et al., 2006). Recently a candidatemale-st即ility gene of a male-st即ile mutant in rice was identified and the functionof this gene is investigated (Shiokai et al.,unpublished).Development of SNP Techniques and Their Application toGenetic Analysis in RiceBased on the teclmique of dot-blot analysis of S haplotypes, we developed anovel teclmique for single nucleotide polymorphism (Sup) analysis, i.e., dot-blot-SNP (Shira.sawa et al., 2006). With t,his method,all the SNPs and short indelswhich had been tested were successfully analyzed･ Since sups are the mostcommon polymorphisms of genomes, they are good DNA markers for QTL analysisand fine mapping Of genes･Althoughthe originalmethod of dot-blot-Sup wascostly in terms of preparation of labeled oligonucleotide probes, a recentlydeveloped bridge hybridization method enables production of new Sup markersat low cost (Shiokai et all, 2009a)･ The dot-blot-Sup teclmique is suitable forgenotyping of a large number of individuals, more than severalthousand, andtherefore applicable to selection of plants in conventionalcross-breeding programsby Sup genotyping of important geneswithknown functions, the number ofRecent Resewch T*ics in the Laborabry of Plant Breeding and Geneths　4lwhich will increase dramatically by genome studies of various crop species. Insuch studies, preparation of a large number of plant DNA samples is labor-intensive･ A recently developed I)NA preparation method, the leaf-punchmethod (Shiokai et all, 2009b), is considered to be an idealI)NA preparationmethod whioh is both simple and cost-e皿cient.For mapping of genes or quantitative trait locus (QTL) analysis usingJaPOnicarice cultivars, DNA markers showing polymorphism between differentcultivars are necessary･ Since JaPOnicarice cultivars are closely related, theyhave few DNA polymorphisms in oommon, even in SNPs･ Soreenlng Of SNPs wascarried out using PCR-RF-SSCP analysis andmismatx)h-cleavage analysis (Shira-sawa et all, 2004, 2007)･ Mutations of the wx gene inrice mutants andglutinOuscultivars were elRciently identified by PCR-RF-SSCPanalysis (Sato and Nishio,2003 ; Horiet all, 2007)･ Mismatch-cleavage analysis using B.rassica petiole.extract wan applied to reverse-genetic selection of mutants from arice populationmutagenizedwith gamma-ray irradiation (Sato et al., 2006).Tolerances to environmentalstresses are important breeding objectives. Wedetected QTLs for tolerances to low temperature at the booもing stage and hightemperature at the seedfilling stage on chromosome 3 and 6, respectively, 1nriceusing Sup markers･ Near-isogenic lines having the QTLs showed significantlyhigher tolerances･ Producing Sup markers in these QTL regions, we are harrow-ing downthe chromosomalreglOnS possibly containing stress-tolerance genes･Genome Study of Brassica and RaphanusSNP teclmiques developed for the study of rice genetics are useful for genomestudy of Brassica･ Producing 300 I)NA markers including 120 EST-based Supmarkers, we constructed a linkage map of 241 DNA markers in B･ raga and usedthese markers for QTL analysis offlowering time and leaf morphologiCaltraits (Liet al･, 2009)･ Synteny analysis of B･ Papa and Arabidopsis thaliana usingsequences of the EST markers enabled identification of candidate genes control-lingflowerlng time and leaf morphologiCaltraits･ We intend to use this linkagemap ln genetic analysis of reproductive traits such as interspecific incompatibility,breakdown of hybrid embryos,andalso self-incompatibility･Receiving generous funding from the Bi0-Oriented Technology ResearchAdvancement Institution (BRAIN), we started a Raphanus genome project in2008･ In this project, we are plannlng tO COnStruCt a linkage map of 3,000EST-ba占ed Sup markersand to isolate BAC clones harboring these SNP markersto cover about 95% of the Raphanus genome. These markers and BAG cloneswill be helpful tools for elucidation of genes controlling unique traits of Ra-phanus.42 T. Nishio and H. KitashibaReference sFukni E, Fujimoto R･ Nishio T (2003) Genomic organization of the S corereglOn and the Sfl&nking reglOnS Of a class-ⅠI S haplotype lnBrMSha rapa. MoI Gbnomぬ鮎t 269 : 361-369.Fujimoto R, Nishio T (2003) Identification of S haplotypes in Brassha by dotblot analysis of SPll alleleG. Them AppI Gbnet 106 : 1433-1437.FujimotD R, Okazaki K, Fukni E, Kusaba M, Nishio T (2006a) Comparison ofthe genome structure of the self-incompatibility (S) locus inintcrspecifiC palrS Of S haplotypesI emetics 173 : 1157-1167･Fujimoto R, Sugimura T, Fukni E, Nishio T (2006b) Suppression of geneexpression of a recessive SPll/SORallele by anuntranscribedSPll/SORalleleinBrassica self-incompatibility. Plant MoI Biolも1 : 577-587.Hinata K, Konno N (1979) Studies on a male sterile strain having the Brassicacampestris nucleus and the Dtplomimuralis cytoplasm･ Ⅰ･ On thebreeding procedure and some charaL!teristics of the male sterile strain.Japan J Breed 29 : 305-311.HoriY, Fujimoto R, Sato Y, Nishio T (2007) A novel wx mutation caused byinsertion of a retrotransposon-like sequence in aglutinous cultivar ofrice (Oryza saliva L).爪のr AppI Gmet 115.･ 2171224.Kimura R, Sato K, Fujimoto R, Nishio T (2002) Recognition specificity ofSelf-incompatibility maintained afbr the divergence of Bragsioaolerαα犯and Brassica Papa. Plant J 29 : 215-223.Kitashiba I, Zhang S-L, Wu J, Kenta Shirasawa K, Nishio T (2008) Sgenotyping and S screenlng utilizing SFB gene polymorphism inJapanese plumand sweet cherry by dot-blot analysis･ MoI Breed21 : 339-349.Li F, Kitashiba I, Inaba K, Nishio T (2009) A Brassica rapa linkage map ofEST-based SNP markers and its application tD QTL analysis forflowenng time and leaf morphologlCaltraitS･ DNA Resewch inPreSS･Nishio T, Kusaba M, Watanabe M, Hinata K (1996) Registration of Sal1elesin Brassica campestris L bythe restriction fragment sizes of SLGs･Tb AppI Gbnet 92 : 388-394.Nishio T, Izumida A, Hanzawa H, Sakamotx) K (2006) Development of StestxBr lines of Brassica oler-, Brassh rapa, and Raphanus sabivusas genetic resources. Acta Hort 706 : 141-144.0knmoto S, Od血ima M, Fujimoto R, SatD Y, Kitashiba H, Nishio T (2007)Self-Compatibility in Brassica napw is caused by independentmutations in S-locus genes. Plant I 50 : 391-400.Sato K, yishio T, Kimura R, Kusaba M, Suzuki T, Eat止eyama K, OckendonDJ, Satta Y (2002) Coevolution of the S-locus genes SRK, SLG,and SPll/SOB in Brassゐα oleⅧ¢α and B. rapa. Gbneths 162:931-940.Sato Y, Fujimoto R, Toriyama K, NiShio T (2003) Commonality of self-recognition specificity of S haplotypes between Brassica oleroM andBrassica raga. Plant MoI Bゐ1 52 : 6171626.Sato Y, Nishio T (2003) Mutation det冶Ction in rice waxy mutant遵by PCR-RF-SSCP. 2%r AppI Gbnet 107 : 560-567.Sato Y, 0knmoto S, Nishio T (2004) I)iversification and alteration of recogni-Recent Research Tbphs in the Labwabry of Plant Breeding and Genetics　43tion specificity of the pollen ligand SPll/SCR in self-incompatibility of Brassica and Raphanus. Plant Cell 16 : 3230-3241.Sato Y, Shirasawa K, Taknhashi Y, Nishimura M, Nishio T (2006) Mutantselection of progeny of gamma-ray-imdiabdrice by I)NA heterodu-plex cleavage using Brassica petiole extract･ Breed Stience 56 :179-183.Shinada T, Kikuchi Y, Fujimotx) R, Kishitani S (2006)Analloplasmic male-sterile line of Brassica olerw harboringthemitochondria fromDi'plotaxis muralis expresses a novel chimeric open reading frame,orf72･ Plant Cell Physゐ1 47 : 5491553.ShiokaiS, Shirasawa K, Sato Y, Nishio T (2009a)lmprovement of the dotrblot-Sup t冶0lmique for e缶cient and cost-effective genotyping･ MoIBreed in pressShiokai S, Kitashiba H, Shirasawa K, Nagano K, Nishio T (2009b)Leaf-punchmethOd to prepare a large number of PCR templates from plants forSNP analyslS. MoI Breding 23 : 329-336.Shirasawa K, Monna L, Kishitani S, Nishio T (2004) Single nucleotidepolymorphisms in randomly selectx)d genes among Japonゐarice(Oryza saliva L･) varieties identified by PCRIRF-SSCP. DNAResewch ll : 275-283.Shirasawa K, Shiokai S, Yamaguchi M, Kishitani S, Nishio T (2006) Dot-blot-SNP analysis for practicalplant breeding and cultivar identificationinrice. Th60r AppI GbnBt 113: 1471155.Shirasawa K, Maeda H, AIonna L, Kishitani S, Nishio T (2007) The number ofgenes having different alleles between rice cultivars estimated bySNP analysis. Theor AppI Gbnet 115 : 1067-1074.Takuno S, Fujimoto R, Sugimura T, Satx) K, Okanoto S, Zhang S-L, Nishio T(2007) Effects of recombination on the hit血hiking diversity inBrassica self-incompatibility locus complex. Gbnetics 177 : 949-958.Taknno S, Oikawa E, Kitashiba H, Nishio T (2009)Assessment of geneticdiverslty Of紙()eSSions in Brassica応eae genetic resources by frequencydistribution analysis of S haplotypes. Submitted.

Recent Research Topics in the Laboratory of Plant Breeding and Genetics

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Recent Research Topics in the Laboratory of Plant Breeding and Genetics

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