Rice Genetics IV - IRRI books - International Rice Research Institute

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primers (G1, G2) and two Tos17-specific primers (T1, T2) were used in separatereactions to detect Tos17 insertions irrespective of their position and orientation. Twoprimer combinations produce PCR products if mutants carry insertions between theT1 and T2 primer sites. If mutants carry insertions outside of this region, only oneprimer combination produces PCR products. To carry out PCR screening efficiently,a two- or three-dimensional DNA-pooling system can be used. To screen a smallpopulation, a two-dimensional system can be used. A mutant of the homeobox gene(OSH15) was found in a small population consisting of 529 lines (Sato et al 1999).Based on the phenotype of the mutant, OSH15 was shown to be involved in the developmentof internodes.Figure 3B shows a three-dimensional pooling system. A population of 960 mutantlines was arrayed in a 96-well format. Seeds of the lines were pooled in a threedimensionalmatrix and DNA was extracted from shoots derived from each pool ofseeds. From nine populations (a total of 8,640 lines), 270 pooled DNAs were extracted.A total of 8,640 lines can be screened by 72 (= 2 × 4 × 9) PCR reactions usinga super pool consisting of four X-pool DNAs. Figure 4 shows one example of screening.Eighteen mutant alleles of OSERECTA were identified. Considering the saturationlevel (see below) of the mutant population used for PCR screening, the OSERECTAgene must be a hot spot for Tos17 insertion. This gene is homologous to ERECTA ofArabidopsis (Torii et al 1996); it is thus named OSERECTA. If a mutant of a targetgene is found, Y- and Z-pool DNAs from the positive population undergo PCR screeningto identify the mutant line. A total of 11,809 lines carrying 84,975 insertions havebeen screened for mutants of 47 genes and mutants of 15 genes have been identified.These include mutants of genes involved in signal transduction and genes for transcriptionfactors. Some mutants are being characterized for their phenotypes. Amongthose, mutants of OSMPK2 (MAP kinase gene), OSDWARF3 homologous to the maizeDWARF3 gene (Winkler and Helentjaris 1995), and OSERECTA showed clear phenotypes.The results described above demonstrate the feasibility of PCR screening of mutants.To make this system generally applicable to any gene of interest, mutationsinduced by Tos17 insertion must be saturated. The success rate (15/47) of PCR screeningsuggests that at least 37,000 lines are required for saturation mutagenesis. The numberof lines required for saturation mutagenesis can also be estimated by using theequation of Clarke and Carbon (1976). By assuming that (1) Tos17 transposes randomlythroughout the chromosomes, (2) the average size of rice genes is 3.0 kb, and(3) the number of transposed Tos17 copies per regenerated plant is eight, then a totalof 58,875 mutant lines are required to have a 99% chance of finding a mutant of anygene. Because Tos17 prefers low-copy, gene-rich regions as integration targets, fewermutant lines may be enough for saturation. Considering the ease of tissue culture andregeneration of rice, saturation mutagenesis with Tos17 is quite feasible.Systematic screening of mutants by sequencing Tos17 insertion sites. AlthoughPCR screening seems to be the most efficient approach for reverse genetics, it maynot be suitable for the analysis of a large number of genes. Considering large-scalefunctional genomics in the postsequencing era, a systematic approach to find mutantsfor a large number of genes is needed. One of the possible approaches is cataloguing286 Hirochika et al
5 kb—G1-T11 kb—5 kb—G1-T21 kb—5 kb—G2-T11 kb—5 kb—G2-T21 kb—Fig. 4. Screening for mutants of OSERECTA. Twenty-two superpools underwent polymerasechain reaction using four combinations of two gene-specific primers (G1, G2) and two Tos17-specific primers (T1, T2). Positive bands (indicated by arrowheads) were detected by DNAblotting with a gene-specific probe. A total of 10,944 lines carrying 72,000 insertions werescreened.Retrotransposons of rice as a tool . . . 287
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Retrotransposons of rice as a tool
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First, the Rice Genetics Cooperativ
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OverviewRice genetics from Mendel t
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Rice genetics from Mendelto functio
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nese cultivar Nipponbare. The chrom
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Gene symbolization in riceIn the ab
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Table 2. Some examples of mapping g
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and YAC libraries, respectively. Th
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gene from wild species is the intro
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ReferencesAbbasi FM, Brar DS, Carpe
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Nagao S. 1951. Genic analysis and l
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Yoshimura S, Yamanouchi U, Katayose
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important traits for which segregat
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trolled by a single recessive gene,
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AromaAromatic rice varieties often
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Submergence tolerance is an interes
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Kinoshita T, Maekawa M. 1986. Genet
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NotesAuthors’ addresses: J.N. Rut
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The Rockefeller Foundation has a lo
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Evolution and implementation of the
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Scientific progress and outputsIn t
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Another salient example is that of
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BOX NO. 1recent international works
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For the purposes of this discussion
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Because of the great diversity (sci
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eeders where final products to enha
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Chen S, Lin XH, Xu CG, Zhang Q. 200
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Toenniessen GH. 1998. Rice biotechn
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Molecular markers,genetic diversity
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Evolution and domestication of rice
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ABCDO. barthiiO. rufipogonO. longis
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Wild(O. rufipogon)Geographical diff
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South Asia (particularly on the wes
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al 1992, Sun et al 1996a,b,c), thou
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wild and cultivated types (Est10, W
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Cai HW, Morishima H. 2000a. Diversi
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Sato YI, Tang SX, Yang LU, Tang LH.
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The solid base laid by classical ri
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The synthesis shown in Figure 1 is
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Arabidopsis, long heralded as a “
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Monocots and eudicots: Is there sti
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Zhang H, Jia J, Gale MD, Devos KM.
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ility of rice productivity (Lu 1999
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Table 1 continuedIntrageneric class
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Molecular markers and evolutionary
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A100100521010078100991009894100O. s
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Two distinct types of sequences wer
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arthii and O. longistaminata, and t
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testing Ka/Ks between the homeologo
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Roschevicz RI. 1931. A contribution
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Miniature inverted repeattransposab
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eau and Wessler 1992, 1994), rice (
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MITEs as a source of allelic divers
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ATIR Olo-D* Olo-CTIRBCas Exp Wan Sn
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Le QH, Wright S, Yu Z, Bureau T. 20
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Microsatellite markers in rice:abun
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make the best SSR markers for rice.
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Predicted frequency100,00080,000Cla
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Relationship between SSRs and genes
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are indicated with RM locus designa
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SSRs have been used to define intro
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provide a bridge for moving rapidly
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Ishii T, McCouch SR. 2000. Microsat
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NotesAuthors’ addresses: S.R. McC
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traits of economic importance (Mack
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Table 2. Tagging and mapping of som
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even at the juvenile stage. Several
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Chen et al (2000) transferred the b
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genome sequence of rice, this will
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Kurata N, Nagamura Y, Yamamoto K, H
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Witcombe JR, Hash CT. 2000. Resista
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QTL mapping in rice:a few critical
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M-QTLsM-QTLs are defined as single
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(genetic/statistical models and cor
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Table 5. The percentage of three ty
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Table 7. Some environment-specific
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ferently to the environments. Simil
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(case 3), one may infer that this g
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Simultaneous QTL introgression and
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Doebley J, Stec A, Gustus C. 1995.
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Visscher PM, Haley CS, Thompson R.
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unavailable until recently with the
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Table 1 continued.Trait QTL a Flank
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the remaining parts of the linkage
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Table 3. QTLs detected for yield an
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Table 6. Correlations of various pa
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The functions of these sequences ne
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Structural and functional genomicsT
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The International Rice GenomeSequen
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the RGP, a PAC (P1-derived artifici
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Progress of sequencing in the RGPWe
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ice genome sequencing to organize a
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Strategies and techniquesfor finish
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uses the quality values generated b
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with the advanced techniques requir
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times contain sufficient data to ma
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amplification of regions that previ
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4. Structure problems—subcloning
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estriction maps or optical maps are
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nal assembly may point to areas tha
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McMurray AA, Sulston JE, Quail MA.
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Sequence-tagged connector/DNA finge
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1999). The development of multiple-
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Transposable elements in the rice H
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ers, maps, mapping populations, and
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Miropeats-OSJNBa0065H03 1.2 cMThres
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NotesAuthors’ addresses: R.A. Win
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Sasaki 1997). Such analysis is ofte
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ABNipponbare × KasalathF 2QTL anal
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were classified into two groups bas
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effective at determining the genoty
Page 248 and 249: addition, some accessions of wild r
Page 250 and 251: Sasaki T, Burr T. 2000. Internation
Page 252 and 253: Mutational analysis has long been t
Page 254 and 255: marked by a deletion/chromosomal re
Page 256 and 257: analysis suggests that the mutation
Page 258 and 259: For drought-response screening, our
Page 260 and 261: Pi-7(t)Xa21Xa10Xa3Xa4Pi-1(t)Pi-k, P
Page 262 and 263: Although mutants with discrete gene
Page 265 and 266: Generation of T-DNA insertionaltagg
Page 267 and 268: Aprobe AEEprobe BpGA1633RBgus Tn p3
Page 269 and 270: Number of loci36%224%170%010 20 30F
Page 271 and 272: Table 2. GUS assay in roots of tran
Page 273 and 274: 1999, Krysan et al 1999, Sato et al
Page 275 and 276: Transposons and functionalgenomics
Page 277 and 278: cesses. To study the interactions a
Page 279 and 280: Constructs were made with the aim o
Page 281 and 282: RBcis-effect of the adjacent strong
Page 283 and 284: Ac knockout mutagenesisThe Ac lines
Page 285 and 286: Table 2 summarizes the transformati
Page 287 and 288: ReferencesBaldwin D, Crane V, Rice
Page 289: NotesAuthors’ addresses: R. Greco
Page 292 and 293: These include T-DNA (Azpiroz-Leehan
Page 294 and 295: polyproteins and two identical 138-
Page 296 and 297: mutant. The mutation in the ent-kau
Page 300 and 301: of insertion mutants by sequencing
Page 302 and 303: Grandbastien M-A, Spielman A, Caboc
Page 304 and 305: NotesAuthors’ addresses: H. Hiroc
Page 306 and 307: human endeavor, greatly contributed
Page 308 and 309: Equally important as the genomic in
Page 310 and 311: Oryzabase is one of the most recent
Page 312 and 313: Database structureA characteristic
Page 314 and 315: types of users. An annotation datab
Page 316 and 317: ReferencesAntonio BA, Fang Z, Sanch
Page 319 and 320: Gene isolation and functionEnhancin
Page 321 and 322: Enhancing deployment of genes forbl
Page 323 and 324: mochi and Tsuyuake (Wu et al 1996).
Page 325 and 326: tive indica rice varieties supports
Page 327 and 328: transcription factor, resulting in
Page 329 and 330: facilitate breeding durable resista
Page 331 and 332: (Inukai et al 1994, Yu et al 1996,
Page 333 and 334: Rossman AY, Howard RJ, Valent B. 19
Page 335 and 336: Molecular signaling in diseaseresis
Page 337 and 338: AOsRac1IED II III IV 214aaOsRac1 KC
Page 339 and 340: Japonica rice variety Kinmaze, whic
Page 341 and 342: Table 1. Characteristics of lesion-
Page 343 and 344: an effective inducer of host resist
Page 345: Takahashi A, Kawasaki T, Henmi K, S
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et al 2000). Sequence analysis of t
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612-amino acid protein, including t
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Genetic dissection of the Xa21-medi
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ReferencesAnderson PA, Lawrence GJ,
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Isolation of candidate genesfor tol
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Materials and methodsPlant material
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Microarray hybridization and data a
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Table 3. Most abundant transcripts
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Microarray technologyThe rapid accu
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view of procedures, pitfalls, and n
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Log(2) ratio1.51.0OC104E01—WCP1OC
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Table 5. Strongly regulated transcr
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ReferencesAdams MD, Soares MB, Kerl
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Acknowledgments: We thank Chris Bor
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y cells separating during tissue de
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(Erwee and Goodwin 1983). The sympl
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tion between the abundance of CDK t
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in a complex of 105 kDa. These resu
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Drew MC, Jackson MB, Giffard S. 197
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Umeda M, Umeda-Hara C, Yamaguchi M,
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asexual reproduction through apomix
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MPManual pollination by breeders×
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Search for apomixis in riceThere ar
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Issues related to achieving synthet
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come and may use imprinting to achi
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Embryo-inducing geneInactive ablati
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M 1 3 3 4 5 6 7 8 9 10 11 12 13 14
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embryos, several genes characterist
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Our search for a rice homologue of
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sequenced the two rice DMC1 genes a
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Gustine DL, Sherwood RT, Huff DR. 1
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Peel MD, Carman JG, Leblanc O. 1997
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TransformationEngineering for virus
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Engineering for virusresistance in
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proteins, (2) the expression of dys
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Antisense and cosuppression RNA. Ex
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Table 1. Transgenic rice with patho
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the disease symptoms. Efforts from
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an ambisense coding strategy (Fig.
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first produced and shown to have vi
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McLean GD, Evans G. 1997. Some pote
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Waterhouse PM, Upadhyaya NM. 1998.
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duction in response to dehydration
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(KIKEKLPG) in the carboxyl terminus
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These three plasmids were used to t
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We now show the effects of the toba
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Since one major goal of plant scien
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Table 4. Copy number of transgenes
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Chan M-T, Chang H-H, Ho S-L, Tang W
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NotesAuthors’ address: Department
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initially fixed by phosphoenolpyruv
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High-level expression of maize C 4-
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The effect of illumination on PPDK
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ate in 2% O 2 can be limited by Pi
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Transgene integration,organization,
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duced by organogenesis, somatic emb
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ing transgene organization. FISH an
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ments and the integration of exogen
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Level 2: activation of local repair
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strongly suggested that plant facto
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other cases it was not. We observed
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Gorbunova V, Levy AA. 1997. Non-hom
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Gene silencing and itsreactivation
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Carbofuran (trade name, Furadan) is
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Akb4xHpaIIJKA52 (R 0)52-6 (R 1)52-9
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ABCDhptuidAmUbi1kb10.08.05.04.13.0F
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Fig. 4. X-gluc staining of germinat
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eflect different causes for silenci
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Table 4. Suppressors of silencing f
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Garrick D, Fiering S, Martin DI, Wh
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Xu Y, Buchholz WG, DeRose RT, Hall
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Rice molecular breeding workshopThi
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the working group can develop a net
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Rice genetics cooperativeA meeting
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