Rice Genetics IV - IRRI books - International Rice Research Institute

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HindIIIHindIIIRB 35S hpt 35S 35S int-uidA nos 5’ mUbi1 intron bar nos LBProbesABC3.1 kbA B CFig. 5. FISH detection of the chromosomal location of T-DNA in JDV92. Denatured chromosomesof R 1 seedlings were hybridized with digoxygenin (DIG)-labeled T-DNA sequences ofpJDV (upper panel). The hybridization signals (yellow arrow) were visualized using the fluorescentdye Cy3 in a background of 4’, 6-diamidine-2’-phenylindole dihydrochloride (DAPI)-stainedchromosomes. FISH analysis revealed two chromosomal transgene integration sites in JDV92(A). Genomic DNA blot analysis of DNA from R 1 seedlings of JDV92, digested with HindIII andprobed with 32 P-labeled hpt (B) or uidA (C), revealed the segregation of two loci (yellow arrowsin panel A), one of which contains multiple copies of the insert and the other a single-copytransgene (arrows in panels B and C). After segregation from the multiple locus, plants bearingthe one-copy locus became GUS positive (see text).Table 3. Epistatic interaction between transgeneloci combined by sexual crossing. Selected 35S/uidA-silenced lines (JDV83, 90, 95) were usedin crosses with functional lines (JDV85, 86); GUSexpression and bialaphos resistance were assayedin the resulting hybrids. Crosses weremade in both directions and 2–3 hybrids wereanalyzed.Hybrid a GUS BARJDV85 × 95 + +JDV86 × 95 + +JDV88* × 95 + +JDV83 × 95 – +JDV85 × 83 – +JDV86 × 83 – +JDV85 × 90 – +JDV86 × 90 – +a* = silenced JDV88 R 1 lines.474 Hall et al
eflect different causes for silencing. For example, the presence of a multicopy locusmay trigger silencing at relatively distant loci such as the super-silencer loci describedby Matzke et al (1994) and Vaucheret (1993), whereas the apparently potently silencedlocus (both uidA and bar are inactive) in JDV95 may have only local (cis)effects. It is noteworthy that the int-uidA gene was preferentially silenced in theseexperiments and it is tempting to attribute this to its being driven by the 35S promoter,which has been implicated in many cases of transgene silencing (Iyer et al 2000).Interestingly, a reactivation of 35S/int-uidA was observed in JDV95 × JDV88.The reactivation of the silenced 35S/int-uidA in JDV88 in the presence of (or by) thetransgene locus in JDV95 is unique in that interaction between two silenced loci resultedin reactivation of the silenced gene. This observation implies that the interactionamong the silenced loci is dependent on distance because the crosses betweenJDV95 and other functional loci (JDV85, JDV86) and silenced loci (JDV83, JDV90)did not show any effect on the reporter gene expression. The remote possibility existsthat the activation observed represents complementation of mutations caused bytransgene insertion. Transgene inactivation resulting from interacting loci has previouslybeen shown to be chromosomal-location-dependent (Schmulling and Rohrig1995). Addressing the physical distance between transgene loci is critical in understandinginactivation and reactivation, but remains a challenge because current FISHtechnology does not allow estimation of the native physical distance between twotransgene loci on different chromosomes. We are examining segregating hybrid linesto determine whether the modification of the functional locus by the multicopytransgene locus is reversible.That transgene silencing is caused by various mechanisms involving modificationsof DNA, RNA, and chromatin structure has now been rigorously established.However, the transgene-silencing phenomena we have observed in rice are complicated,for example, AzaC and TSA treatment reactivated GUS expression in linesJDV90 and JDV92 but not in JDV95. Although multicopy transgene events are mostfrequently silenced (Flavell 1994, Garrick et al 1998, Henikoff 1998, Kumpatla andHall 1999), silencing can occur for a single intact transgene locus (Table 1) and evenfor a single gene flanked by active transgenes at a single locus (JDV88); thus, theremay or may not be epistatic interactions between transgene loci.Alleviating transgene silencingAs silencing of desirable transgenes is a substantial complication in biotechnologicaldevelopment of novel crops, there is considerable interest in avoiding gene silencingor in overcoming its effects. Insight into the mechanisms of both TGS and PTGS israpidly increasing (Cogoni and Macino 1999b, Fagard and Vaucheret 2000, Iyer et al2000) and several mutations (mostly in Arabidopsis) have now been shown to affectgene silencing. Several groups, including our own (Kumpatla et al 1998), are exploringstrategies for evading silencing by introducing sequence heterogeneity into theRCg2 promoter. The suppression of methylation systems and the reduction of opportunitiesfor heterochromatinization, for example, by the insertion of enhancer ele-Gene silencing and its reactivation in transgenic rice 475
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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
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addition, some accessions of wild r
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Sasaki T, Burr T. 2000. Internation
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Mutational analysis has long been t
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marked by a deletion/chromosomal re
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analysis suggests that the mutation
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For drought-response screening, our
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Pi-7(t)Xa21Xa10Xa3Xa4Pi-1(t)Pi-k, P
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Although mutants with discrete gene
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Generation of T-DNA insertionaltagg
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Aprobe AEEprobe BpGA1633RBgus Tn p3
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Number of loci36%224%170%010 20 30F
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Table 2. GUS assay in roots of tran
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1999, Krysan et al 1999, Sato et al
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Transposons and functionalgenomics
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cesses. To study the interactions a
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Constructs were made with the aim o
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RBcis-effect of the adjacent strong
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Ac knockout mutagenesisThe Ac lines
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Table 2 summarizes the transformati
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ReferencesBaldwin D, Crane V, Rice
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NotesAuthors’ addresses: R. Greco
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These include T-DNA (Azpiroz-Leehan
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polyproteins and two identical 138-
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mutant. The mutation in the ent-kau
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primers (G1, G2) and two Tos17-spec
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of insertion mutants by sequencing
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Grandbastien M-A, Spielman A, Caboc
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NotesAuthors’ addresses: H. Hiroc
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human endeavor, greatly contributed
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Equally important as the genomic in
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Oryzabase is one of the most recent
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Database structureA characteristic
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types of users. An annotation datab
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ReferencesAntonio BA, Fang Z, Sanch
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Gene isolation and functionEnhancin
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Enhancing deployment of genes forbl
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mochi and Tsuyuake (Wu et al 1996).
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tive indica rice varieties supports
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transcription factor, resulting in
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facilitate breeding durable resista
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(Inukai et al 1994, Yu et al 1996,
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Rossman AY, Howard RJ, Valent B. 19
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Molecular signaling in diseaseresis
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AOsRac1IED II III IV 214aaOsRac1 KC
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Japonica rice variety Kinmaze, whic
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Table 1. Characteristics of lesion-
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an effective inducer of host resist
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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.
Page 436 and 437: duction in response to dehydration
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Page 444 and 445: Since one major goal of plant scien
Page 446 and 447: Table 4. Copy number of transgenes
Page 448 and 449: Chan M-T, Chang H-H, Ho S-L, Tang W
Page 450 and 451: NotesAuthors’ address: Department
Page 452 and 453: initially fixed by phosphoenolpyruv
Page 454 and 455: High-level expression of maize C 4-
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Page 458 and 459: ate in 2% O 2 can be limited by Pi
Page 461 and 462: Transgene integration,organization,
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Page 465 and 466: ing transgene organization. FISH an
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Page 469 and 470: Level 2: activation of local repair
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Page 473 and 474: other cases it was not. We observed
Page 475 and 476: Gorbunova V, Levy AA. 1997. Non-hom
Page 477 and 478: Gene silencing and itsreactivation
Page 479 and 480: Carbofuran (trade name, Furadan) is
Page 481 and 482: Akb4xHpaIIJKA52 (R 0)52-6 (R 1)52-9
Page 483 and 484: ABCDhptuidAmUbi1kb10.08.05.04.13.0F
Page 485: Fig. 4. X-gluc staining of germinat
Page 489 and 490: Table 4. Suppressors of silencing f
Page 491 and 492: Garrick D, Fiering S, Martin DI, Wh
Page 493: Xu Y, Buchholz WG, DeRose RT, Hall
Page 496 and 497: Rice molecular breeding workshopThi
Page 498 and 499: the working group can develop a net
Page 500: Rice genetics cooperativeA meeting
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