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JARQ 34(3), 153- 158 (2000) htlp://ss.j ircas .affrc.go.jp Visual Detection of Useful Genes on Plant Chromosomes Kiichi FUKUI1* and Nobuko OHMIDO2 Department of Biotechnology, Graduate School of Engineering, Osaka University (Suita, Osaka, 565-0871 Japan) 2 Laboratory of Rice Genetic Engineering, Hokuriku National Agricultural Experiment Station (Joetsu, Niigata, 943-0193 Japan) 1 Abstract In this manuscript the current status of direct visualization of gene location on chromosomes was reviewed by considering 5 aspects as follows: principle of in situ hybridization (ISH) method, historical perspective of mapping genes by in situ hybridization, improvement in the sensitivity of fluorescence in situ hybridization (FISH), visualization of location of useful genes on chromosomes, and prospects for visualization of useful genes. Discipline: Experimental apparatus and method / Biotechnology / Plant breeding Additional key words: physical mapping, rice, FISH Princip l e of in situ hy brid i za t ion (I SH) m eth od lSH is a method for the visualiza tion of the location of nucleotide sequences 011 chromosomcs, nuclei, and even in the tissues. The principle of the method involves the hybridi zation of labeled nucleotide sequences, or probes, directly to DNAs or RNAs with the comp lemen tary sequences in chro111oso111cs, nuclei or tissues. T he location of the labeled probes hybridized to lhc complementary DNA sequences in ch romoso111cs, for exa111plc, is detected by fluorochro111e-l abeled antibodies that recognize the label of the probes under a fl uorescence microscope. T he I Si-i in wh ich fluorescence is used to detect probes is referred to as "fluorescence situ hybridization (FISH)". i,, Histo r ica l p er sp ecti ve of mapping genes b y ISH i n r ice 2> In 1910, the rice chrornosornc 11u111bcr was determined to be 2n=24 by Kuwada 1•>. I( took, however, more than 80 years until all the rice chromoso111cs were identified objectively and a rice ch romosome map was developed by Fukui and liji111a3>using i111aging mcthods 1>. The long time interval from the determination of the r ice chromosome number to the identification of individual chro111oso111cs can be ascribed lo the fact that rice has the smallest genome size of 430 Mb, thus the smallest chromoso111c size, a111ong those of the main cerea ls. A ttempts to vi sualize specific DNA sequences directly on r ice chro111oso111es have been pursued for years w ithout the use of an objective method for identi f'ying rice chromosomes nor a chro111oso111e 111ap at the beginning of the 1980s. f-ukui ct al. 6>succeeded in physically locating I 8S-5.8S-25S r ibosomal RNA genes ( 45S rDNA) loci at the end of a pair of chro111oso111es wi th 12 ; iodine-labelcd ribosoma l RNA probes. T his was the fi rst reproduc ible result of i11 situ hybridization (ISi I) using repeti tive sequences in rice (Fig. la). The location of 4SS rDNA loci in rice was detected by non-radioactive (biotin-labeled) probes by coloration using Japonica r ice in 199J 8l (Fig. lb). FISII was successfully achieved in 1992 for the fi rst ti me and variability in the number of 45S rDNA loci was revealed among the rice species;> (Fig. le). Multicolor FISH using di fTcrcnl fl uorescent co lors simultaneous ly for detecting 5S rDNA and 45S rDNA was developed in 1994 soon after the successful development of FlSH. Presen tly the multicolor FISH is a common method widely utilized to locate different nucle- This work has been supported in pan by the Grant- in-Aid for IRRl/.lapan Sh1111le Research Project and Rice Genome Project, AFFRC, MAFF, Japan. * Corresponding author: fox 1 81- 6- 6879- 744 1, e-mai l k [email protected] Received I 2 November 1999, acceplcd 6 January 2000. 154 JARQ 34(3) 2000 • q Fig. I. Recent advan ces in in situ hybridiza tion (1Sll) for the rias( 15 years n: Detection of ribosomal RNA gene (rDNA) locus with rndioac1ivc ribosoma l RNA. b: Detection of rDNA by n non -RI labeling method. c: Detection ofrDNA loci in lndica rice by FISH. d: Detection of multi-co lor lluorescc:ncc from differcn1probes (tclomerc. green and TrsA, red) simultaneously. fig. 3. Visu31ization of t he location ,or useful genes on rice chron1osomcs n: Ga ll midge resistance gene on chro111oso111e 4. b: Lcafblasl resistance gene on chromosome 2. c: Bacterial blight resistance gene on chromosome 11. d: RFLP murker (X11p247) on chromosome 4. 155 K. F11k11i & N. Ohmido: 1'vfappi11g of Useful Genes by FISI/ otidc sequences si111ultancously, such as telomere sequences (green fluorescence) and rice A genome speci fic tandem repeat sequence, TrsA (red fluorescence) (Fig. ld) 18>. All these efforts have enabled the detection of highly repetitive sequences on ri ce chromosomes reproducibly and elliciently. The devclo11111ent of both the chromo~omc map and the F·1s 1-1 method has contri buted significa ntly to the physical mapping or DNA sequences in rice. Improvement in the sensitivity of FIS H Once reproducible detection of the repet1t1ve sequences on rice ch romosomes using the FISH method was obtained in the earl y 1990s, the main obj ective of f- lSH was Lo enhance th e detection sensi ti vity of the nuorescent signa ls. Even though the v isual detection of repetitive sequences on rice chromosomes had become rout ine work, detection of a unique sequence was considered to be difficult because of the smaller sizes of nucleotide sequences. Fig. 2a shows clones di ffering in si%es, which wcrc:used as probes in the f- lSI I experiments 10 evaluate the sensi tivity of detection of fluorescent signals on rice chromosomes. In the clones wilh insert sizes rang ing -~ from 399 kb lo around I kb, attempts were made to determine whether their nuorescence could be visualized on rice chromosomes after FISl-1 19>. T he largest clones were those of yeast arti fic ial chromosomes ( YAC) w ith 399 and 340 kb of inserted ri ce genomic DNAs. A bacterial arti ficia l chromosome (BAC) with a 180 kb insert, and a cosmicl clone wi th a 35 kb insert were also tested using the f- lSI-I method. T he smallest clones were molecular markers cloned into plasmid vectors with around I kb inserts. To visualize these clones, both the experimental procedu res and the detection equipment were improved. First, the detection sensi ti vity of fai nt nuoresccnt signals from the probe DNAs was enhanced by the introduction of a cooled CCD camera. The camera was directly mounted on top of a microscope. Second, in the FISH protocol, a longer hybridi,.ation period between the labeled probes and chromosomal DNAs was used. The longer hybridization period ensured hybridization between the probes and complementary DNA in the chromosomes. All the clones from nearly 400 to I kb were success fully visualized by the improved f-ISH method. As a result, the detection sensitivity of r!SI I was enhanced by 400 limes throughout the cxperimcnts19l (Fig. 2b). Visualization of location of useful genes on plant chromosomes YAC I) Mapping oft1 resistance gene 10 gall midge, Gm2 399kb BAC 0 180kb Fig. 3 shows the results of the improved FISH Cosmid 0 35kb Plasmid 0 0 0 0 0 0 1.3kb (a) 100kb 400 .-.. CEN .D .. "' t:.. 200 "' c •!:l ~Xa-21 ~NOR ~CEN I 4 ...s:: (b) 0 Fig. 2. Various sizes of probe ONAs used in FISH (a) and improvement of the detection sensitivity of F ISI I (b) (a) Rice chromosome 11 (b) Barley c hromosome 6 Fig. 4. Discrepancy between the chromosome nnd genetic maps for rice chrom osome 11 (a) and barley chromosome 6 (b) 156 method using different DNA clones as probes. All the clones contai ned llSe ful genes such as disease resistance genes. The result of FISH using a YAC c lone (YAC5212) w ith a 340 kb rice genomic DNA was presented, because YAC5212 is Oanking a gall midge (Orsevlia vryzae) resisuincc gene, Gm2. Gall midge is a major diptcrnn insect pest of' rice in India, China, A frica and Sou theast As ia and i ts resistan ce genes arc now being cloned. Fig. 3a shows the YAC signals on the pair ofricc chromosome 4111. Fluorescent signals arc detected in the imerstitial region of long arms indicat ing th e phys ical location of G1112. The posi tion of G1112 was also confirmed by the FISH using 2 RFLP markers genetically I inked to Cm2. 2) Mappi11g o.fa resis1a11ce ge11e to leafblas1, Pi-b The phys ical location of the resistance gene to rice leaf blast was then examined. Rice blast caused by Pyricularia 01:i,zae (Mag11apor1/te grisett (Hebert ) Barr) is the most import<1nt fung,11 diseMc or rice. T he resistance gene. Pi-b was cloned into a BAC clone (BACl23) which has a 180 kb insert of rice genomic DNA. Because the 1.ocation of the genomic DNA on r ice chromosomes was unknown, the FISI I method was employed to physically determine the actual location of the 13/\C clone or the posiiion of the gene. Pi-b. Fig. 3b shows the signals from the BAC clone at the end o f the long arm of r ice chromosome 211l. Haploid rice plan t derived by an ther cultme was used for chromosome rreparations. T hus one single- tagged chromosome shows clear doublet signals wh ich arc essential for the discrimination between genuine signals from noises. Thus the chromosomal location of the Pi-h gene was determined at 2JJ 1.2 according to the rice chromosome mapi 1. We further analyzed the BAC signals on the I 0 chromatids or rice chromosome 2 by the imaging method t<> determine the precise location of the gene on the chromosomes. /\s a result. the Pi-h gene was located at the position of 96. 16 ± 0.9 1 from the end of the short arm w hen the tota l chromosome length of chromosome 2 was I 00.00 171. This method was also employed to determine the 5S ribosomal RNA genes on r ice chromosomes. The 5S rDNA locus was allocated to the posi tion of39.3 ± 2.6 on chromosome 11 ( 11 p 1. 1) from the end of the short arm 1°1• 3) 1'vlt1ppi11g c~/'a resis1a11ce ge11e to bac1eria/ blig/11, Xa-21 The smaller rice genomic DNA o f 35 kb nllcleotide sequences cloned into a cosmid vector, was examined. T he clone contained a resistance gene, Xa-2 / to the r ice disease, ba cterial lcaf'blight (Xa111/to111011as 01:yzae). Bae- JARQ 34(3) 2000 teria l leaf blight is also a m ajor rice disease widely distributed in Asia n countries. T he same improved FISH method was used to locate Xa-2 / on the r ice ch romosome. In th is case, since we used the lndica rice variety JR36, a diploid plant material, the signals were observed in a pair of rice chromosomes. Fig. 3c shows the fluorescent signals fi·om a p<1ir of rice chromosomes. By using the uneven condensation pattern, the rice chromosomes were identi ricd as rice chromosome 11 and the dollblct signals were clearl y observed in the intersti tial region on the long arm (1 lql.3) 191. 4) Mappi11g of lite 1110/ecular marker, Xpn 247 Molecular markers are very useful for the construction of I ink age maps and more than 2,000 molecular markers were developed to construCL the rice linkage map111. The size of the molecular markers vari es and the size o f molecular markers, such as RFLP markers is often less than a few ki lo bascpairs. The RFLP nrnrkers played an importani role in th e constructi on of mt111y linkage maps at the beginning of genome mapping. We applied the f-lSH method to locate a RFLP marker (X11p 247) w ith 1.29 kb of r ice genomic DNA. Fig. 3d shows the signal position from the RFLP marker. The doublet signals were clearly detected at the end of r ice ch romosome 4 (4q2. I ) 1q 1• T he results indicate tha t even a nucleotide sequence w ith about I kb could be successfully detected by the improved FISH method. It also means that practica lly all the fun ctional genes could be directly detected 0 11 th e chromosomes by the improved FISH mcthod 17i . Sel t:incompatibility-related genes of Brassica spp. have already been mapped by using FISI I q· 111. The physical 111a1>ping of the nucleotide sequences also reveals a discrepancy between the physical length o r the chromosomes and the genetic distance ca lcu lated by the recombi nation val ues. Fig. 4 shows 2 examples of the discrepancy detected in rice191 and barley41. Comparison of the chromosomal Jocaiion or Xa-21 determined by FISH iind linkage analyses revealed a remarkable discrepancy between the total length in the 2 maps. The linkage map of chromosome 11 showed a much longer length when the chromosome map and the linkage map were ;,ligned by the 2 landmarks i.e. the centromere and the posi tio11 or Xa-21 now deter111ined 1'1J (Fig. 4a). As demonstrated in the barley chro111oso111c map that was developed by imaging metho<ls"l and linkage map, the length o f the satellite part. that is the terminal region o f the ch romosome 6 was markedly over-estimated in the linkage map. The discrepancy was .igain confirmed by a detailed comparison between the chromosome and dense K. Fukui & N. Ohmido: Mapping ofUscfit! G<!nes b,r FISN 157 linkage maps in barl ey' 21• T he over-estimation of the termi nal length in the linkage map compared to the actua I length o f barley chromosomes determined by FISH and/ or imaging methods suggests th at recombination occurs 2) frequently on ly in the terminal region o f the barley chromosomes. A Ithough the discrepancy between the overall leng th of the chromoso111e and linkage maps was demonstra ted in rice chromosome 11 (Fig. 4a), it remains 10 be determined whether the frequent recombination observed on ly 3) 4) i n the 1cr111inal chrom osoma l regions cou ld al so occur in rice and o ther pl ant species as a general tendency. 5) Prospects for visualization of useful genes 6) V isual ization or mapping of useful genes is i mportant in genetic analyses. Since, not all the plants ha ve as dense linkage maps as rice13) and it is usually time-consuming and laborious to constrnct 7) a dense l i nkage map. FISH i s a useful and effici eni alternative method i n many 8) p lant species. FISH could be appl ied !or the mapping of genes and clones regard less of p lant species. When molccu lar markers that sa nd wich it cenain 9) character ca n be found, the distance between the markers cou ld be obtained by using FISH on chromoso111es and ex tended DNA fibers (EDFs)201• Moreover, it may be a FISI I-orien ted method possible to clone the gene by and not by an ordina ry map-based cloning method. First, 10) 11 ) FISH using the linked 111arker(s) cou ld enab le to dcler111i11c the ehro111oso111al position of the target gene. Then the laser dissection 7, or the ordinary dissection method 12) cou ld be app l ied to collect i hc chromosomal fragments w ith the signals. Finall y. clones in the chromosomal region~ cou ld be obta ined by the degenerated o ligoprimer (DOP)-PCR method using the dissected chromoso111a l frag111en1s as the templates. Direct nmplificati on of riboso111al RNA gc11cs1~> and a-amylase gcncs 15) by the laser-dissection method has been successfully appl ied i 11 some cases. In conclusion, the visualization of uscliil genes is important 1101 onl y 10 determi ne their actual location 011 the chromosomes, bu t also i t 111ay be possible to clone the genes i n conjunc tion w i th the chromosome dissection method even in p lants wi th l imited genome information. It is anticipated that the visual ization method represcntecl by FISH w i ll be m ore uscli1l when it is combincc1 with new methods to manipulate chromosomes and even DNA fibers. Refe rences I ) Pukui. K . ( 1986): Standardiza1ion of karyo1yping plalll 13) chromosomes by a newly developed chromosome image analyzi ng sys1cm (Cl II AS). TheOJ: Appl. G<!net.. 72 , 27 32. Fukui. K. ( 1996): Advances in rice chromosome research. 1990 1995. In Rice Genetics Ill, Proc. 3rd Intl. Natl. Rice Genet. Symp. ed. Khush G. S.. lntcrna1iom1 I Rice Research lns1it111c, Manila, PhilipJ)incs, 117- 130. Fukui, K. & lijima, K. ( 1991 ): Somaiic chro111oso111e map of rice by imaging methods. 711em: Appl. Genet.. 8 I. 589- 596. Fukui, K . & Kakcda , K. ( 1990): QmtnlilHt ivc karyotyping of barley chromosomes by image ana lysis mctl1ods. G<!110111<!, 33, 450-458. Fukui, K .. Ohmido, N. & Khush. Ci. S. ( 1994): Variabil ity in rDNA loci in che genus 01:vza clc1cc1cd 1hrough nuoresccncc in situ hybridization. 71u101: Appl. Genet., 87. 893- 899. Fukui, K. c1 al. { 1987): /11 situ hybridization of 12' 1· l~bclcd rRNA 10 rice chromosomes. /lice G<!net. News1<!11 .• 4, 114- 11 6. Fukui, K. ca al. { 1992): Microdisscc1io11 or plan1 chromosomes by argon-ion laser beam. 77wo,: Appl. Genet., 84, 787- 791. l ijima, K .. Kakcda. K. & Fukui, K. ( 199 1): lclcmi fl cation and charncccriza1io11 or somatic rice chromosomes by imaging methods. 711eo1: Appl. Genet.. 81. 606- 612. lwa110, M. Cl al. (1999): Visualization ofa sclf-imcompaiibi l ity rclnccd SLG gene i11 Bmssica c,111,p<!stri:; L. by multi-color FISH. Tl1M1: Appl. Gener.. 96, 75 1- 757. Kmnisugi, Y. ct al. (1994): Physical mapping of the 5S ribosomal RNA genes on rice chromosome 11. Mo!. G<!u. Ge11et., 245. 133- 138. Kamisugi, Y. c1 al. ( 1998): Visualization or 1hc /Jmssica sclf-i 11compn1ibility S-locus on identified oilseed rape chromosomes. Plaut Mo/. Bini., 38, 1081 - 1087. Kunzel, G., Korzun. L. & Meister. A. (2000): Cy1ologieally intcgra1cd physical RFU' maps for 1hc barley genome based on 1rnnslocmion breakpoints. Gel/<!liCs. I 54. 397-4 12. Ku rata, N. e1 al. ( 1994): A 300 kilobasc in1crval genetic map of rice i ncluding 883 expressed sequences. Nature Genet. , 8. 365- 3 72. 14) Kuwada, Y. ( 19 10): A cy1ological study or O,;v:u .1·a1il'l1 L. Rot. 1Wag. (7i,kyo) 24. 267- 280. 15) Mi1sunaga, S. c1al.: Dircc1 clo11i11g of an o:-amylasc gene from barley chromosome 6 by a microdisscction me1hod (submitted). 16) Nak:unura, M. & Fukui. K. ( 1997): A chro111osomc-oric111cd approach to genome analysis in II woody plant S<!q11oit11/e11tlro11 gigm1t<!11m (Lindi.) Buch holz. /11 Cytogcnc1ic studies of forest Crees and shrub species. eds. 13orzen Z. & Schlarbaum S. E., Univ. Zagreb, Zagreb, 89- 102. 17) Nakamura. S. ct al. ( 1997): Constitution of ;111 800-kb concig in the ncar-ccntromcric region of the rice blast rcsisrnnce gene Pi-11/ using a highly representative rice BAC l ibrnry. 1\1/0/. Ge11. (je11et., 254, 61 1-620. 18) Ohmido, N. & Fukui, K. ( 1997): Visual vcri ficaiion of close disposition between u rice A gc11omc-spccitic ONA sequence (Trs/\J and the tclomcn:: sequence. Pl/111t Mo/. lJiol., 35. 965- 968. 158 19) Ohmido. N.. Akiyama, Y. & Fukui, K. ( 1998): Physical mapping of unique nucleotide sequences on identified rice chromosomes. l'/0111 Mo/. Biol., 38. 1043- 1052. 20) Ohmido, N. ct al. (2000): Qunn1ilication of total genomic DNA and repetitive sequences reveals concu1Tcn1 changes of diftcrent DNA fami lies in ill(/ica andjaponico JARQ 34(3) 2000 rice. 1'vlo/. Gen. Genet., 263. 388- 394. 21) Rajyashri, K. R. ct al. ( 1998): Isolation and PIS! I mapping of yeast artilicial chromosomes (YACs) encompassing an allele of the C1112 gene for gall midge resistance in rice. Theo,: Appl. Genet., 97. 507- 514.