Download Visual Detection of Useful Genes on Plant Chromosomes

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.
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