Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
HIGHLY EFFICIENT METHOD OF GENOME SCANNING
Technical Field
This invention relates to a method for performing high-efficient
electrophoresis of multiple nucleic acid samples to detect nucleic
acids of interest. The invention also relates to an electrophoresis
apparatus used in the method. The method of this invention is useful
particularly in detecting polymorphism in genomic DNAs, genetic
analysis, genetic mapping, and constructing a contig or a physical
map that covers the entire genome of an organism.
Background Art
To detect differences of organisms , such as those between breeds ,
at the nucleic acid level , techniques such as RFLP (Restriction Fragment
Length Polymorphism) and RAPD (Randomly Amplified Polymorphic DNAs)
have been conventionally used. In the RELP method, however, large
quantities of DNA samples are required, and also genomic maps based
on existing RELP markers for the tested organism are necessary to
detect a marker proximal to a particular gene. In addition,
construction of the map requires substantial time, cost and manpower.
Further, only limited organisms have genetic maps that contain KELP
markers at sufficient densities. The detection of polymorphism with
the RAPD method, in contrast, may be applied to a relatively large
number of samples . However, the number of bands stably obtained at
a time is limited. Loosening PCR conditions in attempt to increase
the number of bands tends to deteriorate the reproducibility of the
resulting polymorphic bands.
The AFLP (Amplified Fragment Length Polymorphism) method has
been increasingly used because of its ability to compare a large number
(50 to 100 or more) of bands at a time, low consumption of DNA samples,
and high reproducibility of resulting bands. However, initsoriginal
procedure, the sequencing gel is as large as 40 to 50 cm and the nucleic
acid bands are detected by autoradiograph using isotopes. Thus, the
method requires extensive experience, may be used only in limited
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conditions, takes time for detection, and is not capable of analyzing
a large number of samples (capable to process only up to several dozen
samples at a time).
Recently a method that uses PCR with fluorescent primers and
automatic sequencers has been developed to make the band detection
easier. However, these types of sequences are very expensive
(y10 , 000 , 000 to X20 , 000 , 000 or more) , and an experiment of this
method
would occupy the sequences, which is basically for gene sequencing,
for a considerable period of time. Moreover, since the band detection
in this method assumes that the procedure is performed with a system
using an automatic sequences, the band of interest cannot be isolated
for analysis after the detection step. Thus, there is a major problem
that the detected bands cannot be readily detected as SCAR (Sequence
Characterized Amplified Region) markers by specific primers. In
addition, only 8 to 9 types per set of fluorescent primers are currently
available, allowing merely 64 to 81 combinations of primer pairs at
most.
A known method for detecting polymorphic bands over an entire
genome at a time is RLGS (Restriction Landmark Genome Scanning).
However, this method also uses radioisotope and requires several days
to detect markers of small quantities. Further, it involves handling
of a very large gel (40 x 30 cm) or a long, narrow agarose gel for
every sample, requiring extensive experience as well as muscular
strength. In addition, cleavage of nucleic acids by restriction
endonuclease in the agarose gel requires a large amount of expensive
restriction endonucleases, making the procedure costly.
In RLGS on rice genome (450 MB) , for example, only limited 8-base
restriction endonucleases, such as NotI, may be used to define the
labeled portion. In addition, the theoretical upper limit for the
number of dots obtained from one electrophoresis cycle is 450 MB -
48 = 13 , 700 , and in practice, because of the nature of the electrophoresis
,
the number is 1/3 to 1/6, i.e. 2,000 to 4,000 dots. Moreover, since
sample from each individual is electrophoresed on a separate gel,
electrophoresis patterns of different samples often do not match
completely. Thus, an expensive, large-scale scanner and
two-dimensional electrophoresis software are necessary to compare
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different individuals.
In the early stage in the construction of genomic
libraries in which contiguous clones covering an entire genome
were organized and linked based on overlaps between the
clones, there were attempts to utilize markers in existing
maps such as the RFLP map. However, even in those called
high-density maps there are only about 2,000 markers. Even
in rice, which has a small genome (450 MB), the average
density is merely 200 KB/band or more . Such densities are too
low to construct a contig covering the entire genome. On the
other hand, the search for such a number of RFLP markers would
require vast amount of cost, manpower, and time.
A method used often recently to construct a contig
covering an entire genome is as follows: Component clones of
a library are cleaved by appropriate restriction endonucleases
and the resulting fragments are electrophoresed on a high-
resolution gel; obtained pattern are digitalized and input
into a database; and clones with a common pattern are linked
to each other in computers. However, this method also
requires a vast amount of labor and cost to cleave all the
clones, radiolabel them at their ends, and perform
autoradiography.
Disclosure of the Invention
The present invention has been made in view of the above
mentioned situation. An objective of the invention is to
provide a method for performing electrophoresis of a large
number of nucleic acid samples efficiently and inexpensively
to detect nucleic acids of interest, and an electrophoresis
apparatus used for the method. In the preferred embodiment
of the method, the invention provides a means to detect
polymorphism in genomic DNAs using the method, a means for
genetic analysis, a means for constructing genetic maps, a
means for identifying a genomic clone that corresponds to a
particular band, and a means to construct a group of organized
contigs covering an entire genome.
The present inventors, after conducting extensive studies
to solve the above-mentioned problems, thought that the
electrophoresis using a small-sized gel, which is generally
used in electrophoresis of proteins, could be appropriately
used to perform electrophoresis of a large number of nucleic
acid sample efficiently and detect nucleic acid bands of
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interest.
Thus, the present inventors constructed an
electrophoresis apparatus for electrophoresis of nucleic
acids, where plural 10- to 30-cm square gel plates are
installed on the electrophoresis apparatus at a time and 32
or more nucleic acid samples per gel plate are electrophoresed
with the electrophoresis apparatus simultaneously. Using this
apparatus, the inventors detected nucleic acid markers near
a nonpathogenic gene in Pyricularia oryzae Cavara or near the
brittle culm (kamairazu) mutant gene. As a result, it was
found out that polymorphic bands could be detected at
remarkably higher efficiency compared to that in the
conventional RAPD method.
Linkage analysis on the detected polymorphic bands
revealed that, among the detected polymorphic bands, even
those at particularly proximal position to the target gene
could be obtained by this method at remarkable efficiency
compared to conventional methods.
In addition, the method of the present invention was
found to be useful in isolation and specific amplification of
various important polymorphic bands, such as those detected
by the above method. For example, the present inventors used
this method, thereby isolating several bands that identify
major 10 rice breeds; designed primers according to the
sequence information of the band specific to one of the 10
breeds, Akitakomachi; and performed PCR using genomic DNAs
obtained from the 10 breeds as templates. As a result,
specific PCR-amplification products were found only for
Akitakomachi. Thus, it was revealed that the method of the
present invention could efficiently provide polymorphic bands,
which could be used for easy identification of rice breeds.
Further, the present inventors found out that the method
of the present invention could be used to obtain nucleic acid
markers for constructing a genetic map of an organism or
constructing contigs covering the entire genome of an
organism.
Thus, the present invention relates to a method for
performing electrophoresis and detection of a large number of
nucleic acid samples at high efficiency and low cost, and to
an electrophoresis apparatus used for the method and its use.
More specifically the present invention provides:
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(1) a method for electrophoresis of nucleic acids, said method
comprising the following steps:
a) electrophoresing nucleic acid samples using an
electrophoresis apparatus on which plural 10- to 30-cm square gel
5 plates are installed at a time and with which 32 or more nucleic acid
samples per gel plate are electrophoresed simultaneously, and
b) detecting nucleic acid bands on the gels after the
electrophoresing;
(2) the method according to (1) , wherein the electrophoresing
is performed using gels with discontinuous buffer system;
( 3 ) the method according to ( 1 ) , wherein the nucleic acid samples
are single-stranded DNAs prepared by dissociation of double-stranded
DNAs through denaturation and the electrophoresing is performed using
denaturing gels;
( 4 ) the method according to ( 1 ) , wherein the detecting of the
nucleic acid bands on the gels is performed by fluorescent staining
or silver staining;
( 5 ) the method according to any one of ( 1 ) , ( 2 ) , or ( 4 ) , wherein
the method is performed in order to detect a polymorphism of genomic
DNAs among test individuals;
( 6 ) the method according to (3 ) , wherein the method is performed
in order to detect a polymorphism of genomic DNAs among test individuals ;
( 7 ) the method according to ( 5 ) , wherein the nucleic acid samples
are DNA fragments amplified by AFLP method;
( 8 ) the method according to ( 5 ) , wherein the nucleic acid samples
are heteroduplex DNAs;
(9) a method for preparing DNA fragments comprising a
polymorphism, said method comprising a step of isolating, from gels,
DNA fragments comprising a polymorphism detected by the method
according to any one of (5) through (8);
(10) a DNA fragment comprising a polymorphism among test
individuals, said DNA fragment being isolated by the method according
to (9) ;
( 11 ) the method according to any one of ( 1 ) through ( 8 ) , wherein
the method is performed in order to carry out genetic analysis;
( 12 ) the method according to ( 11 ) , wherein the genetic analysis
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is F2 analysis, RI (recombinant imbred) analysis, or QTL (Quantitative
Traits Loci) analysis;
( 13 ) the method according to any one of ( 1 ) through ( 8 ) , which
is performed to construct a genetic map of an organism;
(14) a genetic map of an organism, said genetic map being
constructed by using, as markers, bands of genomic DNAs comprising
a polymorphism detected by the method according to (13);
(15) a method for selecting, from a genomic DNA library, a clone
corresponding to a particular nucleic acid band on a gel detected
by the method according to any one of (1) through (8), said method
comprising the following steps:
a) dividing a genomic DNA library of a particular organism into
plural sublibraries each of which has a size of 1 or less genome of
the organism;
b) assigning, to all clones included in each of the sublibraries,
a row number, a column number, and a plate number of the sublibrary,
wherein the row, column, and plate are referred to as X coordinate,
Y coordinate, and Z coordinate, respectively;
c) detecting a band by collecting clones representing a
particular row of all plates (X-coordinate clone group), clones
representing a particular column of all plates (Y-coordinate clone
group), and all clones on a particular plate of one sublibrary
(Z-coordinate clone group); by extracting DNAs from each of the
collected clone groups to obtain coordinate samples; by preparing
a genomic DNA from the organism as a control; and by electrophoresing
the coordinate samples and the control in a line using the method
according to any one of (1) through (4);
d) determining a clone in each of the X-coordinate clone group,
the Y-coordinate clone group, and the Z-coordinate clone group, said
clone corresponding to a band with the same mobility on the gel as
that of the nucleic acid of interest in the control; and
e) selecting, from the sublibrary, a clone corresponding to
the determined three-dimensional coordinate;
(16) the method according to (15), wherein the method is
performed in order to construct contigs covering the entire genomic
DNA of a particular organism; and
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(17) an electrophoresis apparatus for electrophoresis of
nucleic acids, wherein plural 10- to 30-cm square gel plates are
installed on said electrophoresis apparatus at a time and 32 or more
nucleic acid samples per gel plate are electrophoresed with said
electrophoresis apparatus simultaneously.
1. ELECTROPHORESIS METHOD AND APPARATUS
The electrophoresis apparatus of this invention is that on which
small-sized polyacrylamide gel plates (10- to 30-cm square, standard
size: 18-cm square) are installed and with which 32 or more (standard:
64) test samples plus several (standard: 2 to 4) size markers per
gel are electrophoresed at a time, as well as with which a large number
of (standard: 256) samples are electrophoresed by using such 2 or
more (standard: 4) gels simultaneously. An example of the
electrophoresis apparatus of this invention is shown in Figs 1 through
4 . In this apparatus , a 1-mm-thick gel prepared with an 18-cm square
glass plate has 66 wells to allow electrophoresis of 64 samples and
2 size standards at a time. The apparatus also allows electrophoresis
of 4 gels at a time. These together allow testing of 256 samples in
one cycle.
The discontinuous polyacrylamide electrophoresis system
(Laemmli , U.K. (1970) Nature 227: 680-685), which is usually used
for electrophoresis of proteins, may be used for the gel of this
invention. The use of thisgelallowshigh-resolution electrophoresis
of as much as 10 u1 of a test sample, even in narrow lanes of 1 mm
in width. It also increases the sensitivity of band detection.
In the electrophoresis of nucleic acids according to this
invention,itispreferable to perform two-layer electrophoresisusing
concentrated gels (Tris-HC1 pH 6. 8, 0.5 M) and isolation gels (Tris-HC1
pH 8.8, 1.5 M) to improve band resolution of the nucleic acids on
the gels . In electrophoresis of nucleic acids , the nucleic acids may
remain in double-strand or denatured into single-strand, depending
on the purpose. In the latter case, denaturing gels (gels containing
6 to 8 . 5 M urea) are used in the electrophoresis . When polymorphism
in nucleic acidsisdetected,electrophoresiswith heteroduplex allows
highly sensitive detection, which can detect minor polymorphisms that
CA 02385943 2002-03-25
may be undetectable by conventional methods.
In the nucleic-acid electrophoresis of this invention, it is
preferable to use silver or fluorescent staining to detect nucleic
acid bands after electrophoresis. Silver staining allows highly
sensitive detection in a short period of time (1 to 2 hours) , requires
less expertise and is safer compared to methods using isotopes . In
addition, the material may be later dried to provide a preservation
sample as well as to increase the sensitivity. With fluorescent
staining, results are obtained in approximately 30 minutes of staining.
With its high recovery rate of nucleic acid, fluorescent staining
is suitable for excision and collection of bands.
In the method of this invention, the efficiency in the step of
loading amplified DNA samples and such on the gel may be improved
drastically, for example, by designing an electrophoresis gel comb
such that 2 or more lanes are loaded at the same interval (9 mm) as
those of the 96 (8 x 12) well microplate.
2. DETECTION OF NUCLEIC ACID MARKERS
To detect polymorphism in nucleic acids using the method of this
invention, combination with AFLP (Amplified Fragment Length
Polymorphism: VosP, et al. (1995) Nucleic Ac idsResearch23: 4407-4414)
can conceivably yield the highest efficiency although combinations
with other forms of detecting nucleic acid polymorphism are also
possible.
The use of AFLP, for example, yields approximately 50
amplification bands per lane from a genome of a size on the order
of that of rice (450 MB), with primers to amplify genome fragments
having a 6-base cleavage site at one end and a 4-base cleavage site
at the other end, when 3-base selective nucleotides are used at each
end (refer to the below-mentioned formula). A system comprising a
standard4-gel 256-sample lane would yield approximately 12,800 bands
in one electrophoresis cycle (Example 2).
Entire genome size - the number of cleavage sections by 6-base
restriction endonuclease - selection rate at both ends of genome
fragment by 3 nucleotides = the number of bands
(450 MB - 46 x 2 - 43 " 2 v 50)
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This number compares to the amount of information obtained from
several gels from RLGS method. In addition, in the method of this
invention lanes to be compared may be placed next to each other,
permitting easy, direct comparison of raw data without the need for
special reading means such as a special reading apparatus.
As shown in Fig. 6 (Example 2) , in actual AFLP experiments where
rice genome DNAs were cleaved by EcoRI and MseI , the number of bands
obtained was close to that estimated with the above formula. However,
band with certain clarity were about half of them.
Performing PCR with 5 to 10 u1 of sample using a 0 . 2-ml microplate
for 96 samples saves costs of nucleotides and such enzymes as
heat-resistance DNA polymerase and increases efficiency by allowing
transfer of 8 samples to the gel at a time using an 8-channel 10-ul
pipette. Thus, one electrophoresis cycle with 4 gels may be easily
performed in one day.
After electrophoresis is completed, the 4 gels may be
silver-stainedsimultaneouslyin one container,whichisan efficient,
low-cost and simple procedure. In general, commercially available
silver-staining kits for protein may be used.
As nucleotide primers for AFLP, any nucleotides corresponding
to selected restriction endonucleases may be used. Further, any
selective sequence with a length of 1 to several bases may be added
to the 3' end. This allows almost infinite combination of primers.
For a plant with a genome of 1 GB or less size, by using EcoRI and
MseI as a 6-base restriction endonuclease and a 4-base restriction
endonuclease, respectively, and by using PCR primers having
3-nucleotide selective sequences, each of which has 64 combinations,
the amplification would be performed in 64 x 64 = 4 , 096 combinations .
This allows search for 10, 000 to 20, 000 polymorphic bands in genetic
analysis between parents with a polymorphism rate of 5 to 10% . This
number is practically sufficient for physical map construction and
exceeds the numbers of makers on conventional genetic maps by an order
of magnitude or more.
Important bands such as those near the target gene, obtained
from bulk analysis (Michelmore RW, et al. (1991) Proc. Natl. Acad.
Sci. USA 88: 9828-9832) or other method, may be cut out after staining
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for further analysis: The cut-out gel is crushed and provided for
extraction in appropriate buffer for another PCR procedure; the PCR
product is then inserted into appropriate plasmid, conjugated, and
thus isolated for sequencing (Example 4).
5 With combinations described above, it is possible to perform
efficient electrophoresis as follows, for example: A set of 4 samples,
2 from breeding parents and 2 from mixtures of approximately 10
individual of F2 homogenous individuals, dominant and recessive each,
are prepared; electrophoresis is performed with 4 lanes / primer-pair;
10 with a standard electrophoresis apparatus (4 gels, 256 lanes),
electrophoresis with 64 primer pairs may be performed at a time. In
this example, tests with all 4,096 combinations of selective primers
are completed in 64 times of electrophoresis (total approximately
2 month). In a breeding combination with approximately 10~
polymorphism, such as indica-japonica crossing, this corresponds to
scanning and searching for 20,000 polymorphic bands in the entire
genome. This efficiency exceeds those of conventional methods by 1
to 2 orders of magnitude and permits gene isolation in a short period
of time, regardless of presence or absence of a genetic map. The cost
for search of all 4 , 096 combinations is also low, being approximately
y400,000.
3. GENETIC ANALYSIS
1) F2 analysis
To determine a genetic distance (or distance on the map) between
a combination of given genes or polymorphism markers, F2 analysis
is most commonly used. The use of F2 analysis in accordance with this
invention even more efficiently determines a distance between 2
polymorphism markers or between a polymorphism marker and a gene.
Like in general F2 analysis, a line (line A) that contains a
given gene and another line (line B) that does not contain the gene
or has distinct differences in traits related to that gene and that
has appropriately small differences in traits from those of A are
selected and crossed to produce a large number of F2. The number of
F2 individuals to be analysed depends on the precision of the analysis .
To achieve 1 CM precision, 50 homogenous individuals (usually recessive
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homogenous individuals) are sufficient to determine the genetic
distance between a gene of interest and a polymorphism marker, by
determining the recombination frequency between the gene and the marker
on100chromosomes. In an analysisofrecessive homogenousindividuals,
if no recombination occurs, all of the individuals should have the
same marker as of the parent with a recessive trait; with the
recombination frequency of 1/100, recombination should be observed
only in 1 chromosome of 1 individual.
Since the standard system of this invention is capable of
analyzing 256 individuals at a time, if such a number of homogenous
individuals are available, recombination of polymorphism markers may
be tested on 512 chromosomes in one electrophoresis cycle, which yields
0.2 CM precision. The use of AFLP for polymorphism markers allows
testing with very small amount of sample DNAs (100 ng or less) ; genomic
DNAs are prepared from each individual and double-digested with EcoRI
and MseI enzymes . Adapters matching with each of the enzymes are then
coupled with the genomic DNA fragments. Usingfirst primers matching
these adapters, the first PCR is performed to amplify the gen~me
fragments. Then second primers are prepared by adding a selected 1-
to 4-base sequence to the 3' end of each of the first primers. Using
these second primers, only a part of the genome fragments which
corresponds to the selected sequences are amplified by PCR. The PCR
products are then separated by molecular weight with electrophoresis
of this invention. After electrophoresis is completed, the gel is
stained by such as silver staining and examined for recombination
in the polymorphism marker of the interest.
In accordance with this invention, F2 analysis with small amount
of DNAs from as much as 256 individuals is completed in one
electrophoresis cycle with a precision of 0.2 CM level. Further, the
process does not require blotting or autoradiography. In addition,
after staining and drying, the 4 gels may be cabinet size and filed
in albums, which permits easy analysis of the results.
2) RI analysis
An RI (Recombinant Inbred) line refers to that obtained by self
fertilization of F2 individuals for several generations which made
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by breeding of different lines. As a result of repeated
self-fertilization, most of lociare homozygous. Because of alimited
number of heterozygotes, the separation ratio of dominant-recessive
traits is 1 : 1 . Thus , genes of interest are homozygous even in dominant
individuals, which makes them applicable to the gene analysis of this
invention . This gene analysis. is more precise than F2 analysis , where
the proportion of heterozygous individuals is 1/2 and the separation
ratio of dominant-recessive traits is 3:1. When an appropriate RI
line is available, gene analysis using genomic DNAs extracted from
a number of the RI line (RI analysis) may be performed in accordance
with this invention in the same manner as in F2 analysis.
3) Narrowing-down of proximal markers
To narrow down, by F2 analysis, proximal markers to the gene
of interest obtained in bulk analysis, and ultimately map the most
proximal markers, electrophoresis of the present invention is useful;
the ability to perform electrophoresis with 256 samples at a time
in the standardmethodprovides very high efficiency in the gene analysis .
With rice (in case of indica-japonica crossing) , as described in 2. ,
given that 20,000 bands of polymorphism markers evenly distribute
over the entire 2,000 CM genome, the number of markers present in
the 20 CM region, 10 CM on each side of the gene obtained from bulk
analysis, is estimated to be 200 bands. To narrow down such a number
of proximal markers to the most proximal markers, l4 recessive
homogenous individuals of F2 generation per marker and DNAs from each
parent are run in 16 lanes as a first step to examine for presence
or absence of recombination between the target gene and the candidate
marker. Thus, recombination frequency for 28 chromosomes per maker
is determined. The resolution in this process is approximately 3.5
CM. Thus, 4 bands of candidate markers are tested per gel, which
corresponds to testing of 16 bands of markers per electrophoresis
cycle. Simultaneously, the individuals having recombination at the
most proximal site to the target gene is identified. After this process ,
using merely 8 lanes for 6 individuals with proximal recombination
and for the parents, testing of the rest 192 markers is completed
in 6 cycles of electrophoresis.
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Given that positions of markers and chromosome recombination
distribute evenly, it is expected that, as a result of above-mentioned
screening, approximately 40 bands of markers be obtained from an 8
CM region, 4 CM on each side of the target gene. The second step is
to map each of the markers remaining from the first screening at 1
CM precision. First, using one gel each, 12 markers are run in 3 cycles
of electrophoresis in order to obtain AFLP products of 62 recessive
homogenousindividualsof F2generation and the parents. Thisproduces
a fine map for the 8 CM region around the target gene, which reveals
individuals having recombination in the region immediately close to
the gene, approximately within 1 CM from the gene. Using such
individuals with proximal recombination, the rest 28 bands are analyzed
to identify their locations on the map. One electrophoresis cycle
of 224 lanes , which is 8 lanes ( 6 individuals with the most proximal
recombination plus parents) times 28 combination, completes the
analysis of the region around the target gene.
Thus, 200 candidate proximal markers selected from 20,000
polymorphic bands by bulk analysis may be narrowed down to most proximal
markers within approximately 1 CM from the target gene by approximately
11 cycles of electrophoresis.
In gene isolation, if the genome size of the organism of interest
is 1 GB or smaller, the average 1 CM in such markers should be 500
kB or less. Therefore, clones proximal to the target gene may be
selected from a genome library of BACs (bacterial artificial
chromosomes) which have approximate average insert size of 150 kB,
to construct a contig.
It is also possible to increase the analysis precision to 0.2
CM level by using 256 recessive homogenous individuals . In this case,
by selecting about 4 bands of appropriate proximal markers and
performing4electrophoresescycles,individualshaving recombination
most proximal to the target gene is identified. Using such individuals
with proximal recombination to check recombination in the presumed
bands most proximal to the gene in the same manner as of the second
step, the most proximal markers are identified easily by 1
electrophoresis cycle.
Herein the principle of the invention has been described with
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two assumptions; bands obtained by electrophoresis distribute at
almost even frequency over the genome or chromosome, and recombination
in chromosome occurs uniformly over the genome. Actually, however,
distribution of bands and recombination sites in a chromosome is not
uniform. Thus, the distance between a proximal marker and the target
gene, where the distance is obtained for the number of F2 individuals
used, tends to be larger than that expected for uniform distribution.
4) Application to marker breeding
A proximal marker for a particular trait gene obtained in the
above described manner may also be used as a highly reliable marker
in conventional breeding by mating. Also, when marker analysis is
performed on a large number of mating offspring, the analysis method
using genome scanning allows easy analysis of a large number of
individuals with small amounts of DNA samples . This would result in
substantial increase in operation efficiency and decrease in cost
compared to conventional techniques using the RFLP method.
5) Application to QTL (Quantitative Traits Loci) Analysis
A QTL is a locus of quantitative trait, where expression of the
trait is not as strong as to be qualitative and, in most cases, several
loci are involved in expression of the trait. QTL analysis is performed
to analyze which loci at which location on the chromosome how much
contribute to expression of the trait.
QTL analysis requires that polymorphisms in a large number of
individual offspring of mating should be tested for a large number
of nucleic acid markers distributed evenly over the entire genorne.
For example, to perform QTL analysis using markers distributing over
a 2,000 CM entire genome at the marker density of approximately 10
CM/marker, 200 markers need to be tested with at least about 50 F2
individuals. Performing thisanalysiswith RFLP markerswould require
200 times of hybridization with membranes blotted with nucleic acids
of 50 individuals. Since one cycle of hybridization requires 2 days,
even if 4 membranes were processed at a time, the whole process would
require 100 days. Even if a membrane could be repeatedly used for
10 times, it would be necessary to collect as much as 100 ~g of nucleic
CA 02385943 2002-03-25
acids from each of 50 individuals to prepare 20 membranes . This would
require enormous labor.
By using AFLP method in the present invention, a few polymorphic
bands are obtained in a lane when an organism with approximately 10~
5 of polymorphism, such as indica-j aponica crosses of rice, is studied.
Therefore, by using appropriate primer pairs, search for 200 markers
is completed by electrophoresis with a little over 50 pairs of primers .
Since 5 primer pairs may be used in 1 electrophoresis cycle (50 x
5 = 250 lanes), 10 cycle (total 10 days) of electrophoresis would
10 complete examination of all the markers for 50 individuals . In addition,
1 ug of DNAs per individual is enough to perform this process.
Specifically, a genome map for the organism with AFLP markers
must be prepared prior to performing this method. In case where such
a map is not available, a map may be readily constructed according
15 to this invention as described in 5 below. Markers are selected from
the map at a desired density. In this case; it is efficient that as
small a number of primer pairs as possible are selected so as to cover
the entire genome.
To achieve an appropriate polymorphic frequency in breeding,
it is recommended to select two genetically distant lines one of which
strongly expresses the quantitative trait of interest and the other
shows very little expression the trait. Breeding of too distant lines
may inhibit smooth isolation of the markers. Using approximately 50
individuals (the number varies depending on purpose of the analysis)
of the F2 or RI line obtained from this breeding, the trait of interest
is quantitatively analyzed. Also, using genomic DNAs prepared from
each individual, the genomic fragments are amplified by AFLP method
as described in 3.1) , and are subjected to electrophoresis using the
system of this invention for examination and recording of polymorphisms
in the marker bands.
To indicate contribution of each locus near each marker to the
trait of interest, the number of traits of interest is multiplied
by the number of polymorphisms in one of the parent in each band,
and the product is plotted for each of the marker bands on the map.
4. APPLICATION TO IDENTIFICATION OF BREEDS AND/OR LINES
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This invention may be used to efficiently search for
necessary markers to identify a particular breed and/or line
of an agricultural or livestock product or various
organisms. AFLP is performed with DNAs obtained from a
large number of breeds to be compared and electrophoresis
is performed simultaneously using the electrophoresis
apparatus of this invention to compare dozens of bands for
dozens to 100 or more breeds at a time. Thus, even with a
large number of breeds, specific recognition bands are
easily selected.
In this application, although combination of n bands
enables identification of up to 2n breeds and/or lines
theoretically (where n is an integer), it may be slightly
less than that practically.
When the breeds and/or lines to be compared are so
close to each other that obtaining polymorphisms by
conventional methods such as AFLP is difficult, products of
AFLP, RAPD, or any other method from genome of the compared
sample may be mixed, heated, and then cooled to provide
heteroduplex; the heteroduplex may be subjected to
electrophoresis for comparison with the bands of interest.
Thus, differences between breeds and/or lines are detected
sensitively, efficiently, and simply.
When a particular band with high identification ability
is identified, the band may be isolated in a manner
described in 7 so that the band may be PCR-amplified with
certain primers. After the PCR amplification, simple
agarose gel electrophoresis enables identification of the
breed within approximately 1 hour. Further, the primers may
be labeled with certain fluorescent labels so that presence
or absence of the band amplified from the primers is
determined by a PCR apparatus equipped with a fluorescent
detector. In this method, 2 to 30 minutes of PCR would
identify the breed and/or line without the need of
electrophoresis.
5. CONSTRUCTION OF A GENETIC MAP OF AN ORGANISM
To construct a genetic map (more accurately, nucleic
acid marker map) covering the entire genome of an organism,
it is necessary, like in QTL analysis, to analyze several
hundreds to thousand or more markers with F2 individuals,
in which the number of F2 individuals depends on the
resolution of the required map. In most higher plants,
CA 02385943 2002-03-25
17
especially in maj or crops , the total length of genome is between 1 , 500
and 2,000 CM. Thus, to construct a genomic map in the density and
precision of about 1 CM with conventional RFLP method, it would be
necessary to prepare membranes from 100 or more F2 individuals and
repeatedly blot the membranes for about 1 , 800 markers . Since 1 cycle
of blotting, including preparation of the probes, requires 4 days,
the whole process would require 1,800 days even if 4 probes were
processed at a time. Also, a very large amount of nucleic acids is
needed for this process ; 1 mg or more DNAs is needed for each F2 individual .
This is one of the reasons that genetic map construction has been
conducted by a~ team comprising dozens of persons over several years .
According to this invention, with breeding parents with
approximately 10% polymorphism, for example, a pair of primers
indicates 4 to 5 polymorphisms on average. Hence, by conducting a
pre-examination to select primer pairs that indicate 6 or more
polymorphisms and by mapping of 128 F2 individuals with the standard
model using the selected primer pairs, 12 or more markers are mapped
in 1 electrophoresis cycle with 2 primer pairs. Thus, a fine map
including 1,800 markers would be completed in total 150 days with
1 person, which is 10 times or more efficient than in conventional
methods. A map containing approximately300markerswould be completed
in approximately 1 month with one person.
Specific procedure is as follows : Two lines at an appropriate
genetic distance are bred to obtain a RI line or F2 individuals to
the number according to the precision of the required map . To minimize
the potential of hybrid sterility and distortion in separation ratio,
the average polymorphism rate between the two lines should be up to
10~ or slightly higher. To achieve the precision of approximately
0.5 to 1 CM, 64 to 128 individuals are generally enough. Genomic DNAs
are prepared from these individuals and amplified by the AFLP method
in the same manner as already described for F2 analysis and RI analysis .
The amplified PCRfragmentsare providedfor electrophoresis,staining,
and analysis by the system of this invention. This is done as follows:
The prepared genomic DNAs are stored in 96-well microplates . A part
of the DNAs (approximately 0.1 ug) are double-digested by EcoRI and
Msel. Adapters are added to the cleavage ends, and pre-amplification
CA 02385943 2002-03-25
18
is performed using primers with the same 5' ends as of the adapters.
Then, using the PCR products of the pre-amplification as templates,
secondary amplification is performed using selective primers with
an appropriate number of selective bases. The amplified products are
appliedfor the electrophoresis apparatus of this invention. The use
of 8- to 12-channel pipettes or 8- to 12-channel microsyringes increases
efficiency of gel loading. When 64 samples are tested, approximately
20 markers may be processed with 4 primer sets in 1 electrophoresis
cycle; when 128 samples are tested, 10 markers with 2 primer sets.
Genetic map construction software such as MAPL or MAPMAKER may be
used for polymorphic band analysis to improve efficiency.
A reference case using a former system of the present invention
is shown herein (Fig. 11) . Eight 15-lane gels were used in this system.
Using 99 RI lines from breeding of two barleys, Azumamugi and Kanto
Nakate Gold, an AFLP map with 227 markers was constructed in
approximately 2 months. This AFLP map was integrated with an
already-existing map including 45 markers to produce a map with 272
markers . In this case, the AFLP map was completed in approximately
2 months by 1 person, which is approximately 40 times higher efficient
than conventional map constructing methods, such as those using STS
markers. The method of this invention is even more efficient than
this former system.
6. APPLICATION TO ORGANISMS WITH LARGER GENOMIC SIZES
The present invention conceivably exerts its potential most when
combined with AFLP. A future of AFLP is in that genome is
double-digested into fragments by EcoRI (which recognizes 6 bases)
and MseI (which recognizes 4 bases); adapters matching the cleaved
sites are added to the fragments; the adapter sequences are used as
PCR primers in the first amplification so that all the fragments are
amplified; selective primers in which 1- to several-base selective
sequence are attached to the 3' end of the first primers are used
as second primers in the second amplification; and thus only genome
fragments having sequences that match the selective primers on both
ends are specifically amplified. In case of an organism with a
relatively small genome size (such as rice: 450 MB) , selective primers
CA 02385943 2002-03-25
19
in which 3-base selective sequences are attached to the EcoRI and
MseI sites on both ends are used as the second primers. Thus, as
described in "2. Detection of nucleic acid markers", approximately
50 bands on average are selectively amplified. With genome of a size
similar to that of filamentous fungi, such as P. oryzae Cavara (40
MB) , applying only 1 base to either of the cleaved sites would produce
50 x (40MB/450MB) x 16 = 70 bands. To organisms with larger genome
sizes, this method may basically be applied simply by increasing the
number of bases of the selective sequence for AFLP primers to 3 or
4.
Alternatively, conditions of electrophoresis may be changed.
Usually, in electrophoresis of PCRproducts , it is convenient to perform
electrophoresis of PCR products under non-denaturing conditions
leaving the material double-stranded. However, with an organism with
a larger genome size (such as barley: 5. 5 GB) , bands may not be clearly
separatedif the PCR productsare double-stranded. Therefore, by using
denaturing gels containing 8.5 M urea and placing the DNA sample in
90°C for 3 minutes in presence of 50~ formamide so that the DNAs are
separated into single strands , and then subj ected to electrophoresis
a large number of bands are clearly distinguished (Example 4).
In practice, as genome size becomes larger, difficulty of AFLP
increases. Thus, it is recommendable to combine the above two
strategies.
7. ISOLATION AND SEQUENCING OF IMPORTANT BANDS FOR PREPARATION OF
SCAR MARKERS
In the embodiment of this invention such as those described in
above sections 3., 4., and 5., bands with information particularly
important in each case are identified: bands proximal to a particular
gene (in section 3. ) ; bands applicable for breed identification (in
section 4.), and band that serve as landmarks on a genetic map (in
section 5.).
These bands may be cut out from the gel to provide SCAR (Sequence
Characterized Amplified Region) markers as follows: DNA material in
the cut-out gel is eluted by crush and extraction, freeze and thaw,
electrophoresis, or other method and provided for PCR amplification
CA 02385943 2002-03-25
using the same primer pair. The PCR products are inserted into
appropriate cloning vectors for sequencing. After the sequence is
determined, the both ends are used as primers of SCAR markers (Example
3). In this case, the use of a fluorescent dye such as cyber green
5 facilitates extraction of nucleic acids. Compared with AFLP method
using sequencer, in which bands are identified but cannot be removed,
the method of this invention is more simple while fully utilizing
the important features of AFLP.
10 8. ISOLATION OF GENOMIC CLONES CONTAINING SPECIFIC BANDS
When bands containing important information are found, such as
illustrated in above section 3 . , 4 . , and 5. , the genomic clones
containing the bands may need to be isolated. Especially, to isolate
a gene with important functions by positional cloning, it is necessary
15 to prepare a series of clone contigs as follows : The most proximal
markers are identified in both sides of the gene by methods such as
2 or 3 ; using these markers , a clone containing the bands are selected
from a genomic library such as the BAC library; using the markers
at both ends of the clone, next contiguous clone is identified, and
20 this process is repeated (walking); then a series of clone contigs
that connect between markers present on both sides of the gene (flanking
markers) is prepared.
When high-density membranes in which component clones of a
genomic library are organized and linked by colony hybridization have
been prepared, the target clone may be picked up by performing
hybridization on the membranesusing amplified bandsasprobes. These
bands may be prepared by direct PCR as described in section 7., or
may be amplified as SCAR markers.
Alternatively, clones containing the band of interest may be
identified asfollows, without the need for hybridization: According
to section 9 . , the genomic library of the target organism is divided
into sub genomic libraries; coordinate markers are prepared by mixing
clone DNAs of the sub genomic libraries such that the clones correspond
to row, column and plate numbers of the microplates; these coordinate
makers are provided for AFLP with same primer pair using genomic DNAs
as control and then for electrophoresis of the present invention;
CA 02385943 2002-03-25
21
thus clones containing the band of interest is identified according
to the coordinates of raw, column, and plate in which the same band
as of the control is.amplified.
9. PREPARATION OF CONTIGS COVERING THE ENTIRE GENOME OF AN ORGANISM
If contiguity status of all of these component clones is unclear
in accordance with a genomic library of an organism, such library
as those based on BAC, contigs are linked together to construct a
larger contig (physical map) covering the entire genome . A physical
map constructed in this manner represents a reproduction of the genomic
structure of the organism. Completion of such a map makes it much
easier to isolate and to identify important functioning genes and
facilitates handling of the genome of the organism, even before the
entire genome is sequenced. In fact, once the entire genome contig
is completed, following genome sequencing may be almost completely
mechanized. Thus far, construction of an entire genome contig has
required a large research group consisting of dozens of persons . With
the present invention, however, a contig covering the entire genome
of an organism whose genome size is close to that of rice (approximately
500 MB) may be constructed in about a year with one person.
Construction of a whole genome contig has been difficult mainly
because it has been difficult to obtain specific labels to mark each
clone composing the genomic library. In this invention, by combining
with AFLP, as many as 30 to 50 highly specific bands per line are
obtained in one cycle, which bands are labeled with two parameters,
3-base sequence on both ends and the number of bases (length) . Each
of these specific bands may be associated with a component clone of
the genomic library and the distribution density of the bands may
be made sufficiently smaller than the length of the BAC clone. Thus,
it is not difficult to link clones having common markers together.
To achieve almost one-to-one correspondence between the AFLP
bands and the library clones, the genomic library for the entire genome,
which generally has several genome equivalents or more, is divided
into several sublibraries of approximately 1 genome equivalent each.
Each component clone of the sublibraries is uniquely identified by
coordinate of row, column, and plate numbers of the microplate.
CA 02385943 2002-03-25
22
Therefore, using row, column, and plate numbers as coordinate
axes, small amounts of DNAs are collected from groups of
clones with a common axis coordinates, and mixed to provide
coordinate samples representing positions on each axis. By
performing genome scanning of the present invention using
these coordinate samples as templates and comparing AFLP
patterns with control lanes, which are prepared by using
whole genome DNA as templates, placed on both sides of the
coordinate lanes, clones corresponding to specific bands from
the genomic DNA templates are readily detected from the
sublibrary. When there are 2 to n corresponding clones in
the sub-group (where n is an integer), 23= 8 to n3 clones
correspond to the bands. In this case, by removing these 8
clones and performing second electrophoresis of the genome
scanning method, the truly corresponding 2 clones are
identified. Specific procedures to prepare coordination
samples of a sublibrary are shown in Example 5.
With a library of a genomic size on the order of that of
P. oryzae Cavara (approximately 40 MB) and of the average
insert size of 120 kB, a sublibrary of approximately 1 genome
equivalent would contain approximately 300 clones, which may
be stored in 8 rows, 6 columns, 6 half-plates. Thus, if one
gel contains 22 lanes, 20 for coordinate samples and 2 for
controls, 2 gels are sufficient to perform electrophoresis of
sublibraries of 6 genome equivalents, i.e., the whole genome
library. In other words, if 1 electrophoresis cycle
identifies and coordinates approximately 60 bands, with 2
primer pairs, 1500 bands are processed in 25 electrophoresis
cycles. This corresponds to 40 MB / 1500 bands = 27 KB/band
density, which is equivalent to identifying 4.4 bands in
average in a BAC clone of an average size of 120 kB. This is
expected to sufficiently cover the whole genome, given that
there is clone redundancy of average 6 genome equivalents
(refer to Example 5).
When the genome size is on the order of that of rice
(450 MB), if the library is 6 genome equivalents with an
average insert size 150 kB, electrophoresis of 6 sublibraries
is completed in 1 cycle by preparing a matrix of 16 rows, 12
columns, 16 x 2 plates using 32 plates, which corresponds to
approximately 1 genome equivalent. This allows identifying
about 25 bands per cycle. Consequently, 200 electrophoresis
cycles would identify 5000 bands, which corresponds
CA 02385943 2002-03-25
23
to 450 MB / 5000 bands = 90 kB/band density. This density should be
sufficient to construct long contigs from a library in which average
150 kB clones.are present at 6 times redundancy.
Brief Description of the Drawings
Fig. 1 is an elevation view and a top view of the main body of
a standard electrophoresis apparatus used in genome scanning, capable
of processing four 18 cm square gels at a time. On each gel, wells
for sample application are made with a comb which can load 66 to 68
samples in 1-mm width. Since 2 to 4 lanes are used for molecule weight
markers or such, 64 samples/gel, i.e. 256 samples on 4 gels are
substantially processed at a time. PCR-amplified samples may be
efficiently applied on the gels by the use of 8-channel micropipettes .
Fig. 2 is a drawing of gel end parts of the standard electrophoresis
apparatus used in genome scanning.
Fig. 3 is a drawing of the comb of the standard electrophoresis
apparatus used in genome scanning.
Fig. 4 is a photograph of a completed standard electrophoresis
apparatus used in genome scanning.
Fig. 5 is an electrophoratogram showing the primary screening
using genome screening for candidates of proximal markers to avrPib,
a nonpathogenic gene of P. oryzae Cavara, obtained through bulk analysis .
Genome scanning combined with AFLP was performed on 4 primer pairs,
A, B, C, and D, which have different proximal marker candidates, using,
from the left, parent lines of avrPib - and +, bulk of avrPib - and
+, and each six F1 lines of avrPib - and +, respectively. The triangle
marks in the figure indicates bands for marker candidates . The primary
screening of the primer pairs A, B, and C indicated coisolation with
the genotype asexpected. On the contrary, recombination wasobserved
in one - line and one + line with the primer pair D (solid triangles) .
Note that each primer pair produced 5 to 60 bands: Three to 4,000
bands may be scanned on one gel. Since the apparatus is capable of
processing 4 gels at a time, 12 to 16,000 bands are scanned in one
cycle . The numbers of selective nucleotides used on these primer pairs
were 3 on the EcoRI side and 1 on the MspI side. The polymorphism
rate between the breeding lines used herein was approximately 5~.
CA 02385943 2002-03-25
24
Since the whole genome size of P. oryzae Cavara is approximately 500
CM, about 500 polymorphic bands are obtained with 256 primer pairs,
i.e., the whole genome is covered with the marker density of 1 CM
/ band.
Fig. 6 is a fine map around avrPib, a nonpathogenic gene of P.
oryzae Cavara, where the map was obtained by mapping proximal markers
to the gene by RAPD method and genome scanning using 125 F1 lines:
As shown in Table l, proximal markers were searched by RAPD method
using 700 primers and by genome scanning using PCR with 251 primer
pairs . The RAPD method required 3 months for the search and mapping
while genome scanning was completed in 1 month. Since only very-clear
bands were counted, the numbers of the counted bands tended to be
small . An: markers obtained bygenome scanning, (Rn) : markers obtained
by RAPD method.
Fig. 7 is an electrophoretogram showing an example of bulk
analysis for search for proximal markers to bc-3, a gene of~Kamairazu,
mutant for cellulose synthesis in rice. For each primer four lanes
are shown: from the left, mutant parent line (M11: japonica) , mutant
(recessive) homogenous bulk, wild-type (dominant) homogenous bulk,
and wild-type parent line (Kasalath: indica) . The arrows in the figure
indicate candidates for proximal markers, which show distinctive
difference between the bulks. There are 20 to 30 bands per lane when
only distinctive bands are counted; when narrower bands are included,
average 50 bands are seen per lane, which reflects the theoretical
value.
Fig. 8 is a photograph of genome scanning performed to search
for genes responsible for the two-rowed spike trait in barley. As
backcrossing hybrids of Azumamugi (six-rowed) and Kanto Nakate Gold
(two-rowed) with Azumamugi (six-rowed) over 7 generations, lines with
two-rowed spike were selected to establish a quasihomogenous genetic
line having the two-rowed spike trait with background of Azumamugi.
This line and the backcross parent Azumamugi are compared by genome
scanning in the photograph of Fig. 8. Differences from 16 primer pairs
were searched for in 32 lanes, and a very limited number of bands
showed differences. The bands with difference are candidates for
polymorphic bands in the regions strongly associated with the two-rowed
CA 02385943 2002-03-25
spike genes.
Fig. 9A shows examples of SCAR (Sequence Characterized Amplified
Region) markers obtained by isolating bands that are specific to a
rice breed, Akitakomachi. The reverse portions are the prepared
5 primers. Fig. 9B is an electrophoratogram showing bands amplified
from nucleic acids of maj or 10 rice breeds by PCR using the primers
shown in Fig. 9A. AspecificbandwasamplifiedonlyfromAkitakomachi.
In "Control 1", a specific band isolated for sequencing was used as
a template.
10 Fig. 10 shows how a clone corresponding to a particular band
is selected directly from a genomic library using genomic scanning.
In this case, the library of P. oryzae Cavara (genome size: 40 MB),
which is 6 genome equivalent with an average insert size 120 kB, is
divided in to 6 sublibraries of approximately 1 genome equivalent
15 each, and clones corresponding to genomic-scan bands are identified
in each sublibrary.
In Fig. 10A, the clone indicated as a dot is expressed as row
C, column 4, and plate a. The coordinate sample representing the
coordinate of row C is prepared by collecting 10 ng each DNAs from
20 clones in all columns in row C on all half plates . In Fig. 10B, one
sublibrary consists of 22 lanes , which include reference genome lanes
and coordinate samples for $ rows , 6 columns , and 6 half plates . Six
genome equivalents are loaded on 2 gels and are processed in 1
electrophoresis cycle. The corresponding clone is identified
25 according to the coordinates which show the same bands as of the control .
Fig. 11 is an example of a gene map of barley constructed by
15-lane gel electrophoresis, from which the genomic scanning was
developed. Among total 272 markers, 227 AFLP markers were mapped by
this method.
Best Mode for Carrying out the Invention
The following Examples are given to further illustrate this
invention; however, the present invention is not intended to be limited
to the specific Examples.
[Example 1] Fine mapping of nucleic acid markers near avrPib, a
CA 02385943 2002-03-25
26
nonpathogenic gene for rice
The nonpathogenic gene in P. oryzae Cavara, avrPib, which
corresponds to the resistant gene in rice, Pi-b, was searched for
nucleic acid markers around the gene and distances between the markers
and the gene were determined. The gene avrPib is a nonpathogenic gene
in P. oryzae Cavara, which corresponds to the resistant gene in rice,
Pi-b; resistance of rice to pathogenic P. oryzae Cavara depends on
recognition of gene products of avrPib by the Pi-b gene in the rice .
Hence, mutation in the avrPib gene disrupts resistance by the Pi-b
gene. Thus, to investigate the cause of mutation as well as
interactions between resistant gene products and nonpathogenic gene
products, it is necessary to isolate the avrPib gene from
resistance-disrupted lines. By crossing with P. oryzae Cavara which
were isolated from regions where disruption of resistance was observed
and were affecting Pi-b, fine mapping of the nonpathogenic gene was
compared between the method of this invention and RAPD method, a
representative conventional method.
Lines retaining avrPib (and thus not capable of affecting
Pi-b-containing rice) and lines without avrPib (and thus affects
Pi-b-containing rice) were crossed to produce a large number of F1
generation lines . These F1 lines were inj ected to rice having Pi-b
to determine presence or absence of avrPib in each line . Then genomic
DNA bulks were prepared from avrPib-present group (+) and avrPib-absent
group (-) , using about a dozen lines for each group. Using 4 lines,
i. e. , 2 from each parent line and 2 from (+) and (-) bulks, AFLP analysis
was performed with 64 x 4 = 256 primer pairs . Since the genome size
of P. oryzae Cavara is approximately 40 MB, which is about 1/10 of
that of rice, the number of primer pairs required to cover the entire
genome is about 1/16 of the case in rice.
For comparison, approximately 540 primers were used for RAPD
(Random Amplified Polymorphic DNAs) amplification. In RAPD method,
144 lanes, for example, may be processed at a time using a large-sized
submarine gel while the bulk method requires 540 x 4 = 2160 lanes.
Hence only stable and distinctive bands were chosen for comparison.
Thus, chosen 1860 bands were compared between the bulks over 15 days,
which resulted in approximately 100 polymorphic bands between the
CA 02385943 2002-03-25
27
parents.
In contrast, the method of present invention compared and
searched for approximately 5700 bands in 4 days, resulting in 304
polymorphic bands (Table 1) . This indicates that the efficiency of
this method is 11 times higher than that of RAPD method (57D0/1860
x 15/4) .
Table 1
Used primers Total Polymorphic bands Average Percentage of
(pairs) bands bands ! polymorophism
obtained primer (pair) (%)
Total Linked to,vrPib
(Closely linked)
RAPD 539 1$61 101 10 (4) 3.5 5.4
AFLP 251 5710 304 86 (41 ) 22.T 5.3
Among these polymorphic bands, 6 bands from RAPD and 41 from
AFLP were considered as candidates for proximal bands to the gene,
in which the bulk method revealed distinctive difference between the
dominant and recessive homogenous groups. These candidate bands were
at fist provided for primary screening, using 12 F1 lines as the
reference (Fig. 5) . The number of F1 lines was increased, and finally,
with 125 F1 lines, a fine map around the avrPib gene was constructed
(Fig. 6) . The RAPD method revealed 2 bands, and the method of present
invention 12 bands, within 20 CM from the target avrPib gene. While
the band closest to the gene detected by RAPD was at 5.3 CM from the
gene, the method of present invention detected a band at 1 . 8 CM, which
was much closer. This distance is considered small enough for
construction of a physical map since 1 CM in P. oryzae Cavara corresponds
to approximately 80 kB.
[Example 2] Fine mapping of bc-3, a Kamairazu (brittle culm) mutant
gene in rice
The bc-3 gene is a causal gene for the mutationwithwhich cellulose
synthesis required for the secondary thickening of cell walls in the
culm of rice is inhibited (Kamairazu or brittle culm) . Nucleic acid
CA 02385943 2002-03-25
28
markers near the bc-3 gene were searched for by genomic scanning.
A japonica bc-3 mutation M11 and an indica wild-type Kasalath were
used as crossing parents for F2 analysis. Ten. mutant homogenous
individualsand eight wild-type homogenousindividualswereidentified
by analysis up to the F3 generation, and their genomic DNA mixtures
as well as those of the parent lines were provided for genomic scanning
bulk analysis in combination with AFLP. The nucleic acids were
double-digested by two enzymes , i . a . , EcoRI (which recognized 6 bases )
and MseI (which recognized 6 bases) . Then, genomic scanning by bulk
analysis was performed with 1430 combination of primers having 3-base
selective nucleotides contiguously to the ends cleaved by the enzymes .
An example of the result is shown in Fig. 7.
As a result, 97 candidates for proximal markers were obtained,
among which 50 were usable to detect recombinant markers in recessive
homogenous individuals. To narrow down the candidates for markers,
each candidate was provided for F2 analysis using 10 recessive
homogenous individuals (20 chromosomes) each. Among them, 24 marker
candidates were analyzed with 32 recessive homogenous individuals,
i . a . , 64 chromosomes , and 1 marker was found to be coisolating with
(at 0 CM distance from) the target gene (Fig. 7).
[Example 3]
When this invention is to be applied by AFLP to an organism with
a very large genome size, such as barley (5.5 GB), if the lengths
of selective primers on the both ends of genome fragments are 3 bases
each, distinctive bands may not be obtained if the DNAs to be tested
remains double-stranded under nondenaturing conditions, which are
used for smaller genome sizes, such as that of rice (450 MB) . In this
case, one approach is to increase the number of selective primers.
Alternatively, however, a sufficient band resolution may be achieved
even with 3-base selective markers by denaturing DNAs into single
strandsupon electrophoresis. To providesuch denaturing conditions,
6 to 8.5 M urea is added to the gel, 50~ formaldehyde is added to
the sample buffer, and the samples are placed in 90°C for 3 minutes
immediately before electrophoresis.
Fig. 8 is a result of genome scanning performed to search for
CA 02385943 2002-03-25
29
genes responsible for the two-rowed spike trait in barley. As
backcrossing hybrids of Azumamugi (six-rowed) and Kanto Nakate Gold
(two-rowed) with Azumamugi (six-rowed) over 7 generations; lines with
two-rowed spike were selected to establish a quasihomogenous genetic
line having the .two-rowed spike trait with background of Azumamugi.
This line and the backcross parent Azumamugi are compared by genome
scanning in the photograph of Fig. 8 . Differences from 16 primer pairs
were searched for in 32 lanes, and a very limited number of bands
showed differences. The bands with difference are candidates for
polymorphic bands in the regions strongly associated with the two-rowed
spike genes.
With barley, average approximately 58 bands are identified under
these conditions . Given that approximately 88 bands are obtained in
average by AFLP of barley using a large-scale sequencer gel (Castilioni
et al. 1998 Genetics 149: 2039-2056) , approximately 67% of the bands
are identified by this method. The method of present invention is
capable of processing 256 lanes per gel per day, which is 5 to 6 times
more efficient than methods using large gels, which processes the
degree of approximately 32 lanes per day.
[Example 4] Isolation of rice-breed distinguishing bands identified
by genomic scanning and designing of specific primers by sequencing
The present inventors developed SCAR markers for easy
identification of breeds among commercially available rice and built
a system with which anyone can readily identify rice breeds by promptly
performing PCR.
Bands that distinguish breeds among 10 major commercially
available rices were searched for with genomic scanning (AFLP) using
55 primers . The search took only 2 days . Among several bands found
suitable for breed identification, a band specific to the breed
"Akitakomachi"wasprovidedfor electrophoresisin a widelane. After
electrophoresis was completed, the lane was stained with a fluorescent
dye (vistra green) , cut out, crushed and extracted in TE buffer, and
providedforPCRamplificationusingprimersforAFLP. ThePCRproducts
were inserted into appropriate plasmids for introduction into E . Coli .
After cultivation and amplification of E. Coli, the plasmids were
CA 02385943 2002-03-25
obtained and the presence of the insert of interest was confirmed
with restriction endonuclease. Then the plasmids were sequenced to
design primers which specifically amplifies the band of interest (Fig.
9A) . When.PCR amplification was performed using DNAs from major 10
5 breeds as templates and using these primers , a specif is band was observed
to be amplified only in Akitakomachi (Fig. 9B).
[Example 5] Selection of a clone of a library corresponding to a nucleic
acid marker
10 To identify a clone that correspond to a particular band obtained
from a genomic library by this method, SCAR markers obtained as in
above Example 3 are usually used to select positive clones by performing
colony hybridization on high-density membranes carrying the library.
However, this approach takes labor and time for a large number of
15 bands. Moreover, inside sequences in the isolated band are not
necessarily unique.
A clone in a library corresponding to a particular band may be
selected directly by genomic scanning as follows, without isolating
marker bands: First, DNAs of genome clones, such as BAC, are extracted
20 from the whole library clones with a plasmid extractor to prepare
plates of the same DNA sequences as of plates of the original clones .
The whole genome library is then divided into several sublibraries
of 1 genome equivalent or smaller size . Thus , each sublibrary contains
average 1 clone corresponding to a particular band. Further, using
25 row, column, and plate numbers as coordinates, 10 ng DNAs each is
collectedfrom clonesonseveralmicroplatesconstituting asublibrary,
which clones have the same coordinate numbers , to obtain coordinate
samplescorresponding to different coordinate positions. For example,
the coordinate sample for row 3 is obtained by collecting 10 ng DNAs
30 each from all clones on row 3 , regardless of plate and column numbers .
All coordinate samples are prepared in the same manner . The genome
scanning is performed in the same manner on these coordinate samples
as well as on the whole genome sample as control. Coordinate sample
DNAs may be prepared without extracting DNAs of all clones in the
genomic library; instead, clones may be cultivated and increased to
about 2 ml, mixed in each of rows, columns, or plates to provide
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coordinate samples, from which mixture DNAs are extracted. Once a
band of interest on the lane amplified from the control whole genome
finds corresponding row, column, and plate bands , the numbers represent
a 3-dimentional coordinate for the target clone, which enables to
pick up the clone from the sublibrary. In case the sublibrary contains
several candidate clones , the candidates may be picked up and provided
again for genome scanning along with control for final determination
(Fig. 10).
This method is particularly powerful for constructing a contig
for entire genome by matching all bands obtained through genome scanning
with library clones so that all constituting clones of a genomic library
are covered. Thus, a whole genome contig may be readily constructed
by one person.
Fig. 10A shows how a particular clone is identified from a genomic
sublibrary consisting of 6 half-plates (3 full-plates), where the
genomic library (average 120 kB, 6 genome equivalents) of P. oryzae
Cavara (genome size: 40 MB) is divided into 6 genomic sublibraries
such that each sublibrary is approximately 1 genome equivalent. In
each genomic sublibrary, the coordinate sample representing row 1
was prepared by collecting 10 ug DNAs from all clones on row 1 on
6 half plates (6 columns x 6 half plates = 36 clones). Therefore,
each genomic sublibrary contained 20 coordinate samples in total
representing 8 rows, 6 columns, and 6 plates, which would identify
8 x 6 x 6 = 288 clones in the entire genomic sublibrary.
As shown in Fig. 10B, electrophoresis was performed by genomic
scanning with 22 lanes at a time, where these coordinate samples were
used in 20 lanes as templates and control whole genome was used in
2 lanes as a template . Thus , clones corresponding to the bands obtained
in the control were readily identified from the sub genomic library.
Since 3 sub genomic libraries may be placed on 1 electrophoresis
gel (66 lanes), only 2 gels are needed to cover the entire genome,
i.e., 6 sublibraries. Thus, with 1 AFLP cycle with l primer pair,
search for clones corresponding to approximately 25 to 40 bands over
the whole genome library, which is 6 genome equivalent, is completed
with 2 gels. Since the standard genomic scanning processes 4 gels
per cycle, clones corresponding to 50 to 80 bands obtained by 2 primer
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pairs may be identified from the whole genomic library in 1
electrophoresis cycle.
Thus, even in conservative estimation, approximately 50 to 80
bands from 2 primer sets are matched with corresponding clones in
1 electrophoresis cycle; approximately 1 , 200 to 2 , 000 bands are matched
with corresponding genome in 25 electrophoresis cycle. Given that
the genome size of P. oryzae Cavara is 40 MB, obtaining 1,500 bands
would yield an average band density of 40 MB / 1,500 bands i 27 kB/band
in the genome, which means that average 4.4 bands would be obtained
in an average clone on 120 KB. Taking the average 6-fold clone
redundancy into consideration, this density should be sufficient for
constructing a genome contig. Thus, where BAC plasmids are already
available, a contig for a whole genomic library consisting of 6 genome
equivalents is completed in approximately 1 month.
Plasmids of an amount for 18 plates may be prepared in about
2 weeks given that an automated plasmid extractor is fully available .
Industrial Applicability
The method and electrophoresis apparatus of this invention has
made it possible to very easily and efficiently detect, identify,
and obtain nucleic acid markers for purposes such as follows:
(1) Development of polymorphism markers to mark a single gene
controlling important functions and/or characters by utilizing
polymorphism of nucleic acids proximal to the gene.
For this purpose, the organism with the gene of interest may
not have a genome map consisting of already-known markers.
( 2 ) Identification and isolation of clones proximal to the target
gene in positional cloning
To isolate and clone a single gene controlling important
functions and/or characters by positional cloning based only on
positional information on the chromosome, markers immediately close
to the gene are searched for, and markers for picking up clones in
the region around the gene from a genome library such as that of a
BAC (bacterial artificial chromosome) are provided. For this purpose,
the organism with the gene of interest may not have a genome map
consisting of already-known markers.
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(3) Gene analysis and high-density mapping
A large number of nucleic acid markers for gene mapping of an
organism are provided efficiently. Linkage analysis of F2 or RI lines
of the organism and a high-density mapping are also performed at high
efficiency. Thus, analysis of quantitative trait loci (QTL) based
on contribution of 2 or more genes is also readily performed.
(4) Identification of breeds
Marker bands for breed identification of commercially available
foods and seeds, including polished rice, are detected efficiently.
Further, obtained bands are isolated and cloned for sequencing in
order to design primer for SCAR (Sequence Characterized Amplified
Region) analysis . Use of such SCAR markers may allow prompt and rapid
breed-identification. Proximal markers to a particular gene may also
be rendered as SCAR markers so that they may be used as more easy-to-use
markers for breeding and so on or be used to pick up BAC clones near
the gene.
(5) Construction a contig covering the whole genome of an organism
Clones composing a genomic library of an organism may be readily
linked together to construct a contig covering the whole genome as
follows: A genomic library of several genome equivalents is divided
into several sublibraries of approximately 1 genome equivalent each.
For each of microplate group composing each sublibrary; the rows,
columns and plates are designated as x, y, and z axes, respectively.
All clone DNAs orthogonal to an axis are grouped together to provide
a coordinate sample. used as template. These coordinate samples and
whole genome DNA as control are placed on an electrophoresis gel as
templates and processed by the genomic scanning. Thus, a clone
corresponding to bands on the whole genome lanes is identified (Fig.
10). Given that bands are obtained at the density of equivalent to
1 band / 20-50 kB, a whole genome contig, which has a clone redundancy
of several folds in average, is completed.
