全 文 ：浙 江 林 学 院 学 报　2004 , 21(3):235 ～ 242
Journal of Zhejiang Forestry College
　　Article ID:1000-5692(2004)03-0235-08
Received date:2004-03-16;Revised date:2004-05-19
Foundat ion item:This research was supported by a grant-in-aid No.07556035 of the Ministry of Education Science and Culture of Japan.
Biography:TANG Ding-qin (1966-), male , born in Dongshan of Fujian Province , received his Ph D degree from the University of Tokyo ,
Japan, and his postdoctoral training was at the Asian Natural Resource &Environmental Center , the University of Tokyo , Japan.Dr
TANG is current ly a specially appointed professor of Zhejiang Forestry College , China.E-mail:tang@zjfc.edu.cn
Genetic analysis of RAPDs in Chamaecyparis
obtusa using partial diallel test
TANG Ding-qin1 ,4 , INOUE Makoto2 , KONDO Akira3 , IDE Yuji4
(1.Key Lab for Modern Silvicultural Technology of Zhejiang Province , Zhejiang Forestry College , Linan 311300 , Zhejiang , China;2.
University Forest in Aichi , The University Forests , Faculty of Agriculture , The University of Tokyo , Tokyo 1 1 3 , Japan ;3 .Shizuoka
Prefecture Forest ry and Forest Products Research Institute , Hamakita 4 3 7 , Negata , Japan ;4 .Graduate S chool of Agricultural and
Life Sciences , The University of Tokyo , Tokyo 113 , Japan)
Abstract:To develop molecular markers for Chamaecyparis obtusa , the inheritance of RAPD fragments
was studied in diploid tissues of 5 parents and 30 diploid controlled F1 progenies in each partial diallel
crosses of these parents.Of 46 primers tested , 18 primers yielded 135 reproducible fragments , of which
42 fragments (31%)were polymorphic among the parents.Fourteen fragments amplified from 7 primers
were selected to test the segregation among controlled F1 progenies.All fragments observed in the parents
were found in the progeny.Segregation of all variable fragments observed in diploid materials fitted the
proportions expected for a dominant Mendelian trait.The segregating fragments were either present or
absent confirming the dominant character of RAPD variation.The usefulness of RAPD fragments as
genetic markers for estimating genetic diversity was also discussed.[ En , 1 fig.2 tab.18 ref .]
Key words:Chamaecyparis obtusa ;diallel crosses;RAPD;segregation analysis
CLC mumber:S718.46;Q943 　　　Document code:A
Random amplified polymorphic DNA (RAPD)is based on the polymerase chain reaction (PCR), using short
arbitrary primers to amplify at random genomic fragments paired with the primers[ 1] .PCR can amplify a very small
amount of DNA with no requirement for laborious cloning , nucleotide sequencing or Southern blotting.The amplified
fragments can be detected by agarose gel electrophoresis within several hours.Despite obvious advantages and the
continuously increasing number of studies employing RAPDs , some problems have been encountered with the use of
RAPD markers.First , there are sometimes large numbers of segregation distortions with RAPD markers.Genetic
studies with RAPDs have implied that only 33%of polymorphic DNA fragments segregated in a Mendelian fashion in
Picea abies[ 2] , approximately 69% in Picea glauca [ 3] , and 86% in Populus[ 4] .In the diallel analysis of F1
progenies of conifers , Carlson et al.[ 5] found that most but not all RAPDs were inherited in the expected dominant
way.Halward et al .[ 6] found that normally dominant F2 segregation occurred and the band patterns in peanut were
often too complex to be used in genetic mapping.These studies indicate that some amplified DNA fragments do not
comply with the simple dominant inheritance patterns.Hence , the employment of RAPD as a genetic marker may
lead to draw a wrong conclusion when all polymorphic DNA fragments are scored as present or absent without regard
to mode of inheritance.Secondly , the banding patterns of RAPD vary extensively under different amplification
conditions , such as the concentration of primer relative to the template and the magnesium concentration (so-called
artifactual variation)[ 7] .Therefore , the standardization of an optimal approach and internal control is necessary if
the amplification products generated by RAPD are expected to be reproducible , scored confidently and resolved
consistently between separate amplification reactions or between studies.The appearance of artifactual variation in
RAPD banding patterns , as well as the segregation distortion , exemplifies the importance of genetic analysis of
RAPD fragments prior to being used as genetic markers.
In Chamaecyparis obtusa , one of the most commonly planted forest tree species in Japan , allozyme has been
used successfully as genetic markers.However , the number of allozyme loci currently studied in C.obtusa are
limited to eleven to fourteen
[ 8] .In contrast , RAPD markers allow direct access to the coding and non-coding
regions of the genome , making their number potentially unlimited.Here , we report results from a study on the
inheritance of RAPD fragments in unrelated individuals of C.obtusa and their F1 progeny from a partial diallel
cross.It was our intention to use this partial diallel design to determine (1)whether the polymorphic RAPD
fragments follow the expected mode of dominant Mendelian traits and (2)whether the polymorphic fragments were
actually observed in every expected F1 progeny.We also discuss the possibility of applying these fragments as
markers to the estimation of genetic diversity in managed C.obtusa forests.
1　Materials and methods
1.1　Plant materials
Partial diallel crosses between five plus-trees of C.obtusa originating from different parts of central Japan
were established in the Forest Technology Center of Shizuoka Prefecture in 1987.Materials for this study came from
these plus-trees and their partially diallel crosses Fl progenies.The controlled F1 progeny materials included six
different crosses and their reciprocal crosses shown as ` ★ in Table 1.Dormant needles were collected from 15
individuals in each cross.All materials were stored at -20 ℃until DNA extraction.
Table 1　List of controlled crosses used for
analysis of RAPD variation
♀/ ♂ Izu 3 Izu 5 Fuji 1 Fuji 5 Fuji 6
Izu 3 - ★ ★ ★ ★
Izu 5 ★ - ★
Fuji 1 ★ - ★
Fuji 5 ★ -
Fuji 6 ★ ★ ★ -
　　　Notes:♀ is female parent;♂is male parent; ★ is F1materials selected for
segregation analysis
1.2　DNA extraction
Total cellular DNA was extracted from each
individual by modified CTAB method[ 9 ,10] .
Approximately 50 mg of frozen tissue was ground
to fine powder in liquid nitrogen.The frozen
powder was homogenized at 4 ℃ in 10 volumes of
pre-cooled isolation buffer [ 100 g · L-1
polyethylene glycol (Mr 6 000), 0.35 mol
sorbitol , 0.1 mol Tris (pH 8.0), 5 g·L-1
spermidine , 5 g ·L-1 spermine.5 g ·L-1 2-
mercaptoehanol] .After centrifuged at 15 000 r·min-1 for 10 minutes at 4 ℃, the supernatant was removed.The
pellet was homogenized with an additional 5 volumes of lysis buffer [ 0.35 mol sorbitol , 0.1 mol Tris (pH 8.0),
5 g·L-1 spermidine , 5 g·L-1 spermine , 50 g·L-12-mercaptoethanol] and 1/10 volume of 100 g·L-1 sarcosine ,
mixed well and then incubated for 10 minutes at room temperature.An equal volume of 2 ×CTAB [ 20 g·L-1
cetyltrimethylammonium bromide , 0.1 mol Tris (pH 9.5), 20 mmol EDTA , 1.4 mol NaCl and 5 g·L-12-
mercaptoethanol] was added and the mixturewas incubated at 65 ℃for 10 minutes.The mixture was emulsified in
236 浙 江 林 学 院 学 报　　　　　　　　　　　　　　　2004 年 9月
an equal volume of chloroform-isoamyl alcohol (24∶1)and centrifuged at 15 000 r·min-1 for 10 minutes at room
temperature.The supernatant was transferred and DNA was precipitated with an equal volume of ice-cold
isopropanol.After centrifuged at 6 000 r·min-1 for 5 minutes at 4 ℃, the DNA pellet was air-dried and re-
dissolved in a small quantity of TE [ 10 mmol Tris (pH 8.0), 1 mmol EDTA ] .Following an additional
purification by phenol/chloroform/ ispropanol alcohol (25∶24∶1)extraction and washing with 700 g·L-1 ethanol ,
the DNA pellet was vacuum-dried and re-dissolved in TE buffer.The concentration of DNA was measured using
fluorometric assay.The template DNA was stored at 5 mg·L-1 for routine RAPD analysis.
1.3　DNA amplification
The PCR protocol was modified from Williams et alsmethod[ 1] .Random 10-nucleotide primers , viz.kit-R ,
kit-T as well as OPA-08 , OPE-08 , OPL-12 , OPP-06 , OPU-06 and OPW-15 from Operon Technologies
(Alameda , Ca USA)were used , among which the last five primers and OPR06 were used for discriminating the
interspecific hybrid clones and horticultural varieties of Chamaecyparis obtusa and C.pisifera[ 11] .Amplification
reactions were performed in a total volume of 25 μL containing 10 mmol Tris-HCl (pH 8.5), 50 mmol KCl , 2
mmol MgCl2 , 0.01 g·L-1 gelatin , 100 μmol dNTP each , 0.5 units of Taq DNA polymerase (Perkin-Elmer
Applied Biosystems), 25 ng of genomic template DNA , and 5 pico-moles of a random primer.PCR amplification
was performed on a Perkin-Elmer DNA Thermal Cycler 9700 programmed at 94 ℃ for the preliminary 5 minutes
followed by a total of 45 cycles of 1 minute at 94 ℃, 2 minutes at 36 ℃ and 2 minutes at 72 ℃, an extension of
7 minutes at 72 ℃ and a soak at 4 ℃.The RAPD products were resolved electrophoretically on 1.5% agarose gels
incorporated with ethidium bromide [ 10 μL (10 g·L-1)·L-1 ] .One control (blank) containing all the
components of a typical PCR reaction but except the template DNA was tested in the preliminary screening of primers
to confirm that amplification products represent amplified genomic DNA and not an artifact of the primer
[ 7] .Sizes of
the amplified products were estimated by using a Marker 4 DNA ladder (Wako , Nippon Gene).After running at
80V for about 5 h , the gel was photographed using the Multi-Analyst system (Bio-Rad Laboratories , CA 94957).
1.4　Genetic analysis of RAPD variants
In the genetic analysis , amplification of DNA was repeated at lest twice.Only the fragments whose presence
and absence was reproducible and unambiguous were retained.Diffuse and/or very weak fragments were not scored
because such fragments have been reported to possess the greatest propensity for poor reproducibility[ 12 ,13] .To study
inheritance of the fragmems in the F1 progeny , 15 individuals were analyzed for each cross.Based on the assumption
that there is a diploid single locus mode of inbheritance , the agreement of the observed segregation ratios of RAPD
fragments with those expected for a dominant Mendelian trait for each set of diallel crosses was calculated using the
χ2-tests.
2　Results
In the preliminary study , we tried to assess several genetic properties of RAPD fragments that are relevant to
their use in the genetic analysis of C .obtusa .To identify primers that detect polymorphism , 46 primers were
screened using DNA from needles of 5 parents shown in Table 1.Of these 46 random primers , 28 primers
(OPA08 , OPP08 , OPR01 , OPR03 , OPR04 , OPR05 , OPR07 , OPR08 , OPR09 , OPR11 , OPR12 , OPR015 ,
OPR17 , OPR18 , OPR19 , OPR20 , OPT01 , OPT05 , OPT09 , OPT10 , OPT13 , OPT14 , OPT15 , OPT16 , OPTI9 ,
OPT20 , OPU06 and OPW15)failed to yield any amplification products.The remaining eighteen primers yielded
135 reproducible fragments and no amplified product was found with these primers in control.Of these 135
fragments, 42 fragments (31%)were polymorphic among the parents.The number of amplified polymorphic
fragments varied from one to four per primer with an average of 2.1 fragments per primer.The size of fragments
ranged from 290 to 1 100 bp.Only the fragments that showed polymorphism in at least one parent analyzed in this
study were considered for the next genetic segregation analysis.Based on these preliminary results , we selected
237第 21卷第3 期 TANG Ding-qin et al.:Genetic analysis of RAPDs in Chamaecyparis obtusa using partial diallel test 　
seven primers (OPR02 , OPT02 , OPT04 , OPT06 , OPT07 , OPT08 and OPT12)which gave the best amplification
products and used them for the analysis of RAPD inheritance in 6 controlled crosses and their reciprocals.
Figure 1 shows the amplification by primer OPT07 in parents and progeny in the cross of Izu 3×Izu 5.The
results from the present analysis of RAPD variation in parent and pooled progeny are summarized in Table 2.
According to the banding patterns of each parent in preliminary amplification , each of 14 scorable and reproducible
fragments amplified with 7 selected primers was identified by segregation of pooled progenies from several crosses.
All the RAPD fragments occurring in parents over all the crosses were referred to 3 categories , in which a dominant
character of the RAPD fragment variation was assumed.
Figure 1　RAPD fragments amplified with the primer OPT07 in parents and progeny
in the cross of Izu 3×Izu 5;M :DAN size marker (Hae Ⅲ cut ΥX174)
　　The first category was those fragments shared by both parents.This was studied only for absent fragments
observed in at least one of the four remaining parents.The absent fragment was found in one of the following three
hypothetical combinations of the parental genotypes:AA ×AA , AA ×Aa or Aa×Aa.The first two combinations
involving a dominant homozygote (AA)were expected to yield monomorphic progeny that shared the parental
fragment.In the third cross between 2 dominant heterozygotes(Aa), the fragment was expected to segregate at 3:1
among the progeny.We observed 19 cases where a fragment was shared by both parents , in eight of which the
fragment occurred in all of the progeny.In the remaining 11 cases involving 7 fragments , each fragment was present
in both parents but showed present/absent polymorphism in their progenies.In all these 11 cases , segregation of the
fragment among the progenies did not deviate significantly (P>0.05 , χ2-test )from the 3∶1 ratio expected for a
dominant Mendelian trait.
The second category was that a fragment was present in one parent but absent in the other.This phenomenon
occurs when one parent possessing a fragment is either a dominant homozygote (AA) or a dominant heterozygote
(Aa)and the other a recessive homozygote (aa).In the former case (AA ×aa), all the progeny were expected
to possess the fragment found in one of the parents.In the latter case (Aa×aa), the fragment was expected to
show present/absent polymorphism , and to segregate at a 1∶1 ratio among the progenies.We found 29 cases where
a fragment was present in one parent but absent in the other.In 4 of these 29 cases , which were OPT04-1080 for
Fuji 1×Fuji 6 and Izu 3×Fuji 1 , OPT06-440 for Izu 5×Fuji 6 andOPT12-610 for Fuji 1×Fuji 6 , the fragment
occurred in all the full-sib progeny.In the remaining 25 cases involving 11 fragments , with the exception of
OPT08-1100 for progeny of Izu 3×Fuji 1 , the fragment present in one parent showed present/absent polymorphism
in the progenies , which did not deviate significantly (P >0.05 , χ2-test) from the 1∶1 ratio expected for a
dominant Mendelian trait.
238 浙 江 林 学 院 学 报　　　　　　　　　　　　　　　2004 年 9月
Table 2　Segregation of RAPD fragments for controlled crosses and the parental genotypes
inferred from sets of controlled crosses
Fragment(1) Cross
Parenes
phenotype(2)
P1 P2
Progeny
Expected Observed
+ - + -
χ2(3)
Inferred parents
genotype
P1 P2
OPR02-1080 Izu 3×Fuji 1 + + 30 0 30 0 Aa AA
OPR02-1080 Izu 3×Fuji 5 + + 22.5 7.5 22 8 0.022 Aa Aa
OPR02-1080 Izu 3×Fuji 6 + + 22.5 7.5 21 9 0.200 Aa Aa
OPR02-850 Izu 3×Fuji 1 + - 15 15 17 13 0.533 Aa aa
OPR02-850 Izu 3×Fuji 5 + + 22.5 7.5 2.5 5 0.556 Aa Aa
OPR02-850 Izu 3×Fuji 6 + + 22.5 7.5 22 7 0.022 Aa Aa
OPR02-800 Izu 3×Fuji 1 + + 22.5 7.5 28 2 2.689 Aa Aa
OPR02-800 Izu 3×Fuji 5 + + 22.5 7.5 24 6 0.200 Aa Aa
OPR02-800 Izu 3×Fuji 6 + + 22.5 7.5 23 7 0.022 Aa Aa
OPT02-1080 Izu 3×Fuji 1 - + 15 15 17 13 0.533 aa Aa
OPT02-1080 Izu 3×Izu 5 - + 15 15 16 14 0.133 aa Aa
OPT02-1080 Izu 5×Fuji 6 + + 22.5 7.5 19 11 1.089 Aa Aa
OPT04-1080 Fuji 1×Fuji 6 + + 30 0 30 0 AA Aa
OPT04-1080 Izu 3×Fuji 1 - + 30 0 30 0 aa AA
OPT04-1080 Izu 3×Fuji 6 - + 15 15 16 14 0.133 aa Aa
OPT06-770 Izu 3×Fuji 5 - + 15 15 17 13 0.533 aa Aa
OPT06-770 Izu 3×Izu 5 - + 15 15 14 16 0.133 aa Aa
OPT06-770 Izu 5×Fuji 6 + - 15 15 13 17 0.533 Aa aa
OPT07-650 Izu 3×Fuji 1 + - 15 15 13 16 0.333 Aa aa
OPT07-650 Izu 3×Izu 5 + - 15 15 17 12 0.867 Aa aa
OPT07-650 Izu 5×Fuji 6 - + 15 15 19 11 2.133 aa Aa
OPT07-550 Izu 3×Fuji 1 - + 15 15 18 11 1.667 aa Aa
OPT07-550 Izu 3×Izu 5 - + 15 15 13 16 0.333 aa Aa
OPT07-550 Izu 5×Fuji 6 + + 30 0 30 0 Aa AA
OPT08-1100 Fuji 1×Fuji 6 - + 15 15 18 12 1.200 aa Aa
OPT08-1100 Izu 3×Fuji 1 + - 15 15 21 9 4.800＊ Aa aa
OPT08-1100 Izu 3×Fuji 5 + - 15 15 15 15 0.000 Aa aa
OPT08-1100 Izu 3×Fuji 6 + + 22.5 7.5 24 6 0.200 Aa Aa
OPT08-1100 Izu 3×Izu 5 + + 30 0 30 0 Aa AA
OPT08-1100 Izu 5×Fuji 6 + + 30 0 30 0 AA Aa
OPT08-550 Fuji 1×Fuji 6 + - 15 15 15 15 0.000 Aa aa
OPT08-550 Izu 3×Fuji 1 - + 15 15 16 14 0.133 aa Aa
OPT08-550 Izu 3×Fuji 5 - + 15 15 13 17 0.533 aa Aa
OPT08-550 Izu 3×Fuji 6 - - 0 30 0 30 aa aa
OPT08-550 Izu 3×Izu 5 - + 15 15 14 16 0.133 aa Aa
OPT08-550 Izu 5×Fuji 6 + - 15 15 16 14 0.133 Aa aa
OPT08-320 Fuji 1×Fuji 6 - - 0 30 0 30 aa aa
OPT08-320 Izu 3×Fuji 1 + - 15 15 16 14 0.133 Aa aa
OPT08-320 Izu 3×Fuji 5 + + 30 0 30 0 Aa AA
OPT08-320 Izu 3×Fuji 6 + - 15 15 13 17 0.533 Aa aa
OPT08-320 Izu 3×Izu 5 + - 15 15 18 12 1.200 Aa aa
OPT08-320 Izu 5×Fuji 6 - - 0 30 0 30 aa aa
OPT12-870 Fuji 1×Fuji 6 + - 15 15 15 15 0.000 Aa aa
OPT12-870 Izu 3×Fuji 1 + + 22.5 7.5 21 9 0.200 Aa Aa
OPT12-870 Izu 3×Fuji 6 + - 15 15 16 14 0.133 Aa aa
OPT12-610 Fuji 1×Fuji 6 + + 30 0 30 0 Aa AA
OPT12-610 Izu 3×Fuji 1 - + 15 15 17 13 0.533 aa Aa
OPT12-610 Izu 3×Fuji 6 - + 30 0 30 0 aa AA
　　Notes:(1)The f ragments is expressed as primer number plus the fragment size.(2) +:fragment present;-:fragment absent.(3)The
criticalχ2at P=0.05 , 1 df , is 3.84.Values marked by an asterisk indicate a signif icant deviation from expected values
239第 21卷第3 期 TANG Ding-qin et al.:Genetic analysis of RAPDs in Chamaecyparis obtusa using partial diallel test 　
　　The third category is the fragment absent from both parents.This was studied only for fragments observed in at
least one of the remaining 4 parents.Two fragments , viz.OPT08-320 and OPT08-550 , were tested for their
segragation.The fragment OPT08-320 was absent in parents such as Fuji 1 , Fuji 6 and Izu 5 , and OPT08-550
was absent in parents such as Izu 3 and Fuji 6.As expected for a cross between two recessive homozygotes ,
fragment OPT08-320 was absent from pooled progenies of Fuji 1×Fuji 6 and Izu 5×Fuji 6 , and OPT08-550 was
absent from progenies of Izu 3 ×Fuji 6.The fragments OPT08-550 and OPT08-320 were also tested in terms of
present fragments of the cross.The segregation of these fragments in the progeny from the parents with these
fragments either did not deviate significantly from the expected 1∶1 ratio or yieldedmonomorphic progeny that shared
the parental fragment.Most early studies concerned with RAPD inheritance in controlled crosses reported results
only for parents and progenies with a fragment.However , assuming the dominant character of the RAPD
polymorphism it appears necessary to test the case where both parents show fragment absence , i.e., both parents
are recessive homozygotes.In addition to the analysis of parents and progenies that showed the presence of a
fragment , the analysis of parental genotypes with some fragment absent also provides relevant information about the
genetic behavior of RAPDs.
In summary , the segregation of all 14 variable fragments found in this study complies with expectedMendelian
inheritance and is consistent with the mode of a dominant single-locus trait.The identification of both the genetic
control of these RAPD variants and the transmission mode of controlled genes from parents to progeny implies the
potential for applying these fragments as genetic markers to further genetic research in Chamaecyparis obtusa .
3　Discussion
Among the primers used in the study , OPA08 , OPE08 , OPL12 , OPP06 , OPR06 , OPU06 and OPW15 have
discriminated C.obtusa from C.pisifera , and successfully identified their horticultural varieties[ 11] .Under the
conditions of the present study , there were not any RAPD products amplified from primers OPA-08 , OPP-08 , OPU-
06 and OPW-15 which amplified the species-specific bands for C.obtusa .Such an unclear reproducibility between
laboratories and even between studies in the same laboratory is one of the major concerns in the use of RAPD
analysis.In general , the RAPD technique utilizes low input substrate and many cycles of amplifcation.The
fragments that are amplified during RAPD analysis probably represent the most successful products among many
competitive candidates for amplification .Therefore , even minor changes in the reaction conditions may alter the
amplifying results , and result in a specific fragment to be amplified in one genetic background but not in another.
This is the artifactual variation characteristic of RAPD and increases the difficulty in the application of this
technique.
Characteristics of dominant Mendelian traits of RAPD fragments and the possibility of applying these fragments
as genetic markers have been confirmed based on the analysis of controlled crosses in C.obtusa of the present
study (Table 2).A similar mode of inheritance of RAPD fragments has been reported in some other forest
species[ 2 ,5 ,11 , 13] .However , published data on RAPD inheritance based on the analysis of controlled crosses is only
available in Picea glauca , Pseudotsuga menziesii [ 5] and Pinus sylvestris[ 13] .The present study tested each RAPD
fragment for its reproducibility of segregation in at least three controlled crosses.Such a multiple cross design could
eliminate the accidental effects of segregating deviations from the expected due to small progeny size[ 2 ,5] , confirm
the parental genotypes from several crosses and infer the parental genotypes of the crosses in which the fragment was
detected in both parents and in all the progeny.Our present results provide additional support for the usefulness of
RAPD fragments as genetic markers.
In terms of application of the RAPD fragments , most of the earlier studies on RAPDs have focused on the use
of this marker for mapping or genetic diagnostics
[ 3 ,5 ,11 ,15] .Moreover , RAPD is particularly useful when vegetative
material is available in minute amounts and when tissues contain high levels of secondary metabolites that inhibit
240 浙 江 林 学 院 学 报　　　　　　　　　　　　　　　2004 年 9月
enzyme activity.An example is Betula alleghaniensis , in which allozymes and RFLPs are of limited value[ 14] .We
are more interested in applying the identified fragments as markers to estimation of genetic diversity fluctuation in
reaforestation .The measurement of genetic diversity fluctuation in managed forests involves accurate measurement in
a large number of organisms.Therefore , this demand to develop rapid and efficient techniques to measure
polymorphisms.RAPD seems to be a right technique.However , the dominant character of RAPD markers implies
that they would be less informative in population analysis than allozymes[ 14] .In conifers , this disadvantage can be
alleviated by using haploid macrogametophytes of the individuals investigated
[ 16] .Such an approach requires the
analysis of multiple samples per individual which increases the amount of work substantially , and is limited to only
seed-producible and seed-analyzable individuals.In Chamaecyparis obtusa , thousand-kernel weight of seeds with
big a pterygium varies from 1.3 to 3.2 g[ 17] so that it is not easy to analyze the macrogametophytes.We
discriminated dominant heterozygotes from homozygotes by testing progenies of many crosses involving one of the
parents with other panents that do not exhibit these specific fragments.These parental genotypes could also be
inferred even without analyzing macrogeametophytes.However , the establishment of diallel or partial diallel crosses
is an indispensable prerequisite for such a multiple cross analysis and it is too laborious to be practically applied in
general.In the other study recently made by us , we used simpler indices (e.g.Jaccards coefficient and Shannons
information measure)computed directly from RAPD phenotype frequencies instead of the expected heterozygosity that
is estimated from allelic frequency by using these identified RAPD markers to evaluate the changes of genetic
diversity among developing seedlings in C.obtusa .The results were concordant with those of a previous study with
the same materials using allozyme markers[ 18] .It seems that the limitations of RAPDs in dominance can be
compensated by large numbers of easily accessible polymorphic markers that provide insights into many unexplored
regions of the genome for detection of genetic diversity fluctuation in C.obtusa reaforestation.
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日本扁柏部分双列杂交试验的 RAPD遗传分析
汤定钦1 , 4 , 井上　诚2 , 近藤　明3 , 井出雄二4
(1.浙江林学院 浙江省现代森林培育技术重点实验室 , 浙江 临安 311300;2.东京大学 爱知演示林 , 东京 113;
3.静冈县林科所 , 静冈 滨北;4.东京大学 农学生命科学研究科 , 东京 113)
摘要:RAPD作为一简易的 DNA分子标记 , 在得以广泛应用的同时 , 存在着随机扩增的 DNA 条带并
不遵行显性遗传模式 , 以及受 PCR反应条件影响大等现象 。以 5个日本扁柏 Chamaecyparis obtusa 优树
无性系及其部分双列杂交的 12组合 (各含 30个体)F1 代为材料 , 探讨 RAPD标记的子代遗传及分离
特征 。研究结果表明:优树无性系中供试的 46个引物中 , 14个引物扩增了 42条多态的片段 , 筛选了
其中 7个引物扩增得来的 14条片段进行分析 , 在子代中均能找到其对应的片段 , 且这些片段符合孟
德尔的遗传分离规律 。说明 RAPD可作为遗传标记在日本扁柏中用于遗传分析 。图 1表 2参 18
关键词:日本扁柏;部分双列杂交;RAPD;分离分析
242 浙 江 林 学 院 学 报　　　　　　　　　　　　　　　2004 年 9月