全 文 ：广 西 植 物 Guihaia 23(2)：149— 154 2003年 3月
利用 RAPD分子标记对番茄杂交
种纯度的鉴定研究
李 丽1，郑晓鹰1，E．Klocke2
(1．北京农林科学 院蔬菜研究 中心 ，北京 100089；2．联邦育种研究 中心 园艺作物 研究所 ，德 国 )
摘 要 ：应用 RAPD(Randomly amplified polymorphic DNA)分子标 记对番茄京丹 1号和毛粉 802的 F1代 杂
交种纯度进行 鉴定 的实验研 究 。该项研究使用 了 1O个 碱基 的单 随机 引物 和 1O个碱 基 的双随机 引物进行扩
增 。在 6O个单引 物扩增反应 中获得 7个京 丹 1号父本特有 的核酸标记 片段 。但在 14个双随机引物对京丹 1
号和毛粉 802杂交组合的扩增反应中获得了 7个京丹 1号 F1代杂交种特有的核酸标记片段和 5个毛粉 802
父本特有的标记带 。实验结果显示 ，双引物 的扩增反应对鉴定双亲亲 缘关系极近 的杂交 种纯度较单 引物扩增
反应更有效。其中，京丹 1号的 14个标记片段在北京蔬菜研究中心，种子纯度检测室又进行 了重复扩增实
验。实验结果为 87 的 RAPD标记可以在使用不同的 PCR仪和不同来源的 Taq酶 的实验条件下得到。
RAPD分子标记技术对鉴定双亲亲缘关系极近的杂交种纯度是真实可靠的。
关键词 ：RAPD；单引物 ；双引物 ；京丹 1号 ；毛粉 802；杂交 种纯 度
中图分类号 ：Q943 文献标识码 ：A 文章编号 ：1000—3142(2003)02—0149—06
Identification of hybrid purity of tomato(Lyco-
persicon esculent um)using RAPD markers
LI LiI，ZHENG Xiao—ying1，E．Klocke
(1．National Engineering Research Centre for Vegetables，Beijing 100089，China：2．Federal Centre for
Breeding Research．Institute of Horticultural Crops，BAZ，Quedlinburg，Germany)
Abstract：To determine the genetic purity of two commercial tomato(Lycopersicon esculentum)hybrids，Jing—
dan No．1 and Maofen 802，randomly amplified polymorphic DNA (RAPD)markers were applied tO discrimi—
nate hybrids from their parental inbred lines．Single primer and two—primer RAPD reactions were assayed．
Sixty single primers were applied tO discriminate the Jingdan No．1 and Maofen 802 hybrids and their parental
inbred lines．Six of them generated polymorphic bands that were different between J ingdan No．1 and its female
line．But no informative products produced between Maofen 802 and its parental lines．J ingdan No．1 and
Maofen 802 were screened with 1 4 two—primer reactions．Three two-primer reactions showed polymorphic
bands discriminated Jingdan No．1 hybrid from its parental inbred lines and Maofen 802 hybrid from its female
inbred lines．The result demonstrate that two-primer RAPD reactions are more effective than the single primer
reactions．Six single primer reactions and three two—primer X04一KI3，KI3一GI2，X04一K1 1 reactions were re—
peated in National engineering research center for vegetable，Seed purity testing laboratory． 87 paternal—
specific markers and hybrid—specific markers in the first experiment，which discriminated Jingdan No．1 hybrid
from his parental were generated on another PCR machine，MJ Research PTC一200 and with a different source
收稿 日期 ：2002—07—08； 修 订 日期 ：2002—10—18
基金项目：中德合作研究项 目资助(99—02)
作者简介：李 丽(1963一)，女 ，湖北安陆县人 ，硕士 ，植物学专业 ，从事种子质量检验及生化 ，分子标记技术实验研究工作。
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Taq polymerase purchased from Katara company．
Key words：Lycopersicon esculentum ；RAPD；two primer；hybrid purity
1 Intr0ducti0n
In Fl seed production，hybrid production is a—
chieved by manual emasculation and pollination．In
such a complicated procedure，there is a chance for
self pollination due to incomplete emasculation．
Thus，it is essential to screen the genetic purity of
Fl hybrid seeds before they are provided to the hy—
brid seed market．
W e determine hybrid purity based on morpho—
logical markers。but it takes a whole season and it
is costly．It is often difficult to find sufficient char—
acteristic morphological traits distinguishing hy—
brids from their parentalinbred lines since the cul—
tivars are sensitive to changes in environmental
conditions．The level of variation among domesti—
cated tomato varieties is very low，especially be-
cause parental inbred lines commonly have close re-
lationships．
Other methods such as isozyme analysis and e—
lectrophoresis of seed storage proteins have been
successfully developed(Bassiri 1976，Ladizinsky
and Hymowits 1979)． However，these techniques
can not differentiate the closely related genotypes
due to limited polymorphism．
M olecular markers such as RFLP，AFLP，
SSR，RAPD have been applied for the discrimina—
tion of the closely related genotypes of parental
lines and genetic purity testing of tomato hybrid
varieties． (Livneh 1989， Hoshizum 1993，Arens
1995，Paran 1998)． Comparing these methods。
RFLP and AFLP are lengthy，costly，and labour in-
tensive． Simple sequence repeat (SSR ) requires
primer information for specific sequence on a crop
special genome． The RAPD technique is advanta—
geous due to its rapidity， simplicity，and need for
small amounts of genomic DNA． Its disadventages
is lack of lab to lab reproducibility．The key factor
is different PCR machines and a different SOUrce of
Taq polymerase． Schierwater and Ender s (1993)
results demonstrated different thermostable DNA
polymerases may amplify different RAPD prod—
ucts．Macpherson et a1．(1993)assay showed that
RAPD assay is likely influenced by multiple fac—
tors，especially that different PCR machines may
produce very different data sets． So for Jingdan
No．1 the experiments were repeated in two 1abor—
tories． The marker generated by K 1 1，X04，X08，
GO9，G12，K13一G12，XO4一K13，which discriminate
hybrid from its parental line also repeated in the
latter labortory．The reaction performanced in two
different PCR machine and two source of Taq poly—
merase． The present study try some way to re—
search the RAPD assays reproducibility．
In the present study，we applied single primer
and two—decamer primer sereening tomato．J．Hu
et a1． (1995)Southern analysis revealed that the
two—primer reactions had no homology to the bands
amplified in single—primer reactions involving the
same primers． Furthermore，these new markers
were not linked to markers amplified with the same
primers in the standard RAPD reactions，sugges—
ting that they were amplified from different ge—
nomic regions．And the two—primer RAPD tends to
amplify more and smaller fragments than the
standard RAPD technique． The present study in—
vestigated how to descriminate purity of hybrid
seeds，and which one is more effective between the
two ’primer reactions and the single primer reac·
tions．The results of this study indicated that the
two—primer products is efficient for this aim．
2 Plant materials and M ethods
2．1 Plant material
Two tomato (L cDper cD esculentum)culti—
vars were commercial hybrid varieties，Jingdan No．
1(T26)and its two parental inbred lines designated
T27(male)，T25 (female)；Maofen 802(T19)and
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2期 李 丽等 ：利用 RAPD分子标记对番茄杂交种纯度的鉴定研究 l5l
its two parental inbred lines designated T2 1
(male)，T20(female)．
The young plants grew in the greenhouse for
forty days，three fresh young leaves were collected
from each single plant of every varieties．The total
weight is about 30——40 mg． DNA from a single
plant was isolated by a rapid method according to
Dorokhov and Klocke (1996)． The concentration
of DNA was estimated by Pharmacia Biotech (Bio—
chrom ) Ltd． Spectrophotom eter Uhrospec 2000．
W e bulked a pool from each single plant of every
cultivars to ensure the uniformity of accessions．
The concentration of a pooled DNA sample made
from five of single plant DNA ’s was the same as
that of each single plant DNA (1 0 ng／／~L．)
2．2 PCR condition
The reactions were performed in a 12．5 L so—
lution containing Appligene l x buffer (50 m M
KCl，l0 mM Tris—H Cl，PH8．8，l Triton X—i00)
1．5 mM M gC12，i00 M of each nucleotide，0．2 M
primer，0．25 Unit Taq DNA polymerase (Appli—
gene Germany)or 1．0 U nit Taq DNA polymerase
(Katara Japan，Sangon China) and 20 ng DNA．
The reactions employing single decamer primer
were conducted in sealed plates covered with plas—
tic cover or 0．2 tLL microtube and incubated for one
cycle of 2 min．at 94 ℃ ，45 cycles of 0．2 min．at 94
℃ ，l min．at 36℃ (down—ramp rates was 50％)，l
min． at 72 ℃ (up—ramp rates was 50 )，at the
end，9 min．at 72 ℃ ，then kept at 4 ℃ ．The reac—
tions using two—decamer primer were incubated for
one cycle of 0．5 min．at 94 ℃ ，45 cycles of 0．5
min．at 94 ℃ ，l min． at 35 ℃ (down-ramp rates
were 50％)，l min．at 72℃ (up—ramp rates were
50 )，4 min．at 72 ℃ ，then kept at 4℃ ．The am—
plification reaction were performed in GeneAmp
PCR System 9 700 (Perkin Elmer Corp．Norwalk．
CT．USA)and MJ Research PTC一200 (MJ Re—
search Inc．，W atertown，M A ，USA )． After ampli—
fication，5 L of loading buffer (0．25 Bromphe—
nol blue，40 sucrose in sterile bidest water)were
added to each sample which was loaded into a
2．0 9，6 agarose gel in 0．5x TBE，pH8．0(0．044 M
Tris，1．25 mM EDTA—Na2，0．044 M boric acid)
buffer submitted to electrophoresis at 3 V／cm．
The amplified fragments were visualized by 0．2
ethidium bromide staining． The gel were saved in
an image system with videocamera and computer
software KS 400 (Zeiss Germany)．First the prim—
ers were used to screen the parental line pools，then
the amplifications which had been scored hybrid—
specific and paternal—specific bands were repeated
at least twice with DNA from the single plant both
from hybrid variety and from single parental lines．
Only bands that were intensely，reproducible，i．
e．，present or absent in both reactions were ana—
lysed．
2．3 oljgOnucleOtide primer
Sixty single primers(K01一K20，X0l—X20，G01-
G20；$37l，$372，$373，$304，$374，$92，$89)were
applied to discriminate Jingdan No．1 hybrid and
parental inbred lines．Twenty single primers(Y0 1-
Y20)were applied to discrim inate M aofen 802 hy—
brid and its parental inbred lines． Fourteen two—
primer (Kl3一Gl2，Kl3一G09，G09-G12，Y06一Y07，
Y0l—Y06，Y0l—Y07，Y0l—Yl0，Kl l—Kl3，X04一Kl3，
X04一G09，X04-G12，Kl l—G09，Kl l—Gl2，Kl l—X04)
were used for determ ining the two tomato hybrid
varieties from their parental inbred lines．Only ten
nucleotides primer were surveyed in this study．
The primers (Roth Company，Germany． Sangon，
Canada)discussed in this study are sited in Table
1． A GeneRulerTM DNA Ladder M ix (Fermen—
tas，Germany)was used．
3 Results and Discussion
M aofen 802 was screened with twenty single
primers，but there were no informative fragments
that refered to paternal·-specific and hybrid—-specific
markers generated
H ybrid l refers to informative bands of hybrid
in the first experiments．H ybrid 2 refers to inform—
ative bands in repeated experiments．
Sixty single primers were used for Jingdan
No．1，primers K11，K12，K14，X04，X08，G09，G12
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generated one paternal—specific markers，respective—
ly． K11—1500，KI2—1000，K14—300，X04—700，X08—
1031，G09—700，G12—800 can distinguish hybrid T26
from female parent T25 (Table 1，Fig．1)．12 of
the primers reactions produced paternal—specific
bands． A total of 1 82 fragments were produced，
only 4 showed polymorphism．
Fourteen two—primer RA PD reactions were
Table 1 Primers used to discriminate two F1 hybrids from their parental inbred lines in two experiments
Prime一) Sequence P P PM PF Hybrid 1 Hybrid 2
K11(S371)一1500
K12(S372)一900
K14(S374)一300
K14(S374)一1000
X04(S304)一700
XO8(S308)一1O31
GO9(S89)一700
G12(S92)一800
S373--S92—1250
K13一G12—135O
S373一S92—135O
X04·-K13·-900
$304一S373-900
X04一K13-820
S304·-S373·-820
X04-K13·-800
X04一K1 1-600
S304一S37 1-600
X04一K11-1O31
S304一S371-1031
K13一G12—12OO
K13一G12—11OO
X04．．K13．-620
X04．K11—600
X04一K11—1031
AATGCCCCAG + 一
TGGCCCTCAC + 一
CCCGCTACAC + 一
CCCGCTACAC 一 一
CCGCTACCGA + 一
CAGGGGTGGA + 一
CTGACGTCAC + 一
CAGCTCACGA + 一
CAGCTCACGA
GGTTGTACCC 一 +
CAGCTCACGA
CCGCTACCGA + 一
GGTTGTACCC
CCGCTACCGA + 一
GGTTGTACCC
CCGCTACCGA 一 +
GGTTGTACCC
CCGCTACCGA + 一
AATGCCCCAG
CCGCTACCGA 一 +
AATGCCCCAG
GGTTGTACCC 一 +
CAGCTCACGA
GGTTGTACCC 一 +
CAGCTCACGA
CCGCTACCGA + 一
GGTTGTACCC
CCGCTACCGA 一 +
AATGCCCCAG
CCGCTACCGA + 一
AATGCCCCAG
+
+
+
+
+
+
Jingdan No．1+
Jingdan No．1+
Jingdan No．1+
Jingdan No．1
Jingdan No．1+
Jingdan No．1+
Jingdan No．1+
Jingdan No．1+
Jingdan No．1+
Jingdan No．1+
Jingdan No．1+
Jingdan No．1+
Jingdan No．1+
Jingdan No．1+
M aofen 802 ——
M aofen 802 ——
M aofen 802 +
M aofen 802 +
M aofen 802 +
Jingdfin No．1+
Jingdan No．1+
JingdanNo．1+
Jingdan No．1+
Jingdan No．1+
JingdanNo．1+
JingdanNo．1+
Jingdan No．1+
Jingdan No．1+
Jingdan No．1+
Jingdan No．1+
Jingdan No．1+
JingdanNo．1一
Jingdan No．1+
JingdanNo．1+
”Primer number refers to Roth Ramdom Primer Kits and Sangon Canada．The number after primers refers to the band size in base pairs．
P 。PM = male parent；P 。PF= female parent．
performed to distinguish the two tomato hybrid va—
rieties from their parental inbred lines． Primer
pairs K13一G12，X04一K13，XO4一K1 1，generated two
hybrid—specific bands，respectively，of sizes are 1250
and 1350 base pairs for K13一G12；540，820 and 900
base pairs for X04一K13；1031 and 1200 base pairs
for X4一K1 1．These fragments were able to distin-
guish hybrid T26 from its parents T27 and T25．
(Table 1，Fig．2)．
Primer pair XO4一K1 3 generated the paternal—
specific bands，X04一K1 3—620，that was different be—
tween M aofen 802 hybrid T1 9 and its female line
T21．(Table 1，Fig．3)．Primer pair X04一K11 gen—
erated the bands．X04一K1 1—600 and X04一K1 1—103 1
that were different between M aofen 802 hybrid
T19 and both parents (T2O，T21)． (Table 1)．
K13一G12—1 100 and K13一G12-1200 can distinguish
Maofen 802 from its female parent(Fig．3)．
+ + + + + + + + +
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2期 李 丽等 ：利用 RAPD分子标记对番茄杂交种纯度的鉴定研究 l53
A total of 46 products performed by two’。prim’-
er RAPD reactions were generated，9 out of these
fragments，i． e． 20 products showed polymor—
phism．22 of the two—primer combination ampli—
fled the informative fragments．
RAPD technique has been developed for more
X 3*T26 2*T27 2*T25 3*T26 2 T27 2*T25 2*T26 4*T27 2*T25
_ l1l
i◇ ¨ll
嚣 《 0
Il吞；一 滥 - ● _■
Fig．1 The profiles generated by primer K1 1，G09，G12(from left to right)in Jingdan No．1．T26：
hybrid，T27：male parent，T25：female parent．M ：GeneRulerTM DNA Ladder
M ix． The nllTlher indacaleS molecular weight of polymorphic bands．
X
1 200—
800·-一
K1 3一G1 2 X04一K1 3
Fig．2 The profiles generated by primer KI3一G12，X04一K13 in Jingdan No．1．T26
hybrid，T27：male parent，T25：female parent．M ：GeneRulerTM DNA Ladder
M ix．The number indicates molecular weight of hybrid—specific bands area．
X
J 200⋯
600 一
X04一k1 3
2 T1 9 4*T20
K1 3一G1 2
2*T21 2*T1 9 5*T20 3*T21
Fig．3 The profiles generated by primer X04一K13，K13一G12 in M aofen 802．T19：hybrid，
T20：male parent，T21：female parent．M ：GeneRulerTM DNA Ladder M ix．The
number indicates molecular weight of polymorphic bands area．
than 10 years．This study further investigated how
to control the reaction condition which guarantee
to get intensely and reliable RAPD markers．M ac-
pherson et a1．(1993)indicated that DNA concen—
trations between 10 ng and 30 ng and a primer con—
centration of 0．2 M provided dependable amplifi—
cation．Different thermal cyclers are generally ca—
pable of generating reliable，reproducible RAPD
patterns．Since the RAPD assay is likely influenced
by complicated factors because 10 oligonucleotide
primers need a low annealing temperature． Differ—
ent machines may produce very different data sets，
SO collaborating laboratories should consider this
added variable when combining data generated u—
sing different machines． Research by Schierwater
and Ender (1 99 3) demonstrated that the outcome
of a RAPD fingerprint pattern may depend on the
type of polymerase used． In our experience，we al一
瑟
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154 广 西 植 物 23卷
SO need a standardization of the program as well as
all of above mentioned factors．The present study
investigated these problems of RAPD reactions in
order to improve the RAPDs reproducibility． All
of the paternal·—specific markers and hybrid·。specific
markers were repeated in National engineering re—
search center for vegetable，Seed purity testing la—
boratory． Prim ers were purchased from Sangon
Company．Taq polymerase was coming from Kat—
ara Company(Japan)．PCRs were performed using
a Thermal Controller(MJ Research Inc．，W ater—
town，M A ，USA)．W e keep all of other factors in—
cluding primers and DNA plates concentration，
M g and dNTPsconcentration and performancing
program were sam e as the first experiments．Prim—
ers X13一G12。XO4一K13，XO4一K11，XO4，XO8，K11，
K14，K12，GO9，G12 RAPD reactions generated
87 informative fragm ents of the first experi—
ments． Only X04一K1 3 reaction failed to produce
hybrid—specific band，X04～K13-800． K14 reaction
produced another paternal—specific band． K1 4—
1000． That m ean collaborating laboratories could
use the RAPD data sets．
The result used single primer suggested that
there was a close relationship between the parental
inbred Iines of the two tomato hybrid varieties．
The single primer amplification did not generate
content informative fragm ents distinguished
M aofen 802 hybrids from their parents．Sixty sin—
gle primer reactions only produced 7 paternal·。spe。
cific bands for J ingdan No．1．
For this reason 1 4 two—primer reactions were
used to amplify DNA fragments determined the hy—
brids from their parental lines and its number of
reactions was achieved by possible combinations of
the eight primers． After screening 1 4 two primers
the reactions generated 7 hybrid—specific bands for
Jingdan No．1 and 5 paternal—specific bands for
M aofen 802． Evidently the two primer reactions
were more effective than the single primer reac。
tions．
Hu et a1．(1995)indicated a lack of hybridza—
tion of single-primer to two—primer amplified prod—
ucts of different sizes suggested a lack of homolo—
gy． So， two—prim er reactions can amplify new ，
smaller，more inform ative fragments，and the a—
mount of products were not increased so much that
we can analysed clearly． Also，the two—primer
RAPD increased the total number of reactions with
a limited number of primers． 4 vs．20 inform—
ative fragm ents indicated that two—primer reactions
were more effective than single primer reaction．It
is worth to develop its effectiveness．
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148 广 西 植 物 23卷
特别是扫描电镜方面提供微形态方面的资料，为系
统学增加 了新依据 。
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