全 文 ：广 西 植 物 Guihaia Jul． 2013，33(4) :460－464 http:/ / journal． gxzw． gxib． cn
DOI:10． 3969 / j． issn． 1000-3142． 2013． 04． 006
刘志文，邹丹，陈温福． 杂草稻叶绿体籼粳分化的多重 PCＲ分析［J］． 广西植物，2013，33(4) :460－464
Liu ZW，Zou D，Chen WF． Multiplex PCＲ analysis of Indica-japonica differentiation of the chloroplast DNA in weedy rice［J］． Guihaia，2013，33(4) :460
－464
Multiplex PCＲ analysis of Indica-japonica
differentiation of the chloroplast DNA in weedy rice
LIU Zhi-Wen1，2，ZOU Dan1，CHEN Wen-Fu2*
(1． College of Bioengineering，Dalian Polytechnic University，Dalian 116034，China;
2． Ｒice Institute，Shenyang Agricultural University，Shenyang 110161，China )
Abstract:The whole chloroplast genomes of the Indica cultivar 93-11 and Japonica cultivar Peiai64S were analyzed and
compared． A multiplex PCＲ marker of the chloroplast DNA OＲF100 and OＲF29-TrnCGCA region was optimized and con-
structed． More than 200 weedy rice，Asian cultivated rice and common wild rice accessions were then tested using the
multiplex PCＲ． The results showed that there were obvious chloroplast DNA indica-japonica differentiations amongst the
weedy，Asian cultivated and common wild rice accessions． Furthermore，the differentiations in weedy rice were correlated
to the collected regions that were in accordance with the south indica type and north japonica type of cultivated rice in
China． It was suggested that the japonica line of weedy rice should evolve from degraded cultivated rice or the japonica
varieties(as the female parent)natural hybridization with other oryza materials for there was no wild form in north Chi-
na．
Key words:weedy rice;chloroplast DNA;Indica-Japonica differentiation;multiplex PCＲ;hybridization
CLC Number:Q941． 3 Document code:A Article ID:1000-3142(2013)04-0460-05
杂草稻叶绿体籼粳分化的多重 PCＲ分析
刘志文1，2，邹 丹1，陈温福2*
(1． 大连工业大学 生物工程学院，辽宁 大连 116034;2． 沈阳农业大学 水稻研究所，沈阳 110161 )
摘 要:通过分析籼稻 93-11和粳稻培矮 64S 的叶绿体全基因组，优化和构建了籼粳分化的叶绿体分子标记
OＲF100和 OＲF29-TrnCGCA 的多重 PCＲ。应用这个多重 PCＲ对 200余份世界各地杂草稻和其它水稻材料进行分
析。结果表明:杂草稻中有明显的叶绿体籼粳分化，表现出明显的地域性，且与传统的中国栽培稻的南籼北粳能
较好的对应。推测粳型杂草稻可能是栽培稻突变或粳型水稻(作母本)与其它类型水稻材料杂交而形成的。
关键词:杂草稻;叶绿体 DNA;籼粳分化;多重 PCＲ;杂交
Weedy rice(Oryza sativa f． spontanea) ，as a com-
petitive weed with cultivated rice(Oryza sativa) ，is
widely distributed in rice-planting filed over the world，
particularly in south and southeast Asia，south and
north America，northern Australia， and southern
Europe，where the direct seeding or no-till technology is
usually applied to rice farming(Briana et al．，2010;
Tang et al．，2011)． The growth of weedy rice in cul-
收稿日期:2013-01-22 修回日期:2013-04-30
基金项目:国家自然科学基金(30671262) ;大连市科学基金(2010J21DW015)
作者简介:刘志文(1972-) ，男，湖北罗田县人，博士，副教授，主要从事杂草稻的起源与进化研究，(E-mail)alzw@ dlpu． edu． cn。
* 通讯作者:陈温福，男，博士，教授，主要从事水稻高产育种与栽培技术，(E-mail)wfchen5512@ yahoo． com． cn。
tured rice fields could cause significant reduction in
rice yield and affect the quality of rice grains because
of its great growing ability and persistency in rice fields
(Carrie et al．，2010;Zuo et al．，2011)． Compared
with other weeds，weedy rice is very difficult to control
in cultivated rice fields because it shares similar mor-
phological features，physiological traits and herbicide
tolerances with the cultivated rice． Furthermore，weedy
rice and cultivated rice can hybridize owing to the ge-
netic similarity，and potential environmental conse-
quences of weedy rice from introgression following the
commercial release of genetically modified rice(Song et
al．，2011;Xia et al．，2011)． To date the general strat-
egy to control weedy rice is to practice preventative，
cultural，mechanical and chemical means，such as crop
rotation and herbicide application． However，the weedy
rice has been considered a promising and useful reser-
voir for genetic variation to rice improvement，because
it has many useful genetic characteristics，such as dis-
ease resistance，drought and chilling tolerance，compet-
itive ability，etc．，and there is no apparent genetic bar-
riers between weedy rice and cultured rice． Moreover，
weedy rice is successfully acclimatized to the natural
growing conditions(Burgos et al．，2011)．
Weedy rice is taxonomically classified and nomi-
nated as Oryza sativa f． spontanea being the same
genus and species as Asian cultivated rice by morpho-
logical characters and physiological traits． Many
studies have demonstrated that high genetic differentia-
tion in rice genomes occurred in Asian cultivated rice
and its wild ancestor Oryza rufipogon，in which the
signicant differentiation is indica-japonica． The
indica-japonica variation occurred both in the nuclear
genome and in chloroplast DNA of Asian cultivated
rice，presumably in the weedy rice due to the conspeci-
ficity of Asian cultivated rice (Xiong et al．，2010;Zuo
et al．，2011)．
In order to effectively utilize the beneficial genes
and facilitate the control strategies，it is of the oretical
and practical significance to fully understand and clarify
the characteristics of indica-japonica differentiation in
the weedy rice． However，the differentiation research in
weedy rice is still limited，especially in the chloroplast
DNA． The most common method for identication of in-
dica and japonica rice varieties is Chen’s index that ex-
amines six key characters of rice samples． However，
those morphological and physiological characters can be
inuenced greatly by the change of environmental condi-
tions． In addition，it is necessary to measure the con-
cerned characters from mature rice plants when this
morphological-based indica and japonica identification
method is used． Therefore，the long time is needed to
generate the results，which has considerably limited its
wide application for indica or japonica rice identication
(Xiong et al．，2010)．
The sequencing of the chloroplast genome of
japonica rice cv． Peiai64(PA64S，AY522331． 1)and
indica rice cv． 93-11(AY522329． 1)has been completed
in 2004． The completion and global availability of total
rice chloroplast genome sequences have made the devel-
opment of specific molecular markers possible using
tools of comparative genomics that are essentially based
on the differences of entire genomic sequences between
indica and japonica rice． Based on the insertion and de-
letion(InDel)fragments obtained in chloroplast DNA
sequences between PA64S and 93-11，the OＲF100
bands of japonica rice lags behind most of indica rice
because there is a 69 bp deletion at 8 549-8 617 bp in
the indica while most japonica cultivars do not have it．
This region has been considered to be an effective
marker for indica-japonica differentiation． Tang et al．
(2004)found that the indica type rice had a 32 bp in-
sertion at 17 709 － 17 740 bp in the OＲF29-TrnCGCA
spacer but the japonica type did not contain such an in-
sertion，leading to a lag of the indica bands related to
the japonica ones． Therefore，OＲF29-TrnCGCA can be re-
garded as another marker to distinguish the indica chlo-
roplast genome from that of japonica(Ou et al．，2009)．
Multiplex-polymerase chain reaction(multiplex PCＲ)is
a novel technique in which several pairs of primers are
used together in one PCＲ reaction leading to simultane-
ous amplification and detection of different regions or si-
zes of DNA fragments． It will be a very useful and effec-
tive protocol for genetic differentiation．
The main objective in this study was: (a)to
develop and assess a new multiplex-PCＲ marker of
1644期 刘志文等:杂草稻叶绿体籼粳分化的多重 PCＲ分析
above two mentioned markers for reliable and low-cost
screening the different rice materials;(b)to investigate
and reveal the differentiation in weedy rice populations
from different localities with the multiplex-PCＲ;and(c)
to explore the origin of weedy rice by indica-japonica
differentiation．
1 Materials and Methods
1． 1 Plant material and DNA extraction
One hundred and fifty-four weedy rice accessions
were collected by Shenyang Agricultural University
(SYAU)between 2005 and 2007 in rice-planting areas
of China in which thirteen regions from Liaoning Prov-
ince，two from Jilin Province，two from Heilongjiang
Province and one from Hubei Province． Moreover，49
weedy rice accessions from abroad，and Hubei and
Jiangsu provinces in China were obtained from Yunnan
Agricultural University，China(YNAU)and China Na-
tional Ｒice Ｒesearch Institute(CNＲＲI)． In total，227
rice accessions，including 203 weedy rice accessions，8
indica rice varieties，8 japonica rice varieties and 8 com-
mon wild rice accessions were selected for this study．
The 93-11 and PA64S were included as a reference，re-
presenting typical indica and typical japonica． DNA was
extracted according to Ou et al．(2009)．
1． 2 Primers design
PCＲ primers were designed by Primer Premier
5 ． 0 based on the whole chloroplast genome
sequence of 93-11 or PA64S． OＲF29-TrnCGCA
marker primer pair amplification products were de-
signed at 17 620-17 980 bp of PA64S and 17 546-
17 938 bp of 93-11 whereas OＲF100 primer pair
designed at 7 897-8 745 bp of PA64S and 7 903-8
682 bp of 93-11 ． Detailed information of the primer
pairs is given in Table 1 ．
1． 3 Multiplex PCＲ analysis
The multiplex PCＲ assays were carried out as the
following reagent concentrations:50 ng genomic template
DNA;2． 0 μL 10 × Taq PCＲ buffer;2． 0 mmol /L of
MgCl2;125 μmol /L of each dNTP;1． 0 U of Taq DNA
Polymerase(MBI Fermentas Inc ． ，USA) ;0 ． 5μmol / L
表 1 引物的序列和目标片段的预期大小
Table 1 Primer sequence，target fragment and their expected product sizes
Primer Orientation Primer sequence(5-3) Target fragment
Size of product (bp)
Indica Japonica
P1 Forward A AGGCTCGGCGATACTG
P2 Ｒeverse GCAGCCCAAGCGAGACT
OＲF29-TrnCGCA 393 361
P3 Forward AGCCGAGGTCGTGGTAA
P4 Ｒeverse AGTCCACTCAGCCATCTCTC
OＲF100 780 849
of each forward and reverse primer pair(Sangon Compa-
ny，Shanghai)． The final reaction volume was adjusted
to 20 μL with the sterilized ultrapure dddH2O．
Amplifications were done using a PTC 225 Peltier
Thermal Cycler(MJ Ｒesearch Inc． USA)． The amplifi-
cation profile was performed with a touchdown PCＲ pro-
gram as following，5 min at 94 ℃;10 cycles of 45 s at 94
℃，45 s at 60 ℃(minus 0． 5 ℃ /cycle) ，1 min at 72 ℃;
25 cycles of 45 s at 94 ℃，45 s at 55 ℃，1 min at 72
℃;and 10 min at 72 ℃ for final extension，4 ℃ for
holding． The PCＲ products were resolved using 1． 5%
agarose gel electrophoresis．
1． 4 Data analysis
Extensive multiplex PCＲ analyses were conducted
to investigate variation patterns of the rice accessions．
Then the indica-japonica differentiation of the
chloroplast DNA in rice would be analyzed and deter-
mined based on the multiplex-PCＲ amplified profile．
The electrophoretic banding patterns of the sequenced
indica rice variety(93-11)and japonica variety rice
(PA64S)were used as a reference for determining the
indica or japonica genotype，respectively． Consequently，
the banding pattern was scored as indica-genotype(I)if
it was identical to that of 93-11，or as japonica-genotype
(J)if it was identical to that of PA64S，or as indica-ja-
264 广 西 植 物 33卷
ponica-genotype(IJ)if the banding pattern was identical
to that of 93-11 and PA64S．
2 Ｒesults and Analysis
2． 1 Analysis of the OＲF29-TrnCGCAand
OＲF100 marker
To validate the effectiveness of chloroplast
OＲF29-TrnCGCA and OＲF100 InDel molecular marker for
the identification of indica and japonica rice varieties，
four traditional indica rice(93-11，IＲ24，Qishanzhan and
Ezao18)and four traditional japonica rice(PA64S，Nip-
ponbare，SN265 and SN6014)samples from different lo-
calities，were analyzed to examine the polymorphisms．
The OＲF100 PCＲ products amplified by the P3+
P4 primer pair revealed that all the indica rice is con-
sistent with the reference indica rice 93-11． In contrast，
the OＲF100 bands of traditional japonica rice lag behind
93-11 and are consistent with the reference japonica rice
PA64S． Additionally，the results from the OＲF29-
TrnCGCA analysis using P1+P2 primer pair are also com-
patible with the OＲF100，and demonstrate that all the
bands of indica rice are the same as the 93-11 but ja-
ponica rice being the same as PA64S． Ｒesults from i-
dentification of the two primer pairs suggested that they
are identical． These results also confirmed the effective-
ness of the two InDel molecular markers，which could be
used to accurately detect and classify the indica-
japonica differentiation in the chloroplast DNA of culti-
vated rice． The partial electrophoresis results were
showed in Figure1．
2． 2 Multiplex PCＲ development
The OＲF29-TrnCGCAPCＲ reaction would yield two
fragments with 393 /361 bp in length for all the rice ac-
cessions whereas OＲF100 with 849 /760 bp． They
showed that there was 399-488 bp difference between
the two markers． In this study，an attempt was made to
develop a multiplex marker system for these two markers
to distinguish the indica-japonica differentiation in the
chloroplast DNA of rice．
Multiplex PCＲ of the OＲF29-TrnCGCA and OＲF100
assay was performed and validated using the same varie-
ties as the single PCＲ． As shown in Fig． 1，the result
from the multiplex PCＲ analysis revealed is absolutely i-
dentical to that from the two single PCＲ assays respec-
tively． The large band of the OＲF100 is combined with
the small band of the OＲF29-TrnCGCA in japonica rice
being contrary to that in indica rice． This would enlarge
the relative difference between the two bands of the
single PCＲ and avoid the negative results for little
difference with 32 bp or 69 bp． The result demonstrated
that the multiplex PCＲ marker would provide an
effective and accurate method for identifying a large
number of rice varieties for their indica and japonica
characteristics．
图 1 叶绿体标记的单重和多重 PCＲ电泳图
Fig． 1 Electrophoresis of single and multiplex PCＲ
products amplified with the chloroplast markers
2． 3 Indica-japonica differentiation of the rice acces-
sions
To evaluate the indica-japonica differentiation
among the rice accessions，extensive multiplex-PCＲ
molecular analyses were conducted to investigate
variation patterns of the 227 rice accessions from
different regions． The results showed that the indica-ja-
ponica differentiation was detected in 16 cultivated rice
materials and identical to the known type previously．
Similarly，out of 8 common wild rice accessions，4 were
indica types，and the other 4 were japonica types．
Ｒesults from this study showed that the cultivated and
wild Oryza rice species or populations had considerable
indica-japonica differentiation in the chloroplast DNA．
Additionally，among the 203 accessions of the ex-
amined weedy rice，most of them，192 accessions from
north China and five foreign countries exhibited typical
japonica type (Fig． 2)． On the contrary，the remaining
11 from south China(Hubei and Jiangsu province)ac-
3644期 刘志文等:杂草稻叶绿体籼粳分化的多重 PCＲ分析
cessions showed typical indica type． The data from the
field experiment also showed that weedy rice from north
China had a similar agronomic performance of the
typical cultivated japonica rice． The data from the field
experiment also showed that the weedy rice from North
China had a similar agronomic performance of the
typical cultivated japonica rice (data not shown)． These
results suggest that the chloroplast DNA of weedy rice
should come from indica or japonica type rice respec-
tively． It is also indicated that the differentiation of the
weedy rice is correlated to the collected regions，which
are in accordance with the south indica north japonica
cultivated rice in China． It is well recognized that indica
rice is generally grown in south China，whereas japonica
rice is exclusively grown in north China indicating that
weedy rice in China should be polyphyletic evolution．
图 2 部分杂草稻材料的多重 PCＲ扩增效果图
Fig． 2 PCＲ patterns amplified by the multiplex
PCＲ in partial weedy rice
3 Conclusion and Discussion
As shown above，this is the first report on a fast
and reliable multiplex chloroplast DNA PCＲ marker
based on the insertion and deletion(InDel)fragments
obtained in DNA sequences between the entire
chloroplast genomes of the typical indica rice variety 93-
11 and japonica variety rice PA64S for the rice indica-
japonica differentiation analysis． It can provide an effec-
tive and accurate method to identify a large number of
rice varieties for their indica and japonica characteristics
in a short time and any laboratory can do the
identication with PCＲ facilities and electrophoresis．
The clear understanding indica-japonica differentiation
of the chloroplast DNA in weedy rice is of significant
benefit to rice breeders．
Despite of the fact that weedy rice is the sample
species complex as the Asian，its evolutionary origin is
still unclear． Asian cultivated forms mainly show two
types，referred as subspecies indica and japonica． In
eco-geographical terms，indica rice varieties are known
to be adapted to the tropical and subtropical
environments at low latitudes or altitudes with warm cli-
mate conditions，but japonica rice varieties are adapted
to the temperate environment at high latitudes or
altitudes with relatively cool conditions． In addition，In-
dica strains are predominantly distributed from south to
southeast Asia，whereas japonica strains are cultivated in
insular regions，mountain areas，and North countries
(Xiong et al．，2010)． It is concluded that the weedy
rice differentiation is associated with the selected
regions． Data from the present experiment suggest that
the japonica line of weedy rice may be evolved from the
degraded cultivated rice or the japonica varieties(as the
female parent)natural hybridization with other Oryza
materials for there is no wild form in north China and
the weedy rice in China may have different origins．
However，the information obtained from this study is an
initial step toward understanding the origin of weedy
rice，and it needs further research for elucidating their
origins． In summary，the weedy rice of the different re-
gions or biotypes may have originated in different ways
(Xia et al．，2011;Zhang et al．，2012)．
Acknowledgements The authors would like to
thank Dr CHEN Li-Juan(YNAU)and WEI Xing-Hua
(CNＲＲI)for kindly providing partial rice materials．
Ｒeference:
Briana LG，Michael Ｒ，Shih-chung H，et al． 2010． Seeing red:the
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(下转第 442页 Continue on page 442 )
464 广 西 植 物 33卷
显著影响 PEG 6000 胁迫下水稻幼根的长度(图 1:
B) ，表明 PEG 6000 胁迫下水稻幼根的生长和过氧
化氢水平没有明确关联。
2． 3 PEG 6000 胁迫下 SHAM对水稻幼根的影响
用 1 mmol·L-1 SHAM(使用浓度参照 Bartoli et
al．，2005) ，预处理水稻幼根并将幼根置于 PEG 6000
下培养后发现(方法同 DMTU) ，SHAM 的处理使得
PEG 6000胁迫下水稻幼根过氧化氢含量进一步上升
(图 2:A)。本研究也发现，SHAM进一步导致了 PEG
6000胁迫下水稻幼根细胞死亡水平的上升和相对含
水量的下降(图 2:C，D) ，表明该抑制剂会降低植物对
PEG 6000所引起的渗透胁迫的耐受。本研究也发
现，SHAM的处理也导致了 PEG 6000 胁迫下水稻幼
根生长的进一步降低(图 2:B)。
3 讨论
SHAM是交替氧化酶的抑制剂，交替氧化酶的
存在能通过降低线粒体电子传递链的过度还原而限
制线粒体中过氧化氢的生成(Viacheslav et al．，
2011;Millenaar et al．，2003) ;尤其作为非光合组织，
线粒体在理论上是根在渗透胁迫下产生 H2O2 的主
要位点(De Carvalho，2008)。因而，本研究认为，
SHAM可能是通过抑制交替氧化酶而进一步刺激了
渗透胁迫下植物 H2O2 的生成。介于以上关于 PEG
6000 胁迫下水稻幼根相对含水量和细胞死亡与过
氧化氢关系的观察，推测 SHAM 对水稻幼根渗透胁
迫耐受性的影响可能也和其刺激了植物过氧化氢的
生成有关。实验结果表明在 PEG 介导的渗透胁迫
下，交替氧化酶也和植物幼根的生长存在着一定的
联系，但其内在机理尚需要进一步研究。
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