全 文 ：第 20 卷　第 3 期　　　　　　　木　　本　　植　　物　　研　　究 2000 年　7 月
Vol.20　No.3　　　　　　　BULLETIN OF BOTANICAL RESEARCH July , 　2000
对 2个源自“水稻×菰”非常规远缘
杂种的优良品系的分子分析
朴亨茂1　赵粉善1　赵基洪1　刘　宝2
(1.通化市农业科学院 ,海龙　135007)
(2.东北师范大学遗传与细胞研究所 ,长春　130024)
摘　要　2个选自“水稻×菰” F1 杂种的自交 18 代(S18)品系整理出多个源自菰的
优良特性 。然而 ,菰遗传物质渐渗给水稻的直接证据尚缺乏 。本文旨在对这 2个优
良品系从分子水平上进行分析 。将分别提取自 2个系及其水稻和菰亲本的基因组
DNA进行 2种内切酶消化后与 10个克隆自菰基因组 DNA 的具多态性的低拷贝或
中度重复序列进行 Southern杂交。结果表明 , 2个系在 20种探针/酶组合中的限制
性片段长度多态性(RFLP)谱带均相同;在其中的 18个探针/酶组合中 ,这 2个系也
均与其水稻亲本(松前)相同;但在其中的 2个探针中 , 2个系明显不同于其水稻亲
本 ,表现在有明显来自供体菰的杂交片段 ,而且伴有水稻亲本杂交谱带的丢失。由
于这 2个系一直经严格自交保持 ,证明其 RFLP 谱带的变异是由于菰中 DNA 渐渗
后再经异源重组和/或相关序列变异所致。
关键词　水稻;菰;杂交渐渗;RFLP 分析
MOLECULAR CHARACTERIZATION OF TWO ELITE RICE
LINES DERIVED FROM A NON-CONVENTIONAL WIDE
HYBRID BETWEEN RICE AND ZIZANIA LATIFOLIA
PIAO Heng-mao1　ZHAO Fen-shan1　ZHAO Ji-hong 1　LIU Bao2
(1.Tonghua Academy of Agriculture ,Hailong 135000 , Jilin Province , China.)
(2.I nstitute of Genetics &Cytology , Northeast Normal University , Changchun 130024 , China.)
Abstract　Tw o S18 elite lines that w ere selected f rom a novel F1 hybrid plant betw een
rice and a perennial wild grass Zizania lati fol ia (Griseb.)Turcz.ex Stapf exhibited
several desirable traits characterist ic of the w ild dono r species Z .lati fol ia.However ,
direct evidence for int rog ression w as lacking .The present study was aimed to charac-

To w hom correspondence should be addressed.
This w ork w as supported by the National Natural Science Foundation of China and the S & T Commission of Jilin
Province.收稿日期:1999-7-29
terize these tw o lines by molecular analysis.Genomic DNA of the two lines and of thei r
rice and Z .lati fol ia parents w ere isolated and digested w ith tw o restriction enzymes.
The blots were then probed w ith 10 diagnostic , low -copy or moderate-repetit ive se-
quences cloned from genomic DNA of Z .lat i folia.I t w as found that RFLP patterns
of the two lines w ere identical in all the 20 probe/enzyme combinat ions.In 18 combi-
nations , the two lines w ere also identical with thei r rice parental cultivar Matsumae.In
tw o probes , how ever , the tw o lines differed distinctively from M atsumae , w ith hy-
bridization fragment(s)apparent ly f rom the w ild dono r species , Z .lat i folia.Mean-
while , loss of the rice parental f ragment(s)was also observed w ith these tw o probes.
Since the tw o lines were maintained by strict selfing , it w as concluded that the
changed RFLP pat terns of the tw o lines w ere the result of Z .lati folia DNA intro-
g ression , follow ed by possible non-homologous recombination and/or sequence modi-
f ication.
Key words　 Rice;Z .lati folia;Int rogression;RFLP analy sis
Introduction
Due to the ex tensive utilization of genetic variabilties within a majo r crop species , it is im-
po rtant to int roduce useful genes f rom w ild relatives of a g iven crop by w ide hybridization.This
has been achieved w ith remarkable success in some majo r crops , such as in w heat.In others ,
e.g., in rice , however , alien gene utilization through w ide hybridization has not been as suc-
cessful due to the existence of interspecif ic or intergeneric sexual hybridization barriers.The re-
cently advanced biotechnologies hold a great promise to introduce any molecularly characterized
useful genes into a given crop wi thout species limitat ions(Jahne et al., 1995).However , most
of the ag ronomically important genes , such as those determining yield , quality and disease re-
sistance , have not yet been isolated and characterized at the molecular level , and hence not a-
menable to manipulation by molecular approaches.We have reported that by using a simple
sexual hybridizat ion procedure to produce a non-conventional intergeneric F1 hybrid plant be-
tween rice(cv.Matsumae)and a perennial w ild species Zizania lati folia (Griseb.)Turcz.ex
S tapf;we provided molecular evidence that int rogression f rom Z .lati folia into the rice
genome had occurred through a non-conventional fashion(Liu et al., 1999).In the present
study , we characterized , by RFLP analysis , tw o elite lines derived f rom this F1 hybrid plant.
These tw o lines exhibi ted several desirable trait s unique to the wild dono r species Z .lati folia ,
such as mult iple disease resistance , improved grain quali ty and higher yielding , and consequent-
ly were released as tw o new cultivars.We present evidence at the DNA level that both of these
lines contain introgressed DNA from Z .lati fol ia.
Materials and Methods
1.Plant material　The tw o lines(Tong 31 and Tong 35)were originally selected f rom a
2613 期　　　　　朴亨茂等:对 2 个源自“水稻×菰”非常规远缘杂种的优良品系的分子分析
ex tensive segregating F2 population derived from a single F1 plant between rice(cv.Matsumae)
and Z .lat i folia , produced by a simple sexual procedure called “repeated pollination”(Liu et
al., 1999).These tw o lines had been selected based on thei r f ield performance and maintained
by st rict selfing f rom S3 to S18.At S18 , the two lines w ere released as two new cultivars and
named Tong 31 and Tong 35 respectively .
2.Methods　To tal genomic DNA from young leaves of the rice parental cultivar Mat-
sumae , of the wild donor species Z .lati folia and of the tw o lines were isolated according to
the CTAB method (Kidwell and Osborn , 1992).All probes used in this study were cloned
from genomic DNA of Z .lat ifolia as described earlier(Liu et al., 1999).Probe labeling , ge-
nomic DNA digestion , Southern blot ting and hybridization followed our previous reports (Liu
et al., 1997;Liu et al., 1999).Hybridized f ilters w ere w ashed at high stringency (0.1 x
SSC , 65℃).This condition requires more than 85%homology to form stable molecular hy-
brids.
Results and Discussion
Genomic DNA of rice(cv.Matsumae), Z .lati fol ia , Tong 31 and Tong 35 was digested
to completion with two restriction enzymes EcoRI and Hin dIII and transferred onto Hybond
N+(Amersham)filters.The blots were then probed w ith each of the 10 low-copy or moder-
ate -repetitive sequences isolated f rom genomic DNA of Z .lati folia.These sequences ,
though not specific to Z .lati folia , were selected because they gave polymorphic RFIP pat-
terns betw een rice and Z .lati folia w ith at least one of the tw o enzyme digests (Liu et al.,
1999).It was found that RFLP pat terns of the tw o lines w ere identical in all the 20 probe/en-
zyme combinations , indicating that the two lines were genetically very similar.In 18 combina-
tions , the two lines were also identical to their rice recipient rice parent(cv.Matsumae).In
tw o probes(each wi th one enzyme digest , the o ther enzyme digest w as monomorphic), how ev-
er , the two lines dif fered distinctively f rom Matsumae , w ith hybridization f ragment(s)appar-
ent ly derived f rom the w ild donor species , Z .lati folia .Also , loss of the rice parental hy-
bridization fragment(s)was observed in bo th of the tw o probes(Fig.1 and data not show n).
Since that the rice parental cultivar Matsumae w as a genetically pure line , and since that all the
putat ive introgression lines were maintained by strict selfing , there could be no other cause for
changes in the RFLP patterns except fo r introg ression.The observed loss of rice parental hy-
bridization f ragment(s)is assumed to be the result of either non-homologous recombination
(inter-genomic recombination)following introgression o r sequence mutat ion caused by int ro-
g ression.Similar observations have been recently reported in t ransgenic studies(Srivasteva et
al., 1996;Takoto et al., 1997).These autho rs found that an int roduced single foreign gene
could cause sequence alterations of the host genome , resulting in changes in RFLP pat terns(in-
cluding sequence and/o r hybridization f ragment loss)of the t ransgeneic plants.Taking into ac-
count the fact that both the F1 hybrid plant and the tw o lines have chromosome numbers identi-
cal w ith the rice parent(2n=24), the int rog ressed DNA fragment(s)of Z .lati fol ia has to be
262 木　本　植　物　研　究　　　　　　　　　　　　　　　20 卷
incorporated into the rice genome.Although the mechanism for the int rogression is not clear ,
both our and other s earlier studies have show n that fertilization without gametic fusion is pos-
sible(Liu et al., 1999;Pandey , 1975).In addi tion , several studies have confirmed that it is
an ef ficient way to introduce foreign genes through the pollen-tube pathw ay (for a review see
Zhou , 1985), which also supports our hypothesis for the non-conventional intergeneric int ro-
g ression(Liu et al., 1999).
Both Tong 31 and Tong 35 have expressed several desirable trait s that are characteristic of
the donor species Z .lati folia , which are otherwise completely lacking in the rice parental cul-
tivar M atsumae.These t raits include multiple disease resistance(such as resistance to blast and
sheath blight)and improved g rain quality.Also the yield of the two lines is higher than Mat-
sumae by more than 20%(Piao et al., unpublished).Based on the present study , it could be
concluded that the ext raordinary performance of the two lines w ere the results of Z .lat ifolia
DNA introg ression , which in turn may have caused non-homologous recombination and/or se-
quence modificat ion , culminated to concerted gene expression follow ing continued pheno typic
select ion.Our results have apparent implications for other wide hybridization studies.We are
currently looking for molecular markers closely linked to these desirable trait s(e.g ., disease
resistance), introg ressed f rom Z .lat i folia and expressed in the two lines , with the hope to
eventually clone some of the underlying genes.
F ig.1 Southern blot hybridization of sequence Zg 5 to Hind II I digested genomic
DNA of rice , cv.Matsumae(lane 1), Z.latifolia (lane 2), the tw o lines Tong
31(lane 3)and Tong35 (lane 4).The long arrow indicates a hybridization frag-
ment in the tw o lines , which is apparently derived from the wild donor species ,
Z.latifolia ;the short arrows refer to tw o fragments that exist in the rice
parental cultivar Matsumae but are absent from the two lines.Sizes in kbp are
g iven on the left side.
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2633 期　　　　　朴亨茂等:对 2 个源自“水稻×菰”非常规远缘杂种的优良品系的分子分析