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小麦-卵穗山羊草衍生后代的SSR分子标记鉴定和白粉病抗性评价(英文)



全 文 :麦类作物学报 2012,32(1):22-27
Journal of Triticeae Crops
Identification of Wheat-Aegilops Ovata Derivatives Using SSR
Markers and Evaluation of Their Powdery Mildew Resistance
WU Hong-po,WANG Ya-juan,WANG Chang-you,JI Wan-quan
(Colege of Agronomy,Northwest A&F University,Yangling,Shaanxi 712100,China)
Abstract:Aegilops species carrying the U or/and M genomes represent a prominent source of useful
genes for wheat breeding.We generated 19wheat-Aegilops ovata derivatives through hybridization
between common wheat and Aegilops ovata.Four hundred SSR (simple sequence repeat)markers
were used to investigate the polymorphisms among parental lines,Chinese Spring,Shaanyou 225and
Aegilops ovata,of which 341markers(85.25%)could amplify bands in Aegilops ovata,and 20of
them(5%)showed specific bands in Aegilops ovata,and were applied to investigate germplasm in-
heritance of Aegilops ovatain wheat-Aegilops ovata derivatives.The results showed that 10markers
could amplify specific bands in the 19derivatives,indicating that al these 19derivatives inherited
germplasms fromAegilops ovata and these 10SSR markers could be used in further identification of
next generations.We also conducted powdery mildew resistance evaluation on the 19derivatives,and
16of them are immune to powdery mildew like Aegilops ovata,one of their parents,but not like the
other two parental lines,Chinese Spring and Shaanyou 225,which are highly susceptible to powdery
mildew,indicating that the powdery mildew resistance in the 16derivatives were explicitly inherited
fromAegilops ovata.
Key words:Wheat-Aegilops ovata derivatives;Powdery mildew resistance;SSR markers
小麦-卵穗山羊草衍生后代的SSR分子标记
鉴定和白粉病抗性评价*
吴红坡,王亚娟,王长有,吉万全
(西北农林科技大学农学院,陕西杨凌712100)
摘 要:山羊草属植物是普通小麦改良过程中重要的有益基因来源,小麦的许多抗病虫、抗逆基因都来
源于山羊草属植物。本研究利用杂交和回交的方法,成功获得了19株小麦-卵穗山羊草衍生后代,实验选取
均匀分布于小麦各条染色体的SSR标记400个,对三个亲本(中国春、卵穗山羊草和陕优225)以及19株衍生
后代进行分子标记特异性分析,结果表明,341个标记可以在卵穗山羊草中扩增出条带,说明SSR标记在山羊
草中位点丰富;20个标记可以在卵穗山羊草中扩增出特异条带,说明在卵穗山羊草中有不同于另外两个亲本
的特异SSR位点;将20个在卵穗山羊草亲本中有特异条带的标记应用于19株衍生后代中,发现有10个标
记可以在衍生后代中扩增出特异条带,说明19株衍生后代中全部含有卵穗山羊草的遗传物质,这些特异引物
可以继续应用于后代的检测中。本实验还对3个亲本材料和19株衍生后代进行了成株期白粉病抗性鉴定,
结果显示,中国春和陕优225都是9级高感,卵穗山羊草是0级免疫,19株衍生后代中有3株(1-1,1-2,1-3)
分别是7、8和8级感病,其余均为0级免疫,说明衍生后代的白粉病抗性完全遗传自卵穗山羊草亲本。
关键词:小麦-卵穗山羊草衍生后代;白粉病抗性;SSR标记
中图分类号:S512.1;S330    文献标识码:A    文章编号:1009-1041(2012)01-0022-06
*收稿日期:2011-08-25   修回日期:2011-10-09
基金项目:国家高技术研究发展计划(863计划)项目(2011AA100103)。
作者简介:吴红坡(1984-),男,硕士研究生,研究方向为分子染色体工程育种。E-mail:whongpo@163.com
通讯作者:吉万全(1963-),男,博士,教授,博士生导师,主要从事染色体工程育种研究。E-mail:jiwanquan2003@126.com
  Aegilops is a genus generaly known as
goatgrass,belonging to the grass family,Poace-
ae.There are about 26species and numerous
subspecies in the genus[1].Most of species in
this genus are able to hybridize with various
types of wheat,and they are sometimes classi-
fied as members of the wheat genus,Tritic-
um[2].
The U or/and M genomes carried by Ae-
gilops species represent a prominent source of
useful genes for wheat breeding.A number of
resistance genes against diverse pathogens and
pests have been derived from Aegilops in com-
mon wheat(Triticum aestivum)[3-5].In addi-
tion,these species have good adaptability to ex-
treme  climatic  conditions, especialy  to
drought[6-8].Induced chromosome pairing of the
U or/and M genome chromosomes to their
wheat homoeologues alows the compensating
transfer of Aegilops genes into cultivated
wheat[9-11],and this is a prevailing and most ef-
fective method for conducting wheat improve-
ment through transfer of alien genes.
Aegilops ovata Roth is a tetraploid(2n=4x
=28)species with the genome formula UgUg-
MgMg,where the Ug genome was derived from
the U genome of the diploid species Aegilops
umbellulata Zhuk.(2n=2x=14,UU)and the
Mg genome originated from the M genome of
Aegilops comosa Sm.(2n=2x=14,MM)[12-14].
Aegilops ovata Roth is a valuable source for dis-
ease and pest resistance[5,15],which can be used
for improving cultivated bread wheat,Triticum
aestivum (2n=6x=42,AABBDD)[16].A lot
of researches have been done in genus Aegilops,
but research related to Aegilops ovata is rela-
tively few.Gil[15]conducted evaluation analysis
of Aegilops species and resistance investigation
of Aegilops ovata to powdery mildew,wheat
leaf rust,Hessian fly,and greenbug in 1985,
and Friebe[11]developed and identified a complete
set of Triticum aestivum-Aegilops ovata chro-
mosome addition lines in 1997.Research related
to Chinese genotype of Aegilops ovatais rare.
Genetic markers are a powerful tool for
mapping and gene cloning,providing a reliable
and efficient approach to trace the specific gene
underlying the interested traits of different spe-
cies[17-21].SSR(Simple sequence repeat)marker
is a kind of genetic markers which shows high
level of polymorphisms in a broad range of
plants,especialy among cereals[22-25]. SSR
marker has been applied into genus Aegilops for
a long period of time,but application of SSR
marker for investigation of Aegilops ovata has
not yet been done.
Here we report the generation and identifi-
cation of 19Wheat-Aegilops ovata derivatives u-
sing SSR markers and evaluation of their pow-
dery mildew resistance.
1 Materials and Methods
1.1 Materials
Plants of the Aegilopsovata (kindly pro-
vided by Dr.Li Lihui at Chinese Academy of
Agricultural Science)were crossed as male par-
ent with Triticum aestivumcv.Chinese Spring
(CS).The F1generation was backcrossed with
Shaanyou 225,a common wheat cultivar which
converged a lot of beneficial agronomic traits.
Two additional backcrossing were performed u-
sing cultivar Shaanyou 225until BC3 were ob-
tained.19 wheat-Aegilops ovata derivatives
were finaly obtained after BC3individuals were
selfed.
1.2 DNA Extraction
Leaves of seven-day old seedlings of paren-
tal lines and derivatives cultured in petri dishes
in the dark were colected and grinded into parti-
cles in plastic tubes after being frozen in liquid
nitrogen.CTAB method was used for genome
DNA extraction[26].
1.3 SSR Analysis
1.3.1 Marker Selection
Four hundred SSR markers(synthesized by
Sangon Biotech)published on Graingenes Data-
base(http://wheat.pw.usda.gov/)which e-
·32·第1期  WU Hong-po,et al:Identification of Wheat-Aegilops Geniculata Derivatives Using SSR……
venly distributed over 21wheat chromosomes,
were applied to investigate polymorphisms a-
mong parental lines,Chinese Spring,Shaanyou
225and Aegilops ovata.
1.3.2 PCR System and Protocols
TenμL PCR system was used in this exper-
iment,containing 1.00μL 10×PCR buffer
(Mg2+),1.00μL of 2mmol·L
-1 dNTPs,0.5
μL of 5μmol·L
-1 each primer,0.1μL 5U·
μL
-1 rTaq polymerase(TAKARA),50~100ng
of genomic DNA,and ddH2O.PCR was per-
formed folowing protocols published on Grain-
genes Database as 5minutes at 94degree;30cy-
cles[94degree for 30seconds;50~60degree
for 30seconds(depending on primers);72de-
gree for 30seconds];10minutes at 72degree.
1.3.3 Polyacrylamide Gel Electrophoresis
PCR products were analyzed through Poly-
acrylamide Gel Electrophoresis with gel concen-
tration of 8%.Silver staining method was ap-
plied and pictures were taken by camera(Olym-
pus C-5050).
1.4 Powdery Mildew Resistance Investigation
The powdery mildew resistance level of pa-
rental lines and their derivatives were investiga-
ted after inoculation with predominant virulent
race Guanzhong 4(kindly provided by Colege of
Plant Protection of Northwest A&F University)
at heading stage in field condition.Levels of re-
sistance were determined according to the 0~9
level standards[28].
2 RESULTS
2.1 SSR Analysis
Among 400pairs of SSR primers selected,
341pairs SSR primers(85.25%)could amplify
bands in Aegiolps ovata,indicating that simple
sequence repeats were quite abundant in the ge-
nome of Aegiolps ovata and SSR markers were
usable in the genetic analysis of Aegilops.
Twenty pairs SSR primers(5%)ilustrated
Aegiolpsovata-specific bands in PAGE compa-
ring to the other two parents,of which 10pairs
SSR primers(Table 1)could amplify specific
bands in 19wheat-Aegilops ovata derivatives(
Fig.1).The SSR marker XWMC479could am-
plify specific bands in line 4and line 8,and SSR
marker CFA2104in line 2and line 19,SSR
marker BARC127in line 6and line 17;SSR
marker BARC87in line 2,line 3and line 14;
SSR marker XWMC405in line 7;SSR marker
BARC99in line 6;SSR marker XWMC453in
lines 1~19;SSR marker XWMC552in lines 1~
10;SSR marker CFA2141in lines 8~11and
lines 13~16and line 19;SSR marker XGDM33
in lines 12~19.The results suggested that al
the 19Wheat-Aegilopsovataderivatives carried
genetic materials from Aegilops ovata,because
specific bands could be amplified in any of the
derivatives by different SSR markers.These re-
sults also indicated that different markers were
only usable to trace genetic flow in different de-
rivative lines except for marker XWMC453,
which could amplify specific bands in al 19de-
rivative lines.
Ten(50%)SSR markers which could am-
plify specific bands in parental line Aegilops
ovatacould not amplify their specific bands in
the derivatives,and the reason might be the loss
of chromosomes or chromosome fragments
where the SSR loci located.
2.2 Powdery Mildew Resistance Evaluation
Result of powdery mildew resistance inves-
tigation ilustrated that the parental line Ae
gilopsovata was immune to powdery mildew,
whereas the other two parental lines Chinese
Spring and Shaanyou 225were highly suscepti-
ble.Derivative lines 1,2and 3showed highly
susceptible symptoms at heading stage,and oth-
er lines were immune (Fig.2and Table 2),
which indicated that the powdery mildew resist-
ance of derivatives was obtained from Aegilops
ovata,and the transfer of disease resistance
genes from Aegilops ovata to common wheat
was quite feasible through direct hybridization.
·42· 麦 类 作 物 学 报                第32卷
Table 1 SSR Makers with specific bands in the derivatives of wheat and Aegilops ovata
Marker name  Primer sequence(5'-3') Specific fragment size/bp
XWMC479 Forward
:gAccTAAgcccAgTgTcATcAg
Reverse:AgAcTcTTggcTTTggATAcgg 196
CFA2104 Forward
:CCTGGCAGAGAAAGTGAAGG
Reverse:AGTCGCCGTTGTATAGTGCC 294
BARC127 Forward
:TGCATGCACTGTCCTTTGTATT
Reverse:AAGATGCGGGCTGTTTTCTA 185
BARC87 Forward
:GCTCACCGGGCATTGGGATCA
Reverse:GCGATGACGAGATAAAGGTGGAGAAC 175
XWMC405 Forward
:gTgcggAAAgAgAcgAggTT
Reverse:TATgTccAcgTTggcAgAgg 218
BARC99 Forward
:CGCATTCTTTCGCATTCTCTGTCATA
Reverse:CGCATACTGTGTCGTGTTCCTGGTTTAGA 243
XWMC453 Forward
:AcTTgTgTccATAAccgAccTT
Reverse:ATcTTTTgAggTTAcAAcccgA 187
XWMC552 Forward
:AcTAAggAgTgTgAgggcTgTg
Reverse:cTcTcgcgcTATAAAAgAAggA 149
CFA2141 Forward
:GAATGGAAGGCGGACATAGA
Reverse:GCCTCCACAACAGCCATAAT 229
XGDM33 Forward
:GGCTCAATTCAACCGTTCTT
Reverse:TACGTTCTGGTGGCTGCTC 247
1-19represent derivative lines 1-1,1-2,1-3,2-1,2-2,3-1,3-2,3-3,4-1,4-2,
5-1,5-2,5-3,5-4,6-1,6-2,6-3,7-1,7-2,respectively.The same are as in the fig.2and table 2.
Fig.1  A-E:Markers with specific bands in 19derivatives of wheat-Aegilops ovata
(Arrows indicate the specific bands)
·52·第1期  WU Hong-po,et al:Identification of Wheat-Aegilops Geniculata Derivatives Using SSR……
CS:Chinese Spring;225:Shaanyou 225;AG:Aegilops ovata
Fig.2  A and B ilustrate powdery mildew resistance levels of parents and the derivative lines
Table 2 Powdery mildew resistance of the derivative lines
Line name or
code
Level of
resistance
(0-9)
Line name or
code
Level of
resistance
(0-9)
Aegilops.ovata  0  9  0
Chinese Spring  9  10  0
Shaanyou 225  9  11  0
1  8  12  0
2  9  13  0
3  7  14  0
4  0  15  0
5  0  16  0
6  0  17  0
7  0  18  0
8  0  19  0
3 DISCUSSION
In this study,nineteen wheat-Aegilops ova-
ta derivatives were successfuly generated by
crossing and backcrossing,and phenotypes of
these derivatives were diverse from each other
and their last generations,which indicated that
those lines stil need a few generations of selfing
for obtaining stable derivative lines for further
investigation.
Ten SSR markers which could amplify spe-
cific bands in both parental line Aegilops ovata
and its progenies confirmed that al the 19deriv-
atives carried genetic materials from Aegilops
ovata and these 10SSR markers could be further
used in tracing the gene flow of Aegilops ovata
in the derived generations.This experiment was
limited by number of SSR markers selected,and
more specific SSR markers could be discovered in
order to trace the gene flow in a relatively accu-
rate manner and to create relations between spe-
cific traits of Aegilops ovataand genetic markers
for example,specific markers for powdery mil-
dew resistance.
Sixteen of 19derivative lines were immune
to powdery mildew and their powdery mildew
resistance gene was explicitly obtained from Ae-
gilops ovata.Further analysis needs to be done
to figure out if genes underlying traits of pow-
dery mildew resistance of Aegilops ovataare dis-
tinct to genes already known.We hope that the
derivatives which were immune to powdery mil-
dew wil play an important role in improving
wheat powdery mildew resistance level.
In this experiment,we identified 19wheat-
Aegilops ovata derivatives using SSR markers in
molecular genetic level,to further identify these
derivatives,cytogenetic methods,for example,
GISH,should be applied.
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