全 文 ：第4 0卷第 5期 上海师范大学学报(自然科学版) Vol ． 40，No． 5
2 0 1 1年 1 0月 Journal of Shanghai Normal University(Natural Sciences) Oct ． ，2 0 1 1
Genetic mapping of a new Arabidopsis gene SPG1
responsible for microspore development
WEI Jia-li，ZHOU Shu-min，YUAN Xiao-jun，ZHANG Wei*
(School of Life Sciences，Shanghai University，Shanghai 20444，China)
Abstract:An Arabidopsis male sterile mutantshrunken pollen grain1，spg1 was obtained through en-
thyl methane sulfona (EMS)mutagenesis strategy． By the observation of the transverse section，the
mutant showed the aberrant microspores and pollen grains compared with wild type． Genetic assay in-
dicated that spg1 phenotype was controlled by a single recessive mutant locus，and this locus was
mapped to the region about 427kb flanked with genetic marker 3 － AL138638 － 6632 and 3 －
AL353865 － 6814 on chromosome 3 by map-based cloning approach． Bioinformatics analysis showed
there were no known male sterile genes in this region． It indicated SPG1 was a new male sterile gene
in Arabidopsis．
Key words:Arabidopsis;anther;male sterile;microspore;map-based cloning
CLC number:Q 344 Document code:A Article ID:1000-5137(2011)05-0522-06
Received date:2011-04-13
Fundation:The Excellent Young Teacher Foundation of Shanghai under Grant(shu08093) ;National Natural Science Foun-
dation of China(30870225) ;Technical Innovation Fund of Shanghai University;Key Project of Shanghai Committee of Science and
Technology
Biology:WEI Jia-li(1976 －) ，female，Doctor，lector，School of life sciences，Shanghai University;ZHANG Wei(1966
－) ，male，Doctor，professor，School of life science，Shanghai University．
* Corresponding author
1 Introduction
In flowering plant，the matured anther exhibited a four-lobe structure containing four somatic cell layers
(i． e．，epidermis，the endothecium，the middle layer，and tapetum)surrounding the pollen mother cell in the
locule center［1］． The pollen mother cell generated haploid microspore after meiosis，which subsequently devel-
oped into pollen grains［1］． A mature pollen grain is covered by a sculptured exine which includes the outer sex-
ine and the inner nexine． It has been known that the exine is made of polymerized sporopollenin，which is com-
prised of phenols and fatty acid derivatives synthesized predominantly in tapetum［2 － 3］． In Arabidopsis，the mu-
tation of many genes involved in the formation of the pollen exine resulted in male sterility，such as ClaS5［7］，
TDF1［8］，MS2［10］，DEX1［11］，NEF1［12］，FLP1［13］，RGP1l［14］．
The formation of matured pollen grains also depends on the coordinated interaction of microspores and the
tapetal cells［4，15 － 16］． Tapetum，a metabolically active sporophytic cell layer，secretes nutrients into locules for
the development of microspores［17］． Abnormal development of tapetum would lead to the formation of the invia-
ble pollen grains． A serial of genes，isolated from the male sterile mutants were identified to be curcial for the
development of tapetum，including EMS1 /EXS［18］，TPD1［19］，SEK1 and SEK2［20］，DYT1［21］，MYB1 03［22］，
第 5 期 韦嘉励，周树敏，袁晓君，等:遗传定位一个花粉发育相关的拟南芥新基因 SPG1
AMS1［23］，MS1［24］．
Here we report that a male sterile Arabidopsis mutant spg1 through screening of EMS － mutagenized eco-
type Columbia． The microspore in this mutant appeared shrunken and was defective in pollination activity． By
genetic analysis，the phenotype in spg1 was controlled by a single recessive gene． In this paper，the mutated
gene locus had been mapped to a region about 427 kb in Chr3． Our work suggests that SPG1 was required for
the normal microspore development in Arabidopsis．
2 Materials and methods
2． 1 Materials
The Arabidopsis mutant spg1 was isolated from EMS － treatment mutant library on A． thaliana ecotype
Clombia produced by our lab． All the plants used in this paper were planted in a 24-hour-light growth room at
22℃ in Shanghai University，China．
2． 2 Methods
2． 2． 1 Map-based cloning
The mutant was crossed to the wild type on Ecotype Landsberg to get F1 progeny． About 300 F2 plants with
sterile phenotype were used for mapping． The protocol for gene mapping was according to the previous descrip-
tion［21］． The molecular markers were based on the markers published on the website amp． genomics． org． cn
2． 2． 2 DNA extracted
The efficient DNA extracted for gene mapping was performed as the method described by Xin，et al［25］．
2． 2． 3 Observation of anther by light microscopy
For tissue section，the inflorescences of wild type and mutant were fixed in the FAA solution at least 24 h
after pumping with the vacuum pump． Then the inflorescences were dehydrated with alcohol solution in a gradi-
ent of concentration from 50% to 100% routinely． After the inflorescences were embedded in paraffin，they
were cut in the sections of 6 μmol·L －1 by using a microtome． Then the sections were stained with 1% Toluid-
ine Blue for 30 min，and photographed under the light Leica DM2500 microscope．
2． 2． 4 Stained the pollen with Alexanda solution
The full open flowers were fixed for 24 h in the stationary liquid (60% methyl hydrate，30% chloroform，
1% 2，4，6 trinitrophenol，1% HgCl2)at room temperature，rehydrated with 70%，50% and 30% alcohol inde-
pendently for 30min and rinsed twice with H2O． Then the materials were stained with Alexander solution
(9． 5% alcohol，25% Glycerol，4% AC，0． 01% Malachite Green，0． 05% acid fuchsin，0． 005% orange G)at
45℃ for 24 h;Finally，the detached anthers were observed and taken photos under a light Leica DM2500 mi-
croscope．
3 Results
3． 1 Isolation of mutant spg1
The mutant spg1 was obtained from the EMS mutant library of Arabisopsis (Columbia ecotype)． The mu-
tant phenotype was inherited stably in the F2 progeny backcrossed to wild type (Columbia ecotype)． Compared
to wild type，the siliques in spg1 were short and empty，and they were completely sterile (Fig． 1 A)． The devel-
opment of vegetable and flower in spg1 mutant was identified to wild type． But，the pollen grains can not adhere
to the stigma in the spg1 mutant after its anthers were dehisced (Fig． 1 B)． It means spg1 can not been pollina-
ted． However，when the mutant was backcrossed with the wild-type pollen grains，its siliques were developed
normally． This result indicated the stigma in spg1 mutant was viable． To see whether the pollen was viable in
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上海师范大学学报(自然科学版) 2011 年
the mutant，the anther was stained with Alexanda solution． In figure 1，the pollen grains in wild type were red
but those in spg1 were green (Fig． 1 C)． It shows that the inviable pollen grains caused the complete sterile
phenotype in the spg1mutant．
A． Wild type (Columbia)plant and spg1plant;B． A WT flower and a spg1 flower;
C． The stained anthers of WT and spg1 with Alexanda solution
Figure 1 Phenotype of WT (wild type)and spg1
3． 2 Development of anther in the spg1 mutant
To further investigate the development of microspore and pollen grains in anther in the spg1 mutant，we
generated the transverse sections of anther in wild type and spg1 mutant． From stage 1 to stage 9，the anther in
spg1 mutant had no significant differences comparing to that in wild type． In Fig． 2A and Fig． 2E，normal anther
structure and tetrads were seen in wild type and spg1 at stage 7． At stage 9，the microspores generated an exine
wall and became vacuolated in both wild type and spg1 mutant (Fig． 2B，F)． At stage 12，the tapetum was de-
generated and the pollen mitotic occurred (Fig． 2C，G)． But，in this stage，the emptiness microspores were seen
in the mutant． Furthermore，some microspores had become flat in shape in spg1 mutant at stage12 (Fig． 2G)．
Until stage 14，the mature pollen grains were released from the anther in wild type (Fig． 2D)． However，all of
the microspores were shrunken to the flat ones in this last stage (Fig． 2H)． This data shows that the develop-
ment of microspore was defective in spg1 mutant．
3． 3 Genetic analysis of spg1mutant
We backcrossed spg1 mutant as the female parent with wild type to generate the F1 progeny． All the F1
progeny showed wild-type phenotype，indicating the mutant phenotype was controlled by recessive gene． In 130
F2progeny，48 plants showed normal phenotype and 17 plants exhibited male sterile phenotype (X
2［3:1］=
0． 039;P ＞ 0． 5)． The result manifests that the male sterile phenotype in spg1 was caused by a single recessive
gene mutation．
3． 4 Mapping of the mutated SPG1 locus
To research the gene responsible for the spg1 mutant phenotype，we isolated the mutant gene by map-
based cloning approach． About 20 markers distributing uniformly on 5 chromosomes in Arabidopsis were ap-
plied for the first mapping，and the mutant locus was linked to marker 3 － AP000606 － 4852 and 3 － AP049862
－ 8052 in Chr3． As shown in Fig． 3 A，there were only 3 recombinants for each using marker 3 － AP000606 －
4852 and 3 － AP049862 － 8052 respectively． For the fine mapping，several markers in this region were used． At
the end，the mutant locus was mapped to a region about 427kb between 3 － AL138638 － 6632 and 3 －
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第 5 期 韦嘉励，周树敏，袁晓君，等:遗传定位一个花粉发育相关的拟南芥新基因 SPG1
Figure 2 Anther development in wild type and spg1(A，B，C，D)wild type;(E，F，G，H)spg1．(A，E)
Stage 7 anthers．(B，F)Stage 9 anthers．(C，G)Stage 12 anthers．(D，H)Stage 14 anthers (Bar = 10 μmol·L －1)
AL353865 － 6814 (Fig． 3)． There were about 120 genes predicted in this region on the database
(www． arabidopsis． org)． And no previously reported male sterile genes were found in this region． It means a
new locus was responsible for the male sterile phenotype in spg1．
Figure 3 A． The result of PCR with two kinds of wild type and individual lines in the mapping population using marker
3 － AP000606 － 4852 and 3 － AP049862 － 8052;C． Columbia ecotype;L，Landsberg erecta ecotype;Line1 － 22，22 individual
mapping lines;B． SPG1 locus in chromosome 3 between marker 3 － AL137079 － 6100 and marker 3 － AL162459 － 7163
(The black cycle indicates the centromeric region;the black square indicates the locus of the SPG1gene)
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4 Discussion
In this work，we characterized a male sterile Arabidopsis mutant spg1，which mutant phenotype was iden-
tified to link to a new gene mutation by map-based cloning approach． The male sterility of spg1 was attributed
to the defective pollen grains，since the pollen appeared abnormal by the paraffin transverse section of the mu-
tant anther． However，the tapetum in spg1 displayed normal development，though it was considered as a nutrient
factory for the formation of microspores． The wall of pollen grain in angiosperm contains two lays:exine and in-
tine［2 － 3］． The tapetal supplies nutrient for microspore to form exine，and the formation of intine mainly depends
on the microspore itself． Thus，the interruption of nutrient synthesis for pollen wall in tapetal and pollen itself
both could result in the formation of the inviable pollen grains． Further，the dex1 mutant indicates that the de-
fect in exine patterning also results in the pollen degeneration，despite the synthesis of sporopollenin occurring
in the mutant［4］． In the ruputured pollen grain1 rpg1)mutant，the change of the exine patterning leads to the
rupture and degeration of microspore［5］． However，we can not elucidate what lead to the shrunken pollen grains
in spg1 mutant in this paper． In our future work，a larger mapping population would be generated for the isola-
tion of SPG1gene，and the function of SPG1 in microspore development was further investigated．
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遗传定位一个花粉发育相关的拟南芥新基因 SPG1
韦嘉励，周树敏，袁晓君，张 卫*
(上海大学 生命科学学院，上海 200444)
摘 要:通过 EMS诱变获得一个拟南芥突变体 shrunken pollen grain1，spg1．根据横切片的观察结果，与野生型相比较，突
变体观察到异常的花粉粒．遗传学的实验表明:spg1 突变现象由一个隐性的单基因控制．通过图位克隆的方法，spg1 突变
位点定位在 3 号染色体的分子标记 3 － AL138638 － 6632 和 3 － AL353865 － 6814 之间大约 427kb 的区间内．生物信息学
的分析表明，在这个区间内没有任何已知的与育性相关的基因． 以上结果说明 SPG1 是一个控制拟南芥雄性不育的新
基因．
关键词:拟南芥;花药;雄性不育;小孢子;图位克隆
(责任编辑:顾浩然)
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