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Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang Dominant Genic Male Sterile Rice
Journal of Genetics and Genomics (Formerly Acta Genetica Sinica) July 2007, 34(7): 616-622
Research Article
Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang Dominant Genic Male Sterile Rice Tingyou Huang1,2,3, Yuping Wang1,2, Bingtian Ma1,2, Yuqing Ma1,2, Shigui Li1,2,① 1. Rice Research Institute of Sichuan Agricultural University, Chengdu 611130, China; 2. Key Laboratory of Crop Genetic Resource and Improvement of Ministry of Education, Sichuan Agricultural University, Ya’an 625014, China; 3. Mianyang Agriculture Science Research Institute, Mianyang 621023, China
Abstract: Pingxiang dominant genic male sterile rice (PDGMSR) was the first dominant genic male sterile mutant identified in rice (Oryza sativa L.), and the corresponding dominant genic male sterile gene was designated as Ms-p. The fertility of PDGMSR can be restored by introduction of a dominant epistatic fertility restoring gene in some rice varieties. In the present study, E823, an indica inbred rice variety, restored the fertility of PDGMSR, and the genetic pattern was found to be consistent with a dominant epistatic model, therefore, the dominant epistatic fertility restorer gene was designated as Rfe. The F2 population from the cross of PDGMSR/E823 was developed to map gene Rfe. The F2 plants with the genotypes Ms-pMs-pRferfe or Ms-pms-pRferfe were used to construct a fertile pool, and the corresponding sterile plants with genotypes Ms-pMs-prferfe or Ms-pms-prferfe were used to construct a sterile pool. The fertility restoring gene Rfe was mapped to one side of the microsatellite markers RM311 and RM3152 on rice chromosome 10, with genetic distances of 7.9 cM and 3.6 cM, respectively. The microsatellite markers around the location of the Ms-p gene were used to finely map the Ms-p gene. The findings of this study indicated that the microsatellite markers RM171 and RM6745 flanked the Ms-p gene, and the distances were 0.3 cM and 3.0 cM, respectively. On the basis of the sequence of rice chromosome 10, the physical distance between the two markers is approximately 730 kb. These findings facilitates molecular marker-assisted selection (MAS ) of genes Ms-p and Rfe in rice breeding programs, and cloning them in the future. Keywords: dominant epistatic fertility restoring gene; dominant genic male sterile; gene mapping; PDGMSR
Male-sterility is a common phenomenon in plants. This phenomenon has been described in over 600 plant species, including several vital crops such as rice (Oryza sativa L.), barley (Hordeum vulgare L.), sorghum (authorization), maize (Zea mays L.), and rape (Brassica napas L.) [1]. Most of the male-sterile rice mutants are recessive genic sterile, including the cytoplasmic male sterility (CMS), used in the three-line hybrid rice and the photo-thermosensitive recessive genic male sterility used in two-line hybrid rice. Few rice mutants display dominant genic male
sterility, and among them only two dominant genic male sterile rice mutants have been reported, known as the temperature sensitive dominant genic male sterile rice mutant[2] and Pingxiang dominant genic male sterile rice (PDGMSR)[3]. The PDGMSR was first described by Yan and colleagues in 1978, and its genic sterile gene was designated as Ms-p[3]. Subsequent studies demonstrated that its fertility was not related to cytoplasmic factors, but was jointly controlled by two pairs of dominant genic genes, the Pingxiang dominant genic sterile gene and the
Received: 2006-09-17; Accepted: 2007-01-08 This work was supported by the Innovation Group Development Project of the Ministry of Education of China (No.IRT0435) and Superexcellence Doctorial Dissertation Fund from Ministry of Education of China (No.200054). ① Corresponding author. E-mail: [email protected]; Tel: +86-28-8272 2661, Fax: +86-28-8272-6875 www.jgenetgenomics.org
Tingyou Huang et al.: Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang…
epistatic fertility-restoring gene. When the two genes were present in one plant, the dominant epistatic gene inhibited the expression of the sterile gene, and the plant was then fertile [4]. The dominant genic sterile gene Ms-p was mapped in the region between RM228 and RM258 on rice chromosome with a distance of 2.6 cM from G2155[5]. However, studies related to fine mapping of Ms-p and its corresponding restoring gene have not been conducted so far. In the present study, E823, an indica rice variety that is able to restore the fertility of PDGMSR, was crossed with PDGMSR, and microsatellite markers were used to map the genic sterile gene Ms-p and the dominant epistatic fertility-restoring gene.
1 1.1
Materials and Methods Plant materials
Pingxiang dominant genic male sterile rice (PDGMSR) and E823, an indica inbred rice variety and the restorer of PDGMS were used to produce, F1, F2, and F3 mapping populations. 1.2
Population construction
PDGMSR was crossed with E823 as the maternal parent. During the heading period, the pollens of all the plants were examined using I2-KI staining method and were categorized based on pollen dyeing rates. Plants with pollen dyeing rates less than or equal to 5% were considered sterile, whereas plants with over 5% pollen dyeing rates were considered fertile. Seeds from F1 plants were collected to develop F2 family. The parent E823, F1 and F2 plants were planted, and 120 plants were raised belonging to F2 family. The fertility classifications for the F2 families were carried out according to the pollen dyeing rates. Seeds of the individual fertile plants in F2 families, which conformed to the ratio of sterile plants to fertile plants of 3:13, were collected to develop F3 family. E823, F1 and F3 plants were planted, and 60 plants were raised belonging to F3 family. The pollen fertilities of all F2 and F3 plants were investigated. www.jgenetgenomics.org
1.3
617
DNA extraction and PCR
DNA extraction and PCR were performed as described by Chen et al [6]. PCR products were separated by electrophoresis on 3% agarose gels and visualized under UV light after staining with ethidium bromide (EB). 1.4
Genetic mapping
The polymorphic markers between the sterile and the fertile pools were used to screen the sterile plants. The banding patterns of the sterile plants, E823, and heterogenous bands were marked with 0, 2, and 1, respectively. Segregating data was analyzed using the software MapMaker3.0[7]. Because the sterile plants had heterozygotic and homozygotic genotypes of Ms-p, both the sterile and the heterogenous banding patterns were marked with 0 when mapping the gene Ms-p.
2 2.1
Results Fertility performances and analyses of the parents and progenies
Both E823 and F1 plants were fertile, and seeds from 63 F1 plants were collected to generate F2 families, the seeds of one F1 plant generated one F2 family. The family in which every plant was fertile was complete fertility family, the others was fertility segregating family. There were a total of 7,560 individual plants, and the fertility of 37 families was segregated with the numbers of sterile plants between 5 and 19. A ratio of 1:1 (χ2 =1.59, P=0.21>0.05) between complete fertile and fertility segregating F2 families were observed. These results indicated that the inbred rice variety E823 harbored the gene for restoring the fertility of PDGMSR. However, among the 37 F2 families segregating for fertility, 14 F2 families did not agree with the ratio of sterile to fertile plants of 3:13, although 23 families did (χ2 =0.49−3.50, P=0.06 to 0.48; data not shown). See the discussion. A total of 443 F2 plants were used for mapping
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analysis. Seeds were collected from 49 randomly selected fertile individuals from the F2 families that exhibited the ratio of 3:13 (sterile plants: fertile plants) to develop F3 family. Among the F3 families, the fertility of 28 families were segregated, the others showed complete fertility, and the ratio of fertility segregating families to the complete fertile families was 6:7 (χ2=1.659, P=0.20), which conformed to the
(Fig.1). Therefore, the inheritance of fertility of PDGMSR showed the dominant epistatic interactive inheritance, and the restoring gene in rice E823, designated as Rfe, is the dominant epistatic fertility restoration gene. The genetic model is shown in Fig. 1.
theoretical ratio of the “dominant epistatic model”
According to the genetic model, the genotypes of
Fig. 1
Fig. 2
2.2
Mapping of dominant epistatic restoring gene
Genetic model of fertility-restoring of PDGMSR
Separation of microsatellite marker RM3152 in sterile population
M: DNA Marker (DL2000); lanes 1−17: sterile plants; S: sterile pool; F: fertile pool; P1: parental E823; P2: parental PDGMSR; *: recombinant plants. www.jgenetgenomics.org
Tingyou Huang et al.: Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang…
sterile plants in the F2 families was Ms-pMs-prferfe or Ms-pms-prferfe, and the genotypes of individual F2 fertile plants that segregated for fertility in F3 families was Ms-pMs-pRferfe or Ms-pms-pRferfe. The same amount of DNA from the extreme sterile plants and fertile plants from F2 plants of both genotypes were used to construct sterile and fertile pools for mapping the dominant epistatic restoring genes Rfe. A total of 313 pairs of microsatellite markers were used to screen parents, and 51 pairs (16.29%) distributed in 12 chromosomes of rice were polymorphic. Further analysis with fertile and sterile pools, and parts of the sterile individual plants revealed that only RM311 on chromosome 10 was linked to gene Rfe. According to the microsatellite markers, a portion of the marker in the region where RM311 is located was used to analyze all of the 443 sterile individual plants, and RM3152 was linked to the dominant epistatic restoring gene Rfe (Fig. 2). According to a local molecular linkage map (Fig. 3A), the dominant epistatic restoring gene Rfe was located on one side of RM311 and RM3152 on chromosome 10 with the genetic distances of 7.9 and 3.6 cM, respectively.
2.3
619
Fine mapping of Ms-p
Microsatellite markers near G2155 were used to analyze the parents and sterile individuals of the F2 population. Polymorphisms in RM171 and RM6745 were detected between the two parents, but the markers RM228 and RM258 were not polymorphic. Analysis of the sterile plants using markers RM171 and RM6745 indicated that the two markers were linked to Ms-p. Three plants among the 443 sterile plants were recombinant for RM171, and 27 plants for RM6745, and all the recombination plants for RM171 and for RM6745 were different, indicating that the two markers flanked the gene, and Ms-p was located between RM171 and RM6745, the distances were 0.3 cM and 3.0 cM, respectively.
3
Discussion
Male sterility is the basis for the utilization of heterosis, and the mechanism has been a focus of research in plant molecular biology. Kihar et al. (1968) suggested that male sterility in plant was only an issue of nucleo-cytoplasmic relation, and genic male-sterility is because of poor maintenance temporarily. PDGMSR is the first dominant genic male sterile rice mutant, and has thermosensitivity characteristic where part of the pollen grains will be fertile over 29℃[8] and then reciprocal crosses can be conducted to show the relationship between sterility and nucleo-cytoplasmid phenomena. The skewed segregation of 14 F2 families (See the results 2.1) may be contributed by the thermosensitivity and it is the characteristic that made Yan and colleagues to demonstrate that its fertility is not related to cytoplasm by the method of reciprocal crosses. Therefore, the discovery of PDGMSR provides a strong evidence for the existence of genic male sterility in plants [3]. There were two hypotheses, related to the ge-
Fig. 3 The partial molecular linkage map of Rfe and Ms-p on chromosome 10 (A) and the electronic-contigs of Ms-p (B)
netic mechanism of fertility restoration in dominant genic male sterility, which are known as the “dominant epistasis” and the “multiple allelism” hypotheses[9]. Conventional genetic method was used to deter-
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mine the genetic mechanism of dominant epistatic [4]
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RM6745 flank Ms-p more closely than RM228 and
interaction . Theoretically, the most reliable method
RM258. Moreover, these markers were located be-
to determine the genetic mechanism for dominant
tween RM228 and RM258. This was consistent with
genic male sterility restoration is gene mapping. If the
the findings of Chen et al. (2000)[5]. According to the
gene responsible for the restoration and genic male
sequence of chromosome 10, RM171 is located in
sterile gene are mapped to the same location, they can
GenBank Accession AC079888, whereas RM6745 is
be considered multiple alleles, and the mechanism of
in GenBank Accession Ac091122. The sequences of
fertility restoration is “multiple allelism”, otherwise it
the contigs between the two markers is approximately
is the “dominant epistasis”[9]. Studies related to the
730 kb, and the electronic contigs in the region where
mapping of the dominant epistatic fertility restoring
Ms-p is located were constructed (Fig. 3 B). This fa-
gene of PDGMSR Rfe have not been carried out. In
cilitates cloning of Ms-p.
the present study, the dominant epistatic restoring
In the region of chromosome 10 that is close to the
gene Rfe was mapped on chromosome 10 where the
loci of Rfe and Ms-p, the rice fertility restoring genes Rf1,
Ms-p was also identified. According to the rice mo-
Rf4, Rf5, and Rf6(t) have also been located
lecular map
[10]
[11−13]
. It
, the position of RM3152 linked to Rfe
seems that the genes involved in rice fertility have
is different from those of RM171and RM6745 linked
been distributed in the form of a cluster and that their
to Ms-p (Table 1), indicating that Rfe and Ms-p were
relationship remains to be elucidated.
not allelic, and their relation is dominant epistatic in-
Although more than 10 genic male sterile genes,
teraction. Therefore, it can be concluded that the ge-
tms4(t), tms5, psm2, tms6, TMS, tms3(t), tms2, pms1,
netic mechanism for fertility restoration of PDGMSR
tms1, ms-h(t), Ms-p, rtms1, and psm3
is dominant epistasis. Because the markers obtained
been mapped by molecular markers, only TMS and
in this study were far away from Rfe, and all the
Ms-p are dominant. Cloning of these genes will help
markers were located on one side, markers at the
to elucidate the mechanism of dominant male sterile
other side should be obtained to fine map and use
in rice and further aid the utilization of rice heterosis.
gene Rfe by MAS in breeding program .
The markers flanking Ms-p obtained in this study are
Using 119 plants, Chen et al.[5] mapped the
[2,14-22]
, have
expected to be useful in conducting such a study.
Pingxiang dominant genic male sterile gene Ms-p to a
About the PDMSR, the heterosis utilization can
position between microsatellite markers RM228 and
be done by using homozygote of PDGMSR to cross
RM258 on chromosome 10, with a distance to G2155
with rice varieties (line) containing Rfe[3]. In addition,
of 2.6 cM. However, fine mapping has not been con-
PDMSR can be used as a bridge parent, and by using
ducted. In this study, using 443 sterile plants obtained
recurrent selection and convergent crosses to accu-
from 7,560 plants of F2 population to finely map Ms-p,
mulate some interesting genes, new rice lines with
we found that microsatellite markers RM171 and
ideal characteristics would be developed.
Table 1
Markers positions (cM) linked with the genes involved in PDGMSR
Marker
Linked
Marker position on maps
gene
JRGP RFLP
Cornell RFLP
IRMI
RM3152
Rfe
17.9−19
6.1−12.6
17.9
RM171
Ms-p
53.9−54.7
50.55−57.8
55
RM6745
Ms-p
55.6−57.5
64.5−68.5
55.3−58.6
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Tingyou Huang et al.: Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang…
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萍乡显性核不育水稻育性相关基因的定位和遗传分析 黄廷友1,2,3, 王玉平1,2, 马炳田1,2, 马玉清1,2, 李仕贵1,2 1. 四川农业大学水稻研究所, 成都 611130； 2. 四川农业大学作物基因资源与遗传改良教育部重点实验室, 雅安 625014； 3. 绵阳农业科学研究所, 绵阳 621023 摘 要: 萍乡显性核不育水稻(Pingxiang Dominant Genic Male Sterile Rice, PDGMSR)是在水稻中首次发现的显性核不育材料, 其育性由两对显性基因互作控制, 一对是萍乡显性核不育基因Ms-p, 另一对是显性上位恢复基因(dominant epistatic fertility restorer gene, Rfe)。两者共同存在时显性上位恢复基因能抑制不育基因的表达, 从而使育性表现可育。本实验用一个对萍乡 显性核不育水稻有恢复能力的水稻品种E823 与萍乡显性核不育水稻配制杂交组合,将(萍乡核不育水稻/E823)F2作为定位群 体, 根据F3株系的育性分离, 选择育性分离株系对应F2单株(基因型为Ms-pMs-pRefrfe和Ms-pms-pRferfe)构建可育池, 用对应 F2株系中的不育单株(基因型为Ms-pMs-prferfe或Ms-pms-prferfe)构建不育池, 将显性上位恢复基因Rfe定位在水稻 10 染色体 RM311 和RM3152 一侧, 遗传距离分别为 7.9 cM和 3.6 cM。根据已有的Ms-p的定位结果, 合成 10 染色体部分微卫星引物, 对 不育单株进行分析, 发现RM171 和RM6745 位于Ms-p的两侧, 距离分别为 0.3 cM和 3.0 cM。根据 10 染色体的测序结果, 将 Ms-p界定在约 730 kb的范围内, 并构建了Ms-p的电子重叠群。植物显性核不育的育性恢复机理存在“复等位基因”和“显 性上位互作”两种假说, 贺浩华等用经典的遗传学方法证明了萍乡显性核不育水稻育性恢复的遗传机理属于“显性上位互 作”。理论上认为, 确定其遗传机理最为有效的方法是基因定位, 如果不育基因和恢复基因位于同一位点, 则其遗传机理属 于“复等位基因”, 否则为“显性上位互作”。本实验将不育基因和恢复基因定位在水稻 10 染色体不同的位点, 用基因定位 的方法证实了萍乡显性核不育水稻育性恢复的遗传机理属于“显性上位互作”。 关键词: 显性上位恢复基因；显性核不育；基因定位；萍乡显性核不育水稻 作者简介: 黄廷友(1969−), 男, 四川人, 副研究员, 博士, 研究方向: 水稻遗传育种及分子生物学
www.jgenetgenomics.org
Research Article
Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang Dominant Genic Male Sterile Rice Tingyou Huang1,2,3, Yuping Wang1,2, Bingtian Ma1,2, Yuqing Ma1,2, Shigui Li1,2,① 1. Rice Research Institute of Sichuan Agricultural University, Chengdu 611130, China; 2. Key Laboratory of Crop Genetic Resource and Improvement of Ministry of Education, Sichuan Agricultural University, Ya’an 625014, China; 3. Mianyang Agriculture Science Research Institute, Mianyang 621023, China
Abstract: Pingxiang dominant genic male sterile rice (PDGMSR) was the first dominant genic male sterile mutant identified in rice (Oryza sativa L.), and the corresponding dominant genic male sterile gene was designated as Ms-p. The fertility of PDGMSR can be restored by introduction of a dominant epistatic fertility restoring gene in some rice varieties. In the present study, E823, an indica inbred rice variety, restored the fertility of PDGMSR, and the genetic pattern was found to be consistent with a dominant epistatic model, therefore, the dominant epistatic fertility restorer gene was designated as Rfe. The F2 population from the cross of PDGMSR/E823 was developed to map gene Rfe. The F2 plants with the genotypes Ms-pMs-pRferfe or Ms-pms-pRferfe were used to construct a fertile pool, and the corresponding sterile plants with genotypes Ms-pMs-prferfe or Ms-pms-prferfe were used to construct a sterile pool. The fertility restoring gene Rfe was mapped to one side of the microsatellite markers RM311 and RM3152 on rice chromosome 10, with genetic distances of 7.9 cM and 3.6 cM, respectively. The microsatellite markers around the location of the Ms-p gene were used to finely map the Ms-p gene. The findings of this study indicated that the microsatellite markers RM171 and RM6745 flanked the Ms-p gene, and the distances were 0.3 cM and 3.0 cM, respectively. On the basis of the sequence of rice chromosome 10, the physical distance between the two markers is approximately 730 kb. These findings facilitates molecular marker-assisted selection (MAS ) of genes Ms-p and Rfe in rice breeding programs, and cloning them in the future. Keywords: dominant epistatic fertility restoring gene; dominant genic male sterile; gene mapping; PDGMSR
Male-sterility is a common phenomenon in plants. This phenomenon has been described in over 600 plant species, including several vital crops such as rice (Oryza sativa L.), barley (Hordeum vulgare L.), sorghum (authorization), maize (Zea mays L.), and rape (Brassica napas L.) [1]. Most of the male-sterile rice mutants are recessive genic sterile, including the cytoplasmic male sterility (CMS), used in the three-line hybrid rice and the photo-thermosensitive recessive genic male sterility used in two-line hybrid rice. Few rice mutants display dominant genic male
sterility, and among them only two dominant genic male sterile rice mutants have been reported, known as the temperature sensitive dominant genic male sterile rice mutant[2] and Pingxiang dominant genic male sterile rice (PDGMSR)[3]. The PDGMSR was first described by Yan and colleagues in 1978, and its genic sterile gene was designated as Ms-p[3]. Subsequent studies demonstrated that its fertility was not related to cytoplasmic factors, but was jointly controlled by two pairs of dominant genic genes, the Pingxiang dominant genic sterile gene and the
Received: 2006-09-17; Accepted: 2007-01-08 This work was supported by the Innovation Group Development Project of the Ministry of Education of China (No.IRT0435) and Superexcellence Doctorial Dissertation Fund from Ministry of Education of China (No.200054). ① Corresponding author. E-mail: [email protected]; Tel: +86-28-8272 2661, Fax: +86-28-8272-6875 www.jgenetgenomics.org
Tingyou Huang et al.: Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang…
epistatic fertility-restoring gene. When the two genes were present in one plant, the dominant epistatic gene inhibited the expression of the sterile gene, and the plant was then fertile [4]. The dominant genic sterile gene Ms-p was mapped in the region between RM228 and RM258 on rice chromosome with a distance of 2.6 cM from G2155[5]. However, studies related to fine mapping of Ms-p and its corresponding restoring gene have not been conducted so far. In the present study, E823, an indica rice variety that is able to restore the fertility of PDGMSR, was crossed with PDGMSR, and microsatellite markers were used to map the genic sterile gene Ms-p and the dominant epistatic fertility-restoring gene.
1 1.1
Materials and Methods Plant materials
Pingxiang dominant genic male sterile rice (PDGMSR) and E823, an indica inbred rice variety and the restorer of PDGMS were used to produce, F1, F2, and F3 mapping populations. 1.2
Population construction
PDGMSR was crossed with E823 as the maternal parent. During the heading period, the pollens of all the plants were examined using I2-KI staining method and were categorized based on pollen dyeing rates. Plants with pollen dyeing rates less than or equal to 5% were considered sterile, whereas plants with over 5% pollen dyeing rates were considered fertile. Seeds from F1 plants were collected to develop F2 family. The parent E823, F1 and F2 plants were planted, and 120 plants were raised belonging to F2 family. The fertility classifications for the F2 families were carried out according to the pollen dyeing rates. Seeds of the individual fertile plants in F2 families, which conformed to the ratio of sterile plants to fertile plants of 3:13, were collected to develop F3 family. E823, F1 and F3 plants were planted, and 60 plants were raised belonging to F3 family. The pollen fertilities of all F2 and F3 plants were investigated. www.jgenetgenomics.org
1.3
617
DNA extraction and PCR
DNA extraction and PCR were performed as described by Chen et al [6]. PCR products were separated by electrophoresis on 3% agarose gels and visualized under UV light after staining with ethidium bromide (EB). 1.4
Genetic mapping
The polymorphic markers between the sterile and the fertile pools were used to screen the sterile plants. The banding patterns of the sterile plants, E823, and heterogenous bands were marked with 0, 2, and 1, respectively. Segregating data was analyzed using the software MapMaker3.0[7]. Because the sterile plants had heterozygotic and homozygotic genotypes of Ms-p, both the sterile and the heterogenous banding patterns were marked with 0 when mapping the gene Ms-p.
2 2.1
Results Fertility performances and analyses of the parents and progenies
Both E823 and F1 plants were fertile, and seeds from 63 F1 plants were collected to generate F2 families, the seeds of one F1 plant generated one F2 family. The family in which every plant was fertile was complete fertility family, the others was fertility segregating family. There were a total of 7,560 individual plants, and the fertility of 37 families was segregated with the numbers of sterile plants between 5 and 19. A ratio of 1:1 (χ2 =1.59, P=0.21>0.05) between complete fertile and fertility segregating F2 families were observed. These results indicated that the inbred rice variety E823 harbored the gene for restoring the fertility of PDGMSR. However, among the 37 F2 families segregating for fertility, 14 F2 families did not agree with the ratio of sterile to fertile plants of 3:13, although 23 families did (χ2 =0.49−3.50, P=0.06 to 0.48; data not shown). See the discussion. A total of 443 F2 plants were used for mapping
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analysis. Seeds were collected from 49 randomly selected fertile individuals from the F2 families that exhibited the ratio of 3:13 (sterile plants: fertile plants) to develop F3 family. Among the F3 families, the fertility of 28 families were segregated, the others showed complete fertility, and the ratio of fertility segregating families to the complete fertile families was 6:7 (χ2=1.659, P=0.20), which conformed to the
(Fig.1). Therefore, the inheritance of fertility of PDGMSR showed the dominant epistatic interactive inheritance, and the restoring gene in rice E823, designated as Rfe, is the dominant epistatic fertility restoration gene. The genetic model is shown in Fig. 1.
theoretical ratio of the “dominant epistatic model”
According to the genetic model, the genotypes of
Fig. 1
Fig. 2
2.2
Mapping of dominant epistatic restoring gene
Genetic model of fertility-restoring of PDGMSR
Separation of microsatellite marker RM3152 in sterile population
M: DNA Marker (DL2000); lanes 1−17: sterile plants; S: sterile pool; F: fertile pool; P1: parental E823; P2: parental PDGMSR; *: recombinant plants. www.jgenetgenomics.org
Tingyou Huang et al.: Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang…
sterile plants in the F2 families was Ms-pMs-prferfe or Ms-pms-prferfe, and the genotypes of individual F2 fertile plants that segregated for fertility in F3 families was Ms-pMs-pRferfe or Ms-pms-pRferfe. The same amount of DNA from the extreme sterile plants and fertile plants from F2 plants of both genotypes were used to construct sterile and fertile pools for mapping the dominant epistatic restoring genes Rfe. A total of 313 pairs of microsatellite markers were used to screen parents, and 51 pairs (16.29%) distributed in 12 chromosomes of rice were polymorphic. Further analysis with fertile and sterile pools, and parts of the sterile individual plants revealed that only RM311 on chromosome 10 was linked to gene Rfe. According to the microsatellite markers, a portion of the marker in the region where RM311 is located was used to analyze all of the 443 sterile individual plants, and RM3152 was linked to the dominant epistatic restoring gene Rfe (Fig. 2). According to a local molecular linkage map (Fig. 3A), the dominant epistatic restoring gene Rfe was located on one side of RM311 and RM3152 on chromosome 10 with the genetic distances of 7.9 and 3.6 cM, respectively.
2.3
619
Fine mapping of Ms-p
Microsatellite markers near G2155 were used to analyze the parents and sterile individuals of the F2 population. Polymorphisms in RM171 and RM6745 were detected between the two parents, but the markers RM228 and RM258 were not polymorphic. Analysis of the sterile plants using markers RM171 and RM6745 indicated that the two markers were linked to Ms-p. Three plants among the 443 sterile plants were recombinant for RM171, and 27 plants for RM6745, and all the recombination plants for RM171 and for RM6745 were different, indicating that the two markers flanked the gene, and Ms-p was located between RM171 and RM6745, the distances were 0.3 cM and 3.0 cM, respectively.
3
Discussion
Male sterility is the basis for the utilization of heterosis, and the mechanism has been a focus of research in plant molecular biology. Kihar et al. (1968) suggested that male sterility in plant was only an issue of nucleo-cytoplasmic relation, and genic male-sterility is because of poor maintenance temporarily. PDGMSR is the first dominant genic male sterile rice mutant, and has thermosensitivity characteristic where part of the pollen grains will be fertile over 29℃[8] and then reciprocal crosses can be conducted to show the relationship between sterility and nucleo-cytoplasmid phenomena. The skewed segregation of 14 F2 families (See the results 2.1) may be contributed by the thermosensitivity and it is the characteristic that made Yan and colleagues to demonstrate that its fertility is not related to cytoplasm by the method of reciprocal crosses. Therefore, the discovery of PDGMSR provides a strong evidence for the existence of genic male sterility in plants [3]. There were two hypotheses, related to the ge-
Fig. 3 The partial molecular linkage map of Rfe and Ms-p on chromosome 10 (A) and the electronic-contigs of Ms-p (B)
netic mechanism of fertility restoration in dominant genic male sterility, which are known as the “dominant epistasis” and the “multiple allelism” hypotheses[9]. Conventional genetic method was used to deter-
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mine the genetic mechanism of dominant epistatic [4]
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RM6745 flank Ms-p more closely than RM228 and
interaction . Theoretically, the most reliable method
RM258. Moreover, these markers were located be-
to determine the genetic mechanism for dominant
tween RM228 and RM258. This was consistent with
genic male sterility restoration is gene mapping. If the
the findings of Chen et al. (2000)[5]. According to the
gene responsible for the restoration and genic male
sequence of chromosome 10, RM171 is located in
sterile gene are mapped to the same location, they can
GenBank Accession AC079888, whereas RM6745 is
be considered multiple alleles, and the mechanism of
in GenBank Accession Ac091122. The sequences of
fertility restoration is “multiple allelism”, otherwise it
the contigs between the two markers is approximately
is the “dominant epistasis”[9]. Studies related to the
730 kb, and the electronic contigs in the region where
mapping of the dominant epistatic fertility restoring
Ms-p is located were constructed (Fig. 3 B). This fa-
gene of PDGMSR Rfe have not been carried out. In
cilitates cloning of Ms-p.
the present study, the dominant epistatic restoring
In the region of chromosome 10 that is close to the
gene Rfe was mapped on chromosome 10 where the
loci of Rfe and Ms-p, the rice fertility restoring genes Rf1,
Ms-p was also identified. According to the rice mo-
Rf4, Rf5, and Rf6(t) have also been located
lecular map
[10]
[11−13]
. It
, the position of RM3152 linked to Rfe
seems that the genes involved in rice fertility have
is different from those of RM171and RM6745 linked
been distributed in the form of a cluster and that their
to Ms-p (Table 1), indicating that Rfe and Ms-p were
relationship remains to be elucidated.
not allelic, and their relation is dominant epistatic in-
Although more than 10 genic male sterile genes,
teraction. Therefore, it can be concluded that the ge-
tms4(t), tms5, psm2, tms6, TMS, tms3(t), tms2, pms1,
netic mechanism for fertility restoration of PDGMSR
tms1, ms-h(t), Ms-p, rtms1, and psm3
is dominant epistasis. Because the markers obtained
been mapped by molecular markers, only TMS and
in this study were far away from Rfe, and all the
Ms-p are dominant. Cloning of these genes will help
markers were located on one side, markers at the
to elucidate the mechanism of dominant male sterile
other side should be obtained to fine map and use
in rice and further aid the utilization of rice heterosis.
gene Rfe by MAS in breeding program .
The markers flanking Ms-p obtained in this study are
Using 119 plants, Chen et al.[5] mapped the
[2,14-22]
, have
expected to be useful in conducting such a study.
Pingxiang dominant genic male sterile gene Ms-p to a
About the PDMSR, the heterosis utilization can
position between microsatellite markers RM228 and
be done by using homozygote of PDGMSR to cross
RM258 on chromosome 10, with a distance to G2155
with rice varieties (line) containing Rfe[3]. In addition,
of 2.6 cM. However, fine mapping has not been con-
PDMSR can be used as a bridge parent, and by using
ducted. In this study, using 443 sterile plants obtained
recurrent selection and convergent crosses to accu-
from 7,560 plants of F2 population to finely map Ms-p,
mulate some interesting genes, new rice lines with
we found that microsatellite markers RM171 and
ideal characteristics would be developed.
Table 1
Markers positions (cM) linked with the genes involved in PDGMSR
Marker
Linked
Marker position on maps
gene
JRGP RFLP
Cornell RFLP
IRMI
RM3152
Rfe
17.9−19
6.1−12.6
17.9
RM171
Ms-p
53.9−54.7
50.55−57.8
55
RM6745
Ms-p
55.6−57.5
64.5−68.5
55.3−58.6
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621
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萍乡显性核不育水稻育性相关基因的定位和遗传分析 黄廷友1,2,3, 王玉平1,2, 马炳田1,2, 马玉清1,2, 李仕贵1,2 1. 四川农业大学水稻研究所, 成都 611130； 2. 四川农业大学作物基因资源与遗传改良教育部重点实验室, 雅安 625014； 3. 绵阳农业科学研究所, 绵阳 621023 摘 要: 萍乡显性核不育水稻(Pingxiang Dominant Genic Male Sterile Rice, PDGMSR)是在水稻中首次发现的显性核不育材料, 其育性由两对显性基因互作控制, 一对是萍乡显性核不育基因Ms-p, 另一对是显性上位恢复基因(dominant epistatic fertility restorer gene, Rfe)。两者共同存在时显性上位恢复基因能抑制不育基因的表达, 从而使育性表现可育。本实验用一个对萍乡 显性核不育水稻有恢复能力的水稻品种E823 与萍乡显性核不育水稻配制杂交组合,将(萍乡核不育水稻/E823)F2作为定位群 体, 根据F3株系的育性分离, 选择育性分离株系对应F2单株(基因型为Ms-pMs-pRefrfe和Ms-pms-pRferfe)构建可育池, 用对应 F2株系中的不育单株(基因型为Ms-pMs-prferfe或Ms-pms-prferfe)构建不育池, 将显性上位恢复基因Rfe定位在水稻 10 染色体 RM311 和RM3152 一侧, 遗传距离分别为 7.9 cM和 3.6 cM。根据已有的Ms-p的定位结果, 合成 10 染色体部分微卫星引物, 对 不育单株进行分析, 发现RM171 和RM6745 位于Ms-p的两侧, 距离分别为 0.3 cM和 3.0 cM。根据 10 染色体的测序结果, 将 Ms-p界定在约 730 kb的范围内, 并构建了Ms-p的电子重叠群。植物显性核不育的育性恢复机理存在“复等位基因”和“显 性上位互作”两种假说, 贺浩华等用经典的遗传学方法证明了萍乡显性核不育水稻育性恢复的遗传机理属于“显性上位互 作”。理论上认为, 确定其遗传机理最为有效的方法是基因定位, 如果不育基因和恢复基因位于同一位点, 则其遗传机理属 于“复等位基因”, 否则为“显性上位互作”。本实验将不育基因和恢复基因定位在水稻 10 染色体不同的位点, 用基因定位 的方法证实了萍乡显性核不育水稻育性恢复的遗传机理属于“显性上位互作”。 关键词: 显性上位恢复基因；显性核不育；基因定位；萍乡显性核不育水稻 作者简介: 黄廷友(1969−), 男, 四川人, 副研究员, 博士, 研究方向: 水稻遗传育种及分子生物学
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