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Ultrastructure and Gene Mapping of the Albino Mutant al12 in Rice (Oryza sativa L.)
遗 传 学 报
Acta Genetica Sinica, December
2006, 33 (12)：1112–1119
ISSN 0379-4172
Ultrastructure and Gene Mapping of the Albino Mutant al12 in Rice (Oryza sativa L.) XIA Jiu-Cheng1,2,3, WANG Yu-Ping1,2, MA Bing-Tian1,2, YIN Zhao-Qing1,2, HAO Ming1,2, KONG De-Wei1,2 , LI Shi-Gui1,2,
①
1. Rice Institute of Sichuan Agricultural University, Wenjiang 611130, China; 2. Key Laboratory of Crop Genetic Resources and Improvement, Ministry of Education, Sichuan Agricultural University, Ya’an 625014, China; 3. Panzhihua University, Panzhihua 617000, China Abstract: Seedling albino mutation resistant to low temperature is an adaptability of rice (Oryza sativa L.) to cold. The mutant, a conditional expression controlled by development and temperature, differs from other albino mutants. The chlorophyll content of the mutant was measured using a portable chlorophyll meter, and the ultrastructure of the chloroplast was observed using a transmission electron microscope. Chlorophyll content was 1.2 SPAD, and the chloroplast did not develop, with only small vesicle-like structures. A segregation analysis of the reciprocal crosses between the albino mutation line with the rice line 9311 demonstrated that the albino trait was controlled by a single recessive gene, which was flanked by SSR markers RM5068 and RM3702 on the short arm of chromosome 8 with a distance of 0.5-1.1 cM and 4.9 cM, respectively. This gene was mapped within a 6 cM interval region and was tentatively referred to as al12. Key words: rice; albino mutant; chloroplast; gene mapping
At present, many albino mutants have been found in rice (Oryza sativa L.). Iwata et al.[1,2] and Maekawak et al.[3] reported 11 mutants, from al1 to al11. Shu et al.[4] reported the albino mutant W25, and YU et al.[5] reported the albino mutant alb21. However, most of these are lethal mutants[1,2,5], having a low value for both theoretical and practical applications. A new mutant (259) was found in our laboratory, which behaves like an albino before the 3-leaf-stage (only with endosperm supply energy) at a low temperature (<24℃), but after the 3-leaf-stage, the mutant gradually turns green and completes the reproduction. It is a conditional expression mutant and is directly related to the adaptation of the plant and can be a unique material for researching the ad-
versity adaptation of rice. By crossing and backcrossing with 9311, the separation for normal and albino plants in the progenies fits well to a ratio of 3:1, suggesting that the mutant is controlled by a recessive gene. The gene was tentatively named al12. In this article, the chlorophyll content was calculated using a portable chlorophyll meter and the ultrastructure change of the chloroplast was observed using a transmission electron microscope. Recently, with the rapid development of the SSR molecular marker technique, many new SSR markers have been exploited constantly. A high-density genetic map was completed[6] based on the SSR markers in rice. Moreover, other molecular markers and a physical map have been constructed; especially the
Received: 2005-12-22; Accepted: 2006-04-03 This work was supported by Program for New Century Excellent Talents in University (No. NCET-04-0907) and Program for Innovative Research Team in University (No.IRT0453). ① Corresponding author. E-mail: [email protected]
XIA Jiu-Cheng et al.: Ultrastructure and Gene Mapping of the Albino Mutant al12 in Rice (Oryza sativa L.)
draft sequence of the rice genome (O. sativa L. ssp. indica and O. sativa L. ssp. japonica) has been completed[7,8]. These make it possible to clone the gene of the rice, resulting in the isolation of many important genes[9 13]. In this study, the gene al12 has been -
mapped and its biological mechanism has been discussed.
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were dehydrated using an ethanol serials and were then embedded with epon812. Specimens were sliced using a microtome LKB-V (LKB, Uppsala, Sweden) and the chloroplast structure of the young leaf was observed using a transmission electron microscope H-600 (Hitachi, Hitachi, Japan)[15]. 1. 2. 5
DNA extraction
DNA was extracted using the CTAB method[16].
1
Materials and Methods
1. 1
1. 2. 6
SSR primers were obtained from http://www.
Materials At the seedling stage, the albino phenomena was
SSR primer synthesis and PCR amplification
gramene.org/microsat/ssr.html; and PCR amplifica-
found in the offspring of a hybrid (D702B ×
tion was performed as described previously[17].
DXiangB), and a stabilized albino mutant line (Line
1. 2. 7
Mapping
259) was developed through seven consecutive gen-
Individual plants in the mapping population
erations of selfing and selection. Both D702B and
(9311×259) were genotyped and the genetic maps
DxiangB are the indica maintenance lines. Line 9311
were drawn using the Mapmaker3.0 software
was used as a control because it is a model indica restorer line and has similar genetic background with the albino mutant under consideration. 1. 2
(Whitehead Institute for Biomedical Research Center, Massachusetts, USA). 1. 2. 8
Methods
1. 2. 1
Temperature for expressing albino
RT-PCR
RNA was extracted from the equiponderant leaves of the mutant and 9311 using BIOZOL (Bio-Flux, Tokyo, Japan) and was then transcribed
Rice seeds were sown in a box and put into an RXZ-280B intelligent climate-control chamber (Jingnan, Ningbo, China). The color changes of the rice seedlings were observed under different temperatures.
1 min, 55℃ for 1 min, and 72℃ for 1 min, 30 cycles;
1. 2. 2
and 72℃ for 6 min. The PCR products were checked
Time for expressing albino
Rice seeds were sown in the field at a temperature ranging between 18℃ and 24 ℃ and the color
using Rever-TraAce (ToYoBo, Osaka, Japan); cDNA was amplified as follows: 94℃ for 5 min; 94℃ for
in 1.5% agaroses and each PCR amplification was repeated thrice.
changes of the seedlings were observed at different stages after sowing.
2
1. 2. 3
2. 1
Chlorophyll content
Results Phenotyping of the albino mutant al12 The leaves of the albino mutant al12 were white
The chlorophyll content of the rice seedling was measured using MINOLTA (chlorophyll meter) SPAD-502 (MINOLTA, Osaka, Japan)[14].
before the 3-leaf-stage (less than 30 d after sowing) at
1. 2. 4
green. The control line 9311 also exhibited a white
Ultrastructure of the chloroplast
The leaves of the rice seedling were cut into small blocks (1 mm × 2 mm) and double fixation with 3% glutaraldehyde containing 0.1 mol /L phosphonic acid buffer (pH 7.3) and 1% osmic acid. These materials
a low temperature (<24℃) and then gradually turned color in the climate-controlled growth chamber when the temperature was lower than 16℃ (Tables 1 and 2, Figs. 1 and 2). The other normal varieties were also white at low temperatures.
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遗传学报
Table 1
Acta Genetica Sinica
Vol.33 No.12 2006
Color change of young leaves of rice under different temperatures
Temperature(℃ )
14
16
18
20
22
24
26
Albino mutant
White
White
White
White
White
White
Green
9311 (CK)
White
White
Green
Green
Green
Green
Green
Table 2
Color change of young leaves of rice after sowing seeds
Days after sowing (d)
10
20
30
40
Albino mutant
White
White
Yellow
Green
9311(CK)
Green
Green
Green
Green
Fig. 1
Phenotype of the albino mutant at the 2-leaf stage
Fig. 2
Phenotype of the 9311 at the 2-leaf stage
2. 2
Chlorophyll content of the albino mutant al12
mutant had also turned into normal (Table 3).
The chlorophyll contents of the base, middle,
Ultrastructure of the chloroplast of the mutant al12
and top leaves of the albino mutant grown in the cli-
The ultrastructure of the mutant was observed at
mate-control chamber were measured. When the mu-
different temperatures (albino phenotype at 24℃ and
tant returned to normal, the chlorophyll content of the
green phenotype at 26℃) on a 2-leaf-stage using a
mutant and 9311, the check entry, were determined. significantly different from that of 9311, the check
transmission electron microscope. According to Figs. 3, 4, and 5, the chloroplast of the mutant did not develop normally, except for the formation of several
entry before the 3-leaf-stage (P< 0.01). There was no
small vesicle-like structures at 24℃. However, the
notable difference in the chlorophyll content between
mutant exhibited a normal chloroplast structure at
the mutant and 9311 (P < 0.01) when the mutant
26℃ in the 2-leaf-stage. The observation showed
turned green, indicating that the chlorophyll content
that the ultrastructure of the mutant would become
of the mutant had reached a normal level and that the
normal at 26℃.
The chlorophyll content of the mutant seedlings were
2. 3
XIA Jiu-Cheng et al.: Ultrastructure and Gene Mapping of the Albino Mutant al12 in Rice (Oryza sativa L.)
Table 3
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Chlorophyll content (CC) of the mutant and 9311 Seedling (2-leaf-stage) Material
Replication
CC(SPAD)
1 2 3 1 2 3
1.2 1.2 1.3 24.3 25.6 26.0
Mutant (259)
9311 (CK) ANOVA. F(0.01)
Normal stage
Average (SPAD) 1.2
25.3
2402.2**
Replication
CC (SPAD)
1 2 3 1 2 3
24.3 25.1 21.3 24.7 25.0 26.3
Average (SPAD) 23.6
25.3
1.2
**: significantly different level.
Fig. 3
Chloroplast of the mutant 259 at 24℃ (2-leaf-stage, × 35 000)
Fig. 4
Chloroplast of the mutant 259 at 24℃ (2-leaf-stage, × 15 000)
2. 4
Inheritance analysis of the albino mutant al12
The albino mutant (259) was crossed and backcrossed with 9311 to bring about F1 at Wenjiang, Chengdu in August, 2004. F2 generation was obtained by self-crossing at Lingshui, Hainan. F 1 behaved green and F2 fitted to the ratio 3:1 (green:white), as 2
determined by the χ -test (Table 4). This proved that the mutant was controlled by a recessive gene.
2. 5
Gene mapping of the albino mutant al12 A total of 44 polymorphic SSR markers between
the mutant parent (259) and the parent 9311 were obtained from 350 pairs of the SSR primer (RM1RM350). Using the bulked segregant analysis (BSA) method[18], it was observed that the SSR markers RM25, RM72, RM152, and RM339 had a linkage to the gene al12 besides the difference between the two DNA pools. In the mapping population (9311×259),
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Fig. 5 Table 4
遗传学报
Vol.33 No.12 2006
Chloroplast of the mutant 259 at 26℃(2-leaf-stage, ×35 000) Separation ratio among the F2 population
Cross
Dominant individual
Recessive individual
Total
(3:1)
259×9311
594
189
783
0.26
9311×259
2116
719
2835
0.17
RM25 had 16 single-crossing recombinants and 1 double-crossing recombinant; RM72 had 26 single-crossing recombinants and 1 double-crossing recombinant; RM152 had 30 single-crossing recombinants; and RM339 had 34 single-crossing recombinants and 1 double-crossing recombinant. The linkage analysis showed that the gene al12 was located on chromosome 8. RM152 and the other three markers were located on the two flanks of the gene al12, respectively. The gene was separated from RM25, RM72, RM152, and RM339 by 12.3 cM, 26.0 cM, 18.4 cM, and 32.0 cM, respectively. Then 33 pairs of the SSR primer between RM25 and RM152 were synthesized[6]. Among these, the primers RM3702, RM5068, and RM8266 showed difference between
Fig. 6
Acta Genetica Sinica
the two parents. RM3702 is a zero allele marker, which can be amplified as a clear band in 9311 but nothing in the mutant (259)[10]. To map exactly, the mapping population was extended to 300 recessive individuals. The primer RM3702 then had 24 single-crossing recombinants, RM5068 had 3 recombinants (zero allele markers cannot tell single-crossing or double-crossing), and RM8266 had 13 single-crossing recombinants and 1 double-crossing recombinant. RM3702 and the other two markers, RM5068 and RM8266, stood on two flanks, separated from the gene by 4.9 cM, 0.5-1.1 cM, and 3.3 cM, respectively (Fig. 6). 2. 6
RT-PCR
According to the result of the mapping gene and the genome sequence of chromosome 8 (http://rgp. dna.affrc.go.jp), two candidate coding sequences (CDS) were found in the entire contigs between the two close SSR markers RM5068 and RM3702 on chromosome 8 related with the mutant phenotype
The part linkage map of the al12 gene on chromosome 8 of rice
XIA Jiu-Cheng et al.: Ultrastructure and Gene Mapping of the Albino Mutant al12 in Rice (Oryza sativa L.)
(albino at low temperature): the putative cytochromosome P450 (CYP) and the putative cold shock protein (CSP). Three pairs of primers were designed according to the sequence of the two CDSs. The internal reference was designed from β-actin (Table 5). The RNA of the albino mutant and 9311 were retrotranscripled into cDNA, and the cDNA were then amplified using PCR. The result showed that the CDS of exhibiting expression difference in the RNA level is the putative cytochromosome P450 (CYP) (Fig. 7). Table 5 Nucleic acid sequence of the four pairs of primers Primers CYP-1 CYP-2 CSP β-actin
Forward (5′→3′) CCCGTCACGCATTCATTC ATTGCATGAGGCGAAGG CACGCACTTATGATGACC GAACTGGTATGGTCAAGGCTG
Reverse (5′→3′) CGCTCGTAGTCGCCAACA GCCCAGCAGCCAGAACA AGTATGGACTACGGAATGT ACACGGAGCTCGTTGTAGAAG
Fig. 7 The result of RT-PCR of the mutant and 9311 with the four pairs of primers Lane M: Marker; Lane 1, 3, 5, 7: mutant (259); Lane 2, 4, 6, 8: 9311.
3
Discussion
Albino of seedling resistant to low temperature, which is an important trait, has high value in biology and breeding. The energy for the development and growth of the young seedlings is provided by the endosperm before the 3-leaf-stage and thereafter by the solar energy. In young leaves of rice, at low temperature, the chlorophyll synthesis and the development of chloroplast may temporarily stop to combat the adverse environment. After the 3-leaf-stage, the mutant resumes to synthesize chlorophyll and construct the chloroplast and thus begins to synthesize energy via photosynthesis. The albino mutant is a positive
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mutation. Except for the mutant al11 that behaves like a variegated albino and a greenable albino mutant W25, the other mutants reported are lethal mutants[1,2,5]. The new albino mutant al12 is similar to W25. Both are conditional expressions associated with the temperature and development stage [4] and are controlled by a recessive gene according to the separation behavior of F1 progeny plants[19]. However, these are different in their origins and preconditions of the albino expression between the line 259 and the line W25. The mutant W25 resulted from artificial mutation through irradiation of the thermosensitive male sterile line 2177s seeds with 300 Gy60Co-γ ray[4]. This mutant behaves like an albino before the 4-leaf-stage under 25℃. However, the mutant al12 originated from a natural mutation in the progenies of the hybrid D702B × DXiangB. Hence, it was considered that the greenable albino mutant can be obtained by artificial and natural methods. Researches on the phenotype, physiology, biochemistry, and microstructure have been carried out in the mutant W25[4,19,20]. In this study, the chlorophyll content of the albino mutant al12 was determined and the chloroplast ultrastructure was observed. It was noted that the chlorophyll content was very low and the chloroplast was abnormal with only many micro-vesicles. Weng et al.[20] believed that the low content of soluble protein and Rubisco, the lazy Rubisco activase, may be responsible for the albino. In addition, the gene al12 was first located between the two close SSR markers RM5068 and RM3702 on chromosome 8 by positional cloning[4,19,20]. Using RT-PCR, a CDS sequence of the putative cytochromosome P450 (CYP) exhibited expression differences in the RNA level between the mutant and 9311. Cytochromosome P450 is a super-family including 10 subfamilies, such as CYP51, CYP71, CYP72, and CYP85, distributed in the whole genome of different plants[21]. This may be the key to the question regarding the existence of many different albino mutants in rice. Using Blast, it was observed that this putative sequence is very similar to the known gene CYP89A2 of Arabidopsis (http://www.
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遗传学报
ncbi.nlm.nih.gov/BLAST/), belonging to the CYP71 subfamily. The gene may associate with iron ion binding, oxygen binding, electron transport, amino acid and derivative metabolism, and lipid-metabolism (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?d b=gene&cmd=search&term=CYP89A2), (http:// www.tigr.org/tigr-scripts/euk_manatee/shared/OR F_infopage.cgi?db=osa1&orf=11674.t00462). On the basis of this information, the mutant behaved albino at low temperatures in the seedling stage because of the change of some base sequences in the open reading frame (ORF) or in the up/down stream of the CDS. However, further researche is necessary for fine mapping, cloning, transferring, and complementation of the gene to confirm the necessity of albino mutation. Moreover, the albino gene al12 can be transferred into other varieties by continuous backcrossing, and new varieties with the albino marker can be bred to remove fake plants at early stages, to purify the seed, and to reduce the field work[22]. References: [1] Iwata N, Satoh H, Omura T. Linkage analysis by use of trisomics in rice(Oryza sativa L.). IV. Linkage groups locating on chromosomes 2 and 10. Japan J Breed, 1981, 31 (Suppl. 1): 66-67. [2] Iwata N, Omura T. Linkage studies in rice (Oryza satival) some albino genes and their linkage relation with marker genes. Sci Bull Fac Agr Kyus Hu Univ, 1978, 33(1): 18. [3] Maekawa K, Rikiishi K, Matsuura T, Noda K. Revertants originated from chlorophyll mutants from the distantly related rice varieties. Japan J Breed Sci, 1996, 46(Suppl. 2): 107. [4] SHU Qing-Yao, WU Dian-Xing, XIA Ying-Wu, LIU Gui-Fu. Study on greenism characteristics of greenable albino mutation line W25 of rice (Oryza sativa L.). Journal of Zhejiang Agricultural University, 1996, 22(2): 219-220 (in Chinese). [5] YU Qing-Bo, JIANG Hua, MI Hua-Ling, ZHOU Gen-Yu, YANG Zhong-Nan. The physiological character and molecular mapping in rice albino21 mutant. Journal of Shanghai NormaUniversity, 2005, 34(1): 70-75(in Chinese with an English abstract). [6] McCouch S R, Teytelman L, Xu Y, Lobos K B, Clare K, Walton M, Fu B, Maghirang R, Li Z, Xing Y Z, Zhang Q F, Kono I, Yano M, Fjellstrom R, DeClerck G, Schneider D, Cartinhour S, Ware D, Stein L. Development and mapping of 2240 new SSR markers for rice (Oryza sa-
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segregant analysis: a rapid method to detect markers in specific genomic regions using segregating populations.
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[20] WENG Xiao-Yan, JIANG De-An, LU Qing. Changes in rubisco activity, Rubisco activase activity and photosynthetics rate in greenable albino mutation line of rice during greening. Acta Phytophysiologica Sinica, 2000,
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水稻(Oryza sativa L.)苗期低温白化突变体 al12 的超微结构 与基因定位 夏九成1,2,3，王玉平1,2，马炳田1,2，殷兆晴1,2，郝 铭1,2，孔德伟1,2，李仕贵1,2 1. 四川农业大学水稻研究所，温江 611130； 2. 作物基因资源与遗传改良教育部重点实验室，雅安 625014； 3. 四川省攀枝花学院, 攀枝花 617000 摘 要: 水稻苗期低温白化突变是水稻在发育早期对低温胁迫的一种适应性，是一种受发育和温度控制的条件表达，它与其 他水稻白化突变有本质的不同。本研究利用便携式叶绿素测量仪测定了白化时期植株的叶绿素含量和用透射电镜观察了叶 绿体的结构变化。结果发现叶绿素平均含量仅为 1.2（SPAD），而叶绿体也不能正常发育仅有囊泡状结构。通过与 9311 的 正反交实验及子代的分离表现证明该性状受一个隐性核基因的控制。另外利用 SSR 分子标记技术将该基因定位在第 8 染色 体上，两侧最近的 SSR 标记 RM5068 和 RM3702 分别距基因 0.5～1.1 cM 和 4.9 cM，基因被定位在约 6 个 cM 的区间内。 我们将该基因暂时命名为 al12。 关键词: 水稻；白化突变体；叶绿体；基因定位 作者简介: 夏九成（1976-），男，四川西昌人，在读博士，研究方向：植物遗传学。E-mail: [email protected]
Acta Genetica Sinica, December
2006, 33 (12)：1112–1119
ISSN 0379-4172
Ultrastructure and Gene Mapping of the Albino Mutant al12 in Rice (Oryza sativa L.) XIA Jiu-Cheng1,2,3, WANG Yu-Ping1,2, MA Bing-Tian1,2, YIN Zhao-Qing1,2, HAO Ming1,2, KONG De-Wei1,2 , LI Shi-Gui1,2,
①
1. Rice Institute of Sichuan Agricultural University, Wenjiang 611130, China; 2. Key Laboratory of Crop Genetic Resources and Improvement, Ministry of Education, Sichuan Agricultural University, Ya’an 625014, China; 3. Panzhihua University, Panzhihua 617000, China Abstract: Seedling albino mutation resistant to low temperature is an adaptability of rice (Oryza sativa L.) to cold. The mutant, a conditional expression controlled by development and temperature, differs from other albino mutants. The chlorophyll content of the mutant was measured using a portable chlorophyll meter, and the ultrastructure of the chloroplast was observed using a transmission electron microscope. Chlorophyll content was 1.2 SPAD, and the chloroplast did not develop, with only small vesicle-like structures. A segregation analysis of the reciprocal crosses between the albino mutation line with the rice line 9311 demonstrated that the albino trait was controlled by a single recessive gene, which was flanked by SSR markers RM5068 and RM3702 on the short arm of chromosome 8 with a distance of 0.5-1.1 cM and 4.9 cM, respectively. This gene was mapped within a 6 cM interval region and was tentatively referred to as al12. Key words: rice; albino mutant; chloroplast; gene mapping
At present, many albino mutants have been found in rice (Oryza sativa L.). Iwata et al.[1,2] and Maekawak et al.[3] reported 11 mutants, from al1 to al11. Shu et al.[4] reported the albino mutant W25, and YU et al.[5] reported the albino mutant alb21. However, most of these are lethal mutants[1,2,5], having a low value for both theoretical and practical applications. A new mutant (259) was found in our laboratory, which behaves like an albino before the 3-leaf-stage (only with endosperm supply energy) at a low temperature (<24℃), but after the 3-leaf-stage, the mutant gradually turns green and completes the reproduction. It is a conditional expression mutant and is directly related to the adaptation of the plant and can be a unique material for researching the ad-
versity adaptation of rice. By crossing and backcrossing with 9311, the separation for normal and albino plants in the progenies fits well to a ratio of 3:1, suggesting that the mutant is controlled by a recessive gene. The gene was tentatively named al12. In this article, the chlorophyll content was calculated using a portable chlorophyll meter and the ultrastructure change of the chloroplast was observed using a transmission electron microscope. Recently, with the rapid development of the SSR molecular marker technique, many new SSR markers have been exploited constantly. A high-density genetic map was completed[6] based on the SSR markers in rice. Moreover, other molecular markers and a physical map have been constructed; especially the
Received: 2005-12-22; Accepted: 2006-04-03 This work was supported by Program for New Century Excellent Talents in University (No. NCET-04-0907) and Program for Innovative Research Team in University (No.IRT0453). ① Corresponding author. E-mail: [email protected]
XIA Jiu-Cheng et al.: Ultrastructure and Gene Mapping of the Albino Mutant al12 in Rice (Oryza sativa L.)
draft sequence of the rice genome (O. sativa L. ssp. indica and O. sativa L. ssp. japonica) has been completed[7,8]. These make it possible to clone the gene of the rice, resulting in the isolation of many important genes[9 13]. In this study, the gene al12 has been -
mapped and its biological mechanism has been discussed.
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were dehydrated using an ethanol serials and were then embedded with epon812. Specimens were sliced using a microtome LKB-V (LKB, Uppsala, Sweden) and the chloroplast structure of the young leaf was observed using a transmission electron microscope H-600 (Hitachi, Hitachi, Japan)[15]. 1. 2. 5
DNA extraction
DNA was extracted using the CTAB method[16].
1
Materials and Methods
1. 1
1. 2. 6
SSR primers were obtained from http://www.
Materials At the seedling stage, the albino phenomena was
SSR primer synthesis and PCR amplification
gramene.org/microsat/ssr.html; and PCR amplifica-
found in the offspring of a hybrid (D702B ×
tion was performed as described previously[17].
DXiangB), and a stabilized albino mutant line (Line
1. 2. 7
Mapping
259) was developed through seven consecutive gen-
Individual plants in the mapping population
erations of selfing and selection. Both D702B and
(9311×259) were genotyped and the genetic maps
DxiangB are the indica maintenance lines. Line 9311
were drawn using the Mapmaker3.0 software
was used as a control because it is a model indica restorer line and has similar genetic background with the albino mutant under consideration. 1. 2
(Whitehead Institute for Biomedical Research Center, Massachusetts, USA). 1. 2. 8
Methods
1. 2. 1
Temperature for expressing albino
RT-PCR
RNA was extracted from the equiponderant leaves of the mutant and 9311 using BIOZOL (Bio-Flux, Tokyo, Japan) and was then transcribed
Rice seeds were sown in a box and put into an RXZ-280B intelligent climate-control chamber (Jingnan, Ningbo, China). The color changes of the rice seedlings were observed under different temperatures.
1 min, 55℃ for 1 min, and 72℃ for 1 min, 30 cycles;
1. 2. 2
and 72℃ for 6 min. The PCR products were checked
Time for expressing albino
Rice seeds were sown in the field at a temperature ranging between 18℃ and 24 ℃ and the color
using Rever-TraAce (ToYoBo, Osaka, Japan); cDNA was amplified as follows: 94℃ for 5 min; 94℃ for
in 1.5% agaroses and each PCR amplification was repeated thrice.
changes of the seedlings were observed at different stages after sowing.
2
1. 2. 3
2. 1
Chlorophyll content
Results Phenotyping of the albino mutant al12 The leaves of the albino mutant al12 were white
The chlorophyll content of the rice seedling was measured using MINOLTA (chlorophyll meter) SPAD-502 (MINOLTA, Osaka, Japan)[14].
before the 3-leaf-stage (less than 30 d after sowing) at
1. 2. 4
green. The control line 9311 also exhibited a white
Ultrastructure of the chloroplast
The leaves of the rice seedling were cut into small blocks (1 mm × 2 mm) and double fixation with 3% glutaraldehyde containing 0.1 mol /L phosphonic acid buffer (pH 7.3) and 1% osmic acid. These materials
a low temperature (<24℃) and then gradually turned color in the climate-controlled growth chamber when the temperature was lower than 16℃ (Tables 1 and 2, Figs. 1 and 2). The other normal varieties were also white at low temperatures.
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Table 1
Acta Genetica Sinica
Vol.33 No.12 2006
Color change of young leaves of rice under different temperatures
Temperature(℃ )
14
16
18
20
22
24
26
Albino mutant
White
White
White
White
White
White
Green
9311 (CK)
White
White
Green
Green
Green
Green
Green
Table 2
Color change of young leaves of rice after sowing seeds
Days after sowing (d)
10
20
30
40
Albino mutant
White
White
Yellow
Green
9311(CK)
Green
Green
Green
Green
Fig. 1
Phenotype of the albino mutant at the 2-leaf stage
Fig. 2
Phenotype of the 9311 at the 2-leaf stage
2. 2
Chlorophyll content of the albino mutant al12
mutant had also turned into normal (Table 3).
The chlorophyll contents of the base, middle,
Ultrastructure of the chloroplast of the mutant al12
and top leaves of the albino mutant grown in the cli-
The ultrastructure of the mutant was observed at
mate-control chamber were measured. When the mu-
different temperatures (albino phenotype at 24℃ and
tant returned to normal, the chlorophyll content of the
green phenotype at 26℃) on a 2-leaf-stage using a
mutant and 9311, the check entry, were determined. significantly different from that of 9311, the check
transmission electron microscope. According to Figs. 3, 4, and 5, the chloroplast of the mutant did not develop normally, except for the formation of several
entry before the 3-leaf-stage (P< 0.01). There was no
small vesicle-like structures at 24℃. However, the
notable difference in the chlorophyll content between
mutant exhibited a normal chloroplast structure at
the mutant and 9311 (P < 0.01) when the mutant
26℃ in the 2-leaf-stage. The observation showed
turned green, indicating that the chlorophyll content
that the ultrastructure of the mutant would become
of the mutant had reached a normal level and that the
normal at 26℃.
The chlorophyll content of the mutant seedlings were
2. 3
XIA Jiu-Cheng et al.: Ultrastructure and Gene Mapping of the Albino Mutant al12 in Rice (Oryza sativa L.)
Table 3
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Chlorophyll content (CC) of the mutant and 9311 Seedling (2-leaf-stage) Material
Replication
CC(SPAD)
1 2 3 1 2 3
1.2 1.2 1.3 24.3 25.6 26.0
Mutant (259)
9311 (CK) ANOVA. F(0.01)
Normal stage
Average (SPAD) 1.2
25.3
2402.2**
Replication
CC (SPAD)
1 2 3 1 2 3
24.3 25.1 21.3 24.7 25.0 26.3
Average (SPAD) 23.6
25.3
1.2
**: significantly different level.
Fig. 3
Chloroplast of the mutant 259 at 24℃ (2-leaf-stage, × 35 000)
Fig. 4
Chloroplast of the mutant 259 at 24℃ (2-leaf-stage, × 15 000)
2. 4
Inheritance analysis of the albino mutant al12
The albino mutant (259) was crossed and backcrossed with 9311 to bring about F1 at Wenjiang, Chengdu in August, 2004. F2 generation was obtained by self-crossing at Lingshui, Hainan. F 1 behaved green and F2 fitted to the ratio 3:1 (green:white), as 2
determined by the χ -test (Table 4). This proved that the mutant was controlled by a recessive gene.
2. 5
Gene mapping of the albino mutant al12 A total of 44 polymorphic SSR markers between
the mutant parent (259) and the parent 9311 were obtained from 350 pairs of the SSR primer (RM1RM350). Using the bulked segregant analysis (BSA) method[18], it was observed that the SSR markers RM25, RM72, RM152, and RM339 had a linkage to the gene al12 besides the difference between the two DNA pools. In the mapping population (9311×259),
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Fig. 5 Table 4
遗传学报
Vol.33 No.12 2006
Chloroplast of the mutant 259 at 26℃(2-leaf-stage, ×35 000) Separation ratio among the F2 population
Cross
Dominant individual
Recessive individual
Total
(3:1)
259×9311
594
189
783
0.26
9311×259
2116
719
2835
0.17
RM25 had 16 single-crossing recombinants and 1 double-crossing recombinant; RM72 had 26 single-crossing recombinants and 1 double-crossing recombinant; RM152 had 30 single-crossing recombinants; and RM339 had 34 single-crossing recombinants and 1 double-crossing recombinant. The linkage analysis showed that the gene al12 was located on chromosome 8. RM152 and the other three markers were located on the two flanks of the gene al12, respectively. The gene was separated from RM25, RM72, RM152, and RM339 by 12.3 cM, 26.0 cM, 18.4 cM, and 32.0 cM, respectively. Then 33 pairs of the SSR primer between RM25 and RM152 were synthesized[6]. Among these, the primers RM3702, RM5068, and RM8266 showed difference between
Fig. 6
Acta Genetica Sinica
the two parents. RM3702 is a zero allele marker, which can be amplified as a clear band in 9311 but nothing in the mutant (259)[10]. To map exactly, the mapping population was extended to 300 recessive individuals. The primer RM3702 then had 24 single-crossing recombinants, RM5068 had 3 recombinants (zero allele markers cannot tell single-crossing or double-crossing), and RM8266 had 13 single-crossing recombinants and 1 double-crossing recombinant. RM3702 and the other two markers, RM5068 and RM8266, stood on two flanks, separated from the gene by 4.9 cM, 0.5-1.1 cM, and 3.3 cM, respectively (Fig. 6). 2. 6
RT-PCR
According to the result of the mapping gene and the genome sequence of chromosome 8 (http://rgp. dna.affrc.go.jp), two candidate coding sequences (CDS) were found in the entire contigs between the two close SSR markers RM5068 and RM3702 on chromosome 8 related with the mutant phenotype
The part linkage map of the al12 gene on chromosome 8 of rice
XIA Jiu-Cheng et al.: Ultrastructure and Gene Mapping of the Albino Mutant al12 in Rice (Oryza sativa L.)
(albino at low temperature): the putative cytochromosome P450 (CYP) and the putative cold shock protein (CSP). Three pairs of primers were designed according to the sequence of the two CDSs. The internal reference was designed from β-actin (Table 5). The RNA of the albino mutant and 9311 were retrotranscripled into cDNA, and the cDNA were then amplified using PCR. The result showed that the CDS of exhibiting expression difference in the RNA level is the putative cytochromosome P450 (CYP) (Fig. 7). Table 5 Nucleic acid sequence of the four pairs of primers Primers CYP-1 CYP-2 CSP β-actin
Forward (5′→3′) CCCGTCACGCATTCATTC ATTGCATGAGGCGAAGG CACGCACTTATGATGACC GAACTGGTATGGTCAAGGCTG
Reverse (5′→3′) CGCTCGTAGTCGCCAACA GCCCAGCAGCCAGAACA AGTATGGACTACGGAATGT ACACGGAGCTCGTTGTAGAAG
Fig. 7 The result of RT-PCR of the mutant and 9311 with the four pairs of primers Lane M: Marker; Lane 1, 3, 5, 7: mutant (259); Lane 2, 4, 6, 8: 9311.
3
Discussion
Albino of seedling resistant to low temperature, which is an important trait, has high value in biology and breeding. The energy for the development and growth of the young seedlings is provided by the endosperm before the 3-leaf-stage and thereafter by the solar energy. In young leaves of rice, at low temperature, the chlorophyll synthesis and the development of chloroplast may temporarily stop to combat the adverse environment. After the 3-leaf-stage, the mutant resumes to synthesize chlorophyll and construct the chloroplast and thus begins to synthesize energy via photosynthesis. The albino mutant is a positive
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mutation. Except for the mutant al11 that behaves like a variegated albino and a greenable albino mutant W25, the other mutants reported are lethal mutants[1,2,5]. The new albino mutant al12 is similar to W25. Both are conditional expressions associated with the temperature and development stage [4] and are controlled by a recessive gene according to the separation behavior of F1 progeny plants[19]. However, these are different in their origins and preconditions of the albino expression between the line 259 and the line W25. The mutant W25 resulted from artificial mutation through irradiation of the thermosensitive male sterile line 2177s seeds with 300 Gy60Co-γ ray[4]. This mutant behaves like an albino before the 4-leaf-stage under 25℃. However, the mutant al12 originated from a natural mutation in the progenies of the hybrid D702B × DXiangB. Hence, it was considered that the greenable albino mutant can be obtained by artificial and natural methods. Researches on the phenotype, physiology, biochemistry, and microstructure have been carried out in the mutant W25[4,19,20]. In this study, the chlorophyll content of the albino mutant al12 was determined and the chloroplast ultrastructure was observed. It was noted that the chlorophyll content was very low and the chloroplast was abnormal with only many micro-vesicles. Weng et al.[20] believed that the low content of soluble protein and Rubisco, the lazy Rubisco activase, may be responsible for the albino. In addition, the gene al12 was first located between the two close SSR markers RM5068 and RM3702 on chromosome 8 by positional cloning[4,19,20]. Using RT-PCR, a CDS sequence of the putative cytochromosome P450 (CYP) exhibited expression differences in the RNA level between the mutant and 9311. Cytochromosome P450 is a super-family including 10 subfamilies, such as CYP51, CYP71, CYP72, and CYP85, distributed in the whole genome of different plants[21]. This may be the key to the question regarding the existence of many different albino mutants in rice. Using Blast, it was observed that this putative sequence is very similar to the known gene CYP89A2 of Arabidopsis (http://www.
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ncbi.nlm.nih.gov/BLAST/), belonging to the CYP71 subfamily. The gene may associate with iron ion binding, oxygen binding, electron transport, amino acid and derivative metabolism, and lipid-metabolism (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?d b=gene&cmd=search&term=CYP89A2), (http:// www.tigr.org/tigr-scripts/euk_manatee/shared/OR F_infopage.cgi?db=osa1&orf=11674.t00462). On the basis of this information, the mutant behaved albino at low temperatures in the seedling stage because of the change of some base sequences in the open reading frame (ORF) or in the up/down stream of the CDS. However, further researche is necessary for fine mapping, cloning, transferring, and complementation of the gene to confirm the necessity of albino mutation. Moreover, the albino gene al12 can be transferred into other varieties by continuous backcrossing, and new varieties with the albino marker can be bred to remove fake plants at early stages, to purify the seed, and to reduce the field work[22]. References: [1] Iwata N, Satoh H, Omura T. Linkage analysis by use of trisomics in rice(Oryza sativa L.). IV. Linkage groups locating on chromosomes 2 and 10. Japan J Breed, 1981, 31 (Suppl. 1): 66-67. [2] Iwata N, Omura T. Linkage studies in rice (Oryza satival) some albino genes and their linkage relation with marker genes. Sci Bull Fac Agr Kyus Hu Univ, 1978, 33(1): 18. [3] Maekawa K, Rikiishi K, Matsuura T, Noda K. Revertants originated from chlorophyll mutants from the distantly related rice varieties. Japan J Breed Sci, 1996, 46(Suppl. 2): 107. [4] SHU Qing-Yao, WU Dian-Xing, XIA Ying-Wu, LIU Gui-Fu. Study on greenism characteristics of greenable albino mutation line W25 of rice (Oryza sativa L.). Journal of Zhejiang Agricultural University, 1996, 22(2): 219-220 (in Chinese). [5] YU Qing-Bo, JIANG Hua, MI Hua-Ling, ZHOU Gen-Yu, YANG Zhong-Nan. The physiological character and molecular mapping in rice albino21 mutant. Journal of Shanghai NormaUniversity, 2005, 34(1): 70-75(in Chinese with an English abstract). [6] McCouch S R, Teytelman L, Xu Y, Lobos K B, Clare K, Walton M, Fu B, Maghirang R, Li Z, Xing Y Z, Zhang Q F, Kono I, Yano M, Fjellstrom R, DeClerck G, Schneider D, Cartinhour S, Ware D, Stein L. Development and mapping of 2240 new SSR markers for rice (Oryza sa-
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水稻(Oryza sativa L.)苗期低温白化突变体 al12 的超微结构 与基因定位 夏九成1,2,3，王玉平1,2，马炳田1,2，殷兆晴1,2，郝 铭1,2，孔德伟1,2，李仕贵1,2 1. 四川农业大学水稻研究所，温江 611130； 2. 作物基因资源与遗传改良教育部重点实验室，雅安 625014； 3. 四川省攀枝花学院, 攀枝花 617000 摘 要: 水稻苗期低温白化突变是水稻在发育早期对低温胁迫的一种适应性，是一种受发育和温度控制的条件表达，它与其 他水稻白化突变有本质的不同。本研究利用便携式叶绿素测量仪测定了白化时期植株的叶绿素含量和用透射电镜观察了叶 绿体的结构变化。结果发现叶绿素平均含量仅为 1.2（SPAD），而叶绿体也不能正常发育仅有囊泡状结构。通过与 9311 的 正反交实验及子代的分离表现证明该性状受一个隐性核基因的控制。另外利用 SSR 分子标记技术将该基因定位在第 8 染色 体上，两侧最近的 SSR 标记 RM5068 和 RM3702 分别距基因 0.5～1.1 cM 和 4.9 cM，基因被定位在约 6 个 cM 的区间内。 我们将该基因暂时命名为 al12。 关键词: 水稻；白化突变体；叶绿体；基因定位 作者简介: 夏九成（1976-），男，四川西昌人，在读博士，研究方向：植物遗传学。E-mail: [email protected]