- Email: [email protected]
Analysis of the Phylogenetic Relationships Among Several Species of Gramineae Using ACGM Markers
遗 传 学 报
Acta Genetica Sinica, December
2006, 33 (12)：1127–1131
ISSN 0379-4172
Analysis of the Phylogenetic Relationships Among Several Species of Gramineae Using ACGM Markers LU Yong-Quan1,2, YE Zi-Hong3, WU Wei-Ren1,① 1. Department of Agronomy, College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310029, China; 2. Biotechnology Research Center, Heilongjiang Academy of Agricultural Sciences, Haerbin 150086, China; 3. College of Life Science, China Jiliang University, Hangzhou 310018, China Abstract: To study the transferability of rice (Oryza sativa L.) genome data, we used amplified consensus genetic markers to analyze the phylogenetic relationships among several species and genera in Gramineae. Ten accessions representing five grass genera (Oryza, Zea, Setaria, Triticum, and Phyllostachys) were used. According to the genetic distances, a cluster tree was constructed. The relationships among the five genera could be simply described as ((Oryza + (Zea + Setaria)) + Triticum) + Phyllostachys. The results suggest that the genetic distance between rice and maize (Z. mays L.) or rice and millet (Setaria italica L.) is closer than that between rice and wheat (Triticum aestivum L) or rice and bamboo. Key words: ACGM markers; phylogenetic relationship; Gramineae; genome; transferability
Botstein et al.[1] were the first to use restriction fragment length polymorphisms as genetic markers to construct a human genetic map. Since then, many new molecular marker techniques have been developed, such as random amplified polymorphic DNA[2], amplified fragment length polymorphism[3], microsatellite or simple sequence repeat[4,5], sequence-related amplified polymorphism[6], and single-nucleotide polymorphism[7]. Good molecular marker systems are very useful tools for genetic research (e.g., constructing genetic maps, mapping genes or quantitative trait loci) and breeding (e.g., marker-assisted selection). Draft genome sequences of two rice (Oryza sativa L.) cultivars, 93-11[8] and Nipponbare[9], representing indica and japonica subspecies, respectively, have been completed. A set of over 28 000 full-length cDNA sequences from Nipponbare has been released[10]. Complete sequences of chromosomes 1, 4, -
and 10 of Nipponbare have been published[11 13]. In addition, a tentative assembly of all chromosomes of
rice has been released (The Institute of Genomic Research, TIGR; http://www.tigr.org). These data provide an opportunity to systematically search for DNA polymorphisms and to exploit DNA markers on a large scale in rice. Gramineae is a major family among the angiosperms, consisting of five to six subfamilies, 60-80 tribes, 720-765 genera, and more than 10 000 species[14]. Most food crops, e.g., wheat (Triticum aestivum L.), rice, and maize (Zea mays L.), and forage plants belong to this family. It is impossible to sequence the genome of each species. Comparative genomics studies have shown that linear organization exists among different genomes in Gramineae[15]. Therefore, the information of rice genome could be used to study the genetic basis of other plants in Gramineae. The closer the genetic relationship of a species to rice, the better the utilization of the information. Amplified consensus genetic marker (ACGM) is a polymerase chain reaction (PCR)-based marker
Received: 2005-11-14; Accepted: 2006-04-10 This work was supported by the China National Programs for High Technology Research and Development (863 Program) (No. 2003AA207160). ① Corresponding author. E-mail: [email protected]; Tel: +86-571-8697 1910
遗传学报
1128
with primers designed in conservative regions of coding sequences[16]. Therefore, ACGM would be quite useful for phylogenetic studies. In this study, we applied the ACGM markers to analyzing the phylogenetic relationships among several species and genera in Gramineae. Our purposes were to examine the feasibility of using ACGM markers exploited from rice for the phylogenetic study in Gramineae and to provide reference for the use of rice genome data to the genetic study of other species in Gramineae.
1
Materials and Methods
1. 1
Plant materials
Ten accessions representing five grass genera were used (Table 1), including japonica rice variety Nipponbare and indica rice variety 93-11; two maize inbred lines F683 and F743 (bred by Zhejiang University); two millet (Setaria italica L.) lines 4a and 21; two wheat species T. macha and T. spelta (collected from the Plant Garden of Zhejiang University); and two bamboo species Phyllostachys atrovaginata C. and P. propinqua C. (collected from the Plant Garden of Hangzhou). Table 1 No. 1 2 3 4 5 6 7 8 9 10
1. 2
Plant materials used in this research Plant materials Oryza sativa ssp. japonica var. Nipponbare O. sativa ssp. indica var. 93-11 Zea mays var. F683 Z. mays var. F743 Setaria italica var. 4a S. italica var. No.21 Triticum macha T. spelta Phyllostachys atrovaginata P. propinqua
Acta Genetica Sinica
Vol.33 No.12 2006
containing 50 ng of template DNA, 0.5 μmol/L of each primer, 200 μmol/L of each dNTP, 1.5 mmol/L of MgCl2, 0.1% of Triton X-100, 1 unit of Taq polymerase, and 1.5 μL of 10× PCR reaction buffer. A touchdown-PCR[19] program was used: 5 min at 94℃; 10 cycles of 30 s at 94℃, 30 s at 59℃ minus 0.3℃/cycle, 1 min at 72℃; 20 cycles of 30 s at 94℃, 30 s at 56℃, 1 min at 72℃; and 5 min at 72℃ for a final extension. For primer pairs that did not generate good amplification results, we adjusted the initial annealing temperature from 55℃ to 60℃. Each of the primer pairs was tested twice to confirm the repeatability of the observed bands in each genotype. PCR products were separated on 6% nondenaturing polyacrylamide gel electrophoresis (80 volts, 2.5 h). Gels were silver stained for visualizing DNA bands, following the procedure of Xu et al[20]. 1. 3
Statistical analysis of phylogenetic relationship
Bands were scored as 1 (presence) or 0 (absence). Genetic distances between accessions were calculated according to Nei and Li[21]. The distance matrix was used to construct the Unweighted PairGroup Method Using Arithmetic averages (UPGMA) dendogram using the NEIGHBOR model of the PHILIP Version 3.6c[22].
Genome 2n=2x=24
2 2n=2x=24 2n=2x=20 2n=2x=20 2n=2x=18 2n=2x=18 2n=6x=42 2n=6x=42 2n=2x=48 2n=2x=48
Results
All the primers used could yield stable PCR products in each accession (Fig. 1). In general, the
Detection of ACGM
Thirty-eight pairs of ACGM primers[17] were used in the experiment. Modified CTAB (cetyltrimethylammonium bromide) method[18] was used to extract DNA from young leaves of each plant material. PCR was performed in a 15 μL reaction mixture
Fig. 1 PCR products obtained from primer pair GA24 in different accessions The lane codes 1-10 are consistent with the accession codes shown in Table 1.
LU Yong-Quan et al.: Analysis of the Phylogenetic Relationships Among Several Species of Gramineae Using ACGM Markers
Fig. 2
The UPGMA tree of the 10 grass accessions based on 38 ACGM markers
distances between different genera were relatively large whereas those within genera were relatively small. According to the genetic distances, a cluster tree was constructed (Fig. 2). It was as expected that accessions of a same genus formed a subgroup before being grouped with other genera. The relationship among the five genera could be simply described as (((Oryza+(Zea+Setaria))+Triticum)+Phyllostachys).
3
1129
Discussion
In this study, we have analyzed the genetic evolutionary relationships among five major genera belonging to the grass family (i.e., Oryza, Zea, Setaria, Triticum, and Phyllostachys) using newly developed ACGM markers[17]. The clustering result is consistent -
with those obtained in previous studies[23 25]. For example, according to traditional morphological classification, Zea and Setaria belong to the family Paniceae[23]. In this study, Zea and Setaria are classified together prior to other genera. In the early classification system of the grass family, Oryza was included into Bambusoideae[24]. However, we found that there was a certain distance between Oryza and Phyllostachys, which is consistent with the modern classification system[25]. Paterson et al.[26] drew a systematic evolutionary tree for phanerogam based on the study of whole-genome duplication of crops in grass family. In their evolutionary tree, Zea, Sorghum, Oryza, and Hordeum cluster together, which can be simply described
as: Hordeum + (Oryza + (Sorghum + Zea)). In this study, Triticum was selected instead of Hordeum, but both genera belong to Pooideae Triticeae. Therefore, the result of this study is similar to that they obtained. Another remarkable conclusion of this work is that Phyllostachys is at the base of the cluster tree. Bamboo has a significant position in evolution. Since the 1950s, wooden bamboo or some herbaceous communities with biological characters similar to bamboo have been considered to be the original community of the grass family[23]. Recently, new knowledge about Bambusoideae was gained and corresponding experimental supports were found with the development of molecular biology[23]. On the basis of nucleic acid sequence analysis, Clark et al.[27] reported that Bambusoideae and Pooideae are closely related, which form a sister group of Oryza. This is consistent with our result. References: [1] Botstein D, White R L, Skolnick M, Davis R W. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet, 1980, 32(3): 314-331. [2] Williams J G, Kubelik A R, Livak K J, Rafalski J A, Tingey S V. DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res, 1990, 18(22): 6531-6535. [3] Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res, 1995, 23(21): 4407-4414.
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[4] Becker J, Heun M. Barley microsatellites: allele variation and mapping, Plant Mol Biol, 1995, 27(4): 835-845. [5] Becker J, Vos P, Kuiper M, Salamini F, Heun M. Combined mapping of AFLP and RFLP markers in barley. Mol Gen Genet, 1995, 249(1): 65-73. [6] Li G, Quiros C F. Sequence-related amplified polymorphism (SRAP) a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theor Appl Genet, 2001, 103(3): 455-461. [7] Kruglyak L, Nickerson D A. Variation is the spice of life. Nat Genet, 2001, 27(3): 234-236. [8] Yu J, Hu S, Wang J, Wong G K, Li S, Liu B, Deng Y, Dai L, Zhou Y, Zhang X, Cao M, Liu J, Sun J, Tang J, Chen Y, Huang X, Lin W, Ye C, Tong W, Cong L, Geng J, Han Y, Li L, Li W, Hu G, Huang X, Li W, Li J, Liu Z, Li L, Liu J, Qi Q, Liu J, Li L, Li T, Wang X, Lu H, Wu T, Zhu M, Ni P, Han H, Dong W, Ren X, Feng X, Cui P, Li X, Wang H, Xu X, Zhai W, Xu Z, Zhang J, He S, Zhang J, Xu J, Zhang K, Zheng X, Dong J, Zeng W, Tao L, Ye J, Tan J, Ren X, Chen X, He J, Liu D, Tian W, Tian C, Xia H, Bao Q, Li G, Gao H, Cao T, Wang J, Zhao W, Li P, Chen W, Wang X, Zhang Y, Hu J, Wang J, Liu S, Yang J, Zhang G, Xiong Y, Li Z, Mao L, Zhou C, Zhu Z, Chen R, Hao B, Zheng W, Chen S, Guo W, Li G, Liu S, Tao M, Wang J, Zhu L, Yuan L, Yang H. A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science, 2002, 296(5565): 79-92. [9] Goff S A, Ricke D, Lan T H, Presting G, Wang R, Dunn M, Glazebrook J, Sessions A, Oeller P, Varma H, Hadley D, Hutchison D, Martin C, Katagiri F, Lange B M, Moughamer T, Xia Y, Budworth P, Zhong J, Miguel T, Paszkowski U, Zhang S, Colbert M, Sun W L, Chen L, Cooper B, Park S, Wood T C, Mao L, Quail P, Wing R, Dean R, Yu Y, Zharkikh A, Shen R, Sahasrabudhe S, Thomas A, Cannings R, Gutin A, Pruss D, Reid J, Tavtigian S, Mitchell J, Eldredge G, Scholl T, Miller R M, Bhatnagar S, Adey N, Rubano T, Tusneem N, Robinson R, Feldhaus J, Macalma T, Oliphant A, Briggs S. A draft sequence of the rice genomes (Oryza sativa L. ssp. japonica). Science, 2002, 296(5565): 92-100. [10] Kikuchi S, Satoh K, Nagata T, Kawagashira N, Doi K, Kishimoto N, Yazaki J, Ishikawa M, Yamada H, Ooka H, Hotta I, Kojima K, Namiki T, Ohneda E, Yahagi W, Suzuki K, Li C, Ohtsuki K, Shishiki T, Otomo Y, Murakami K, Iida Y, Sugano S, Fujimura T, Suzuki Y, Tsunoda Y, Kurosaki T, Kodama T, Masuda H, Kobayashi M, Xie Q, Lu M, Narikawa R, Sugiyama A, Mizuno K, Yokomizo S, Niikura J, Ikeda R, Ishibiki J, Kawamata M, Yoshimura A, Miura J, Kusumegi T, Oka M, Ryu R,
遗传学报
Acta Genetica Sinica
Vol.33 No.12 2006
Ueda M, Matsubara K, Kawai J, Carninci P, Adachi J, Aizawa K, Arakawa T, Fukuda S, Hara A, Hashizume W, Hayatsu N, Imotani K, Ishii Y, Itoh M, Kagawa I, Kondo S, Konno H, Miyazaki A, Osato N, Ota Y, Saito R, Sasaki D, Sato K, Shibata K, Shinagawa A, Shiraki T, Yoshino M, Hayashizaki Y, Yasunishi A. Collection, mapping, and annotation of over 28 000 cDNA clones from japonica rice. Science, 2003, 301(3631): 376-379. [11] Sasaki T, Matsumoto T, Yamamoto K, Sakata K, Baba T, Katayose Y, Wu J, Niimura Y, Cheng Z, Nagamura Y, Antonio B, H Kanamori, Hosokawa S, Masukawa M, Arikawa K, Chiden Y, Hayashi M, Okamoto M, Ando T, Aoki H, Arita K, Hamada M, Harada C, Hijishita S, Honda M, Ichikawa Y, Idonuma A, Iijima M, Ikeda M, Ikeno M, Ito S, Ito T, Ito Y, Ito Y, Iwabuchi A, Kamiya K, Karasawa W, Katagiri S, Kikuta A, Kobayashi N, Kono I, Machita K, Maehara T, Mizuno H, Mizubayashi T, Mukai Y, Nagasaki H, Nakashima M, Nakama Y, Nakamichi Y, Nakamura M, Namiki N, Negishi M, Ohta I, Ono N, Saji S, Sakai K, Shibata M, Shimokawa T, Shomura A, Song J, Takazaki Y, Terasawa K, Tsuji K, Waki K, Yamagata H, Yamane H, Yoshiki S, Yoshihara R, Yukawa K, Zhong H, Iwama H, Endo T, Ito H, Hahn J, Kim H, Eun M, Yano M, Jiang J, Gojobori T. The genome sequence and structure of rice chromosome 1. Nature, 2002, 420(6915): 312-316. [12] Feng Q, Zhang Y, Hao P, Wang S, Fu G, Huang Y, Li Y, Zhu J, Liu Y, Hu X, Jia P, Zhang Y, Zhao Q, Ying K, Yu S, Tang Y, Weng Q, Zhang L, Lu Y, Mu J, Lu Y, Zhang L, Yu Z, Fan D, Liu X, Lu T, Li C, Wu Y, Sun T, Lei H, Li T, Hu H, Guan J, Wu M, Zhang R, Zhou B, Chen Z, Chen L, Jin Z, Wang R, Yin H, Cai Z, Ren S, Lv G, Gu W, Zhu G, Tu Y, Jia J, Zhang Y, Chen J, Kang H, Chen X, Shao C, Sun Y, Hu Q, Zhang X, Zhang W, Wang L, Ding C, Sheng H, Gu J, Chen S, Ni L, Zhu F, Chen W, Lan L, Lai Y, Cheng Z, Gu M, Jiang J, Li J, Hong G, Xue Y, Han B. Sequence and analysis of rice chromosome 4. Nature, 2002, 420(6913): 316-320. [13] Yu Y, Rambo T, Currie J, Saski C, Kim H, Collura K, Thompson S, Simmons J, Yang T, Nah G, Patel A, Thurmond S, Henry D, Oates R, Palmer M, Pries G, Gibson J, Anderson H, Paradkar M, Crane L, Dale J, Carver M, Wood T, Frisch D, Engler F, Soderlund C, Palmer L, Tetylman L, Nascimento L, Bastide M, Spiegel L, Ware D, O’Shaughnessy A, Dike S, Dedhia N, Preston R, Huang E, Ferraro K, Kuit K, Miller B, Zutavern T, Katzenberger F, Muller S, Balija V, Martienssen R, Stein L, Minx P, Johnson D, Cordum H, Mardis E, Cheng Z, Jiang J, Wilson R, McCombie W, Wing R, Yuan Q, Ouyang S, Liu J, Jones K, Gansberger K, Mof-
LU Yong-Quan et al.: Analysis of the Phylogenetic Relationships Among Several Species of Gramineae Using ACGM Markers
fat K, Hill J, Tsitrin T, Overton L, Bera J, Kim M, Jin S, Tallon L, Ciecko A, Pai G, Van S, Utterback T, Reidmuller S, Bormann J, Feldblyum T, Hsiao J, Zismann V, Blunt S, Vazeilles A, Shaffer T, Koo H, Suh B, Yang Q, Haas B, Peterson J, Pertea M, Volfovsky N, Wortman J, White O, Salzberg S, Fraser C, Buell C, Song R, Fuks G, Llaca V, Kovchak S, Young S, Bowers J, Paterson A, Johns M, Mao L, Pan H, Dean R. The rice chromosome 10 sequencing consortium. In-depth view of structure, activity, and evolution of rice chromosome 10. Science, 2003, 300(5625): 1566-1569. [14] http://www.nju.edu.cn/cps/site/NJU/njuc/plantsweb [15] Gale M D, Devos K M. Comparative genetics in the grass. Proc Natl Acad Sci USA, 1998, 95(5): 1971-1974. [16] Fourmann M, Barret P, Froger N, Baron C, Charlot F, Delourme R, Brunel D. From Arabidopsis thaliana to Brassica napus: development of amplified consensus genetic markers (ACGM) for construction of a gene map.
S. ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res, 1991, 19(14): 4008-4008. [20] Xu S, Tao Y, Yang Z, Chu J. A simple and rapid method used for silver staining and gel preservation. Hereditas (Beijing), 2002, 24(3): 335-336 (in Chinese with an English abstract). [21] Nei M, Li W H. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA, 1979, 76(10): 5269-5273. [22] Felsenstein J. PHILIP-Phylogeny inference package. Cladistics, 1989, 5(1): 164-166. [23] Wu Z, Lu A, Tang Y, Chen Z, Li D. The families and genera of angiosperms in China (A comprehensive analysis). In: Gramineae. Beijing: Science Press, 2003, 322-349 (in Chinese). [24] Chapman G, Peat W. An introduction to the Grassea (including bamboos and cereals). Beijing: Science Press,
Theor Appl Genet, 2002, 105(8): 1196-1206. [17] Lu Y, Wang X, Huang W, Xiao T, Zheng Y, Wu W. Development of amplified consensus genetic markers in Gramineae based on rice intron length polymorphisms.
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1996, 30-49 (in Chinese, translation by Wang Y). [25] http://www.ncbi.nlm.nih.gov. [26] Paterson A H, Bowers J E, Chapman B A. Ancient poly-
Scientia Agricultura Sinica, 2006, 39(3): 433-439 (in
ploidization predating divergence of the cereals, and its
Chinese with an English abstract). [18] Murray M G, Thompson W F. Rapid isolation of highmolecular- weight plant DNA. Nucleic Acids Res, 1980,
consequences for comparative genomics. Proc Natl Acad Sci USA, 2004, 101(26): 9903-9908. [27] Clark L G, Kobayashi M, Mathews S, Spangler R E,
8(19): 4321-4325. [19] Don R H, Cox P T, Wainwright B J, Baker K, Mattick J
Kellogg E A. The Puelioideae, a new subfamily of poaceae. Syst Bot, 2000, 25(2): 181-187.
应用 ACGM 标记分析禾本科几个物种间的系统发生关系 卢泳全1,2，叶子弘3，吴为人1 1. 浙江大学农业与生物技术学院，杭州 310029； 2. 黑龙江省农业科学院生物技术研究中心，哈尔滨 150086； 3. 中国计量学院，杭州 310018 摘 要: 为了验证水稻基因组数据的通用性，利用 ACGM 标记分析了禾本科几个不同种属植物的亲缘关系。选用 10 份材料， 它们分别代表禾本科的 5 个属(Oryza, Zea, Setaria, Triticum, 和 Phyllostachys)。根据遗传距离建立了一个聚类树。这 5 个属 的亲缘关系可以简单地表示为：((Oryza + (Zea + Setaria)) + Triticum) + Phyllostachys。研究结果表明，水稻与玉米或水稻与 粟之间的遗传距离比水稻和小麦或水稻与竹子之间的遗传距离近。 关键词:
ACGM 标记；遗传关系；禾本科；基因组；通用性
作者简介: 卢泳全（1974-），女，黑龙江人，博士，研究方向：植物分子生物学。E-mail: [email protected]
Acta Genetica Sinica, December
2006, 33 (12)：1127–1131
ISSN 0379-4172
Analysis of the Phylogenetic Relationships Among Several Species of Gramineae Using ACGM Markers LU Yong-Quan1,2, YE Zi-Hong3, WU Wei-Ren1,① 1. Department of Agronomy, College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310029, China; 2. Biotechnology Research Center, Heilongjiang Academy of Agricultural Sciences, Haerbin 150086, China; 3. College of Life Science, China Jiliang University, Hangzhou 310018, China Abstract: To study the transferability of rice (Oryza sativa L.) genome data, we used amplified consensus genetic markers to analyze the phylogenetic relationships among several species and genera in Gramineae. Ten accessions representing five grass genera (Oryza, Zea, Setaria, Triticum, and Phyllostachys) were used. According to the genetic distances, a cluster tree was constructed. The relationships among the five genera could be simply described as ((Oryza + (Zea + Setaria)) + Triticum) + Phyllostachys. The results suggest that the genetic distance between rice and maize (Z. mays L.) or rice and millet (Setaria italica L.) is closer than that between rice and wheat (Triticum aestivum L) or rice and bamboo. Key words: ACGM markers; phylogenetic relationship; Gramineae; genome; transferability
Botstein et al.[1] were the first to use restriction fragment length polymorphisms as genetic markers to construct a human genetic map. Since then, many new molecular marker techniques have been developed, such as random amplified polymorphic DNA[2], amplified fragment length polymorphism[3], microsatellite or simple sequence repeat[4,5], sequence-related amplified polymorphism[6], and single-nucleotide polymorphism[7]. Good molecular marker systems are very useful tools for genetic research (e.g., constructing genetic maps, mapping genes or quantitative trait loci) and breeding (e.g., marker-assisted selection). Draft genome sequences of two rice (Oryza sativa L.) cultivars, 93-11[8] and Nipponbare[9], representing indica and japonica subspecies, respectively, have been completed. A set of over 28 000 full-length cDNA sequences from Nipponbare has been released[10]. Complete sequences of chromosomes 1, 4, -
and 10 of Nipponbare have been published[11 13]. In addition, a tentative assembly of all chromosomes of
rice has been released (The Institute of Genomic Research, TIGR; http://www.tigr.org). These data provide an opportunity to systematically search for DNA polymorphisms and to exploit DNA markers on a large scale in rice. Gramineae is a major family among the angiosperms, consisting of five to six subfamilies, 60-80 tribes, 720-765 genera, and more than 10 000 species[14]. Most food crops, e.g., wheat (Triticum aestivum L.), rice, and maize (Zea mays L.), and forage plants belong to this family. It is impossible to sequence the genome of each species. Comparative genomics studies have shown that linear organization exists among different genomes in Gramineae[15]. Therefore, the information of rice genome could be used to study the genetic basis of other plants in Gramineae. The closer the genetic relationship of a species to rice, the better the utilization of the information. Amplified consensus genetic marker (ACGM) is a polymerase chain reaction (PCR)-based marker
Received: 2005-11-14; Accepted: 2006-04-10 This work was supported by the China National Programs for High Technology Research and Development (863 Program) (No. 2003AA207160). ① Corresponding author. E-mail: [email protected]; Tel: +86-571-8697 1910
遗传学报
1128
with primers designed in conservative regions of coding sequences[16]. Therefore, ACGM would be quite useful for phylogenetic studies. In this study, we applied the ACGM markers to analyzing the phylogenetic relationships among several species and genera in Gramineae. Our purposes were to examine the feasibility of using ACGM markers exploited from rice for the phylogenetic study in Gramineae and to provide reference for the use of rice genome data to the genetic study of other species in Gramineae.
1
Materials and Methods
1. 1
Plant materials
Ten accessions representing five grass genera were used (Table 1), including japonica rice variety Nipponbare and indica rice variety 93-11; two maize inbred lines F683 and F743 (bred by Zhejiang University); two millet (Setaria italica L.) lines 4a and 21; two wheat species T. macha and T. spelta (collected from the Plant Garden of Zhejiang University); and two bamboo species Phyllostachys atrovaginata C. and P. propinqua C. (collected from the Plant Garden of Hangzhou). Table 1 No. 1 2 3 4 5 6 7 8 9 10
1. 2
Plant materials used in this research Plant materials Oryza sativa ssp. japonica var. Nipponbare O. sativa ssp. indica var. 93-11 Zea mays var. F683 Z. mays var. F743 Setaria italica var. 4a S. italica var. No.21 Triticum macha T. spelta Phyllostachys atrovaginata P. propinqua
Acta Genetica Sinica
Vol.33 No.12 2006
containing 50 ng of template DNA, 0.5 μmol/L of each primer, 200 μmol/L of each dNTP, 1.5 mmol/L of MgCl2, 0.1% of Triton X-100, 1 unit of Taq polymerase, and 1.5 μL of 10× PCR reaction buffer. A touchdown-PCR[19] program was used: 5 min at 94℃; 10 cycles of 30 s at 94℃, 30 s at 59℃ minus 0.3℃/cycle, 1 min at 72℃; 20 cycles of 30 s at 94℃, 30 s at 56℃, 1 min at 72℃; and 5 min at 72℃ for a final extension. For primer pairs that did not generate good amplification results, we adjusted the initial annealing temperature from 55℃ to 60℃. Each of the primer pairs was tested twice to confirm the repeatability of the observed bands in each genotype. PCR products were separated on 6% nondenaturing polyacrylamide gel electrophoresis (80 volts, 2.5 h). Gels were silver stained for visualizing DNA bands, following the procedure of Xu et al[20]. 1. 3
Statistical analysis of phylogenetic relationship
Bands were scored as 1 (presence) or 0 (absence). Genetic distances between accessions were calculated according to Nei and Li[21]. The distance matrix was used to construct the Unweighted PairGroup Method Using Arithmetic averages (UPGMA) dendogram using the NEIGHBOR model of the PHILIP Version 3.6c[22].
Genome 2n=2x=24
2 2n=2x=24 2n=2x=20 2n=2x=20 2n=2x=18 2n=2x=18 2n=6x=42 2n=6x=42 2n=2x=48 2n=2x=48
Results
All the primers used could yield stable PCR products in each accession (Fig. 1). In general, the
Detection of ACGM
Thirty-eight pairs of ACGM primers[17] were used in the experiment. Modified CTAB (cetyltrimethylammonium bromide) method[18] was used to extract DNA from young leaves of each plant material. PCR was performed in a 15 μL reaction mixture
Fig. 1 PCR products obtained from primer pair GA24 in different accessions The lane codes 1-10 are consistent with the accession codes shown in Table 1.
LU Yong-Quan et al.: Analysis of the Phylogenetic Relationships Among Several Species of Gramineae Using ACGM Markers
Fig. 2
The UPGMA tree of the 10 grass accessions based on 38 ACGM markers
distances between different genera were relatively large whereas those within genera were relatively small. According to the genetic distances, a cluster tree was constructed (Fig. 2). It was as expected that accessions of a same genus formed a subgroup before being grouped with other genera. The relationship among the five genera could be simply described as (((Oryza+(Zea+Setaria))+Triticum)+Phyllostachys).
3
1129
Discussion
In this study, we have analyzed the genetic evolutionary relationships among five major genera belonging to the grass family (i.e., Oryza, Zea, Setaria, Triticum, and Phyllostachys) using newly developed ACGM markers[17]. The clustering result is consistent -
with those obtained in previous studies[23 25]. For example, according to traditional morphological classification, Zea and Setaria belong to the family Paniceae[23]. In this study, Zea and Setaria are classified together prior to other genera. In the early classification system of the grass family, Oryza was included into Bambusoideae[24]. However, we found that there was a certain distance between Oryza and Phyllostachys, which is consistent with the modern classification system[25]. Paterson et al.[26] drew a systematic evolutionary tree for phanerogam based on the study of whole-genome duplication of crops in grass family. In their evolutionary tree, Zea, Sorghum, Oryza, and Hordeum cluster together, which can be simply described
as: Hordeum + (Oryza + (Sorghum + Zea)). In this study, Triticum was selected instead of Hordeum, but both genera belong to Pooideae Triticeae. Therefore, the result of this study is similar to that they obtained. Another remarkable conclusion of this work is that Phyllostachys is at the base of the cluster tree. Bamboo has a significant position in evolution. Since the 1950s, wooden bamboo or some herbaceous communities with biological characters similar to bamboo have been considered to be the original community of the grass family[23]. Recently, new knowledge about Bambusoideae was gained and corresponding experimental supports were found with the development of molecular biology[23]. On the basis of nucleic acid sequence analysis, Clark et al.[27] reported that Bambusoideae and Pooideae are closely related, which form a sister group of Oryza. This is consistent with our result. References: [1] Botstein D, White R L, Skolnick M, Davis R W. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet, 1980, 32(3): 314-331. [2] Williams J G, Kubelik A R, Livak K J, Rafalski J A, Tingey S V. DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res, 1990, 18(22): 6531-6535. [3] Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res, 1995, 23(21): 4407-4414.
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应用 ACGM 标记分析禾本科几个物种间的系统发生关系 卢泳全1,2，叶子弘3，吴为人1 1. 浙江大学农业与生物技术学院，杭州 310029； 2. 黑龙江省农业科学院生物技术研究中心，哈尔滨 150086； 3. 中国计量学院，杭州 310018 摘 要: 为了验证水稻基因组数据的通用性，利用 ACGM 标记分析了禾本科几个不同种属植物的亲缘关系。选用 10 份材料， 它们分别代表禾本科的 5 个属(Oryza, Zea, Setaria, Triticum, 和 Phyllostachys)。根据遗传距离建立了一个聚类树。这 5 个属 的亲缘关系可以简单地表示为：((Oryza + (Zea + Setaria)) + Triticum) + Phyllostachys。研究结果表明，水稻与玉米或水稻与 粟之间的遗传距离比水稻和小麦或水稻与竹子之间的遗传距离近。 关键词:
ACGM 标记；遗传关系；禾本科；基因组；通用性
作者简介: 卢泳全（1974-），女，黑龙江人，博士，研究方向：植物分子生物学。E-mail: [email protected]