全 文 ：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

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: wuwr@zju.edu.cn; Tel: +86-571-8697 1910
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 devel-
oped, such as random amplified polymorphic DNA[2],
amplified fragment length polymorphism[3], microsa-
tellite 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., construct-
ing genetic maps, mapping genes or quantitative trait
loci) and breeding (e.g., marker-assisted selection).
Draft genome sequences of two rice (Oryza sa-
tiva L.) cultivars, 93-11[8] and Nipponbare[9], repre-
senting indica and japonica subspecies, respectively,
have been completed. A set of over 28 000 full-length
cDNA sequences from Nipponbare has been re-
leased[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 Re-
search, TIGR; http://www.tigr.org). These data pro-
vide 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 angio-
sperms, consisting of five to six subfamilies, 60-80
tribes, 720-765 genera, and more than 10 000 speci-
es[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
1128 遗传学报 Acta Genetica Sinica Vol.33 No.12 2006
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 phy-
logenetic 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 Uni-
versity); 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 Plant materials used in this research
No. Plant materials Genome
1 Oryza sativa ssp. japonica var.
Nipponbare
2n=2x=24
2 O. sativa ssp. indica var. 93-11 2n=2x=24
3 Zea mays var. F683 2n=2x=20
4 Z. mays var. F743 2n=2x=20
5 Setaria italica var. 4a 2n=2x=18
6 S. italica var. No.21 2n=2x=18
7 Triticum macha 2n=6x=42
8 T. spelta 2n=6x=42
9 Phyllostachys atrovaginata 2n=2x=48
10 P. propinqua 2n=2x=48


1. 2 Detection of ACGM
Thirty-eight pairs of ACGM primers[17] were
used in the experiment. Modified CTAB (cetyl-
trimethylammonium bromide) method[18] was used to
extract DNA from young leaves of each plant mate-
rial. PCR was performed in a 15 μL reaction mixture
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 poly-
merase, 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 re-
peatability 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 relation-
ship
Bands were scored as 1 (presence) or 0 (ab-
sence). Genetic distances between accessions were
calculated according to Nei and Li[21]. The distance
matrix was used to construct the Unweighted Pair-
Group Method Using Arithmetic averages (UPGMA)
dendogram using the NEIGHBOR model of the
PHILIP Version 3.6c[22].
2 Results
All the primers used could yield stable PCR
products in each accession (Fig. 1). In general, the

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 1129


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 Discussion
In this study, we have analyzed the genetic evo-
lutionary relationships among five major genera be-
longing 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 classi-
fied together prior to other genera. In the early classi-
fication system of the grass family, Oryza was in-
cluded 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- ge-
nome duplication of crops in grass family. In their
evolutionary tree, Zea, Sorghum, Oryza, and Hor-
deum 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 cor-
responding experimental supports were found with
the development of molecular biology[23]. On the ba-
sis 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.
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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: luyongquan@126.com