Plant Science 169 (2005) 976–984

www.elsevier.com/locate/plantsci

A preliminary assessment of the genetic relationship

between Erianthus rockii and the ‘‘Saccharum complex’’
using microsatellite (SSR) and AFLP markers
Q. Cai a, K.S. Aitken b,*, Y.H. Fan a, G. Piperidis c, P. Jackson d, C.L. McIntyre b
a
Yunnan Sugar cane Research Institute, Academy of Agriculture Sciences, Eastern Lingquan Road 363, Kaiyuan, Yunnan 661600, China
b
CSIRO Division of Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, Qld 4067, Australia
c
David North Plant Research Centre, Bureau of Sugar Experiment Stations, 50 Meiers Rd, Indooroopilly, Brisbane, Qld 4068, Australia
d
Davies Laboratory, CSIRO Division of Plant Industry, University Drive, Townsville, Qld 4814, Australia
Received 9 May 2005; received in revised form 6 July 2005; accepted 7 July 2005
Available online 1 August 2005

Abstract

Erianthus rockii is a drought and cold tolerant wild relative of sugar cane from China that is currently being used in sugar cane
introgression programs. It has not been described outside of China. Although classified as an Erianthus species using morphological
characters, its relationship with other Erianthus species and other species in the Saccharum complex is not well understood. Genotypes
representing seventeen species from five genera in the Saccharum complex were evaluated with approximately 200 microsatellite (SSR) and
amplified fragment length polymorphism (AFLP) markers and similarity matrices constructed. Principal component analysis (PCA) was
undertaken separately with the AFLP and SSR data. The results from both data sets were very similar and suggested that E. rockii was distinct
from other Erianthus and Saccharum species. E. rockii clustered with E. fulvus and two Miscanthus species, mid-way between the major
Saccharum and Erianthus clusters.
# 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: AFLP; Erianthus rockii; Saccharum complex; SSR

1. Introduction robustum in Hawaii [3] and Miscanthus spp. in Taiwan [4].

The ‘‘Saccharum complex’’ consists of five species of the
genus Saccharum plus closely related genera including
Erianthus sect. Ripidium, Miscanthus sect. Diandra Keng,
Narenga and Sclerostachya [1]. Most modern sugar cane
cultivars (Saccharum spp.) are derived principally from
hybrids of interspecific crosses between a small number of
Saccharum officinarum and S. spontaneum clones [2]. This
restricted genetic base could limit progress in sugar cane
breeding programs. Consequently, introgression of genes
from wild species is being used to increase the genetic
variability in sugar cane. The focus in most breeding programs
has generally been on using S. spontaneum but other species
of the ‘‘Saccharum complex’’ have been used, including S.
* Corresponding author. Tel.: +61 07 3214 2360; fax: +61 07 3214 2950.
E-mail address: karen.aitken@csiro.au (K.S. Aitken).
More recently, interest has increased in using Erianthus spp.
[5,6], due largely to desirable phenotypic characters in this
genus such as vigour, drought tolerance, waterlogging
tolerance, and disease resistance. To date, only a very few
intergeneric hybrids have been reported. These include a
hybrid between Saccharum spp. and Miscanthus spp. [7] and
several hybrids between S. officinarum and E. arundinaceus
[7–10].
Erianthus rockii Keng [11], a wild species originating in
the Yunnan, Sichuan and the Tibetan regions of China, is of
interest to sugar cane breeders in China as it is drought and
cold tolerant. E. rockii has been classified as one of eight
species in Erianthus [12], however, it is not mentioned in
other classification systems of sugar cane [13]. Attempts to
introgress desirable traits from E. rockii began in the early
1990’s at the Yunnan Sugar cane Research Institute (YSRI)
in Kaiyuan, China, and hybrids between E. rockii and a

0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2005.07.002
 978 Q. Q.
CaiCai
et et
al.al.
/ Plant
/ Plant
Science
Science
169169
(2005)
(2005)
976–984
976–984 977

commercial sugar cane variety (Saccharum spp.) were Table 1

studied, their source and chromosome numbers [21] (where known)

on morphological characters and agronomic data, for use as a

No. Accession Species Name S Ch. No.

parents in further crossing programs, but there has been no
molecular verification of these hybrids to date. Despite the 1 Q96 Variety B
2 Q117 Variety B
strong interest in using this species for introgression, 3 TROJAN Variety B
especially in China, the relationship between E. rockii and 4 NCO310 Variety B
other Erianthus species or other species in the Saccharum 5 66N2008 Variety B
complex is not well understood. A study on the phyloge- 6 POJ 2878 Variety B
netics of Miscanthus, Saccharum and related genera using 7 Red Luohan S. officinarum Y

8 Badila S. officinarum B 80
sequence data from the ITS region clustered E. rockii with N. 9 Korpi S. officinarum B 80
porphyrocoma and E. fulvus [15]. 10 Luohanzhe S. officinarum Y
A number of different DNA markers have been used 11 Songxu Bainianzhe S. sinense Y
to determine levels of genetic variation and phylogenetic 12 Guangxi Zhuzhe S. sinense Y
relationships in the ‘‘Saccharum complex’’ including 13 Guilin Zhuzhe S. sinense Y
14 Qianwei Luzhe S. sinense Y
RAPDs [16,17], 5S rRNA intergenic spacer region 15 Sichuan Luzhe S. sinense Y
[18,19], the internal transcribed spacer region of rDNA 16 Uba S. sinense Y
[20], RFLPs [21,22], SSR’s from maize [23] and AFLP’s 17 Nagans S. barberi Y
[24,25]. One of the major advantages of AFLP markers [26] 18 Pansahi S. barberi Y
is that they allow the simultaneous analysis of large numbers 19 Nargori S. barberi Y 116
20 Mungo S. barberi Y
of marker loci throughout the genome. This feature is 21 IJ73-414 S. robustum B
especially useful in sugar cane and its relatives as almost all 22 IJ76-352 S. robustum B
species of Saccharum and Erianthus are highly polyploid 23 IJ76-411 S. robustum B
(Saccharum: 6x ! 12x; Erianthus: 2x ! 6x), with large 24 IJ76-416 S. robustum B
chromosome numbers [21,27]. AFLP’s have already been 25 YN 75-1-7 S. spontaneum Y 80
26 Longchuan16 S. spontaneum Y
successfully used for the analysis of genetic diversity within 27 Burma S. spontaneum B 96
and between Erianthus species [24] and between sugar cane 28 Coimbatore S. spontaneum B 64
varieties [25]. They have also been used to determine genetic 29 Djatiroto S. spontaneum B 112
diversity in a number of other plant species [28,29]. More 30 Glagah-1286 S. spontaneum B 112
recently, SSR’s have become increasingly popular for 31 IK76-6 S. spontaneum B
32 Mandalay S. spontaneum B 96
molecular genetic studies due to their reproducibility, ease of 33 YN 97-3 E. fulvus Y
scoring and fast throughput compared to other marker 34 YN 97-4 E. fulvus Y
techniques. They have also been found to be highly 35 Rockii82-93 E. rockii Y
polymorphic [30,31]. More than 200 SSR’s have now been 36 Rockii83-224 E. rockii Y
isolated from sugar cane [32] and have been used to map the 37 Rockii95-19 E. rockii Y
38 Rockii95-20 E. rockii Y
sugar cane genome [33]. 39 YN 82/53 E. arundinaceus Y
There is worldwide interest in introgression of new genes 40 YN 82/69 E. arundinaceus Y
from wild species into sugar cane. As this species has not 41 YN 82/85 E. arundinaceus Y
been described outside China, we were interested in studying 42 YN 82/118 E. arundinaceus Y 40
the similarity between E. rockii and other Erianthus and 43 YN 82/133 E. arundinaceus Y
44 YN 82/143 E. arundinaceus Y 60
Saccharum species. Does E. rockii appear to be distinct from 45 YN 82/156 E. arundinaceus Y
other Erianthus and Saccharum species or is it similar to 46 YN 95/21 E. arundinaceus Y
other well-characterised species, but known by a different 47 HN 92/84 E. arundinaceus Y
name in China? In this study, we have used both SSR’s and 48 HN 92/85 E. arundinaceus Y
AFLP’s to undertake a preliminary assessment of the 49 HN 92/109 E. arundinaceus Y
50 IJ76-476 E. arundinaceus B 60
relationship between E. rockii and other members of the 51 IK76-22 E. arundinaceus B 60
Saccharum complex. 52 IK76-48 E. arundinaceus B 60
53 IK76-79 E. arundinaceus B 60
54 IS76-126 E. arundinaceus B 60
2. Materials and methods 55 SES305 E. elephantinus B 20
56 SES309 E. procerus B
57 US67-8-1 E. ravennae B 20
2.1. Plant material for the diversity analysis 58 Bengalense E. bengalense B
59 Sarpet E. sarpet B 30
Sixty-nine clones representing 17 species from five 60 YN 95-9 M. sinensis Y
genera were used in this study (Table 1). Of the 69 plant 61 YN 95-10 M. sinensis Y
62 YN 95-11 M. sinensis Y
samples, 41 were taken from the National Nursery of Sugar
978 Q. Q.
CaiCai
et et
al.al.
/ Plant
/ Plant
Science
Science
169169
(2005)
(2005)
976–984
976–984 977

T able 1 (Continued )
a
No. Accession Species Name S Ch. No.
63 YN 95-17 M. sinensis Y 60
64 Wujiemang 82–122 M. floridulus Y
65 YN 82/102 N. porphyrocoma Y
66 YN 83/199 N. porphyrocoma Y exposed to Kodak X-ray film for 3–4 days.
67 GD 25 N. porphyrocoma Y 30
68 GD 64 N. porphyrocoma Y 30
69 YN 82-21 P. schumach Y AFLP analysis [26] was carried out using 300–500 ng of
a
The source of the germplasm, B is BSES, Australia and Y is Yunnan
Sugar cane Research Institute, China.
cane Germplasm Resources (NNSGR) at the Yunnan Sugar
cane Research Institute (YSRI) in China and 28 were
obtained from the Bureau of Sugar Experiment Stations
(BSES) in Australia. All genera in the ‘‘Saccharum
complex’’ except Sclerostachya, were included in the
diversity study. Where possible, accessions were selected
to encompass a range in the geographical distribution and
chromosome number of a species. Six cultivars were also
included to represent commercially grown sugar cane. The
17 species surveyed included five species from Saccharum
(S. officinarum, S. sinense, S. barberi, S. robustum, S.
spontaneum), eight species from Erianthus (E. arundina-
ceus, E. elephantinus, E. procerus, E. ravennae, E.
bengalense, E. sarpet, E. fulvus, E. rockii), two from
Miscanthus (M. floridulus, M. sinensis), Narenga porphyr-
ocoma and Pennisetum schumach (Table 1). Depending on
available germplasm from 1 to 16 individuals per species
were included in the analysis (Table 1).
2.2. DNA isolation
Young leaves were sampled from the field, freeze-dried,
then ground to a powder and stored at 20 8C. Genomic
DNA was extracted following the CTAB method [34]. The
quality and quantity of the DNA was checked on a 0.75%
agarose gel and diluted appropriately for PCR.
2.2.1. Selection of SSR’s and screening of diversity
samples
SSR primers from the sugar cane microsatellite
consortium collection were screened across samples from
three species (S. officinarum, S. spontaneum and E.
arundinaceus) and 10 robust and polymorphic SSR’s were
selected for the diversity study (Table 2). PCR reactions
were carried out in a total of 20 ml containing 25 ng template
DNA, 0.2 mM of forward and reverse primers, 0.2 mM of
each dNTP, 3.0 mM MgCl2 and 0.4 U Tth plus Taq
polymerase (Biotech International) and accompanying
buffer. During PCR, the SSR products were labelled with
a 33P dCTP (3000 Ci/mmol). Reactions were run on a Gene
Amp1 PCR System 2700 thermal cycler. Cycling conditions
were 3 min at 94 8C followed by 35 cycles of 1 min at 94 8C,
2 min at the appropriate annealing temperature (ranged from
50 to 56 8C depending on SSR), 1 min at 72 8C and a final
extension step of 5 min at 72 8C. The amplified products
were mixed with an equal volume of loading dye, denatured
at 95 8C for 5 min, and 3.6 ml was run on a denaturing 5%
polyacrylamide (20:1) gel at 90 W for 2 h. The gels were
subsequently dried using a gel dryer for 25 min at 80 8C and
2.2.2. AFLP analysis of diversity samples
genomic DNA which was double-digested with EcoRI and
MseI and linked to the specific adapters. Primers with one
selective nucleotide were used for preamplification. EcoRI
primers with three selective nucleotides were end-labelled
with g-33PATP and mixed with unlabelled MseI primers for
hot selective amplification. All PCR reactions were carried
out in a Gene Amp1 PCR System 2700. PCR products
were separated as described above, but gels were exposed
to high resolution Kodak Biomax MR X-ray film for 3–4
days.
2.2.3. PCA analysis of diversity data
As almost all species within the Saccharum complex are
polyploid and given the lack of segregation data for sugar
cane species and relatives, no assumptions on the genetic
allelism of SSR fragments could be made. Therefore, both
SSR and AFLP data were scored for the presence (1) or
absence (0) of bands. The data was transformed to a matrix
of similarity coefficients using Jaccard’s coefficient,
Gsij = a/(a + b + c), where Gsij is the measure of genetic
similarity between individuals i and j, a is the number of
polymorphic bands present in both individuals, b is the
number of bands present in i and absent in j, and c is the
number of bands present in j and absent in i. This definition
of similarity excludes bands that are absent in both
individuals from the calculation. The reason for this is
the lack of an AFLP band in two genotypes may not be due
to a common evolutionary event and the presence of multiple
alleles at SSR loci inflates genetic similarity if 0, 0 matches
are treated as informative. Principal component analysis was
performed using the eigen computational modules in
NTSYS [35].
3. Results
3.1. Genetic similarity measured using AFLP’s
Two AFLP primer combinations were used and detected
a total of 173 bands of which only one was monomorphic
across all 17 species. Table 2 summarises the number of
markers scored for each species studied. Using AFLP’s, each
primer pair generated from 24 to 37 markers for Saccharum
spp., 22–50 markers for Erianthus spp., 12–33 for
Miscanthus spp. and 19–35 for Narenga sp. Overall,
Saccharum produced the highest number of AFLP markers
(56–74) and Pennisetum the lowest (22) (Table 2). The S.
sinense accessions had the most AFLP markers of any
Table 2
The number of markers detected by both AFLP and SSR primers in each species
Species AFLP’s SSR’s
a b
ACC/CTC ACG/CTG T Av SSCIR 11 SSCIR 12 SSCIR 19 SSCIR 21 SSCIR 26 SSCIR 33 SSCIR 36 SSCIR 54 SMC 1608cl SMC 1825la T Av
Varieties 29 34 63 31.5 11 10 7 16 12 8 15 14 9 7 109 10.9
S. officinarum 24 32 56 28 9 5 8 14 6 5 13 10 9 5 84 8.4
S. sinense 37 37 74 37 11 4 12 18 11 7 11 15 7 6 102 10.2

Q. Cai et al. / Plant Science 169 (2005) 976–984

S. barberi 36 35 71 35.5 11 7 12 16 8 8 15 17 9 7 110 11.0
S. robustum 36 33 59 34.5 13 7 12 18 5 8 11 16 7 6 103 10.3
S. spontaneum 29 32 61 30.5 12 5 24 20 17 10 19 16 7 7 137 13.7
E. fulvus 12 13 25 12.5 3 1 2 14 2 1 3 9 2 2 39 3.9
E. rockii 50 22 72 36 14 2 5 14 10 5 10 19 5 3 87 8.7
E. arundinaceus 31 26 57 28.5 3 1 3 1 16 3 15 13 4 3 62 6.2
E. elephantinus 21 15 36 18 1 1 1 1 2 3 1 5 2 1 18 1.8
E. procerus 27 17 44 22 2 1 1 1 4 2 2 5 4 1 23 2.3
E. ravennae 23 15 38 19 1 1 2 1 4 3 2 4 4 1 23 2.3
E. bengalense 27 17 44 22 1 1 2 1 5 2 3 6 1 1 23 2.3
E. sarpet 21 18 39 19.5 2 1 3 1 5 3 1 5 1 1 23 2.3
M. sinensis 33 16 49 24.5 1 2 2 10 12 4 6 9 3 2 51 5.1
M. floridulus 20 12 32 16 2 2 1 4 6 2 5 9 2 2 35 3.5
N. porphyrocoma 35 19 54 27 10 1 4 7 11 2 5 13 4 2 59 5.9
P. schumach 15 7 22 11 1 2 2 2 4 1 3 3 1 1 20 20
Total 108 65 173 25.2 22 16 43 28 21 16 30 31 23 11 241 24.1
a
Total number of bands.
b
Average numbers of bands.

979
 980
Table 3
Intra- and inter-specific similarities (Jaccards coefficient) using (a) AFLP and (b) SSR data
(a)
V S.o S.si S.b S.r S.s E.f E.r E.aC E.aI E.e E.p E.ra E.b E.s M.s M.f N.p
Variety 0.81
S. officinarum 0.77 0.82
S. sinense 0.68 0.61 0.83
S. barberi 0.70 0.62 0.74 0.78
S. robustum 0.61 0.57 0.59 0.64 0.73
S. spontaneum 0.46 0.37 0.51 0.55 0.49 0.68
E. fulvus 0.17 0.16 0.17 0.19 0.21 0.25 0.88
E. rockii 0.18 0.16 0.17 0.18 0.17 0.21 0.16 0.48
E. arundinaceus C 0.11 0.12 0.10 0.11 0.12 0.11 0.14 0.16 0.79
E. arundinaceus I 0.11 0.11 0.09 0.09 0.10 0.10 0.13 0.17 0.79 0.93
E. elephantinus 0.10 0.10 0.09 0.08 0.10 0.10 0.13 0.16 0.60 0.72
E. procerus 0.12 0.12 0.10 0.11 0.12 0.10 0.13 0.16 0.77 0.80 0.70

Q. Cai et al. / Plant Science 169 (2005) 976–984

E. ravennae 0.12 0.13 0.10 0.12 0.14 0.10 0.15 0.15 0.60 0.65 0.76 0.71
E. bengalense 0.10 0.10 0.10 0.11 0.12 0.11 0.13 0.17 0.75 0.68 0.57 0.76 0.61
E. sarpet 0.12 0.13 0.10 0.12 0.12 0.10 0.17 0.17 0.71 0.73 0.63 0.77 0.67 0.69
M. sinensis 0.13 0.11 0.14 0.14 0.14 0.14 0.11 0.21 0.11 0.10 0.11 0.12 0.12 0.13 0.13 0.64
M. floridulus 0.09 0.09 0.11 0.11 0.11 0.10 0.10 0.16 0.12 0.09 0.10 0.12 0.13 0.15 0.13 0.58
N. porphyrocoma 0.14 0.12 0.13 0.14 0.14 0.13 0.15 0.23 0.20 0.19 0.23 0.18 0.17 0.21 0.21 0.18 0.15 0.76
P. schumach 0.06 0.06 0.06 0.08 0.07 0.09 0.11 0.14 0.15 0.15 0.16 0.16 0.15 0.16 0.15 0.15 0.18 0.21

(b)
V S.o S.si S.b S.r S.s E.f E.r E.aC E.aI E.e E.p E.ra E.b E.s M.s M.f N.p
Variety 0.52
S. officinarum 0.44 0.49
S. sinense 0.38 0.39 0.59
S. barberi 0.47 0.43 0.45 0.42
S. robustum 0.33 0.29 0.33 0.34 0.36
S. spontaneum 0.29 0.23 0.31 0.29 0.29 0.32
E. fulvus 0.21 0.23 0.22 0.23 0.22 0.20 0.31
E. rockii 0.19 0.19 0.19 0.20 0.18 0.16 0.12 0.29
E. arundinaceus C 0.08 0.07 0.09 0.08 0.05 0.08 0.10 0.08 0.48
E. arundinaceus I 0.06 0.04 0.08 0.05 0.04 0.06 0.09 0.06 0.40 0.65
E. elephantinus 0.04 0.04 0.04 0.05 0.05 0.04 0.07 0.08 0.28 0.30
E. procerus 0.04 0.05 0.05 0.04 0.03 0.04 0.07 0.05 0.43 0.37 0.37
E. ravennae 0.06 0.06 0.06 0.06 0.04 0.05 0.11 0.07 0.34 0.35 0.52 0.44
E. bengalense 0.06 0.05 0.07 0.05 0.05 0.05 0.11 0.07 0.37 0.40 0.32 0.39 0.28
E. sarpet 0.07 0.07 0.09 0.07 0.05 0.08 0.10 0.09 0.48 0.43 0.34 0.45 0.41 0.30
M. sinensis 0.11 0.11 0.11 0.12 0.08 0.10 0.08 0.16 0.16 0.10 0.11 0.15 0.17 0.12 0.14 0.51
M. floridulus 0.10 0.08 0.11 0.11 0.07 0.09 0.04 0.16 0.18 0.14 0.18 0.18 0.21 0.18 0.15 0.46
N. porphyrocoma 0.10 0.09 0.12 0.10 0.10 0.11 0.09 0.14 0.11 0.11 0.07 0.09 0.11 0.07 0.13 0.09 0.09 0.35
P. schumach 0.10 0.09 0.11 0.11 0.10 0.10 0.10 0.10 0.08 0.10 0.03 0.08 0.05 0.08 0.10 0.14 0.10 0.14
 981 Q. Q.
CaiCai
et et
al.al.
/ Plant
/ Plant
Science
Science
169169
(2005)
(2005)
976–984
976–984 981

Fig. 1. Principal component analysis using both AFLP and SSR data: (a) AFLP data Components 1 and 2; (b) AFLP data Components 1 and 3; (c) SSR data
Components 1 and 2; (d) SSR data Components 1 and 3. (*): variety, (~): S. officinarum, ( ): S. spontaneum, ( ): S. robustum, (*): S. barberi, (~): S. sinense,
(&): E. arundinaceus (Indonesia), (&): E. arundinaceus (China), (‘): E. procerus, ( ): E. rockii, ($): E. fulvus, (§): E. elephantinum, ( ): E. ravenae, (^): E.
bengalense, (^): E. sarpet, ( : M. sinensis,( ): M. floridulus,( ): N. porphyrocoma, ( ): P. schumach.

species (74), followed by E. rockii (72), with E. fulvus (24)
and P. schumach (22) having the least (Table 2).
Using AFLP’s, the genetic similarity coefficients varied
from a minimum of 0.06 to a maximum of 0.93 with an
average of 0.3 (Table 3). Within species similarities ranged
from 0.93 for the E. arundinaceous clones from Indonesia
to 0.48 for the four E. rockii clones. One of the E. rockii
clones, Rockii95-19 was much less similar to the other
three clones, and removal of this clone increased the genetic
similarity coefficient to 0.84. Erianthus procerus and E.
arundinaceus (China) were the most similar species at 0.80,
closely followed by the E. arundinaceus clones from
Indonesia and China (0.79) and E. sarpet and E. procerus
(0.77) and E. procerus and E. arundinacues (China) (0.77).
Erianthus fulvus and E. rockii were least similar to other
Erianthus species. Within Saccharum, the S. officinarum
and S. sinense clones were the most similar at 0.82 and
0.83, respectively. Saccharum officinarum compared to the
varieties was the most similar at 0.79 but compared to S.
spontaneum was the least similar at 0.37. Erianthus rockii
was most similar on average to N. porphyrocoma (0.23)
and M. sinensis (0.21) and least similar to P. schumach
(0.14).
3.2. Genetic similarity measured using SSR’s
All 10-primer pairs successfully amplified multiple
fragments in each of the species analysed. All SSR’s were
polymorphic between species and within the Saccharum
species studied. A total of 241 markers were generated using
the SSR’s. Using SSR’s, the average numbers of markers/
SSR ranged from 8.4 to 13.7 for Saccharum spp., 1.8–8.7 for
Erianthus spp., 3.5–5.1 for Miscanthus spp., and 5.9 for
Narenga spp. Each SSR generated from 11 to 43 bands
across the species studied, with Saccharum species having
the highest number of markers overall and Miscanthus
species having the lowest (Table 2). Saccharum spontaneum
was the species with the highest number of markers (84–
110) and E. elephantinus (18) was the species with the least.
Using the SSR data, the Jaccard’s genetic similarity
coefficients ranged from a minimum of 0.04 between S.
officinarum and E. arundinaceus to a maximum of 0.52
between E. elephantinus and E. ravennae and averaged 0.18
(Table 3). Within Erianthus species similarities ranged from
0.64 for the Indonesian E. arundinaceus to 0.28 for E. rockii.
Again Rockii95-19 did not cluster with the other E. rockii
species tested and, if this was removed from the group the
982 Q. Q.
CaiCai
et et
al.al.
/ Plant
/ Plant
Science
Science
similarity increased to 0.41. Erianthus ravennae was most
similar to E. elephantinus (0.52) and E. fulvus and E. rockii
were most dissimilar to other Erianthus species. Within the
Saccharum species, S. sinense had the highest similarity at
0.59 and S. spontaneum the lowest at 0.32. Saccharum
barberi was most similar to the varieties (0.47), closely
followed by S. officinarum and the varieties (0.44), with S.
officinarum and S. spontaneum the most dissimilar (0.23).
From the similarity matrix E. rockii was most similar to S.
barberi and least similar to E. procerus.
3.3. Genetic relationships within and between the
Saccharum and Erianthus genera
PCA analyses were carried out using the similarity
matrices generated with both the AFLP and the SSR markers.
The first two components of the PCA analysis using the AFLP
data explained 20.4 and 14.3% of the total variation (Fig. 1a).
Three major clusters were observed. One cluster contained the
Saccharum species and another all of the Erianthus species
apart from E. fulvus and E. rockii. The third cluster was
located between the other two clusters and contained E. rockii,
E. fulvus, the two Miscanthus species and N. porphyrocoma,
as well as P. schumach. The third PCA component explained
8.5% of the variation and further separated the genera in
cluster 3 above (Fig. 1b). In this component, the four E. rockii
accessions clustered with three of the five Miscanthus
accessions, and not with E. fulvus or N. porphyrocoma. The
PCA performed with the SSR data gave similar results
although the clustering was less tight (Fig. 1c and d).
Components 1 and 2 explained 13.6 and 4.9% of the variation,
respectively. In this analysis, four loose clusters were
observed, namely Erianthus spp. (excluding E. fulvus and
E. rockii), Miscanthus spp. and Narenga sp., E. rockii and E.
fulvus, and Saccharum spp., with E. rockii positioned between
the Miscanthus/Narenga cluster and the Saccharum spp.
cluster (Fig. 1c). The third PCA component explained 3.3% of
the variation and separated the Saccharum spp.
4. Discussion
4.1. Intra-specific diversity measured using AFLP and
SSR markers
The two primer combinations used for the AFLP analysis
generated a total of 173 bands. They were able to distinguish
between all but two of the accessions in this study. The two
indistinguishable accessions, Guangxi Zhuzhe and Guilin
Zhuzhe, are both S. sinense but from different regions of
China. In contrast to the AFLP assay, the 10 SSR’s screened
across the 69 accessions from 17 species revealed a total of
241 bands, and enabled each accession to be uniquely
identified. Overall, more markers were identified in
Saccharum spp. than in other species. The larger chromo-
some numbers, higher ploidy levels, and extent of
169169
(2005)
(2005)
976–984
976–984 982
interspecific hybridization within the Saccharum species
may explain why this genus had more alleles detected than
the other species.
A diversity study using RAPDs [16], found S. sinense to be
the most diverse Saccharum species, followed by S.
spontaneum, with S. officinarum and S. barberi the least
diverse Saccharum species. However, a similar study using
RFLP [21] and SSR markers from maize [23], reported that S.
spontaneum was the most diverse Saccharum species and S.
officinarum the least; our results obtained using SSR’s from
sugar cane and AFLP’s are in agreement with the results using
RFLP and SSR markers [21,23] with S. spontaneum being the
most diverse. As a species, S. officinarum has undergone
prolonged directional selection for sucrose content; cane
thickness and low fibre, which could explain its low level of
variation compared to the other Saccharum species.
4.2. Erianthus rockii versus Saccharum spp. and
Erianthus spp
Both SSR and AFLP PCA analyses were similar in
structure. In all cases, the Saccharum species clustered
together and away from the non-Saccharum species.
Erianthus rockii accessions formed a distinct cluster,
although one accession (Rockii95-19) consistently clustered
with the Miscanthus spp., suggesting that this clone may be
mislabelled in the YSRI collection or that E. rockii and
Miscanthus are diverse species. Analysis of a larger number
of accessions of E. rockii is needed to determine this.
Morphologically, however, this accession is indistinguish-
able from the other E. rockii accessions (Cai and Fan,
unpublished data). PCA analysis clustered E. rockii, E.
fulvus, Miscanthus spp., N. porphyrocoma and P. schumach
together between the Saccharum spp. and remaining
Erianthus spp. clusters. From both the SSR and AFLP
data, E. rockii appears to be distinct from other Erianthus
species and from Saccharum species and is positioned
equally distant from the two major species clusters,
Saccharum spp. and the other Erianthus species. It appears
to be a distinct species, more closely related to E. fulvus,
Miscanthus species and Narenga species, than to either other
Erianthus species or Saccharum species, and would be
included in the ‘‘Saccharum complex’’. A phylogenetic
study of Miscanthus, Saccharum and related genera [15]
using sequence data from the ITS region clustered E. rockii
with N. porphyrocoma and E. fulvus similar to this study. In
contrast M. floridulus and M. sinense did not cluster with E.
rockii. These results could relate to the difference in data
used for the analysis, sequence from a single gene compared
to hundreds of random markers in this study.
4.3. Species-relationships within Saccharum and
Erianthus genera
Although only 32 individuals from the five Saccharum
species and sugar cane varieties were studied, the results
 983 Q. Q.
CaiCai
et et
al.al.
/ Plant
/ Plant
Science
Science
obtained from this small group were similar to previous
studies using RAPD [16], RFLP [21] and SSR’s isolated
from maize [23]. In the present study, S. spontaneum
accessions were well separated from the other Saccharum
species, the varieties were closely associated with S.
officinarum accessions and S. sinense and S. barberi
accessions formed a group between S. officinarum and S.
spontaneum. Other studies using RAPDs [16], RFLPs [21]
and maize SSR’s [23] also observed the separation between
S. spontaneum accessions and the other Saccharum species.
The association between the varieties and S. officinarum has
also been noted in previous studies [21]. The latter
association is to be expected, as the varieties are developed
from initial hybrids between S. officinarum and S.
spontaneum followed by a series of backcrosses to either
S. officinarum or varieties with a large component of S.
officinarum [2]. The positioning of S. sinense and S. barberi
as a group between S. officinarum and S. spontaneum has
also been seen with the maize SSR, RFLP and RAPD data
and confirms the taxonomic view that S. sinense and S.
barberi are of interspecific and intergeneric hybridization
between these two species [36]. Saccharum robustum
accessions also clustered and the cluster lies between the
S. sinense/S. barberi cluster and the S. spontaneum cluster.
These results are consistent with the traditional taxonomic
schemes derived from the morphological and biochemical
data [36].
Both SSR and AFLP data identified E. fulvus and E.
rockii as the most closely related Erianthus species to the
Saccharum complex. Using both analysis methods E.
arundinaceus, E. sarpet, E. procerus, E. begalense, E.
elephantnium and E. ravennae form a closely related group.
This is in accordance with RFLP data [22], RAPD data [16],
maize SSR data [23] and AFLP data [24]. Erianthus
arundinaceus collected from Indonesia had the lowest level
of variability of all the species tested. Using PCA with the
AFLP data, three groups formed, one containing most of the
Erianthus species, one the Saccharum species and the third
group placed between the other two groups contained the
two Miscanthus species, the Narenga species, E fulvus and
E. rockii. The distance between most of the Erianthus
species and the Saccharum complex has also been seen with
RAPD data [16] and chloroplast data [37]. The SSR data
placed Narenga closer to Saccharum than Erianthus as did
both the chloroplast and RAPD data.
4.4. Comparison of AFLP and SSR data for assessing
genetic diversity
As in many systems studied, in the present study SSR’s
revealed a higher level of polymorphism than AFLP’s
[31,30]. There was good correlation between the AFLP and
SSR data although the AFLP data revealed a better
resolution of genetic relationships than the SSR data; this
also has been seen in a number of other studies of other
species [38,39]. It has been reported in other studies that
169169
(2005)
(2005)
976–984
976–984 983
SSR’s were well correlated with AFLP’s at the interspecies
level, however at the intraspecies level the correlation
disappeared [31]. The reason for differences seen with SSR’s
could be due to the constraint on allele size and rapid and
complex mutational processes within the SSR and flanking
regions that result in size homoplasy [40]. The high level of
genetic diversity revealed with SSR’s may also contribute to
the lack of correlation, as genetic similarity is lower with
SSR’s than with other marker systems as seen in sugar cane
using a range of markers types [21,22,16,23] and in other
crops [31], which could reduce the statistical power to define
relationships. This could be over come by the use of larger
numbers of SSR’s. These results suggest that E. rockii is
different to the other species studied. Successful crosses
between E. rockii and sugar cane would introgress novel
germplasm and assist in the broadening of the genetic base
of sugar cane.
References
[1] S.K. Mukherjee, Origin and distribution of Saccharum, Bot. Gaz. 119
(1957) 55–61.
[2] N. Berding, B.T. Roach, Germplasm collection, maintenance, and use,
in: D.J. Heinz (Ed.), Sugarcane Improvement through Breeding,
Elsevier, Amsterdam, 1987, pp. 143–210.
[3] D.J. Heinz, Wild Saccharum species for breeding in Hawaii, Proc. Int.
Soc. Sugar Cane Technol. 12 (1967) 1037–1043.
[4] C.C. Lo, Y.-H. Chen, Y.-J. Huang, S.C. Shih, Recent progress in
Miscanthus nobilization process, Proc. ISSCT 19 (1986) 514–521.
[5] P.Y.P. Tai, J.D. Miller, Phenotypic characteristics of the hybrids of
sugar cane related grasses, J. Am. Soc. Sugar Cane Technol. 8
(1988) 5–11.
[6] B.L. Legendre, Use of feral germplasm for sugar cane improvement in
Louisiana, Proc. ISSCT 20 (1989) 883–891.
[7] K. Alix, F. Paulet, J.C. Glaszmann, A. D’Hont, Inter-Alu-like species-
specific sequences in the Saccharum complex, Theor. Appl. Genet. 99
(1999) 962–968.
[8] A. D’Hont, P.S. Rao, P. Feldmann, L. Grivet, N. Islam-Faridi, P.
Taylor, J.C. Glaszmann, Identification and characterisation of sugar
cane intergeneric hybrids, Saccharum officinarum Erianthus arun-
dinaceus, with molecular markers and DNA in situ hybridisation,
Theor. Appl. Genet. 91 (1995) 320–326.
[9] P. Besse, C.L. McIntyre, D.M. Burner, C.G. de Almeida, Using
genomic slot blot hybridisation to assess intergeneric Sacchar-
um Erianthus hybrids (Andropogonae–Saccharinae), Genome 40
(1997) 428–432.
[10] G. Piperidis, M.J. Christopher, B.J. Carroll, N. Berding, A. D’Hont,
Molecular contribution to selection of intergeneric hybrids between
sugar cane and the wild species Erianthus arundinaceus, Genome 43
(2000) 1033–1037.
[11] Keng, Erianthus rockii Keng, Sinensis 10 (1939) 291.
[12] S.-L. Chen, Gramineae (5), in: Chinese Academy of Sciences com-
mittee (Ed.), Botanical Gazetteer of China 10(2), Sciences publisher,
China, 1997, pp. 48.
[13] J.E. Irvine, Saccharum species as horticultural classes, Theor. Appl.
Genet. 98 (1999) 186–194.
[14] J.-Y. Huang, J.-X. Liao, Intergeneric hybrisation of Saccharum L. with
Narenga porphyrocoma, Miscanthus floridulus and Erianthus rockii,
the morphology and isozyme analysis of their hybrid F1 clones,
Southwest China J. Agr. Sci. 10 (1997) 92–98.
[15] T.R. Hodkinson, M.W. Chase, M.D. Lledó, N. Salamin, S.A.
Renvoize, Phylogenetics of Miscanthus, Saccharum and related
984 Q. Q.
CaiCai
et et
al.al.
/ Plant
/ Plant
Science
Science
genera (Saccharinae, Andropogoneae, Poaceae) based on DNA
sequences from ITS nuclear ribosomal DNA and plastid trnL intron
and trnL-F intergenic spaces, J. Plant Res. 115 (2002) 381–392.
[16] N.V. Nair, S. Nair, T.V. Sreenivasan, M. Mohan, Analysis of genetic
diversity and phylogeny in Saccharum and related genera using RAPD
markers, Gen. Res. Crop Evol. 46 (1999) 73–79.
[17] Y.-H. Fan, H. Chen, X.W. Shi, Q. Cai, M. Zhang, Y.-P. Zhang, RAPD
analysis of S. spontaneum from different ecospecific colonies in
Yunnan, Acta Bot. Yunnanica 23 (3) (2001) 298–308.
[18] P. Besse, C.L. McIntyr, N. Berding, Ribosomal DNA variations in
Erianthus, a wild sugar cane relative (Andropogoneae–Saccharinae),
Theor. Appl. Genet. 92 (1996) 733–743.
[19] Y.B. Pan, D.M. Burner, B.L. Legendre, An assessment of the phylo-
genetic relationship among sugar cane and related taxa based on the
nucleotide sequence of 5S rRNA intergenic spacers, Genetica 108
(2000) 285–295.
[20] H. Chen, Y.-H. Fan, J.-G. Xiang-Yu, Q. Cai, Y.-P. Zhang, Phylogenetic
relationship of Saccharum and related species inferred from sequence
analysis of the rDNA ITS region, Acta Agron. Sinica 29 (3) (2003)
379–385.
[21] Y.H. Lu, A. D’Hont, D.I.T. Walker, P.S. Rao, P. Feldmann, J.C.
Glaszmann, Relationships among ancestral species of sugar cane
revealed with RFLP using single copy maize nuclear probes, Euphy-
tica 78 (1994) 7–18.
[22] P. Besse, C.L. McIntyre, N. Berding, Characterisation of Erianthus
sect. Ripidium and Saccharum gerplasm (Andropogoneae–Sacchar-
inae) using RFLP markers, Euphytica 93 (1997) 283–292.
[23] A. Selvi, N.V. Nair, N. Balasundaram, T. Mohapatra, Evaluation of
maize microsatellite markers for genetic diversity analysis and fin-
gerprinting in sugar cane, Genome 46 (2003) 394–403.
[24] P. Besse, G. Taylor, B. Carrol, N. Berding, D. Burner, C.L. McIntyre,
Assessing genetic diversity in a sugar cane germplasm collection using
an automated AFLP analysis, Genetica 104 (1998) 143–153.
[25] M.L.A. Lima, A.A.F. Garcia, K.M. Oliveira, S. Matsuoka, H. Ari-
zono, C.L. de Souza Jr., A.P. de Souza, Analysis of genetic similarity
detected by AFLP and coefficient of parentage among genotypes
of sugar cane (Saccharum spp.), Theor. Appl. Genet. 104 (2002)
30–38.
[26] P. Vos, R. Hogers, M. Bleeker, M. Reijans, T. Van Der Lee, M. Hornes,
AFLP: a new concept for DNA fingerprinting, Nucleic Acids Res. 23
(1995) 4407–4414.
[27] G. Piperidis, A. D’Hont, Chromosome composition analysis of various
Saccharum interspecific hybrids by genomic in situ hybridisation
(GISH), Int. Soc. Sugar Cane Technol. Congr. 11 (2001) 565.
169169
(2005)
(2005)
976–984
976–984 984
[28] A. Angiolillo, M. Mencuccini, L. Baldoni, Olive genetic diversity
assessed using amplified fragment length polymorphisms, Theor.
Appl. Genet. 98 (1999) 411–421.
[29] L. Amsellem, J.L. Noyer, T. Le Bourgeois, M. Hossaert-McKey,
Comparison of genetic diversity of the invasive weed Rubus alceifolius
Poir. (Rosaceae) in its native range and in areas of introduction, using
amplified fragment length polymorphism (AFLP) markers, Mol. Evol.
9 (2000) 443–455.
[30] K.-S. Wu, S. Tanksley, Abundance, polymorphism and genetic map-
ping of microsatellites in rice, Mol. Gen. Genet. 241 (1993) 225–235.
[31] W. Powell, M. Morgante, C. Andre, M. Hanafey, J. Vogel, S. Tingey,
A. Rafalski, The comparison of RFLP, RAPD, AFLP and SSR
(microsatellite) markers for germplasm analysis, Mol. Breeding 2
(1996) 225–238.
[32] G.M. Cordeiro, G.O. Taylor, R.J. Henry, Characterisation of micro-
satellite markers from sugar cane (Saccharum sp.), a highly polyploid
species, Plant Sci. 155 (2000) 161–168.
[33] K.S. Aitken, P.A. Jackson, C.L. McIntyre, A combination of AFLP and
SSR markers provides extensive map coverage and identification of
homo(eo)logous linkage groups in a sugar cane cultivar, Theor. Appl.
Genet. 110 (2005) 789–801.
[34] D.A. Hoisington, Laboratory protocols: CIMMYT Applied Molecular
Genetics Laboratory, Mexico, DF, CIMMYT, 1992.
[35] F.J. Rohlf, NTSYS-PC: numerical taxonomy and multivariate analysis
system. Version 2.1, Applied Biostatistics, New York, 1997.
[36] J. Daniels, B.T. Roach, Taxonomy and evolution, in: D.J. Heinz (Ed.),
Sugarcane Improvement Through Breeding, Elsevier, Amsterdam,
1987, pp. 143–210.
[37] B.W.S. Sobral, D.P.V. Braga, E.S. LaHood, P. Keim, Phylogenetic
analysis of chloroplast restriction enzyme site mutations in the Sac-
charinae Griseb. Subtribe of the Andropogoneae Dumort. Tribe,
Theor. Appl. Genet. 87 (1994) 843–853.
[38] J.R. Russell, J.D. Fuller, M. Macaulay, B.G. Hatz, A. Jahoor, W.
Powell, R. Waugh, Direct comparison of levels of genetic variation
among barley accessions detected by RFLPs, AFLP’s, SSR’s and
RAPDs, Theor. Appl. Genet. 95 (1997) 714–722.
[39] J. Jakše, K. Kindlhofer, B. Javornik, Assessment of genetic variation
and differentiation of hop genotypes by microsatellite and AFLP
markers, Genome 44 (2001) 773–782.
[40] R. Peakall, S. Gilmore, W. Keys, M. Morgante, A. Rafalski, Cross-
species amplification of soybean (Glycine max) simple sequence
repeats (SSR’s) within the genus and other legume genera: implica-
tions for the transferability fo SSR’s in plants, Mol. Biol. E 15 (10)
(1998) 1275–1287.