Preliminary Study Using ISSRs to Differentiate Imperata Taxa (Poaceae: Andropogoneae) Growing in the US
Rodrigo Vergara, Marc C. Minno, Maria Minno, Douglas E. Soltis,
and Pamela S. Soltis
Southeastern Naturalist, Volume 7, Number 2 (2008): 267–276
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2008 SOUTHEASTERN NATURALIST 7(2):267–276
Preliminary Study Using ISSRs to Differentiate Imperata
Taxa (Poaceae: Andropogoneae) Growing in the US
Rodrigo Vergara1,2,*, Marc C. Minno3, Maria Minno3, Douglas E. Soltis1,
and Pamela S. Soltis2
Abstract - Imperata cylindrica (cogongrass) is an invasive weed long established
in the southeastern US, and considerable effort is devoted to its control. Two
native species, I. brevifolia (California satintail) and I. brasiliensis (Brazilian
satintail), also occur in the US, and the latter is sympatric to cogongrass. Certain
Imperata morphotypes growing in the field are difficult to identify. To clarify
their identity, inter-simple sequence repeats (ISSRs) were used to assess genetic
differentiation among eight populations in the US representing Brazilian
satintail, California satintail, three potential morphotypes of cogongrass, and
three unknowns. Samples preserved in 95% ethyl alcohol and silica-gel did not
produce repeatable band patterns, so DNA from fresh leaves was extracted and
analyzed by polymerase chain reaction (PCR) amplification. Results indicate
that California satintail (D = 0.67), a commercial cogongrass cultivar (D = 0.66),
and a short-hairy morphotype of cogongrass (D = 0.65) were the most distinctive
operational taxonomic units (OTUs) compared. The unweighted pair group
method with arithmetic mean (UPGMA) dendrogram showed two well-supported
clusters of taxa containing Brazilian satintail (Bootstrap value = 96%) and
the tall morphotypes of cogongrass (Bootstrap value = 83%), respectively. Among
the morphotypes of cogongrass analyzed, the tall-hairy and tall-glabrous plants
formed a cluster from which the short-hairy morphotype and the cultivar were genetically
divergent. Our results refute taxonomic arrangements placing Brazilian
satintail as a synonym of cogongrass.
Imperata cylindrica (L.) (cogongrass) is an invasive weed from Asia
that has been established in the southeastern US for nearly 100 years (Tabor
1952a, b). Considerable effort is devoted toward controlling cogongrass, not
only in the US, but also throughout its range (Byrd and Bryson 1999, Coile
and Shilling 1993, Dozier et al. 1998, Tanner and Werner 1986, Van Loan
et al. 2002). Despite the invasiveness of cogongrass, plant nurseries in the
US have been propagating and selling ornamental cultivars of the species,
commonly called Japanese blood grass, throughout much of the US for many
years. In addition, two species occur natively in the US (Hitchcock 1950):
Imperata brevifolia Vasey (California satintail, in the southwestern US) and
Imperata brasiliensis Trinius (Brazilian satintail, in South Florida), which
1Department of Botany, University of Florida, Gainesville, FL 32611. 2Florida Museum
of Natural History, University of Florida, Gainesville, FL 32611. 3600 NW 35th
Terrace, Gainesville, FL 32607. *Corresponding author - rodver@ufl .edu.
268 Southeastern Naturalist Vol.7, No. 2
also is invasive in central Florida and Louisiana (Allen et al. 1991; Wunderlin
and Hansen 2003, 2006). We surveyed several populations of Imperata
species growing wild in the US and observed differences in biological and
morphological characteristics of these plants. Specifically, we found shorthairy,
tall-hairy, and tall-glabrous morphotypes. We were uncertain if these
variants were all cogongrass or if more than one species was represented.
The purpose of this study was to preliminarily analyze and compare the
species and morphotypes of Imperata present in the US using inter-simple
sequence repeats (ISSRs) in order to clarify the identity and distribution of
the various Imperata taxa.
ISSRs were chosen for this study because of their suitability in identifying
cultivars, varieties, and hybrids of cultivated plants (Wolfe and Liston
1998). ISSRs are dominant molecular markers generated by polymerase
chain reaction (PCR) amplification using primers developed from within
simple sequence repeats (SSRs). Each primer can potentially produce
multiple random fragments from across the entire genome, yielding highly
polymorphic bands. Such bands are interpreted as diallelic loci considering
only band presence or band absence (Wolfe and Liston 1998). Analyses were
conducted at the Laboratory of Molecular Systematics and Evolutionary Genetics,
FLMNH, University of Florida, Gainesville, FL.
Table 1. Imperata operational taxonomic units (OTUs) growing in the southern US analyzed
using ISSR fragments. Cogongrass = I. cylindrica, Brazilian satintail = I. brasiliensis, and
California satintail = I. brevifolia.
IDA Species Morphotype Origin State: County Code
088 1 Cogongrass Short, hairy Japan AL: Mobile cyl (sh)
(pubescent over the
whole leaf sheath)
048 2 Cogongrass Tall, hairy Philippines FL: Levy cyl (th)
(pubescent over the
whole leaf sheath)
047 3 Cogongrass Cultivar (red leaves) Japan NC: Iredell cyl (cv)
005-2 4 Brazilian Typical (tall, glabrous) Native FL: Miami-Dade bra
028 5 I. sp.B Tall, glabrous Unknown MS: Pearl River unk (MS)
054 6 California Typical (glabrous) Native CA: Ventura bvf
052 7 I. sp.B Wide blade Unknown FL: Collier unk (SFL)
097 8 I. sp.B Tall, glabrous Unknown FL: Hillsborough unk (NFL)
ASample identification from original collection.
BPutative Brazilian satintail population.
2008 R. Vergara, M.C. Minno, M. Minno, D.E. Soltis, and P.S. Soltis 269
We collected, analyzed, and compared samples of Imperata from
eight populations typified as eight different operational taxonomic units
(OTUs) found in Alabama, California, Florida, Mississippi, and North
Carolina (Table 1). In working with Imperata taxa from throughout the
range of the genus in the US, we observed plants growing wild at numerous
sites, including the original sites of introduction of cogongrass in the
Mobile area of Alabama, at the Mississippi Experiment Station in McNeil,
around Gainesville, FL, and at the US Department of Agriculture station
near Brooksville, FL. For most populations, inflorescences were not
available at the time of our visit, and we focused on vegetative characteristics.
Plants of cogongrass from along the Gulf Coast, including eastern
Louisiana, southern Mississippi, southern Alabama, and the western Florida
Panhandle had hairy leaf sheaths and were shorter in height than most
populations from peninsular Florida. Populations of California satintail
from California, Arizona, and Nevada, as well as Brazilian satintail from
Homestead (Miami-Dade County), FL, and plants of the Japanese blood
grass cultivar from California, Maryland, and North Carolina were glabrous,
except for hairs on the margins of the leaf sheaths in the vicinity of
the ligule. We hypothesized that an unidentified specimen from Picayune
State Forest in South Florida (Collier County), that was tall with very
wide leaves and bulbous culm bases, was Brazilian satintail. Lastly, a tallglabrous
taxon that we thought may also be Brazilian satintail was found
growing at the Mississippi Experiment Station in McNeil, at one site in
DeSoto National Forest in Mississippi, near Romar Beach in Alabama,
and at many locations in peninsular Florida.
DNA extraction and purification
Although fresh leaves are the best material for DNA extractions,
silica-gel dried samples are, in general, considered a suitable alternative
for extracting high-quality DNA (Chase and Hills 1991). In studies using
ISSRs, RAPDs, and AFLPs with some other members of Andropogoneae,
most of the extractions employed fresh leaf tissue (Hodkinson et al. 2002,
Nair et al. 1999, Pan et al. 2000) and, in a few cases, freeze-dried tissue
(Besse et al. 1998). Apparently, silica-gel or alcohol-dried tissue stored
at room temperature is not frequently used to extract DNA for ISSRs
and other similar genetic markers in this group of plants. The extraction
of DNA from silica-dried material has been shown to be problematic in
certain Poaceae because of the reactivation of DNases after the tissues
are re-hydrated in the DNA extraction process, and the buffers used there
are not effective (Adams et al. 1999). These authors also indicate that
alcohol-dried tissue seems to overcome this problem by irreversibly denaturing
In our study, we tried extracting DNA from samples preserved in both
alcohol and silica gel. Leaves of living plants from each population were
270 Southeastern Naturalist Vol.7, No. 2
cut into small pieces and stored in coded plastic vials containing either
of two kinds of preservatives for over a year before processing. One set
of samples was preserved with 95% ethyl alcohol, and a second set was
dried and stored in silica gel. Approximately 20 mg of leaf tissue from
each sample was ground in a mortar and pestle with liquid nitrogen and
sand and processed using both a CTAB DNA extraction protocol modified
from Doyle and Doyle (1987) and Cullings (1992) and the Promega
Wizard DNA Extraction Kit. Preliminary ISSR runs revealed that there
were no differences in DNA quality between extractions from samples
preserved in alcohol or silica gel; in both cases, DNA quality was poor.
Therefore, fresh leaves from eight available populations were used for
the final analysis. DNA was extracted from ground fresh leaves using the
QIAGEN DNeasy® Plant Mini Kit, obtaining clean DNA. Electrophoresis
was used to check DNA quality in 1.2% agarose gels, which were stained
with ethidium bromide, exposed to ultraviolet light, and photographed using
an EDAS 290 Kodak camera.
Five ISSR primers chosen at random were obtained from the University
of British Columbia Biotechnology Laboratory: UBCBL-set #9:
810, 815, 825, 830, and 841 (Table 2). The primers were optimized and
further tested using DNA samples extracted from fresh leaves. PCR reactions
were carried out in an Eppendorf Mastercycler thermocycler using
the cycle profile described by Huang and Sun (2000). Optimization was
made for each primer, testing two concentrations of formamide (1% and
2%) and two concentrations of MgCl2 (1.7 and 2.5 mM) in a factorial test
through a temperature gradient using an Eppendorf Mastercycler gradient
thermocycler. The PCR reaction also included Taq buffer (Mg-free),
dNTP, Taq polymerase, genomic DNA, and the ISSR primer, completing
a 15-μl reaction volume. The five primers were tested using the respective
optimized ISSR reactions. After optimization, we performed three
replications of each PCR reaction, for each primer/sample combination.
Table 2. ISSR primers optimized for annealing temperature, MgCl2 concentration, formamide
concentration, and tested for polymorphism and repeatability. Primers were obtained randomly
from primer set #9, UBCBL (University of British Columbia Biotechnology Laboratory).
Annealing analyzed Size Polymorphism
Primer Sequence (5’–3’) temperature (C°) fragments range (bp) (%)
810A GAGAGAGAGAGAGAGA-T 41.8 – – –
815A CTCTCTCTCTCTCTCT-G 41.8 – – –
825 ACACACACACACACAC-T 41.1 16 540–1370 94
830 TGTGTGTGTGTGTGTG-G 41.8 18 380–1250 72
841 GAGAGAGAGAGAGAGA-YC 42.8 19 300–1260 95
ADiscarded because of low repeatability.
2008 R. Vergara, M.C. Minno, M. Minno, D.E. Soltis, and P.S. Soltis 271
PCR products were separated by electrophoresis in 2% agarose gels using
a Sigma 100-bp ladder to assess band size.
Band scoring and data analysis
Bands were scored on agarose gels by first using a Kodak camera system
(1D 3.5.4 USB, DC290 Capture) to assign the size of the fragments and then
counting and matching bands among samples and replicates by eye in order
to account for uneven migration in the gels. Bands coinciding in molecular
weight and mobility were regarded as equal fragments. Bands representing
fragments greater than 1500 bp were ignored, because they were not repeatable
and weak. The presence of a band was coded as “1” and the absence as
“2.” After bands were scored, the data matrix was analyzed using the TFPGA
1.3 software (Miller 1997) to obtain modified Rogers’ genetic distances
(Wright 1978) and dendrograms generated by unweighted pair group method
with arithmetic mean (UPGMA). Support of nodes was obtained using bootstrapping
with 1000 replicates.
Results and Discussion
Quality of DNA extractions and ISSR reactions
In this study, samples preserved in silica-gel and alcohol yielded poorquality
DNA, and the amplification of ISSR products was low and highly
unrepeatable, regardless of the extraction protocol used. In contrast, satisfactory
genomic DNA was obtained from fresh material extracted with the
QIAGEN DNeasy® Plant Mini Kit. This DNA was of high molecular weight
and it provided repeatable results in the amplification of PCR products.
The ISSR optimization experiments using the genomic DNA obtained
from fresh material indicated that, for all five primers, the best concentrations
for MgCl2 and formamide were 2.5 mM and 1%, respectively. The
annealing temperature varied depending upon the primers (Table 2), and
the final formula for the PCR reactions was: 5.05 μl H2O, 1.5 μl Taq buffer
(Mg-free), 1.5 μl 25 mM MgCl2, 1.2 μl 2.5 mM dNTPs, 0.15 μl Formamide
Table 3. Wright’s (1978) modification of Rogers’ genetic distances among Imperata operational
taxonomic units (OTUs) based on 53 ISSR loci. D is the average genetic distance for each
OTUA 1 2 3 4 5 6 7 8 D
1 cyl (sh) – 0.63 0.66 0.67 0.57 0.71 0.63 0.64 0.65
2 cyl (th) – 0.67 0.66 0.34 0.73 0.67 0.31 0.52
3 cyl (cv) – 0.66 0.67 0.61 0.64 0.69 0.66
4 bra – 0.63 0.60 0.24 0.64 0.59
5 unk (MS) – 0.70 0.64 0.36 0.56
6 bvf – 0.61 0.71 0.67
7 unk (SFL) – 0.66 0.59
8 unk (NFL) – 0.57
AFor details regarding OTUs see Table 1.
272 Southeastern Naturalist Vol.7, No. 2
(SLS), 0.5 μl 10 μM primer, 0.1 μl Taq polymerase (Promega), and 5 μl
DNA (1/50 dilution).
After optimization, only primers 825, 830, and 841 showed polymorphic,
clear, and repeatable band patterns. Therefore, only these primers
were scored and analyzed. Scoring them conservatively on the gels (i.e.,
bands from different samples that appear at similar migration distances were
scored as having the same band), the three primers yielded a total of 53 fragments
or loci. The size of the analyzed fragments ranged from 300 to 1370
bp, and 87% of the loci were polymorphic (Table 2).
Similarity among OTUs
The genetic distances obtained among OTUs (Table 3) show that California
satintail (bvf) from California, Japanese blood grass (cogongrass
cultivar, cyl [cv]) and the short-hairy morphotype of cogongrass (cyl
[sh]) collected in Alabama near the site of first introduction of cogongrass
into the US from Japan, were the most distinctive OTUs compared, with
average genetic distances (D) of 0.67, 0.66, and 0.65, respectively, from
all other samples. The UPGMA dendrogram obtained from the genetic
distances (Fig. 1) shows the formation of two well-supported clusters
(bootstrap values >75%), which do not include any of the three OTUs
above. The first cluster includes a known Brazilian satintail population
Figure 1. UPGMA dendrogram comparing eight Imperata operational taxonomic
units (OTUs; see Table 1) based on Wright’s (1978) modification of Rogers’ genetic
distances and using ISSR markers. Numbers above branches are bootstrap percentages.
Thick lines indicate clusters strongly supported by bootstrapping (bootstrap
values >75%). Relative branch lengths indicate relative genetic distances between
2008 R. Vergara, M.C. Minno, M. Minno, D.E. Soltis, and P.S. Soltis 273
(bra) and an unknown wide-bladed plant that we thought was Brazilian
satintail from south Florida (unk [SFL]). The second cluster is composed
of the tall-hairy morphotype of cogongrass (cyl [th]) as well as the two
tall-glabrous unknowns from northern Florida (unk [NFL]) and Mississippi
(unk [MS]). The two tall morphotypes (hairy and glabrous) are
common throughout peninsular Florida, but were found at only a few places
in Mississippi and Alabama, including the second site of introduction
of cogongrass at the Mississippi Experiment Station in McNeil. Although
it would be useful to examine phylogenetic relationships among species
within the genus using gene sequence data, these two highly supported
clusters suggest that the ISSR markers employed in this study are able to
discriminate between cogongrass and Brazilian satintail, and that the two
species are therefore genetically divergent. Based on morphological traits
and interbreeding, Hall (1998) and Ward (2004) thought Brazilian satintail
to be the same species as cogongrass, but the genetic divergence between
them shown by our ISSR analysis does not agree with this concept,
suggesting parallel morphological evolution. Figure 2 illustrates the differences
in electrophoresis band patterns between the two well-supported
clusters, as well as the consistency of band patterns among replicates.
Figure 2. Gel illustrating PCR-amplified fragments from ISSR analysis using
primer UBCBL 841. First lane (L) is the Sigma 100-bp ladder (standard band sizes
are shown). The following lanes are the band patterns of the Imperata operational
taxonomic units (OTUs) studied with three replications. 1 = cyl (sh), 2 = cyl (th),
3 = cyl (cv), 4 = bra, 5 = unk (MS), 6 = bvf, 7 = unk (SFL), 8 = unk (NFL) (see
Table 1). Arrows show the two highly consistent clusters as indicated by bootstrapping
(see Fig. 1). Arrows pointing upward indicate the cluster representing typical
and wide-bladed morphotypes of Brazilian satintail (I. brasiliensis). Arrows pointing
downward indicate the cluster representing tall-hairy and tall-glabrous morphotypes
of cogongrass (I. cylindrica).
274 Southeastern Naturalist Vol.7, No. 2
Our results also indicate that the unknown wide-bladed population
from south Florida (Picayune State Forest in Collier County) was Brazilian
satintail and the other unknown populations from northern Florida and Mississippi
were cogongrass. These tall-glabrous plants clustered strongly with
tall-hairy cogongrass. The most common types of cogongrass in the US have
hairs on the leaf sheaf, unlike California satintail and Brazilian satintail, and
this character should help with the identification of non-fl owering Imperata
plants. However, we demonstrate here that a plant lacking hairy leaf sheaths
can still be cogongrass. Based on the literature, we believe that the shorthairy
morphotype of cogongrass represents its original introduction from
Japan, while one or both of the tall morphotypes are originally from the
Philippines (Tabor 1949, 1952a, 1952b).
Overlap of populations of Brazilian satintail with cogongrass in Florida
and Louisiana as well as with different variants of cogongrass in Alabama,
Mississippi, and Florida may set the stage for hybridization to occur. Interbreeding
of different species and variants may potentially produce hardier,
more aggressive plants that reproduce well from seed. Such hybrids could
be very problematic to control. The tall-glabrous morphotype of cogongrass
may represent such a hybrid because it shares characteristics of Brazilian
satintail (e.g., lack of hairs on the leaf sheath). However, additional analysis
of Imperata taxa using ISSRs or other genetic markers is needed, especially
using more primers and comparing more populations in the US, to look for
hybridization. Analysis of all ten species of Imperata using additional primers
is also necessary in order to understand better the genetic similarities
among these grasses.
We thank Richard Reardon (US Forest Service) for providing funding for this
project, the US Park Service for granting permission to collect on federal lands, Dr.
Ashley Morris (University of South Alabama) for her advice on ISSR methods, and
Dr. Pablo Speranza (Universidad de la República, Uruguay) for his help with DNA
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