An assessment of genetic diversity within and between populations of Phalaris minor using ISSR markers

Abstract

Intra- and inter-sample similarities for four populations of the annual grass weed Phalaris minor from Haryana state, India, were examined using inter-simple sequence repeat (ISSR) DNA markers. Levels of polymorphism within and between populations were low in comparison with values reported from other grassy weed species. Analysis of inter-population similarities allowed a partial differentiation of the four populations and of pairs of populations classified by cropping system. Analysis of the intra-population similarity data showed a weak but consistent and statistically significant negative correlation between the molecular similarity of seedlings and the physical distance between their mother plants over distances up to 40 m (the maximum separation tested) in all four populations. The consistency of the observed relationship between molecular similarity and physical separation, and the differences in cultivation practices at the four sites, suggested that the relationship may be a result of localized out-crossing, rather than an effect of localized seed rain. The results of the analyses are discussed in relation to the potential for evolution of multiple traits in the weed in response to changes in the wheat production system in the region.

Full text

An assessment of genetic diversity within and between
populations of Phalaris minor using ISSR markers
N McROBERTS*, W SINCLAIR*,AMcPHERSON*,ACFRANKE*à,
R P SAHARAN§, R K MALIK§, S SINGH§ & G MARSHALL*
*Scottish Agricultural College, Crop & Soil Management Research Group, Edinburgh, UK, School of Biological & Environmental Sciences,
Central Queensland University, Rockhampton, Qld, Australia, àInternational Institute of Tropical Agriculture, Oyo Road, Ibadan, Nigeria, and
§CCS Haryana Agricultural University, Agronomy Department, Hisar, India
Received 19 November 2002
Revised version accepted 12 August 2005
Summary
Intra- and inter-sample similarities for four populations
of the annual grass weed Phalaris minor from Haryana
state, India, were examined using inter-simple sequence
repeat (ISSR) DNA markers. Levels of polymorphism
within and between populations were low in comparison
with values reported from other grassy weed species.
Analysis of inter-population similarities allowed a par-
tial differentiation of the four populations and of pairs
of populations classified by cropping system. Analysis of
the intra-population similarity data showed a weak but
consistent and statistically significant negative correla-
tion between the molecular similarity of seedlings and
the physical distance between their mother plants over
distances up to 40 m (the maximum separation tested) in
all four populations. The consistency of the observed
relationship between molecular similarity and physical
separation, and the differences in cultivation practices at
the four sites, suggested that the relationship may be a
result of localized out-crossing, rather than an effect of
localized seed rain. The results of the analyses are
discussed in relation to the potential for evolution of
multiple traits in the weed in response to changes in the
wheat production system in the region.
Keywords: Phalaris minor, herbicide resistance, genetic
diversity, ISSR markers.
M
C
R
OBERTS
N, S
INCLAIR
W, M
C
P
HERSON
A, F
RANKE
AC, S
AHARAN
RP, M
ALIK
RK, S
INGH
S&M
ARSHALL
G (2005)
An assessment of genetic diversity within and between populations of Phalaris minor using ISSR markers. Weed
Research 45, 431–439.
Introduction
Weeds are a persistent problem in world agriculture
despite intensive efforts to eradicate them or bring their
populations under control. Phenotypic and genotypic
diversity are commonly cited as central features in the
successful survival of weed populations in the face of
concerted control operations by farmers. Genotypic and
phenotypic plasticity confer the ability to adapt and
survive by exploiting niches which are unsuitable for
other plants, particularly crop species (Dekker, 1997).
Phalaris minor (Retz.) is a diploid, predominantly
in-breeding, annual grass weed which is widely distri-
buted on the Indo-Gangetic plains. It may cause yield
losses of up to 50% in wheat crops following rice. It has a
high competitive ability with wheat. This, combined with
its high fecundity, has resulted in the grass colonizing 16
million hectares of wheat cropping land in the region. Its
presence in the irrigated rice–wheat system is an ongoing
threat to India’s sustainability in food grain production
(Singh et al., 1997, 1999; Franke et al., 2003). Although
P. minor management in wheat has been problematic in
this region for several decades, the situation dramatically
worsened with the development of resistance to the
commonly used phenyl urea herbicide isoproturon (IPU)
in some intensively farmed areas of Haryana, Punjab and
Uttar Pradesh in the early 1990s (Malik & Singh, 1993).
By 1999, it was estimated that herbicide-resistant
biotypes had infested around 1 million hectares in these
three states (Yaduraju, 1999). With the introduction in
Correspondence: N McRoberts, Scottish Agricultural College, Crop & Soil Research Group, Edinburgh EH9 3JG, UK. Tel: (+44) 1292 520331; Fax:
(+44) 1292 525052; E-mail: neil.mcroberts@sac.ac.uk
2005 European Weed Research Society Weed Research 2005 45, 431–439

1998 of three herbicides with alternative modes of action
against P. minor, sulfosulfuron, fenoxaprop-P-ethyl and
clodinafop-propargyl, the control of P. minor in the areas
hit by IPU resistance improved. However, farmers in
areas of the plains that have not been infested by resistant
biotypes have a low awareness of the dangers of resistance
development, and do not make use of the alternative
herbicides unless they are confronted clearly with reduced
IPU efficacy (Franke et al., 2003).
Herbicide-resistant biotypes have been found in geo-
graphically separated regions of the Indo-Gangetic
plains. The level of resistance widely varies among
biotypes and increases if IPU use is continued (Malik
et al., 1998). In Haryana State, resistant biotypes were
initially found predominantly on heavy soils in the north-
east (Fig. 1), where rice–wheat is the dominant crop
rotation and P. minor has dominated the wheat weed flora
for a long time (Singh et al., 1995). However, in recent
years, P. minor has adapted to other rotations, such as
cotton–wheat and millet–wheat, on lighter soils, and IPU
use is increasing in drier southern areas. As a result of this
increase in use in new areas and farmersÕreluctance to
adopt more expensive and unfamiliar alternative herbi-
cides in already-infested areas, the infestation area of
IPU-resistant biotypes is moving southwards.
Our understanding of how P. minor will respond to
management regimes currently in place is limited and it
would therefore be useful to characterize the nature of
the genetic variation contained within selected popula-
tions of the species, providing a baseline against which
to compare future assessments of population structure.
A number of previous studies have utilized isozyme
analysis to characterize weed diversity, particularly
where herbicide resistance is known to occur (Warwick
& Marriage, 1982; Mouemar & Gasquez, 1983; Barnett
& Shore, 1990; Lefol et al., 1995; Sterling & Hou, 1997).
However, isozyme analysis of genetic variation is often
limited by comparatively low levels of detectable
variation, particularly in studies on small spatial scales
(Holt, 1994; Piquot et al., 1999). In recent years,
molecular techniques have become increasingly import-
ant (Penner, 1995). They are now used routinely to
investigate and characterize patterns of plant diversity,
population history and structure and evolution with an
increasing body of work where molecular approaches
have been applied to weedy species (Colosi & Schaal,
1997; Moodie et al., 1997; McRoberts et al., 1999;
Meikle et al., 1999; Ash et al., 2003; Ye et al., 2003).
Of the molecular techniques utilized for the study of
population genetic structure, PCR-based DNA finger-
printing using single oligonucleotide primers is currently
one of the most common when studying natural plant
populations. The primers utilized to demarcate the
regions of DNA amplified by the PCR are commonly
based on random DNA sequences (RAPDs) (Welsh &
McClelland, 1990; Williams et al., 1990) or repeat
sequence DNA, as used in directed amplification of
minisatellite DNA (DAMD) and inter-simple sequence
repeats (ISSR) (Heath et al., 1993; Zietkiewicz et al.,
1994). All these techniques provide anonymous DNA
markers capable of detecting DNA polymorphisms
between individuals. ISSRs are amplified from single-
primer PCR reactions where the primer is designed from
di- or tri-nucleotide repeat motifs with a random
anchoring sequence of one to three nucleotides. ISSR
markers, representing the nucleotide sequence between
two microsatellite sites, have been shown to be useful for
DNA fingerprinting in natural plant populations (Gupta
et al., 1994; Wolfe & Liston, 1998; Wolfe et al., 1998;
Esselman et al., 1999; Aga et al., 2005; Zhang et al.,
2005).
In this paper our specific aims were to apply ISSR
markers in P. minor with the purpose of making an
initial estimate of the extent of inter- and intra-popula-
tion genetic variation in P. minor in Haryana. We aimed
to relate the results on genetic diversity in the weed to
the agricultural production systems within the state.
Materials and methods
Study area and sampling
Seeds of P. minor were collected at four sites, one each in
the districts of Karnal, Lalhoda, Hisar and Jhajjar in
Haryana State, India. The sampled populations were all
Delhi
H
L
K
J
Fig. 1 An outline map of Haryana state showing the approximate
locations of four sites where Phalaris minor populations were
sampled: H, Hisar; J, Jhajjar; K, Karnal; L, Lalhoda.
432 N McRoberts et al.
2005 European Weed Research Society Weed Research 2005 45, 431–439

within 150 km of each other (Fig. 1) on typical village
farms. The populations from Karnal and Lalhoda
districts were from areas where the soil type was clay
loam, the cropping pattern was dominated by rice–
wheat and IPU-resistant biotypes were ubiquitous. The
sampled sites in Hisar and Jhajjar had sandy soils, a
more diverse cropping pattern with either summer
fallow (common at Jhajjar) or cotton (Hisar) instead
of rice, no history of IPU use and no known incidence of
herbicide-resistant biotypes.
At each site, before the wheat harvest in early April
2000, P. minor ear heads were bagged prior to seed shed
and removed intact from 15 mature plants along a line
transect at an inter-plant distance of c. 4 m. Ten plants
in a continuous sequence from each transect were
subsequently used for ISSR analysis. From each of
these 10 plants, five seeds from the main seed head were
selected at random and germinated under controlled
conditions in a growth chamber (Conviron, Controlled
Environments Limited, Winnipeg, MB, Canada) at 18C
with a 9 h photoperiod. The resulting seedlings were
harvested after 30 days for DNA isolation. Although
the sampling procedure was intended to generate a
sample of 200 individual fingerprints (i.e. 50 individuals
from each of four populations) a final sample size of 189
fingerprints was obtained (see Results) because of
difficulties in obtaining germination from seed samples,
particularly in the case of seed harvested at Jhajjar.
Repeat germinations were attempted until the seed from
the selected plants was exhausted.
DNA isolation and inter-simple sequence repeat
amplification
For each seedling, 0.1 g of fresh leaf material per plant
was harvested and stored frozen ()70C) for subsequent
DNA extraction using the commercially available
NucleonPhytoPure kit (Tepnel Life Sciences plc,
Manchester, UK) following the procedure provided with
the kit. After determination of their concentrations,
DNA samples were stored frozen ()70C) and used to
prepare stock solutions for subsequent molecular ana-
lysis.
DNA amplifications were carried out using 5¢-
anchored oligonucleotide primers obtained from the
University of British Columbia primer sets and from
Operon Technologies (Almeda, CA, USA) (Table 1).
These primers were selected in previous studies of
molecular variation in wild and weedy plant species in
which they had been proven to generate reliable and
reproducible markers (McRoberts et al., 1999). Ampli-
fication reactions for individual samples were carried out
in 25 lL volumes of a reaction mixture in thin-walled
microtubes (0.65 mL; Anachem, Luton, Bedfordshire,
UK). Each reaction mixture contained 10 ng of template
DNA, 0.5 units of Amplitaq DNA polymerase (Applied
Biosystems, Warrington, Cheshire, UK), 2 m
M
magnes-
ium chloride (Applied Biosystems), 2.5 lL of 10X Taq
buffer (Applied Biosystems), 100 m
M
each dNTP (Ap-
plied Biosystems), 50 pmol primer and sterile, distilled
H
2
O. The reaction mixture was overlayed with mineral
oil and subjected to the following amplification protocol,
in a GeneAmp 480 thermal cycler (Applied Biosystems):
an initial incubation at 95C for 5 min, followed by 35
cycles of 1 min at 95C, 1 min at 55C and 2 min at 72C
and then a final incubation at 72C for 10 min.
Gel electrophoresis and analysis of DNA fingerprints
Amplification products were resolved by electrophoresis
on 1.5% Agarose (Seakem LE, Flowgen, Lichfield,
Cheshire, UK) gels. Electrophoresis was conducted for
3 h, at 120 V using 0.5X Tris–Borate–EDTA (Sigma, St
Louis, MO, USA) as the running buffer. The gels were
Table 1 Primer identities, sequences and number of markers per primer, the proportion of polymorphic markers per primer and the
proportion of ears with polymorphism for ISSR markers from four populations of Phalaris minor sampled in wheat crops in Haryana state,
India
Ref. code Sequence
Number of
markers
Population
MeanHisar Lalhoda Karnal Jhajjar
UBC 809 AGAGAGAGAGAGAGg 6 0.167 0.333 0.500 0.167 0.29
UBC 810 GAGAGAGAGAGAGAGAT 8 1.000 0.500 0.875 0.625 0.75
UBC 812 GAGAGAGAGAGAGAGAA 6 0.500 0.500 0.500 0.667 0.54
2245 CACACACACACACACAR*G 8 0.375 0.250 0.250 0.375 0.31
2246 CACACACACACACACARC 7 0.429 0.429 0.857 0.429 0.54
Mean 7 0.49 0.40 0.60 0.45
Ear
1.0 0.9 1.0 0.78à
*R ¼purine.
The proportion of 10 ears with polymorphisms between seeds.
àBased on nine ears only.
Genetic diversity in Phalaris populations 433
2005 European Weed Research Society Weed Research 2005 45, 431–439

then stained in ethidium bromide (0.5 lgmL
)1
in 0.5X
TBE buffer) before visualization under UV light using a
gel documentation system (GeneGenius; Syngene, Cam-
bridge, UK). The gels were digitally photographed and
stored as separate images in the documentation system.
For each primer utilized in the study, all DNA samples
were examined for each of the populations. For each
primer, an individual plant was identified which had all
reproducible markers present and the incidence of mark-
ers in all other individuals was then scored relative to this
individual. The primers were chosen following testing for
reproducibility of the fingerprints obtained. This was
assessed by examination of the fingerprints from both
replicate PCR amplifications of DNA samples and from
the amplification of replicate DNA extracts from the
same original plant, to ensure the fidelity and reproduc-
ibility of the technique. Only bands that produced clearly
scoreable markers were recorded; faint or ill-defined
bands were disregarded; monomorphic and polymorphic
bands were included in the data set for analysis.
Statistical analysis
Binary data for the presence/absence (incidence) of
markers from each plant were used to construct
similarity matrices using either Jaccard’s coefficient or
the simple matching coefficient (SMC). The similarity
coefficients differ in the way in which they account for
the absence of markers when calculating the similarity
between individuals. For two individuals iand jthe
similarity, S
ij
, with respect to any marker xis:
Jaccard If x
i
¼x
j
¼1, S
ij
¼1, otherwise S
ij
¼0
SMC If x
i
¼x
j
,S
ij
¼1, otherwise S
ij
¼0
Analysis of inter-population and inter-cropping
system similarities
The analysis of differences among populations and
between cropping systems was carried out sequentially.
In the first step, similarity matrices for all 189 individ-
uals were calculated using both similarity coefficients, as
described above. The mean within-population and
between-population similarity matrices for each coeffi-
cient were extracted from these and used to assess the
correspondence between the results from the two coef-
ficients. The correlation between the two sets of corre-
lation coefficients was 0.98 and the remainder of the
analyses were carried out only on the similarity matrix
constructed from the SMC because the use of this
coefficient is logically consistent with the assumptions
required for population-genetic analysis, whereas Jac-
card’s coefficient is not.
In the second step of the analysis, a principal
coordinates (PCO) analysis was used to reduce the
similarity matrix for the 189 individuals to a set of
variables which contained the majority of the informa-
tion on inter-individual similarity (Payne et al., 2002).
The derived variables were then analysed by multiva-
riate analysis of variance (
MANOVA
) to test for differ-
ences between populations and between cropping
systems. The results from the
MANOVA
s were interpreted
using a combination of test statistics and graphical
investigation by canonical variates analysis (CVA)
(Krzanowski, 1990). Finally, in the case of the inter-
population comparisons, the relationship among popu-
lations derived from the CVA of the ISSR data was
compared with the physical distances between the
populations using Procrustes analysis (Digby & Kemp-
ton, 1987; Digby et al., 1989). The data for the
Procrustes analysis consisted of the longitude and
latitude for each population and the set of coordinates
for the mean positions for the populations on the first
two CVA axes.
Analysis of intra-population variation in similarity
The four within-population similarity matrices were
extracted from the full similarity matrix. For each
within-population similarity matrix, mean similarities
within and between groups of seedlings from the
mother plants were used to examine the relationship
between physical separation along the transect and
molecular similarity using a Mantel test (Mantel,
1967).
The Mantel procedure randomly permutes the
columns of the observed data matrices and calculates
a test statistic for each permutation (the standard
product–moment correlation coefficient in the present
case). The value of the test statistic from the observed
data is compared with the sampling distribution from
the data permutations to obtain an empirical signifi-
cance test. In this case, the value of the correlation
coefficient calculated from the observed data was
compared with both the upper and lower tails of the
sampling distribution, as the theoretical range of the
correlation coefficient is from )1 to 1, and values
toward either end of the range indicate a stronger
correlation than would be expected to occur at
random. A large negative value would indicate that
molecular similarity decreased with distance more than
would be expected at random, while a large positive
value would indicate that molecular similarity
increased with distance more than would be expected
at random.
The statistical analyses and calculations were carried
out in Genstat 6.1 (Payne et al., 2002).
434 N McRoberts et al.
2005 European Weed Research Society Weed Research 2005 45, 431–439

Results
General results
Several basic results are summarized in Table 1. The five
primers produced a total of 35 reliable markers, with a
range from 6 to 8 and a mean of 7 markers per primer.
The highest proportion of polymorphic markers was
produced by UBC 810 (0.75 averaged over the four
populations) and the lowest by UBC 809 (0.29 averaged
over populations). The highest incidence of polymorph-
ism occurred within the Karnal population where 60%
of markers were polymorphic and the least in Lalhoda,
where 40% of markers were polymorphic. The propor-
tion of ears with polymorphisms between seeds was
generally high for an inbreeding species (Table 1)
ranging from 1.0 (Hisar and Karnal) to 0.78 (Jhajjar).
Across all four populations only three cases were found
where all five seeds from an ear had identical genotypes
(one ear from Lalhoda and two from Jhajjar). The mean
number of genotypes per ear for each site was (SEM in
parentheses): Hisar, 4.2 (0.29); Lalhoda, 3.8 (0.39);
Karnal, 4.2 (0.29); Jhajjar, 2.6 (0.38).
Comparison of inter-individual similarity (S)
The mean intra- and inter-population similarities, S,
based on all 189 individuals are shown in Table 2.
Seedlings from Jhajjar showed the highest mean intra-
population similarity (90.1%), while those from Karnal
had the lowest (88.1%). Mean Sbetween individuals
from different sites was lowest between Hisar and
Lalhoda (79.9%) and highest between Karnal and
Jhajjar (86.6%).
In the data reduction step using PCO, the first six
PCO axes captured 72% of the variation in Samong the
189 individuals. The percentage variance accounted for
by the first six axes was, in order from axis 1 to 6: 28.7%,
13.1%, 10.6%, 8.2%, 5.8%, 5.3%. Figure 2 shows the
ordination of the individuals, identified by population,
on the first two PCO axes.
Significant differences among populations were found
in the PCO coordinates in the
MANOVA
analysis [approxi-
mate v
2
¼380.86, P< 0.001 (18 d.f.), approximate
F¼30.78, P< 0.001 (18, 510 d.f.)]. The first two CVA
axes captured 92% of the variance in the six PCO axes
and thus 66% (0.92 ·0.72 ¼0.66) of the variance in the
original similarity matrix. Figure 3 shows the ordination
of the 189 individuals on the first two CVA axes and the
positions of the population mean points with their 95%
confidence regions. The first CVA axis principally
Table 2 Mean intra- and inter-population similarities* for Phalaris
minor plants sampled from four populations in Haryana state
based on ISSR markers. Within population similarities are shown
in bold type on the lower diagonal, figures in parentheses are the
standard errors of the mean
Hisar Lalhoda Karnal Jhajjar
Hisar 89.1 (0.20) 79.9 (0.39) 85.3 (0.41) 83.9 (0.38)
Lalhoda 89.1 (0.19) 84.6 (0.30) 85.5 (0.33)
Karnal 88.1 (0.20) 86.6 (0.39)
Jhajjar 91.1 (0.20)
*Based on SMC values.
–0.5 –0.2 0.1 0.4
First
p
rinci
p
al axis (28.7% variance)
–0.4
–0.1
0.2
0.5
Second principal axis (13.1% variance)
Hisar
Lalhoda
Karnal
Jhajjar
Fig. 2 The ordination of 189 Phalaris minor plants resulting from a
principal coordinates analysis of a similarity matrix generated from
35 ISSR markers using the simple matching coefficient.
H
L
K
J
–5 –4 –3 –2 –1 0 1 2 3 4
First canonical axis (76.3% variance)
–3
–2
–1
0
1
2
3
Second canonical axis (16.1% variance)
Hisar
Lalhoda
Karnal
Jhajjar
Fig. 3 An ordination of 189 Phalaris minor seedlings obtained
from seed heads sampled at four sites in Haryana. The ordination
was obtained by canonical variates analysis of the coordinates for
the plants along six principal axes extracted from a similarity
matrix based on ISSR marker data. The large open circles are the
mean points for each population. The diameter of the circles gives
the approximate 95% confidence area for the means.
Genetic diversity in Phalaris populations 435
2005 European Weed Research Society Weed Research 2005 45, 431–439

differentiated the Hisar and Lalhoda samples, while the
second axis separated the Jhajjar from the other three
populations.
The Procrustes analysis of the concordance between
the geographical and molecular similarity ordinations
(using the coordinates for the population means on the
CVA axes) indicated only a weak relationship between
the geographical proximity of populations and their
molecular similarity. Overall, fitting the CVA configur-
ation of the populations to their geographical configur-
ation accounted for 32% of the sums of squares (SS) for
the geographic and molecular configurations around
their joint centroid. The partitioning of the residual SS
among populations suggested that the correspondence
between the geographical and molecular data was
relatively good in the case of Lalhoda and Jhajjar
(which accounted for 10% and 18% of the residual SS
respectively) and poor in the case of Hisar and Karnal
(37% and 35% of the residual SS respectively). The
correspondence between the geographical and molecular
configurations of the four populations is shown in
Fig. 4. Comparison of Fig. 4 with Fig. 1 allows a
qualitative assessment of how well the configuration of
distances among the populations on the basis of the
ISSR markers matches their geographical separation.
Examination of the PCO data for differences between
cropping systems suggested that, overall, mean Sfrom
populations under the rice–wheat system (Karnal and
Lalhoda) was lower (88.6%) than plants from the
populations under cotton–wheat or fallow–wheat (Hisar
and Jhajjar) (89.9%). The
MANOVA
of differences
between cropping systems indicated that the PCO
analysis identified significant differences between crop-
ping systems [approximate v
2
¼138.25, P< 0.001 (6
d.f.), approximate F¼33.97, P< 0.001 (6, 182 d.f.)].
Within-population variation in similarity with distance
The results of the Mantel test procedure, to examine
the association between physical separation of the
mother plants and molecular similarity of seedlings
arising from them, are summarized in Table 3. In all
populations, the observed correlation coefficient was in
the lower 95% tail of the values calculated by the
permutation procedure. These results indicate that
there was a significantly stronger negative correlation
between molecular similarity and physical separation
than would be expected by chance alone. The observed
correlation coefficients were between )0.43 (Lalhoda)
and )0.49 (Jhajjar).
Discussion
Phalaris minor has become established as the major
annual weed affecting wheat production in the rice/
wheat system of the Indo-Gangetic plains. A period of
relatively good weed control, based on the use of
isoproturon, ended in the early 1990s when IPU-
resistant populations appeared. Recent weed control
strategies have focussed on the integrated use of
alternative herbicides (clodinafop-propargyl, sulfosulfu-
ron or fenoxaprop-P-ethyl) with alternative tillage
methods such as zero tillage (Hobbs, 2002). While these
changes in husbandry practices have been successful in
reducing losses from P. minor infestation, adaptation of
the weed to its new environment can be expected and
sustainable control of P. minor will be improved with a
better understanding of its basic biology.
H
L
K
J
H
L
K
J
–0.6 –0.4 –0.2 0 0.2 0.4 0.6
Second
p
rinci
p
al axis
–0.4
–0.3
–0.2
–0.1
0
0.1
0.2
0.3
0.4
First principal axis
Physical
Molecular
20.2
14.7
15.5
15.0
15.9
13.3
Fig. 4 A joint ordination from a Procrustes analysis of four
Phalaris minor populations from wheat crops in Haryana, India
using the geographical configuration (obtained from the latitude
and longitude of the sites) and a molecular configuration (obtained
from mean inter-population similarities). The values next to the
lines connecting points in the molecular configuration are the
molecular distances [¼100 )(mean between population similarity)]
between populations. Populations: H, Hisar; L, Lalhoda; J, Jhajjar;
K, Karnal.
Table 3 Estimated intra-population
correlations between molecular similarity
and inter-parent plant distance in four
Phalaris minor populations from wheat
crops in Haryana state, India
Hisar Lalhoda Karnal Jhajjar
Correlation* of parent plant
separation and seedling
molecular similarity (P)
)0.44 (0.05) )0.43 (0.05) )0.45 (0.01) )0.49 (0.01)
*Product-moment correlation coefficient
Empirical probability that the absolute value of observed coefficient greater than random
expectation based on Mantel permutation test
436 N McRoberts et al.
2005 European Weed Research Society Weed Research 2005 45, 431–439

Based on pervious experience with a range of wild
and weedy species (Moodie et al., 1997; McRoberts
et al., 1999; Meikle et al., 1999), we have successfully
demonstrated in this study that ISSR markers can be
used for assessing genetic diversity in P. minor. The
proportion of monomorphic markers (40–60% of mark-
ers sampled), and consequently the levels of similarity
observed between individuals within and between pop-
ulations, were generally higher than those found in some
other wild grass species (McRoberts et al., 1999) and are
probably indicative of the predominantly inbreeding
behaviour of P. minor (Anderson, 1961).
Some evidence of differentiation between populations
was apparent in the data. Thus, the P. minor populations
analysed here could be partially differentiated on the
basis of either geographic location or on the basis of
cropping system. The biggest difference between indi-
vidual populations was between Hisar and Lalhoda,
although these are the two closest populations geo-
graphically. Historically, the area around Lalhoda has
had one of the most intensive rice–wheat rotations in
Haryana, even though it lies outside the main rice–wheat
region (centred around Karnal) and was one of the
locations where IPU-resistant P. minor was detected
earliest. In contrast, the area around Hisar has little
rice–wheat cropping. Without a more extensive analysis
of population structure across the region it is not
possible to decide whether these differences are a
reflection of local adaptations or founder effects. The
relatively high similarity between the Karnal population
and the other three (particularly Hisar and Jhajjar) may
reflect the importance of the Karnal area as a centre of
wheat production and trade. It is known that seed lots
contaminated with P. minor are regularly traded among
farmers (Yaduraju, 1999) and thus the Karnal area may
act as a source of P. minor for other areas of the state.
However, direct trade in wheat seed among farmers
tends to occur on a fairly localized basis; the exact role
of human activity in mixing local populations of
P. minor remains unclear and requires further investi-
gation in the context of sustainable weed management in
the region. There was some evidence that the diversity of
P. minor plants sampled in this study was highest in
populations (Lalhoda and Karnal) where the dominant
rice–wheat cropping system is particularly well-suited to
reproduction by the weed.
The differentiation observed among the P. minor
populations on the basis of cropping system is con-
founded both by geographic and, possibly, herbicide
tolerance effects. Thus, the split between cropping
systems also reflects a north-and-east to south-and-west
split across the state (combining the rice–wheat popu-
lations of Karnal and Lalhoda, and the dry summer
rotations of Hisar and Jhajjar). IPU resistance is nearly
ubiquitous in rice–wheat regions, but still largely absent
from other areas (Franke et al., 2003). A more detailed
analysis of these effects, carried out over a wider range
of populations, would be needed to fully determine the
factors underlying the partitioning of variation in
P. minor. However, our results have shown that
variation exists between different populations, that
some of the variation can be understood in terms of a
combination of cropping system and location. There is
no evidence that populations from areas where IPU
resistance is common are any less variable than those
from other populations. This observation may have
consequences for the development of adaptations by the
weed to novel production approaches, such as the use of
zero tillage. The use of zero-till under Haryana condi-
tions inhibits P. minor germination and growth through
a combination of factors including high seedbed tem-
perature and low seedbed moisture (resulting from
earlier drilling) and physical inhibition resulting from
the formation of a hard soil surface crust. Thus,
selection pressure on the P. minor population in
response to the use of zero-till is for individuals with
different temperature and moisture requirements and
greater physical strength. While selection for such a
combination of characteristics is likely to take longer
than development of herbicide tolerance, there is
evidence from other species that response to selection
for multiple traits can be rapid (Jordan, 1989a,b). The
rate at which the weed may be able to adapt to such
combinations of changes in its niche will be determined
in part by the available variation within individual
populations and in the population as a whole.
The level of variation within and between popula-
tions of P. minor will be determined partly by its
breeding system. Although the weed is considered to be
an inbreeder, this study provided some evidence that
out-crossing can also occur. First, in all four popula-
tions sampled, the level of polymorphism between
seedlings from individual seed heads was high; for
example, in the Hisar and Karnal populations, all of the
sampled seed heads had some level of polymorphism
between seedlings. In a completely inbreeding species
seeds from a single ear head should be identical.
Second, in all four sampled populations the similarity
of seedlings declined with distance between their mother
plants. A number of mechanisms might generate such a
trend. For example, in undisturbed conditions a seed
rain with a steep dispersal curve, and a mean dispersal
distance in the order of 1 to a few metres, might give rise
to a seedbank in which relatedness declined with
distance over the spatial scale examined in this study.
However, soil cultivation has a large influence on the
pattern of seed dispersal, with different cultivation
methods giving rise to different amounts of seed
Genetic diversity in Phalaris populations 437
2005 European Weed Research Society Weed Research 2005 45, 431–439

displacement (Brain & Marshall, 1999; Marshall &
Brain, 1999). The cultivation methods used in the
different cropping systems sampled in this study would
be expected to have different effects on the distribution
of seed in the soil and therefore on the observed
correlation between physical separation and molecular
similarity. The preparation of the wheat seedbed
typically involves ploughing and planking. Planking
involves pulling a weighted baton over the soil to obtain
a flat, even surface. The procedure causes considerable
lateral displacement of soil and would be expected to
disrupt any spatial pattern of seeds established by seed
rain. Furthermore, at the sites with a rice–wheat
rotation (Karnal and Lalhoda), the summer rice crop
is planted into a paddy prepared from the previous
wheat crop stubble. Preparing a paddy involves flooding
the field and churning up the soil either by driving a
tractor or draught animals through it, before allowing it
to settle to form the paddy into which the rice is
transplanted. Again, movement of water and machinery
during these operations would be expected to result in
considerable redistribution of seeds from the positions at
which they were shed. At the sites where either cotton or
millet was planted, or a summer fallow was used, no
paddying would occur, or no tillage would take place.
Despite these obvious differences in the potential for
seed re-distribution in the four populations, a fairly
robust correlation was observed between the physical
distance between mother plants and the molecular
similarity of their seeds.
Gathering these factors together it is apparent that
there are two basic results in the data to be accounted
for. First, a high level of polymorphism within seed
heads of an apparently inbreeding species. Second, a
fairly stable correlation between the physical distance
between plants which germinated after the current
season’s tillage operations, which varied among sites,
and the average similarity among seeds on those plants
at each site. A plausible mechanism which would
account for both of these observations is localized out-
crossing in P. minor, as pollen dispersal with an
intrinsic dispersal decay curve would provide a mech-
anism for generating a correlation between physical
separation and molecular similarity, which is also
independent of the tillage regime used. Further work
is required to test this hypothesis, but the presence of
out-crossing in the weed would have implications for
its ability to combine useful traits in response to
changes in the production system, particularly if
human activity provides it with a means of rapid
long-distance seed dispersal. The combination of these
effects may have serious long-term implications for the
development of sustainable, intensive wheat production
in the region.
Acknowledgements
The work reported in this paper was carried out as part
of a project funded by the UK Government Department
for International Development (DFID, Project no.
R7331). SAC receives financial support from the Scot-
tish Executive Environment and Rural Affairs Depart-
ment (SEERAD).
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