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Genome Size Analysis of Weedy Amaranthus Species
A. Lane Rayburn,* R. McCloskey, Tatiana C. Tatum, German A. Bollero,
Mark R. Jeschke, and Patrick J. Tranel
ABSTRACT is the agricultural land of the Midwest. Given the intense
farming practices used and the nearly constant changes
Weedy Amaranthus species pose a serious threat to agriculture.
in Best Management Practices, agrichemicals used, etc., One of the areas that causes the greatest concern is development of
herbicide resistance and subsequent transfer of herbicide resistance the landscape is in a continual state of flux. If interspe-
genes among the species. To determine the potential impact of inter- cific hybridization does occur in Amaranthus, introgres-
specific hybridization, one must first be able to detect such hybrids.
sive hybridization could be very important in the evolu-
To determine if genome size could be useful in the detection of inter-
tion and adaptation of these weedy species.
specific hybrids among the weedy Amaranthus species, the genome
Murray (1940) reported that hybrids could be made
sizes of the weedy Amaranthus species sympatric in Illinois were ana-
among various monoecious and dioecious pigweed spe-
lyzed. In a series of experiments, the genome sizes of these species
cies. Wetzel et al. (1999) and Franssen et al. (2001) re-
were determined by flow cytometric analysis. A significant variation
ported the production of interspecific hybrids between
was observed with respect to nuclear DNAcontent in the eight species
two dioecious pigweed species. Tranel et al. (2002) re-
examined. The genome sizes ranged from 0.95 pg in A. palmeri to
ported interspecific hybridization between a monoe- 1.4 pg in A. tuberculatus. Overlap of genome sizes among the species
does exist; however, due to the reproductive biology of the species, cious and a dioecious pigweed species. In all the above
this overlap does not preclude the detection of interspecific hybrids. reports, the hybrids were made under controlled, iso-
Any dioecious weedy Amaranthus plant in Illinois that has a genome
lated conditions. Trucco et al. (2005) demonstrated in-
size between 1.3 pg and 1.1 pg is probably a hybrid. Thus, the potential
terspecific hybrids between A. hybridus and A. tubercu-
for genome size determination to reveal interspecific hybrids among
latus under field conditions. Thus, it is possible that
weedy Amaranthus species has been demonstrated.
hybrids between pigweeds could occur naturally in agro-
nomic fields.
Jeschke et al. (2003) and Wetzel et al. (1999) indicated
W
eedy Amaranthus species (pigweeds) have been
that a major problem with assessing natural hybridiza-
and continue to be a major problem in agronomic
tion in pigweeds is the ability to identify such hybrids.
production. A major contributor to the noxious nature
Jeschke et al. (2003) suggested that genome size could
of these weedy species is their ability to efficiently adapt
be used to identify hybrids between A. hybridus and
to changes in agricultural management practices that
A. tuberculatus. Using flow cytometry to detect hybrid
are specifically designed to control and prevent coloni-
plants has become common practice (Barre et al., 1998;
zation. For example, numerous populations of pigweeds
Williams et al., 2002; Sisko et al., 2003; Nimura et al.,
have evolved herbicide resistance (Heap, 2003). Impor-
2003; Horita et al., 2003). Critical to the usefulness of
tant for the control of these weeds is a basic understand-
genome size in identifying hybrids is the DNA content
ing of how these plants adapt to constantly changing
of the parental types. To identify hybrids, the parents
agricultural practices. An important question is if a char-
need to differ significantly with respect to nuclear ge-
acteristic such as herbicide resistance arises in one spe-
nome size. Such is the case with respect to A. hybridus
cies of pigweed, what is the potential of that characteris-
and A. tuberculatus, the two most common pigweeds in
tic being transferred into related species?
Illinois. The difference in genome size is enough that the
One way in which genetic material could be exchanged
genome size of the hybrid is significantly different from
is by introgressive hybridization. Reports have indicated
both parents (Jeschke et al., 2003). Tranel et al. (2002)
that, in plants, interspecific hybrids occur in nature (Steb-
determined that flow cytometry could be used to defini-
bins, 1950). Although this evolutionary importance has
tively identify hybrids between A. hybridus and A. tuber-
been debated for decades, given the right circumstances,
culatus made under laboratory conditions, and Trucco
interspecific hybrids could play a crucial role in the evo-
et al. (2005) demonstrated that flow cytometry could be
lution of many plant species, especially those labeled
used to detect such hybrids under controlled field con-
noxious weeds. The greatest potential for introgressive
ditions. However, under agronomic field conditions, not
hybridization to have an impact in the evolution of a
only are A. hybridus and A. tuberculatus present but other
species is in areas of much-disturbed habitats (Steb-
pigweeds are also sympatric. Thus, the potential of using
bins, 1950). One of the most continually disrupted areas
genome size in assessing natural hybrids is dependent
on the genome size of all the pigweeds in a given locale.
The purpose of this study was to determine the genome Dep. of Crop Sciences, Univ. of Illinois at Urbana-Champaign, 320
ERML, 1201 W. Gregory, Urbana, IL 61801. Received 23 Feb. 2005. size of pigweed species found in Illinois. Flowcytometry
*Corresponding author (arayburn@uiuc.edu).
was used to assess the genome size of several popula-
tions of each species to document what, if any, genome
Published in Crop Sci. 45:25572562 (2005).
size variation exists among these species and, if it does Crop Ecology, Management & Quality
doi:10.2135/cropsci2005.0163
Crop Science Society of America Abbreviations: FI, fluorescence inhibitor; RBGKew, Royal Botanical
Gardens, Kew, UK. 677 S. Segoe Rd., Madison, WI 53711 USA
2557
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2558 CROP SCIENCE, VOL. 45, NOVEMBERDECEMBER 2005
iment was run in a similar fashion, except that three plants exist, to determine if it is useful in identifying interspe-
per accession per species were analyzed.
cific pigweed hybrids.
In both experiments, the factor species and accessions were
considered fixed. Accessions were nested within species, and
MATERIALS AND METHODS
two and three replications were used for experiment 1 and
experiment 2, respectively. The linear model used in both Several accessions of six species were analyzed for genome
size. These species, together with A. hybridus and A. tubercula- experiments was
tus, are the most common pigweed species in the United States
y
i j k

i

j (i)

i j k
[1]
and are the only pigweeds found in Illinois. The accessions
are reflective of the wide U.S. geographic distribution of each where y
i j k
is an observation, is the population mean,
i
is
species (Table 1). An accession of A. tuberculatus (LW 22) the effect of i th species,
j (i)
is the effect of the jth accession
and an accession of A. hybridus (LW 1-1) were obtained from within the i th species, and
i j k
is the error term. The statistical
the weed science collection, University of Illinois. In addition, analysis was done with the MIXED procedure of SAS (SAS,
two pigweed species were obtained fromRoyal Botanical Gar- 2003). Least significant differences were calculated based on
dens, Kew, UK (RBG Kew). These two species, A. hybridus appropriate degrees of freedom and standard errors for the
(accession no. 44460) and A. retroflexus (accession no. 20301) differences of means.
were used as reference standards. To compare data among studies, a third experiment was
In the first experiment, two plants per accession per species run. The two accessions of A. hybridus were analyzed. Two ac-
were analyzed. The plants were grown as described by Jeschke cessions of another species, A. retroflexus (MH 84 and an ac-
et al. (2003). Briefly, the plants were planted in a mixture of cession from RBG KEW) were also analyzed. Four plants of
perlite, peat, and Drummer silty clay loam supplemented with eachaccessionwere grown, andnuclei were isolatedandexam-
a controlled-release fertilizer. The plants were grown in a ined as described above. W22 was used as an internal standard.
greenhouse until large enough for analysis. During each of 4 d, Afourthexperiment comparing the genome size of A. tuber-
individual plants fromeach species were randomly selected for culatus and A. palmeri was performed. Seeds were planted and
analysis. Leaf tissue was removed, and nuclei were isolated, grown as previously described. After approximately 6 weeks,
stained, and analyzed as described by Jeschke et al. (2003). three leaves were taken from four plants per species. The leaves
The maize line W22 was used as an internal standard for each from each plant were analyzed on the flow cytometer, and
sample. Rolled leaf portions above the growing point of maize genome size was determined as described previously. In addi-
seedlings were co-chopped with the pigweed leaf sample. The tion, A. tuberculatus and A. palmeri leaves were chopped and
samples were analyzed on a Coulter EPICS XLflowcytometer ground together. The samples containing the mixed nuclei were
with an excitation wavelength of 488 nm. Five thousand nuclei analyzed as described previously. The differences between the
were analyzed per sample. The amount of DNA of the experi- two species DNA contents were compared using ANOVA
mental species was determined relative to the internal stan- and LSD tests using the SAS statistical program.
dard. The pg per 2C nucleus was established by running the The chromosome number was thendetermined for A. tuber-
maize standardwith chickenredbloodcells. The secondexper- culatus and A. palmeri. Root tips were rinsed with deionized
water, placed in 8-hydroxyquinoline for 2 h, and fixed in Car-
Table 1. Species and location of origin of the accessions analyzed. noys solution. Root tips were obtained and stained with
aceto-orcein, and root tip squashes were performed. A total
Accession number
of 12 spreads per species were analyzed for chromosome num-
Species Experiment 1 Experiment 2 Origin
ber. The chromosomes were counted using an Olympus BX61
microscope. A. retroflexus PT 42 PT 42 Henry Co., IL
PT 55 PT 55 Champaign Co., IL
MH 25 MH 25 Carbon Co., MT
MH 72 MH 72 North Dakota State Univ. RESULTS
MH 84 MH 84 Dona Ana, NM
A. powelli PT 37 PT 37 Grundy Co., IL The 2C genome size of chicken is reported by Tiersch
PT 41 Henry Co., IL
and Wachtel (1991) to be 2.5 pg per 2C nucleus. Upon
PT 68 PT 68 Champaign Co., IL
comparing maize line W22 with the chicken nuclei, the
MH 32 MH 32 Centre Co., PA
MH 234 MH 234 Moses Lake, WA genome size of W22 was estimated to be 5.33 pg per 2C
MH 237 Seneca, NY
nucleus. This number is in good agreement with McMur-
A. palmeri PT 63 Champaign Co., IL
phy and Rayburn (1991), who estimated the genome MH 251 MH 251 Univ. of Arkansas
MH 252 MH 252 Stoneville, MS
size to be 5.35 pg per 2Cnucleus. Given the almost iden-
MH 262 MH 262 Maricopa, AZ
tical estimates of DNA, all the weedpopulations werecon-
MH 277 MH 277 Beckham, OK
MH 254 Brazos, TX verted using 5.35 pg per 2C nucleus for W22 to maintain
A. albus PT 66 PT 66 Champaign Co., IL
consistency with other data published using W22 as an
PT 106 PT 106 Morgan Co., IL
internal standard.
MH 40 MH 40 Dona Ana, NM
MH 49 MH 49 Stoneville, MS The genome size of the pigweed species ranged from
MH 55 MH 55 Centre Co., PA
0.96 to 1.21 pg per 2Cnucleus in experiment 1 and 0.95 to
A. blitoides MH 215 MH 215 Whitman Co., WA
1.23 pg per 2C nucleus in experiment 2 (Table 2). In MH 219 Franklin Co., MD
MH 206 Harrow, Ontario
both experiments, species were significantly different
MH 213 Dona Ana, NM
with respect to genome size (p 0.001). Accessions
MH 224 Carbon Co., MT
A. spinosus PT 117 PT 117 Cass Co., IL within species were not significant at 0.05. The order
MH 200 MH 200 Charleston, SC
of genome size was similar between the experiments with
MH 271 MH 271 University of Hawaii
the only exception being that A. blitoides had a slightly
MH 276 MH 276 Champaign Co., IL
MH 201 Stoneville, MS larger genome size than A. albus in experiment 1 and
MH 204 Stoneville, MS
a slightly smaller genome size in experiment 2.
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RAYBURN ET AL.: GENOME SIZE VARIATION IN WEEDY AMARANTHS 2559
Table 3. Statistical analysis of Amaranthus tuberculatus and Table 2. Genome sizes of the six Amaranthus species used in
this study. Amaranthus palmeri genome sizes.
Mean 2C genome size Previously
Mean 2C reported 2C Species Plant number (pg) LSD grouping
genome size genome size
A. tuberculatus 1 1.44 0.01 A
Species N (pg) (pg)
2 1.42 0.03 A
3 1.42 0.02 A Experiment 1
A. retroflexus 10 1.21 0.02 1.70 4 1.42 0.02 A
A. palmeri 1 0.92 0.01 B A. blitoides 4 1.18 0.03
A. albus 10 1.16 0.02 2 0.92 0.02 B
3 0.91 0.01 B A. powelli 10 1.11 0.03 1.20
A. spinosus 10 1.04 0.02 1.90 4 0.90 0.01 B
A. palmeri 10 0.96 0.04
Mean represents the average picograms of three leaves per plant
LSD 0.03 ( 0.05)
standard deviation.
Experiment 2
Plants with the same letter are not significantly different at 0.05.
A. retroflexus 15 1.23 0.03 1.70
A. albus 15 1.18 0.03
A. blitoides 12 1.15 0.02
was 0.91 0.01 per 2C nucleus (p 0.0001). When co- A. powelli 15 1.14 0.05 1.20
A. spinosus 15 1.10 0.02 1.90
chopped, the variation in genome size between the two
A. palmeri 15 0.95 0.02
species was 52% (Fig. 1). Both species were observed
LSD 0.02 ( 0.05)
to have 32 chromosomes, with the chromosomes of
Mean standard deviation.
A. palmeri appearing slightly smaller (Fig. 2).
Estimates taken from www.rbgkew.org.uk/cval/homepage.html.
Consistent in both experiments was that A. palmeri had
DISCUSSION
the smallest genome size, whereas A. retroflexus had the
The first six pigweed species examined in this study
largest genome size. The genome sizes of these two spe-
had 27% variation in genome size. Substantiating the
cies were consistent between the two experiments (Ta-
reproducibility of the genome size data is that in both
ble 3). The only species that had significant genome size
experiments, only one minor change in the order of the
variation between the two experiments was A. spinosus.
species was observed and that the actual picogram
Because of inconsistencies in published reports of
amounts were very similar. In addition, the coefficient
genome size of A. retroflexus, the third experiment was
of variation (CV) of the measured G1 peaks averaged
performed. Comparing the A. retroflexus accession
5.0%. This mean CV is higher than recommended by
MH-84 with the accession from RBG KEW revealed
Rayburn (1993). Rayburn (1993) used maize and sor-
that both had similar genome sizes. MH-84 had 1.21 pg
ghum in describing the flow cytometric technique of
per 2C nucleus, consistent with the previous experi-
DNA analysis. In the Amaranthus species, it is much
ments, whereas the RBG KEW accession was observed
more difficult to obtain CVs of 3%. However, Dolezel to have 1.16 pg per 2C nucleus. With respect to A.
and Bartos (2005) recommended that in difficult plant hybridus, the accession LW 1-1 and the RBG KEW
species, CVs around 5.0% are acceptable. accessions had nearly identical genome sizes of 1.06 and
Although such genome size variation has been pre- 1.08 pg per 2C nucleus, respectively.
viously reported in some of the species examined in this In the fourth experiment, cuttings from A. tubercula-
study (Bennett et al., 2000), two major discrepancies tus and A. palmeri were analyzed. There was no signifi-
occur between the previous study and the results pre- cant difference among plants within each species (Ta-
sented in this study. As described by Rayburn (1993) ble 2). A statistically significant difference was observed
and Greilhuber et al. (2005), inconsistency exists with between groups, with the overall average genome size
respect to how genome sizes are reported. To facilitate of A. tuberculatus being 1.43 0.01 per 2C nucleus,
whereas the overall mean genome size of A. palmeri comparisons among studies, genome sizes in this article
Fig. 1. Flow cytometric histogram of relative DNA content of nuclei from A. tuberculatus and A. palmeri co-chopped leaves. The relative
difference between the mean of the G1 peak of A. palmeri to A. tuberculatus was 52%.
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2560 CROP SCIENCE, VOL. 45, NOVEMBERDECEMBER 2005
Fig. 2. Mitotic chromosomes of A. tuberculatus (A) and A. palmeri (B), with both having 2n 32 chromosomes. Bar 5 m.
refer to the amount of DNA found in G1 diploid cells was correct. Upon comparing the chicken nuclei with
Xenopus laevis nuclei defined as having 6.3 pg per 2C and are expressed as the amount of pg per 2C nucleus.
The first discrepancy between the two studies is the nucleus (Freeman and Rayburn, 2004), the chicken nu-
clei were determined to have a 2C genome size of 2.5 pg total pg amounts reported for the species. Bennett et al.
(2000) reported that A. retroflexus and A. spinosus had (data not shown), which is within the reported range of
chicken nuclei (Johnston, 1999). The recalculated ge- 1.7 and 1.9 pg per 2C nucleus, respectively. In the pres-
ent study, A. retroflexus and A. spinosus were observed nome size of W22 using chicken red blood cells was
5.33 pg, which is nearly identical to the 5.35 pg pre- to have 1.22 and 1.07 pg per 2C nucleus, respectively.
In addition, Bennett et al. (1998) reported that A. hy- viously reported. To obtain the numbers observed by
Bennett et al. (2000), the chicken nuclei would have to bridus had 1.4 pg per 2C nucleus, whereas Tranel et al.
(2002) and Jeschke et al. (2003) have reported A. hy- have had a genome size of 4 pg, which would be well
outside the reported range of chicken genome size and bridus to have 1.12 and 1.04 pg per 2C nucleus. One
possible explanation of these differences is that the ac- the newly recalibrated chicken nuclei used in this study.
Therefore, the lower values for the pigweed species re- cessions observed by Bennett et al. (2000) were different
from the accessions of the same species used in the U.S. ported here seem to be correct. However, absolute ge-
nome size is not the only incongruity between the two studies and as such have more DNAper genome. To test
this hypothesis, accessions of A. hybridus and A. retro- studies.
According to Bennett et al. (2000), A. spinosus was re- flexus used by Bennett et al. (2000) were obtained from
RGB KEW and analyzed in conjunction with accessions ported to have the largest genome size of the pigweed
species examined (1.9 pg per 2C nucleus). In this study, of the same two species used in this study. When the
present study was repeated, the A. retroflexus used in A. spinosus was observed to have one of the smallest
genome sizes (1.07 pg per 2C nucleus). This is not a the present study was found to have a genome size of
1.21 pg per 2C nucleus, consistent with the previous re- result of an incorrect standard. Amiscalculated standard
may result in an error of absolute DNA content, but the sults of this study, whereas the accession obtained from
RGB KEW was observed to have 1.16 pg per 2C nu- overall relative ranking of the species would be main-
tained. Therefore, another problem must exist. Another cleus, not the previously reported 1.7 pg (Table 2). The
two species of A. hybridus had nearly identical genome difference between the Bennett et al. (2000) study and
this study is the method used to calculate genome size. sizes of 1.07 pg per 2C nucleus, again not consistent
with the previously reported 1.4 pg per 2C nucleus. Thus, Bennett et al. (2000) used microdensitometry on nuclei
from fixed root tips, whereas flow cytometry of leaf tis- in both cases, the pigweed species genome sizes seem
to be overestimated in Bennett et al. (2000). sue was used in the present study. Although both meth-
odologies are extensively used, they require extreme One possible explanation of these observations is that
in Tranel et al. (2002), in Jeschke et al. (2003), and in care due to technical errors that can occur during analy-
sis (Greilhuber, 1997; Price et al., 2000). the present study, the maize line W22 was used as the
internal standard. W22 is an inbred line of maize that The major concern when using flow cytometry is the
presence of fluorescence inhibitors (FIs) (Price et al., has been reported to have 5.35 pg of DNA per 2C nu-
cleus (Biradar and Rayburn, 1993). Bennett et al. (2000) 2000). Although an internal standard was used during
the flow cytometric analysis to compensate for FIs, the used multiple standards, and the exact standard used
in the pigweed determinations is not clear. Critical to lower values observed in this study may cause concern
that this compensation is not complete. However, Tra- absolute genomes size determinations is the choice of
the reference standard (Johnston, 1999). The reference nel et al. (2002), using flowcytometric analysis, observed
that the F
1
hybrid between two pigweed species had standard should be well defined. Because all the studies
that reported the lower genome size for the pigweed an intermediate genome size between the two parental
genome sizes. The probability that in the F
1
hybrid the species used W22 as a standard, chicken red blood cell
nuclei were used to ensure that the 5.35 pg of W22 exact amount of FIs would be present that would inter-
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RAYBURN ET AL.: GENOME SIZE VARIATION IN WEEDY AMARANTHS 2561
act with the intermediate DNA present in the hybrid to et al. (2003) observed A. tuberculatus to have a genome
size of 1.4 per 2C nucleus. Thus, this hybrid represents give a fluorescence almost exactly half way between the
two parent fluorescence intensities is low. Also, given the the hybridization of the species with the widest range
of genome size. To ensure that the 1.4 pg per 2C nucleus fact that an internal standard was used, such an exact
interaction would be relatively rare. One might suspect estimate for A. tuberculatus was correct and that the ge-
nome sizes between the two dioecious species were that that the maize internal standard used in this study and in
Tranel et al. (2002) could react differently than pigweed distinct, the genome size of A. tuberculatus and A. palmeri
were reassessed. The genome size of A. palmeri was as nuclei to FIs and thus produce abnormal results. The co-
chopping of the A. tuberculatus and A. palmeri leaves expected from the previous experiments, whereas A. tu-
berculatus had 1.4 pg per 2Cnucleus. Thus, any potential gave the same relative difference between the two spe-
cies. Thus, the difference seems real and is not a result hybrid between these two species should have 1.15 pg
per 2C nucleus. This genome size is similar to the ge- of using a maize internal control.
The lower estimate of genome size in A. spinosus in nome size of several pigweed species; however, because
this cross is between to two dioecious parents, the hybrid this study is also supported by recent phylogenic data.
One might expect that the two dioecious species would would also be dioecious. Because the remaining species
are monoecious, the hybrid would be morphologically be more closely related and therefore the genome sizes
of these two species to be similar. However, recent re- distinct and easily distinguished from the surrounding
species. ports have suggested that A. palmeri (a dioecious spe-
cies) and A. spinosus (a monoecious species) may be In interspecific hybridization between dioecious and
monoecious species, an interesting phenomenon occurs. closely related. Franssen et al. (2001) noted that the
pollen morphology of A. palmeri was more similar to Murray (1940) reported that, in dioecious by monoecious
pigweed crosses, dioeciousness is epistatic to monoe- A. spinosus than any other pigweed species. Wasson
and Tranel (in press) using AFLP analysis observed that ciousness. Simply put, F
1
hybrids between monoecious
and dioecious pigweeds are dioecious. Therefore, any phylogenetic analysis and principal coordinate analysis
resulted in a clustering of A. palmeri, with A. spinosus dioecious pigweed plant that has a genome size of less
than 1.3 pg of DNA per 2C nucleus and more than indicating close genetic similarities. Given the apparent
close genetic relationship and almost identical pollen 1.1 pg of DNA per 2C nucleus is a potential hybrid.
Although the exact parentage may be unknown, the morphology, the observation that these species would
have similar genome sizes is not totally unexpected. amount of pigweed-interspecific hybridization in a given
field population may be estimated by observing the Thus, the low genome size observed in A. spinosus is
real and is not an artifact of the methodology. number of dioecious pigweeds with intermediate ge-
nome sizes. Given the wide range of genome sizes found in the
Illinois pigweed species (50%), the initial appearance In conclusion, genome sizes in pigweed species found
in Illinois range from 0.95 to 1.4 pg of DNA per 2C is that genome size would not be effective in identifying
hybrids betweenpigweed species. For instance, if A. retro- nucleus. The two species at the extreme range of the spec-
trum are the two dioecious species. Because both species flexus would hybridize with A. spinosus, the expected
genome size of the hybrid would be 1.13 pg per 2C nu- have 32 chromosomes, this variation is due to intrachro-
mosomal variation. Given the reproductive biology of cleus, similar to the average genome size of A. powelli,
which is 1.13 pg per 2C nucleus. Therefore, genome size the Amaranthus species, the dominance of dioeciousness,
and the wide range of genome size variation between the would be of little use for the random screening of F
1
pigweed hybrids in the field. However, an interesting dioecious species, genome size determinations have the
potential to be useful in addressing questions concern- dichotomy exists with respect to reproduction among
the pigweed species. A. tuberculatus and A. palmeri are ing the amount of interspecific hybridization within the
genus Amaranthus under field conditions. dioecious species, whereas the remaining pigweeds in
Illinois are monoecious. The monoecious plants have a
propensity to self-fertilize due to the proximity of the
ACKNOWLEDGMENTS
male flowers to the female flowers (Franssen et al., 2001).
We thank Mike Horak for providing much of the seed used
In addition, the pollen of the monoecious species does
in this study. We also thank Dr. B. Pilas of the Flow Cytometry
not seem to be as aerodynamic as the pollen from dioe-
Facility, a resource of the University of Illinois Biotechnology
cious species. Therefore, interspecific hybridizationamong
Center, for her assistance. Funding for this research was pro-
monoecious pigweeds would be relatively limited.
vided inpart by the Illinois Soybean ProgramOperating Board
With interspecific hybridization being skewed toward and by the USDA under Award No. 2001-35320-11002.
dioecious by dioecious or dioecious by monoecious spe-
cies, the use of genome size to identify interspecific hy- REFERENCES
brids becomes apparent. For example, if the two dioe-
Biradar, D.P., and A.L. Rayburn. 1993. Heterosis and nuclear DNA
cious species sympatric in Illinois, A. tuberculatus and
content in maize. Heredity 71:300304.
Barre, P., M. Layssac, A. DHont, J. Louarn, A. Charrie, S. Hamon, A. palmeri, were to hybridize, the F1 hybrid would theo-
and M. Noirot. 1998. Relationship between parental chromosomic
retically have an intermediate DNA content between
contribution and nuclear DNA content in the coffee interspecific
the two species. Interestingly, A. palmeri was observed
hybrid C. pseudozanguebariae C. liberica var dewevrei. Theor.
in this study to have the lowest genome size of the weedy
Appl. Genet. 96:301305.
Bennett, M.D., P. Bhandol, and I.J. Leitch. 2000. Nuclear DNA Amaranthus, whereas Tranel et al. (2002) and Jeschke
R
e
p
r
o
d
u
c
e
d

f
r
o
m

C
r
o
p

S
c
i
e
n
c
e
.

P
u
b
l
i
s
h
e
d

b
y

C
r
o
p

S
c
i
e
n
c
e

S
o
c
i
e
t
y

o
f

A
m
e
r
i
c
a
.

A
l
l

c
o
p
y
r
i
g
h
t
s

r
e
s
e
r
v
e
d
.
2562 CROP SCIENCE, VOL. 45, NOVEMBERDECEMBER 2005
amounts in angiosperms and their modern uses807 new estimates. Murray, M.J. 1940. The genetics of sex determination in Amarantha-
ceae. Genetics 25:409431. Ann. Bot. 86:859909.
Nimura, M., J. Kato, M. Mii, and K. Motioka. 2003. Unilateral compat- Dolezel, J., and J. Bartos. 2005. Plant DNAflowcytometry and estima-
ibility and genotypic difference in crossability in interspecific hy-
tion of nuclear genome size. Ann. Bot. 95:99110.
bridization between Dianthus caryophyllus L. and Dianthus japoni-
Franssen, A.S., D.Z. Skinner, K. Al-Khatib, and M.J. Horak. 2001.
cus Thunb. Theor. Appl. Genet. 106:11641170.
Pollen morphological differences in Amaranthus species and inter-
Price, H.J., G. Hodnett, and J.S. Johnston. 2000. Sunflower (Helian-
specific hybrids. Weed Sci. 49:732737.
thus annus) leaves contain compounds that reduce nuclear propid-
Freeman, J.L., and A.L. Rayburn. 2004. Metamorphosis in Xenopus
ium iodide fluorescence. Ann. Bot. 86:929934.
laevis is not associated with large-scale nuclear DNA content varia-
Rayburn, A.L. 1993. Comparative studies of genome content.
tion. J. Exp. Biol. 207:44734477.
p. 204212. In E.A. Zimmer, T.J. White, R. L. Cann, and A.C.
Greilhuber, J., J. Dolezel, M.A. Lysa k, and M.D. Bennett. 2005. The
Wilson (ed.) Methods in enzymology. Vol. 224. Molecular evolu-
origin, evolution, and proposed stabilizaton of the terms genome
tion: producing the biochemical data. Academic Press, Boston.
size and C-value to describe nuclear DNA contents. Ann. Bot.
Sisko, M., A. Ivancic, and B. Bohanec. 2003. Genome size analysis
95:255260.
in the genus Cucurbita and its use for determination of interspecific
Greilhuber, J. 1997. The problem of variable genome size in plants
hybrids obtained using the embryo rescue technique. Plant Sci.
(with special reference to woody plants). p. 1334. In Z. Borzan
165:663669.
and S.W. Schlarbaum (ed.) Cytogenetic studies of forest trees and
Stebbins, G.L. 1950. Variation and evolution in plants. p. 262286,
shrub species. Croatian Forests, Inc., Faculty of Forestry, Univer- 312. Columbia University Press, New York.
sity of Zagreb, Croatia. Tiersch, T.R., and S.S. Wachtel. 1991. On the evolution of genome
size of birds. J. Hered. 82:363368. Heap, I. 2003. Citing online sources: The international survey of herbi-
Tranel, P.J., J.J. Wassom, M.R. Jeschke, and A.L. Rayburn. 2002. cide resistant weeds. Available at www.weedscience.com (accessed
Transmission of herbicide resistance from a monoecious to a dioe-
18 April 2003).
cious weedy Amaranthus species. Theor. Appl. Genet. 105:674679.
Horita, M., H. Morohashi, and F. Komai. 2003. Production of fertile
Trucco, F., M.R. Jeschke, A.L. Rayburn, and P.J. Tranel. 2005. Amara-
somatic hybrid plants between Oriental hybrid lily and Lilium x
rthus hybrids can be pollinated frequently by A. tuberculatus under
formolongi. Planta 217:597601.
field conditions. Heredity 94:6470.
Jeschke, M.R., P.J. Tranel, and A.L. Rayburn. 2003. DNA content
Wasson, J.J., and P.J. Tranel. 2005. Amplified fragment length poly-
analysis of smooth pigweed (Amaranthus hybridus) and tall water-
morphism-based genetic relationships among weedy Amaranthus
hemp (A. tuberculatus): Implications for hybrid detection. Weed
species. J. Hered. 96:410416.
Sci. 51:13.
Wetzel, D.K., M.J. Horak, and D.Z. Skinner. 1999. Use of PCR-based
Johnston, J.S. 1999. Reference standards for determination of DNA
molecular markers to identify weedy Amaranthus species. Weed
content of plant nuclei. Am. J. Bot. 86:609613.
Sci. 47:518523.
McMurphy, L.M., and A.L. Rayburn. 1991. Genome size variation in
Williams, C.G., K.L. Joyner, L.D. Aukland, S. Johnston, and H.J.
maize populations selected for cold tolerance. Plant Breed. 106: Price. 2002. Genomic consequences of interspecific Pinus spp. hy-
bridization. Biol. J. Linnean Soci. 75:503508. 190195.