Weed Science, 47:310-315. 1999

Molecular markers indicate intraspecific variation in the control of Echinochloa spp. with quinclorac Go to table of contents

Nuria Lopez-Martinez Corresponding author. Departamento de Quimica Agricola y Edafologia. E.T.S.I.A.M. Apdo. 3048, 14080-Cordoba, Spain; [email protected]

Antonio Pujadas Salvá

Departamento de Ciencias y Recursos Agrícolas y Forestales. E.T.S.I.A.M. Apdo. 3048, 14080Cordoba, Spain

Robert P. Finch George Marshall Scottish Agricultural College, Auchincruive, Ayr KA6 5HW, Scotland, U.K.

Some species of the genus Echinochloa are troublesome weeds in rice fields. The taxonomy of this genus leads to confusion in many cases due to its great morphological diversity. Because of the differential sensitivity to the herbicide quinclorac shown by Echinochloa spp., it was necessary to assess the botanical and molecular characterization of this weed. Echinochloa colonum, E. oryzoides, and E. oryzicola were very susceptible to quinclorac treatment; by contrast, E. crus-galli and E. hispidula showed some degree of natural tolerance. Physiological and molecular results agreed with the botanical classification of the genus Echinochloa in Flora Europea. The importance of these results is due to yield losses produced by the infestation of Echinochloa and the need for a strategy for Echinochloa management depending on the distribution of the Echinochloa species. Nomenclature: Quinclorac; ECHSP, Echinochloa spp.; base pair; Echinochloa crusgalli, barnyard grass; ECHCG; Echinochloa. Key words: RAPD-PCR.

Genetic diversity, herbicide resistance, molecular markers, quinclorac,

The genus Echinochloa belongs to tribe Paniceae R. Br. subfamily Panicoideae A. Br., family Gramineae Juss (= Poaceae Barnh.). There is disagreement about the species that constitute Echinochloa. The genus may include 20 to 50 species that are widely represented in tropical and warm temperate regions of the world (Clayton and Renvoize 1986; Gould et al. 1972; Michael 1983). Biological taxonomy is a process by which humans organize diversity in the living world; such organization is essential if generalizations are to be made about nature. The taxonomic process of species recognition and delimitation is' a prerequisite for giving names to plants, and an organism s name is the key to literature citations. A name based on problematic taxonomy may cause confusion (McNeill 1982). For example, the discovery that weeds introduced in North America under the name Cardaria draba are really three different species. Not only do these species have different ecological tolerances and geographical distributions in North America, but they show differential responses to 2,4D (Mulligan and Findlay 1974). Similarly, the misidentification of plants of Amaranthus powellii S. Wats. (Powell amaranth) and A. hybridus L. (smooth pigweed) as A. retroflexus L. (redroot pigweed) has led to confusion in the accounts published on atrazine resistance in Amaranthus (Warwick and Weaver 1980). An awareness of the confusion that has existed and the adoption of a more accurate taxonomic treatment are essential for other weed genera, such as Echinochloa spp. and for clear understanding of the different response of species to quinclorac (Lopez-Martinez et al. 1995). The genus Echinochloa contains numerous integrating forms, its diversity apparently stemming from self-pollination combined with adaptation to a wide range of aquatic and ruderal habitats. There is uncertainty as to how this complex should be divided into species and no reliable key to distinguish species within Echinochloa (Clayton and Renvoize 1986).

The taxonomic complexity of Echinochloa spp. in the Iberian Peninsula has led to different classifications. Coutinho (1939) identified only E. crus-galli (L.) Beauv. (subsp. Panicum crus-galli L.); Paunero (1962) recognized two species, E. colonum and E. crus-galli; and Clayton (1980) recognized three species, E. colonum, E. crus-galli, and E. oryzoides. Carretero (1981) detailed five different taxa E. colonum, E. crusgalli, E. hispidula, E. oryzoides, and E. oryzicola. Devesa (1987) gave a different treatment to the genus, showing only four taxa in the southwest of the Iberian Peninsula: Echinochloa colonum, E. crus-galli, E. crus-galli subsp. hispidula, and E. oryzicola. Correct identification is agronomically and economically important because all Echinochloa spp. are aggressive invaders and difficult to control. Crop yield losses depend on the density of Echinochloa spp. and on the duration of the interference. For example, a density of 20 plants m- 2 will reduce rice yield over 80% (VanDevender et al. 1997). The identification of Echinochloa spp. is difficult because of the morphological diversity shown by the genus. Different herbicide susceptibility between species showed the need to establish adequate methods to characterize the genus (Carretero 1989; Lopez-Martinez et al. 1995, 1997). Accordingly, we employed molecular techniques to make a genetic comparison of Echinochloa spp. based on randomly amplified polymorphic DNA (RAPD) analysis together with classic morphological taxonomy. Isozymes have been used to characterize the genus Echinochloa (Gonzalez-Andres et al. 1996), although a low level of polymorphism was detected. To circumvent this problem, RAPD techniques may be an appropriate way to monitor diversity in plant populations (Anderson and Fairbanks 1990; Dawson et al. 1993; Waugh and Powell 1992). RAPD polymerase chain reaction (RAPD-PCR) assay procedures are technically simple, requiring only small

Rafael De Prado

Departamento de Quimica Agricola y Edafologia. E.T.S.I.A.M. Apdo. 3048, 14080-Cordoba, Spain

310 •

Weed Science 47, May June 1999

Reference number, scientific classification, origin, habitat, and herbarium reference of Echinochloa spp. biotypes of this study. TABLE 1.

Scientific name

Origin

Habitat

Herbarium COA

crus-galli oryzoides hispidula hispidula oryzoides oryzoides oryzoides oryzoides crus galli crus galli crus galli crus galli crus galli hispidula hispidula crusgalli hispidula hispidula hispidula hispidula oryzoides hispidula oryzicola E colonum E colonum

Sevilla Tarragona Sevilla Sevilla Badajoz Badajoz Badajoz Badajoz Badajoz Badajoz Badajoz Badajoz Badajoz Sevilla Sevilla Tarragona Sevilla Tarragona Tarragona Sevilla Tarragona Sevilla Tarragona Cordoba Sevilla

Rice Rice Rice Rice Maize Rice Rice Rice Maize Maize Maize Maize Maize Rice Rice Rice Rice Rice Rice Rice Rice Rice Rice Garden grass Rice

22885 22866 22890 22879 22870 22869 22868 22867 22886 22889 22888 22884 22883 22878 22877 22882 22875 22872 22887 22874 22865 22873 22871 22880 22881

Biotype 1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E.

amounts of crude DNA that can be extracted easily. DNA amplification is carried out using random primers (Welsh and McClelland 1990; Williams et al. 1990). This technique has the relative simplicity and rapidity needed for generating molecular markers that can be used in a range of applications because it does not require prior knowledge of a plant 's genetics and can be used to analyze total genomic DNA (Moodie et al. 1997). This work was initiated because of the different response to quinclorac treatment shown by populations of Echinochloa thought to be E. crus galli. Botanical analyses and molecular techniques were carried out in order to clarify taxonomic confusion. In this study, the RAPD-PCR technique was used to assess the amount of interspecific variation in the genus Echinochloa. In our study, DNA amplification before digestion with restriction enzyme analysis was investigated in order to find the polymorphism between species and subspecies of Echinochloa that RAPD analyses were not able to detect.

Materials and Methods Plant Material and Growing Conditions Twenty-five different populations of the genus Echinochloa were analyzed. Plant material was collected at several places in the Iberian Peninsula, including 18 locations from Oryza sativa L. (rice) fields where tolerance to quinclorac had been observed. Testimonies of these biotypes were kept in vouchers in the Herbarium of the Unidad de Botanica, Escuela Tecnica Superior de Ingenieros Agronomos y de Montes, Universidad de Cordoba, sited in Jardin Botanico de Cordoba, Spain (Table 1). Seeds were placed in petri dishes containing filter paper

L-1

KNO 3 solution and were germimoistened with 2 g nated under continuous illumination of 350 Rmol m- 2 s- 1 photosynthetic photon flux (PPF) at 25 C and 80% relative humidity. Five pregerminated seeds were planted in each pot in a 1:2 peat soil mixture (sandy loamy mixed). Plants were placed in a growth chamber with a 16 h photoperiod of 350 s-1 PPE Day/night temperatures were 25/18 C Rmol m-2 and relative humidity was a constant 80%. Plants were watered as required.

Botanical Identification Botanical identification was carried out following Carretero 's (1981) keys on plants grown at the site of origin. Because some characters are highly variable, the primary distinguishing characteristics used were length and width of spikelets, glume upper length, length of upper lemma, length and width of caryopsis, and external marks on the embryo. Botanical identification was carried out in eight different plants of each biotype.

Herbicide Treatment Five pots containing five plants per pot were placed in a growth chamber. At the three-leaf stage, plants were treated with 0.01 to 4.0 kg ai ha- 1 quinclorac (Facet SC ®) using a laboratory track sprayer fitted with a Tee-Jet 8001 flat-fan nozzle delivering 200 L ha- 1 at 250 kPa (De Prado et al. 1992). Plants were kept 15 d in a growth chamber under the same conditions described earlier. After this time, plants were harvested and growth was evaluated by measuring shoot fresh weight. The quinclorac dose causing a 50% reduction of shoot fresh weight (ED 50 ) was calculated by representing the shoot fresh weight vs. the logarithm of herbicide concentration (De Prado et al. 1989). Treatments were replicated three times.

Molecular Assessment DNA Extraction DNA was isolated from leaf tissue belonging to nine plants of each biotype using a standard phenol : chloroform : isoamyl alcohol procedure (Poulsen et al. 1993), except that the centrifugation of ethanol-precipitated DNA was avoided prior to washing because coprecipitants are known to inhibit RAPD reactions. In each case, the DNA samples were mixed in equal quantities to give pooled samples for analysis of genetic variation. To determine the optimum concentration of DNA for a PCR, 0.1, 1, 5, 25, and 100 ng of genornic DNA from a single extraction were used in PCR; 25 ng gave the most consistent results.

PCR Amplification Polymerase chain reactions were carried out in 25- R 1 volumes containing the following: 25 ng sample DNA, 2.5 Rl 10X reaction buffer, l 2 Rl MgCl 2 (25 mM), 2.0 Rl of each dNTP 1 (1.25 mM), 5 pmols of each primer, 2 0.1 Ill of Taq DNA polymerise, l and the balance of water. Mixtures were covered with mineral oil s prior to being placed in a Perkin Elmer Cetus Model 480 thermal cycler.' The reaction mix was heated at 94 C for 5 min to denature the template DNA, which was amplified during 45 cycles of 1 min at 92 Lopez-Martinez et al.: Intraspecific variation in Echinochloa



311

Spikelet characters of 25 different biotypes of Echinochloa spp. Data are the mean of eight different plants of each biotype ± standard error.

TABLE 2. Biotype

Scientific name

1 2

E. crus galli E. oryzoides E. hispidula E. hispidula E. oryzoides E. oryzoides E. oryzoides E. oryzoides E. crus-galli E. crus galli E. crus galli E. crus galli E. crus-galli E. hispidula E. hispidula E. crus-galli E. hispidula E. hispidula E. hispidula E. hispidula E. oryzoides E. hispidula E. oryzicola E. colonum E. colonum

Length of spikelet

Width of spikelet

Length of upper glume

Length of upper lemma

± ± ± ± ± ±

2.63 ± 0.05 3.67±0.08 3.13 ± 0.04 3.03 ± 0.10 3.63 ± 0.09 3.57 ± 0.06 3.43 ± 0.06 3.47 ± 0.11 2.95 ± 0.07 2.58 ± 0.06 2.45 ± 0.08 2.80 ± 0.09 2.40 ± 0.06 3.43 ± 0.12 3.62 ± 0.15 2.20 ± 0.06 3.17 ± 0.15 3.23 ± 0.12 3.33 ± 0.06 3.20 ± 0.03 3.33 ± 0.14 3.47-!-0.03 3.77 ± 0.05 1.86 10.09 2.02 ± 0.11

Length of caryopsis

Width of caryopsis

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.20 ± 0.05 2.05 ± 0.11 1.73 ± 0.04 1.65 ± 0.03 1.90 ± 0.03 1.60 ± 0.04 1.95 1 0.04 1.90 ± 0.09 1.40 ± 0.04 1.50 ± 0.03 1.37 ± 0.05 1.47 ± 0.05 1.27 ± 0.05

External mark of embryo

mm

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

2.95 4.47 3.50 3.52 4.13 4.03 4.03 4.08 3.02 3.30 2.88 3.17 2.53 3.85 3.72 2.87 4.07 3.90 3.65 4.16 3.92 3.83 4.07 2.05 2.03

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.08 0.04 0.07 0.07 0.04 0.05 0.07 0.06 0.06 0.07 0.04 0.07 0.03 0.08 0.07 0.03 0.09 10.05 ± 0.05 ± 0.09 ± 0.03 ± 0.07 ± 0.05 ± 0.07 ± 0.05

1.68 2.05 1.95 1.85 2.23 2.20 2.30 2.20 1.70 1.93 1.68 1.90 1.63 2.03 2.00 1.77 1.93 1.88 1.90 2.23 1.95 2.00 2.20 1.20 1.08

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.05 0.06 0.05 0.03 0.03 0.07 0.04

0.08 0.05 0.03 0.07 0.03 0.03 0.07 0.05 0.04 0.05 10.03 ± 0.03 ± 0.07 ± 0.03 ± 0.05 ± 0.05 ± 0.04 ± 0.03

1.17 2.13 1.24 1.32 1.93 2.29 1.98 1.47 1.84 1.65 1.43 1.03 1.03

0.03 0.04 0.05 0.04 0.03 0.03 -- 0.05 ± 0.03 ± 0.05 ± 0.06 ± 0.05 ± 0.06 ± 0.03 1.57 ± 0.03 1.55 ± 0.03 1.30 -1- 0.04 2.03 ± 0.03 1.42 ± 0.05 1.80 ± 0.04 1.66 ± 0.06 1.62±0.04 2.00 ± 0.07 2.15 ± 0.06 1.03 ± 0.05 0.93 ± 0.02

C, 1.5 min at 35 C, and 2 min at 72 C. The amplification cycles were followed by a final extension step at 72 C for 5 min. Reactions were stopped with 5 µl gel loading buffer (0.005% [wt/v] bromophenol blue, 0.005% [wt/v] xyleno gylanol, 0.05 M EDTA-Na). Approximately 60 primers of kits OP-A, -B, and -R2 were screened for their suitability for the generation of reproducible DNA profiles. Eighteen RAPD primers gave clear amplification products that could be scored readily. RAPD primers used were OP-A (1, 4, 7, 9, 11, 12), OP-B (2, 3, 4, 7, 10, 12, 20), and OP-R (2, 3, 10, 16, 20). To detect intraspecific variation, we developed more sensitive methods. After DNA amplification with OP-A4, digestions with the restriction enzymes EcoRI and HindIII were carried out. Five microliters of DNA amplification products were used for this assay, with 12 µl of 10X buffer 1 and 1 µl enzyme (1 unit 0- ). Samples were maintained at 37 C for 1 h. Products generated by PCR amplifications of DNA were separated by electrophoresis according to size on 1.5% (wt/ v) SeaKem LE 3 agarose gels. The gels ran at approximately 150 V for 1.5 h. Gels were stained with ethidium bromide and visualized by illumination with ultraviolet light. Statistical Analyses Statistical analysis of morphological characters was carried out by setting up a matrix from the measurements obtained. Twenty-five different biotypes were studied with complete linkage of the Euclidean distance using seven different characters. For the statistical analysis of genome diversity, a similarity 312 • Weed Science

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1.40 2.45 2.20 2.10 2.50 2.44 2.40 2.43 1.65 1.78 1.70 1.90 1.68 1.97 2.20 1.77 2.13 2.03 2.05 2.22 2.38 2.13 2.40 1.17 1.40

0.05 0.05 0.08 0.03 0.03 0.04 0.05 0.03 0.03 0.05 0.05 0.06 0.05 0.05 0.06 0.05 0.09 0.06 0.11 0.08 0.09 0.07 0.06 0.05 0.05

1.85 1.58 1.33 1.60 1.53 1.70 1.78 1.65 1.70 1.85 9.00 9.70

± ± ± ± ± ± ± ± ± ± ± ±

0.11 0.08 0.03 0.05 0.08 0.09 0.05 0.03 0.03 0.06 0.04 0.05

0.90 1.90 1.85 1.55 2.00 1.77 1.85 1.90 1.23 1.67 1.30 1.37 1.25 1.43 1.53 1.15 1.50 1.30 1.55 1.58 1.70 1.63 1.90 0.75 0.93

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.04 0.03 0.03 0.06 0.05 0.03 0.05 0.03 0.04 0.03 0.05 0.05 0.03 0.03 0.05 0.03 0.03 0.05 0.03 0.04 0.03 0.04 0.02 0.03

matrix was constructed from presence/absence data scored from RAPD profiles using Jaccard 's coefficient (Gower 1985). A hierarchical cluster analysis was performed using a furthest neighbor clustering algorithm. A principal coordinates analysis was conducted to provide a graphical summary of similarities between samples. Interspecific variation was assessed using pooled DNA samples prepared from eight Echinochloa spp. biotypes. These were analyzed with the selected primers and the resulting DNA profiles were scored. Presence/absence data were recorded for 238 loci. Experiments were repeated at least twice.

Results and Discussion Botanical Identification A morphological classification of biotypes based on growth in different environments showed the difficulty of classifying Echinochloa spp. Often the characters overlapped and more precise methods were required. Biotypes were studied following Carretero 's (1981) keys. Data on characters of the spikelet are presented in Table 2. Statistical analysis of the data from 25 biotypes showed four distinguishable groups linking E. colonum with E. crusgalli and E. hispidula with E. oryzoides. The biotype of E. oryzicola was clustered with E. oryzoides with over 95% morphological similarity. The main distinguishing character of the species was the length of the upper glume, but differences were not enough to form a separate group. Echinochloa colonum was clearly different in all characters and was related to E. crus-galli at a similarity level of 79%. Echinochloa his-

TABLE 3. ED 50

Morphological similarity (%) 100

95

90

85

80

75

70

65

of different populations of Echinochloa spp. 15 d after quinclorac treatment. Data are mean of three replicates ± standard error. Biotype

Biotypes

24 25 1

9 10 11 12 13 16 3

4 14 15 17 18 19 20 22 23 2 5 6 7 8 21

FIGURE 1. Dendrogram based on the Euclidean distance from the mean of eight replicates from the morphological characteristics of 25 biotypes of Echinochloa spp.: E. colonum (24, 25), E. crus-galli (10, 12, 9, 13, 16, 11, 1), E. hispidula (20, 22, 19, 17, 18, 15, 14, 4, 3), E. oryzoides (6, 8, 7, 5, 2, 21), and E. oryzicola (23).

ED 5 o

Scientific name

E. colonum E. colonum E. crus galli E. crus galli E. crus galli E. crus galli E. crus galli E. crus-galli E. crus galli E. hispidula E. hispidula E. hispidula E. hispidula E. hispidula E. hispidula E. hispidula E. hispidula E. hispidula E. oryzicola E. oryzoides E. oryzoides E. oryzoides E. oryzoides E. oryzoides E. oryzoides

± 0.02 ± 0.03 ± 0.08 0.49 ± 0.04 0.31 ± 0.06 0.46 ± 0.07 0.39 ± 0.09 0.90 ± 0.15 0.32 ± 0.02 0.75 ± 0.10 0.70 ± 0.11 2.61 ± 0.21 0.84 ± 0.09 0.41 ± 0.08 0.76 ± 0.11 0.45 ± 0.05 0.53 ± 0.08 0.44 ± 0.11 0.12 ± 0.01 0.12 ± 0.02 0.11 ± 0.02 0.09 ± 0.01 0.11 ± 0.02 0.13 ± 0.02 0.08 ± 0.01 0.18 0.19 0.51

sence data were recorded for 238 loci. Principal coordinate analysis of RAPD data revealed that the five Echinochloa spp. formed only three discrete groups: E crus-galli and E hispidula, E oryzoides and E oryzicola, and E. colonum. It was not possible to distinguish between E crus-galli and E hispidula or between E oryzoides and E oryzicola with the primers used. The biotypes least sensitive to quinclorac were confined to group I (Figure 2).

od (2) od (5). 0 • ol (23)

pidula and E. oryzoides were similar at the 84% level, but the relation between E. crus-galli and E. hispidula was only correlated at about 60% (Figure 1).

Herbicide Treatment The biotypes responded differently to quinclorac treatment. Echinochloa colonum was very susceptible, as were E. oryzoides and E. oryzicola with ED 50 values close to 0.1 kg ai ha- 1 . By contrast, E. hispidula showed some degree of tolerance to quinclorac, especially in the highly tolerant number 14. ED 50 values ranged from 0.5 to 2.6 kg ai ha- 1 . Echinochloa crus-galli was less sensitive than E. hispidula, with an ED 50 range from 0.4 to 0.9 kg ai ha- 1 . ED 50 values from E. crus-galli and E hispidula overlapped in many cases (Table 3).

cg (13) cg (16) . . is (15) • his (14) col (25)

principal coordinate 1 FIGURE 2. Principal coordinate analysis of genetic similarity of eight pop-

Molecular Studies Interspecific variation was assessed using eight pooled DNA samples from each Echinochloa species. Presence/ab-

ulations representative of the five species described for the genus Echinochloa in southeastern Europe based on analysis of 238 loci generated by 18 RAPD primers. od, E. oryzoides; ol, E. oryzicola; cg, E. crus galli, col, E. colonum; his, E. hispidula.

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313

Levels of similarity (%)

rise to a different profile (Figure 4). Digestion with restriction enzymes did not provide any evidence of polymorphism between closely related species such as E. crus galli and E. hispidula. In this study, RAPD markers showed three different groups, although the current taxonomy for Echinochloa classification in Spain recognizes five different species (Carretero 1981). Because PCR analyses have been shown to be a useful tool for monitoring diversity in plant populations, these molecular results together with the different sensitivity to the herbicide quinclorac seem to indicate that Clayton 's (1980) classification of the genus Echinochloa is more in accordance with our results.

Sources of Materials 1

Perkin Elmer Cetus, 61 Main Avenue, Norwalk, CT 06859. Operon kits A, B, and R. Operon Technologies Inc., 1000 Atlantic Avenue, Suite 108, Alameda, CA 94501. 3 SeaKem LE, agarose gels. FMC Bioproducts, Rockland, ME 04841. 2

3. Dendrogram constructed from furthest neighbor cluster analysis of 238 loci generated by RAPD amplification, based on percent genetic si milarity. These analyses refer to the same eight biotypes of Figure 2. FIGURE

A dendrogram constructed from furthest neighbor cluster analyses showed three discrete groupings of Echinochloa spp. based on levels of genetic similarity and revealed that the biotypes belonging to E. crus galli and E. hispidula were closely related with 70% similarity. Biotypes of E. oryzoides and E. oryzicola were less correlated, with only about 50% similarity. Echinochloa colonum (25) presented a genetic similarity with E. crus galli and E. hispidula of 65% and with E. oryzicola at 25% (Figure 3). These results were correlated with the physiological behavior of the genus after treatment with quinclorac. All of our results to date indicated that primer OP A4 (AATCGGGCTG) may help to classify Echinochloa spp. In particular, it should be noted that a major 800-bp product appears in E. crus galli and E. hispidula, and a 1,600-bp product appears in E. oryzicola and E. oryzoides. Echinochloa colonum, which is readily identified by its morphology, gives

FIGURE

Acknowledgments The authors thank BASF for supplying seeds and chemicals, Neil McRoberts for computational assistance, and R. L. Zimdahl for his review of the manuscript. Financial support for this study was provided by CICYT (project AGF 97-1230). The Scottish Agricultural College receives financial support from the Scottish Agriculture and Fisheries Department.

Literature Cited Anderson, W. R. and D. J. Fairbanks. 1990. Molecular markers: important tools for plant genetic resource characterization. Diversity 6:51–53. Carretero, J. L. 1981. El genero Echinochloa en el Suroeste de Europa. Anales J. Bot. Mad. 38:91–108. Carretero, J. L. 1989. Variacion en la sensibilidad al propanil del genero Echinochloa de los arrozales valencianos (España). Pages 407–411 in Proceedings of the 4th EWRS Mediterranean Symposium. Clayton, W. D. 1980. Echinochloa. Pages 261–262 in T. G. Tutin, V. H. Heywood, N. A. Burges, D. M. Moore, D. H. Valentine, S. M. Wal-

4. Amplified loci from RAPD primer OP-A4. Lane M = 100-bp marker. The number of each biotype is at the beginning of each pattern. Lane

1, E. colonum (biotype 25); lane 2, E. oryzicola (biotype 23); lanes 3–6, E. oryzoides (biotypes 2, 5, and 7); lanes 7–9, E. crus galli (biotypes 1, 13, and

16); lanes 10–12, E. hispidula (biotypes 14, 15, and 17). The experiment was replicated three times.

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Received October 12, 1998, and approved April 3, 1999.

Lopez-Martinez et al.: Intraspecific variation in Echinochloa



315

Introduction Material and methods Botanical identification Plant Material and Growing Conditions

Herbicide treatment Molecular assessment DNA extraction PCR Amplification

Statistical analysis Results and discussion Botanical identification Herbicide treatment Molecular studies Sources of materials Acknowledgments Literature cited

Molecular markers indicate intraspecific variation in the

amplified polymorphic DNA (RAPD) analysis together with classic morphological ..... Statistical analysis of the data from 25 biotypes showed four distinguishable ...

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