Phragmites australis

Weed Science 2011 59:366–375

Genetic Diversity of Iranian Clones of Common Reed (Phragmites australis) Based on Morphological Traits and RAPD Markers Marjan Diyanat, Ali A. S. Booshehri, Hassan M. Alizadeh, Mohammad R. Naghavi, and Hamid R. Mashhadi* The genetic diversity of 39 clones of common reed originating from different geographical areas of Iran were evaluated using morphological and RAPD analyses. High level of morphological variation was observed among clones. The 16 primers used in this study amplified 149 scorable RAPD loci among which 123 were polymorphic (83.1%). A dendrogram was prepared on the basis of a similarity matrix of RAPD data using the unweighted pair-group method with arithmetic averages (UPGMA) algorithm and separated the 39 clones into four groups, which mainly were in accordance with geographical origins. The results of the morphological comparison mostly corresponded with the results of RAPD analysis. It is possible that these variations among clones will affect successful management of common reed using chemical or the other methods of control. Nomenclature: Common reed, Phragmites australis (Cav.) Trin. ex Steud. Key words: Morphological traits, RAPD, diversity, cluster analysis.

Common reed is a perennial grass and is the most widespread flowering plant in the world (Clevering and Lissner 1999). It is found on every continent except Antarctica and is common throughout Asia, North America, and Europe (Saltonstall 2002). Common reed enlarges its population by clonal growth through rhizomes and is a typical plural clonal plant species (Dong 1996). It now is found in many provinces in Iran and causes problems in some provinces (e.g., Ardebil). In the last few years, the field of molecular biology has provided new tools for studying population structure. Clonal diversity and evolutionary processes in wetland species (such as cattain [Typha] and cordgrass [Spartina]) were studied for the first time, using allozyme polymorphisms (McNaughton 1975; Raybould et al. 1991; Silander 1985). Since the 1980s, new perspectives in how to study evolutionary processes and population dynamics in common reed became available with the development of molecular markers (de Kroon and van Groenendael 1997; Jackson et al. 1985). However, our knowledge of the underlying evolutionary processes in determining the clonal diversity still is limited. Significant morphological differences have been found both among different populations of common reed and different clones within the same populations, irrespective of site conditions (Bjo¨rk 1967; Clevering 1999; Clevering et al. 2001; Hansen et al. 2007; Pauca˜-Comañescu et al. 1999; Rolletschek et al. 1999). Part of the clone-specific variability in common reed can be attributed to differences in chromosome number. A euploid range of 3x, 4x, 6x, 7x, 8x, 10x, 11x, and 12x (with x 5 12) has been found for this species, with tetraploid (2n 5 48) and octoploid (2n 5 96) being the most frequently observed (Clevering and Lissner 1999). Shoots of octoploid generally are longer and thicker and have larger leaves than those of tetraploid (Clevering et al. 2001; Hanganu et al. 1999; Pauca˜-Comañescu et al. 1999). This relationship between euploidy level and morphology is common because the most immediate and universal effect of polyploidy is an increase in cell size. However, polyploidy does not always lead to an overall increase in the plant size, DOI: 10.1614/WS-D-10-00163.1 * Assistant Professor, Associate Professor, Associate Professor, Associate Professor, and Professor, Department of Agronomy and Plant Breeding, University of Tehran, Karaj, Iran. Current address of first author: Islamic Azad University, Science and Research Branch, Tehran, Iran. Corresponding author’s E-mail: [email protected] and [email protected].

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because a common effect of polyploidy also is a reduction in the number of cell divisions during development (Stebbins 1971). It has been suggested that genetic variation among populations from different geographic regions has arisen as a result of growth in different climatic conditions (Clevering et al. 2001). Along a latitudinal gradient, gradual change occurs in day length, and the same happens in the amount of solar radiation and air temperature. Common reed populations, originating from different geographic regions along a latitudinal gradient from Northern Sweden to Spain, differed in time of cessation of growth, shoot morphology, and biomass allocation. Under the same environmental conditions, common reed originating from higher latitudes started growing earlier than southern populations, but finished the growth early in the season. The southern populations failed to complete the whole growth cycle before the first frost and did not develop mature seeds (Clevering et al. 2001). Morphological and physiological traits often are influenced by the environment, but genetic markers are not affected by the environmental factors, so they might present the most reliable method of distinguishing different reed populations (Saltonstall 2003a). Different molecular techniques have been employed to detect clonal diversity in common reed, such as allozymes (Clevering et al. 2001; Clevering and Lissner 1999; Hauber et al. 1991; Pellegrin and Hauber 1999), random amplified polymorphic DNAs (RAPDs) (Curn et al. 2007; Keller 2000; Koppitz and Ku¨hl 2000; Ku¨hl et al. 1999; Neuhaus et al. 1993), restriction fragment length polymorphisms (RFLPs) (Koppitz et al. 1997; Saltonstall 2003b) and amplified fragment length polymorphisms (AFLPs) (Lambertini et al. 2006), microsatellite (SSR) analysis, and chloroplast DNA sequencing (Saltonstall 2002, 2003a). The randomamplified polymorphic DNA technique was developed in 1990 (Williams et al. 1990): it has become recognized as a more accessible technique, and it is relatively low-cost and requires very small quantities of genomic DNA (Ragot and Hoisington 1993; Russell et al. 1997). It is a fairly simple method, and has been used in many studies for the clarification of nomenclature (Keil and Griffin 1994), identification of herbarium accessions (Khadari et al. 1995), or elucidation of genetic relationships (Russell et al. 1997). The probability of success for management strategies will increase with understanding of the nature and extent of genetic diversity among reed clones. If genetically diverse

Figure 1. The geographic origin of the 39 clones of common reed used in this study.

Iranian clones of common reed evolve, they might respond differentially to weed management strategies, including herbicides. Published studies of population genetic variation in common reed have focused on geographically localized populations in Europe and the United States (Djebrouni 1992; Hansen et al. 2007; Koppitz et al. 1997; Ku¨hl and Neuhaus 1993; McKee and Richards 1996; Neuhaus et al. 1993; Pellegrin and Hauber 1999; Zeidler et al. 1994). This study investigates the genetic diversity and morphological differences among clones of common reed in Iran. Materials and Methods

Plant Material. The rhizomes of 39 clones of common reed used in this study were collected from different regions of Iran in March 2005. The sampling areas ranged from 39u339N., 47u469E (Moghan) to 31u549N, 47u269E (Shoosh) (Figure 1). For the purpose of this study, these plant clones are identified by name and abbreviation of their location of origin (Table 1). The minimum distance between locations was more than 10 km. At each location, more than 50 rhizome pieces were collected, and the stand of common reed sampled was considered to be one clone (rhizomes collected from a single plant). A day after collection, the rhizomes were divided into small portions with two or three visible, well-formed buds and transplanted into an experimental plot. Before transplanting, the soil was plowed and harrowed. After transplanting was completed, the plots were immediately irrigated. The rhizomes of clones were grown in plots (3 m by 3 m, with 2-m spacing from the adjacent plot) at Research Farm of Pardise of Agriculture and Natural Science, University of Tehran, Karaj, Iran (35u449N, 51u109E, altitude 1300 m) using a randomized complete block design with three replications. The soil was a fine sandy-loam. Cultural operations, including frequent irrigation (every 2 d) and manual elimination of other weeds were done during season.

Plots were watched closely and repeated efforts were made to maintain the purity of individual clones. A 2-yr field study was conducted using rhizomes and methods were the same in both years. In the first year, ambient climatic conditions were measured at a nearby weather station; the average air temperatures in April, May, June, July, August, September, and October were 11, 17, 25, 25, 28, 25, and 19 C, respectively. Morphological Study. At maximum biomass, the time when shoot length did not increase further, the ten tallest shoots per replicate were harvested by clipping them at ground level. The shoots were wrapped in large plastic bags before being transported to the laboratory (Hansen et al. 2007). In order to facilitate comparison between the clones, special attention was paid to the tallest shoots within each sample, which had originated from the apical rhizome buds. Therefore, the shoots were of similar age and, because they all belonged to the first spring cohort of shoots, they probably were little-affected by intraspecific competition (Cosentino et al. 2006). In the laboratory, the length of each shoot was measured from the clipped base to the uppermost leaf. Basal diameter (cm) was measured between the two lowest nodes. Number of internodes (per shoot) was counted before the shoot was divided into leaf, stem (with leaf sheath), and panicle. Panicle length (cm) was determined for each shoot. Length and width (cm) of the sixth leaf above the ground was measured for each shoot. Leaf area (cm2) was measured with leaf area meter.1 Aboveground dry weight (g) was determined after drying to constant weight at 80 C for 48 h. Aboveground dry weight was obtained by adding the dry weights of the stem, leaf, and panicle. At the end of the growing season, the percentage of flowering shoots was determined. Time of panicle appearance and senescence (days after planting) also were recorded. Diyanat et al.: Genetic diversity of common reed

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Table 1. Collection codes and origin sites of sampled clones of common reed. No.

Origin site

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39

Moghan Moghan Moghan Moghan Moghan Moghan Mesgaran Mashhad Nazarie Dezfool Dezfool Ojirob Motahhari-shahrak Shoosh Shoosh Sarabeyavari Sarabeyavari Bisotoon Mahidasht Shahre-Ray Shahrake-Sinamayi Varamin-Goltape Varamin-Gharchek Varamin-Gharchek Varamin-Gharchek Dolat Abad Dolat Abad Mohammad Shahr Mohammad Shahr Beheshtmasoome Beheshtmasoome Beheshtmasoome Sari Sari Gorgan Gonbad Gonbad Gorgan Gorgan

Province Ardebil Ardebil Ardebil Ardebil Ardebil Ardebil Khorasan Khorasan Khorasan Khozestan Khozestan Khozestan Khozestan Khozestan Khozestan Kermanshah Kermanshah Kermanshah Kermanshah Tehran Tehran Tehran Tehran Tehran Tehran Tehran Tehran Tehran Tehran Qom Qom Qom Mazandaran Mazandaran Golestan Golestan Golestan Golestan Golestan

RAPD Assay. Common reed leaf samples were taken and stored at 220 C until preparation. DNA for polymerase chain reaction (PCR) assay was isolated according to the CTAB method of Rogers and Bendich (1985) and stored in 13 TRIS-EDTA buffer solution at 4 C. The RAPD analysis was performed using a set of 16 random primers2 (Table 2) on all common reed clones. PCR was carried out in a 25 ml reaction mixture containing 1.9 mM MgCl2, 0.5ml primer, 1.253 PCR Buffer, 0.2 mM of each deoxyribonucleotide triphosphate (dNTP), approximately 100 ng genomic DNA, and 1 U Taq DNA polymerase.3 Amplifications were performed in a thermal cycler programmed as follows: 1 min at 92 C, 1 min at 35 C, 2 min at 72 C, and finally at 72 C for 4 min. Amplified products were separated by gel electrophoresis in 1.5% agarose and tris-acetate-EDTA (TAE) buffer. Amplification products were separated in 1.5% agarose gels run in 13 TAE buffer and detected by staining with ethidium bromide. RAPD fingerprints were amplified repeatedly (the same results were obtained in three independent PCR experiments). The clear and distinct banding pattern indicated suitability of this method. Statistical Analyses. Quantitative analyses of morphological traits were carried out using SAS software.4 Pearson’s coefficients were used to determine the degree of associations among the traits. Euclidean distances were estimated for all 368

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Codes A-MO1 A-MO2 A-MO3 A-MO4 A-MO5 A-MO6 KO-ME KO-MA KO-NA KZ-D1 KZ-D2 KZ-OJ KZ-MO KZ-S1 KZ-S2 KE-S1 KE-S2 KE-BI KE-MA T-SHR T-SHS T-VGO T-VG1 T-VG2 T-VG3 T-DO1 T-DO2 T-MO1 T-MO2 Q-BE1 Q-BE2 Q-BE3 MA-S1 MA-S2 G-GR1 G-GO1 G-GO2 G-GR2 G-GR3

Latitude N 39u339 39u309 39u319 39u339 38u159 38u509 36u479 36u209 36u089 32u159 32u149 32u129 32u169 31u549 31u459 34u299 34u399 34u229 34u199 35u339 35u279 35u039 35u099 35u139 35u209 35u469 35u409 35u509 35u579 34u309 34u399 34u599 36u379 36u299 36u079 37u109 37u479 36u309 36u519

Longitude E 47u469 47u449 48u019 48u039 47u549 47u049 55u439 55u069 55u259 48u269 48u279 48u299 48u229 47u269 46u359 46u569 46u199 47u269 47u529 51u229 51u139 51u309 51u369 51u409 51u479 51u109 51u029 51u069 51u149 51u169 51u189 51u309 52u569 53u049 54u169 54u439 54u519 54u059 54u289

pairs of clones using standardized values of the traits and the formula pffi Xn dxy ~ ~1(Xi {yi )2 , ½1 i~1 where xi and yi are the ith characters measured on two clones and n is the number of characters (Romesburg 1984). The data matrix usually is standardized to recast the units of measurement as dimensionless units by standardizing the data; all traits become equally important in determining the distances (Manly 1986). Euclidean distance coefficients measure the literal distance between two objects when they are viewed as points in the two-dimensional space formed by their attributes (Romesburg 1984), in this case morphological traits. Cluster analysis was then performed using the distance coefficients (Swofford and Olsen 1990). The contribution of the measured morphological traits to the overall differences between the clones was explored using principal components analysis (PCA). For RAPD analysis, polymorphic RAPD fragments were scored as 1 for the presence and 0 for the absence of a DNA band for each clones. The data matrix was entered into the NTSYS program (Rohlf 1998) and analyzed using the qualitative routine to generate Dice similarity index as in Nei and Li (1979). A dendrogram showing genetic relationships of the 39 clones was constructed using the unweighted pair-group method with arithmetic averages (UPGMA).

Table 2. RAPD primers used, their sequence of nucleotides, and the percent of polymorphic bands produced by each primer. Marker no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Average

Primer name

Nucleotide sequence 59R39

Number of total bands

Percent of polymorphic bands

UBC 1 UBC 3 UBC5 UBC9 UBC 13 UBC16 UBC64 UBC 66 UBC76 UBC77 UBC82 UBC 84 UBC89 UBC95 UBC96 UBC100

CCT GGG CTT C CCT GGG CTT A CCT GGG TTC C CCT GCG CTT A CCT GGG TGG A GGT GGC GGG A GAG GGC GGG A GAG GGC GTG A GAG CAC CAG T GAG CAC CAG G GGG CCC GAG G GGG CGC GAG T GGG GGC TTG G GGG GGG TTG G GGC GGC ATG G ATC GGG TCC G

15 8 9 9 9 8 9 10 8 9 8 10 8 10 10 9 149 9.31

92 87.5 80 80 73.3 88 85 75.6 90 80 75 90 83.3 80.9 86.9 83.3

Results Variability of Morphological Traits. The results showed that clones of common reed were significantly different in all evaluated morphological traits (Table 3), suggesting that selection for these traits could be possible.

80.98

When grown under the same environmental conditions, common reed originating from higher latitudes began their growth earlier than those of southern clones, but finished growth early in the season. Shoot length ranged from 157.0 to 237.6 cm. The southern clones KZ-OJ and KZ-MO had the

Table 3. Variation in different morphological traits among common reed clones (6 standard error). Clone A-MO1 A-MO2 A-MO3 A-MO4 A-MO5 A-MO6 G-GO1 G-GO2 G-GR1 G-GR2 G-GR3 KE-BI KE-MA KE-S1 KE-S2 KO-MA KO-NA KO-ME KZ-D1 KZ-D2 KZ-MO KZ-OJ KZ-S1 KZ-S2 MA-S1 MA-S2 Q-BE1 Q-BE2 Q-BE3 T-DO1 T-DO2 T-MO1 T-MO2 T-SHR T-SHS T-VG1 T-VG2 T-VG3 T-VGO LSD

Shoot length (cm)

Shoot diameter (cm)

Number of internodes (per shoot)

Leaf length (cm)

Leaf width (cm)

179.0 6 6.67 185.3 6 6.33 167.0 6 6.83 170.3 6 7.77 170.6 6 5.01 172.3 6 8.10 188.6 6 9.16 192.0 6 8.81 180.3 6 5.47 178.0 6 8.26 177.0 6 5.53 176.6 6 8.24 170.6 6 8.16 181.0 6 8.54 189.0 6 9.05 157.0 6 4.50 173.3 6 8.32 161.0 6 3.41 206.0 6 8.68 214.6 6 4.33 230.3 6 9.47 237.6 6 4.08 221.6 6 5.90 228.3 6 6.08 171.0 6 6.51 193.0 6 8.14 187.0 6 7.40 173.0 6 8.34 184.0 6 9.85 185.6 6 5.40 180.3 6 11.46 195.0 6 9.84 181.0 6 12.0 195.0 6 9.93 200.0 6 7.38 190.6 6 6.86 179.6 6 11.29 184.3 6 4.82 173.0 6 6.8 9.32

0.45 6 0.05 0.48 6 0.04 0.50 6 0.03 0.47 6 0.02 0.45 6 0.03 0.51 6 0.04 0.38 6 0.04 0.41 6 0.04 0.41 6 0.04 0.38 6 0.04 0.41 6 0.03 0.45 6 0.02 0.54 6 0.02 0.50 6 0.04 0.48 6 0.03 0.32 6 0.02 0.45 6 0.02 0.30 6 0.04 0.45 6 0.03 0.45 6 0.02 0.53 6 0.05 0.53 6 0.03 0.60 6 0.02 0.60 6 0.05 0.60 6 0.03 0.51 6 0.05 0.47 6 0.02 0.48 6 0.05 0.54 6 0.03 0.45 6 0.05 0.45 6 0.04 0.48 6 0.04 0.48 6 0.03 0.42 6 0.03 0.44 6 0.05 0.50 6 0.02 0.47 6 0.05 0.44 6 0.03 0.48 6 0.03 0.02

16.67 63.15 16.33 62.37 16.00 62.32 16.67 62.32 18.33 63.15 16.67 63.89 16.83 63.33 18.50 61.33 16.83 62.11 18.33 63.44 18.50 61.15 15.00 61.50 16.17 62.89 14.00 62.37 14.17 62.29 15.00 62.80 18.33 63.35 14.17 62.03 21.00 62.67 21.17 62.89 21.83 62.48 20.00 62.23 23.50 63.23 23.67 61.91 20.17 63.58 16.17 62.77 16.67 62.94 19.17 63.70 16.50 62.02 14.17 62.47 13.50 62.08 15.00 62.82 16.00 61.37 15.17 61.54 15.33 62.70 15.83 61.91 16.00 62.65 15.17 63.40 14.33 61.73 3.12

36.98 6 2.23 36.42 6 2.25 37.38 6 5.00 39.80 6 5.25 40.83 6 5.69 39.83 6 4.55 52.75 6 3.52 49.73 6 7.88 55.45 6 4.33 52.42 6 2.52 51.80 6 4.70 40.45 6 3.61 40.78 6 3.48 42.10 6 4.33 42.05 6 1.46 40.08 6 2.07 43.05 6 2.13 38.82 6 4.06 52.45 6 3.74 50.68 6 2.64 51.62 6 5.08 51.50 6 2.51 50.42 6 3.46 46.95 6 3.27 36.75 6 3.05 43.12 6 3.48 34.05 6 1.69 35.07 6 2.25 34.05 6 3.50 43.40 6 4.15 43.43 6 2.92 44.75 6 2.88 44.47 6 2.47 44.82 6 4.24 45.00 6 2.54 43.78 6 2.71 44.40 6 3.44 44.98 6 2.94 42.73 6 2.81 4.53

2.83 6 0.14 3.03 6 0.17 2.97 6 0.25 2.87 6 0.21 2.87 6 0.12 2.80 6 0.42 2.54 6 0.46 2.70 6 0.18 2.73 6 0.05 2.30 6 0.19 2.40 6 0.20 2.54 6 0.45 2.70 6 0.16 3.21 6 0.29 3.10 6 0.20 2.21 6 0.39 2.90 6 0.25 2.14 6 0.11 3.21 6 0.50 3.34 6 0.44 3.60 6 0.18 3.35 6 0.10 3.70 6 0.23 3.81 6 0.51 4.21 6 0.27 3.40 6 0.40 2.10 6 0.16 1.83 6 0.12 2.04 6 0.35 3.64 6 0.45 3.51 6 0.55 3.13 6 0.10 3.10 6 0.21 3.11 6 0.32 2.90 6 0.20 3.23 6 0.25 3.33 6 0.19 3.30 6 0.29 3.30 6 0.23 0.33

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Table 4. Variation in different morphological traits among common reed clones (6 standard error). Clone A-MO1 A-MO2 A-MO3 A-MO4 A-MO5 A-MO6 G-GO1 G-GO2 G-GR1 G-GR2 G-GR3 KE-BI KE-MA KE-S1 KE-S2 KO-MA KO-NA KO-ME KZ-D1 KZ-D2 KZ-MO KZ-OJ KZ-S1 KZ-S2 MA-S1 MA-S2 Q-BE1 Q-BE2 Q-BE3 T-DO1 T-DO2 T-MO1 T-MO2 T-SHR T-SHS T-VG1 T-VG2 T-VG3 T-VGO LSD

Leaf area (per plant) (cm2)

Aboveground dry weight (g)

Percentage of flowering shoots

Panicle length (cm)

571.0 6 11.83 556.0 6 15.22 563. 0 6 16.04 539.0 6 6.45 558.0 6 15.22 560.0 6 28.35 522.0 6 15.22 529.0 6 24.64 520.0 6 9.31 538.0 6 12.99 531.0 6 24.39 329.0 6 16.64 334.0 6 27.17 321.0 6 6.23 333.0 6 15.76 330.0 6 22.97 361.0 6 28.30 321.0 6 5.12 641.0 6 10.49 664.0 6 7.78 619.0 6 15.64 621.0 6 15.45 595.0 6 6.27 585.0 6 11.56 486.0 6 9.69 521.0 6 13.32 443.0 6 13.49 641.0 6 11.76 463.0 6 6.07 440.0 6 9.94 431.0 6 28.66 465.0 6 16.93 476.0 6 4.36 497.0 6 10.90 484.0 6 16.88 484.0 6 18.95 467.0 6 9.22 323.0 6 9.18 497.0 6 4.44 18.45

9.62 6 0.55 9.66 6 1.43 9.48 6 0.68 9.51 6 0.92 9.93 6 0.93 9.74 6 1.56 9.27 6 0.45 10.35 6 0.61 8.48 6 0.60 9.02 6 1.26 9.07 6 1.64 8.27 6 0.65 8.09 6 0.49 7.86 6 0.87 8.01 6 0.72 8.57 6 0.61 8.93 6 0.25 8.62 6 0.38 11.56 6 0.26 12.23 6 0.42 12.79 6 0.50 12.83 6 0.53 12.59 6 1.43 12.57 6 0.57 9.38 6 0.95 9.42 6 1.22 8.36 6 0.83 8.48 6 0.35 8.83 6 1.09 8.36 6 0.78 8.53 6 0.61 8.43 6 0.62 8.46 6 1.08 9.53 6 1.02 9.38 6 .066 8.74 6 .036 8.48 6 0.65 8.38 6 0.43 8.57 6 1.31 1.01

85.0 6 1.61 84.0 6 1.80 89.0 6 1.89 90.0 6 0.76 80.0 6 2.08 82.0 6 0.76 70.0 6 5.51 74.0 6 3.28 69.0 6 3.91 75.0 6 1.04 80.0 6 1.53 17.0 6 1.04 29.0 6 0.87 20.0 6 1.04 15.0 6 0.87 75.0 6 7.55 76.0 6 1.61 85.0 6 1.76 30.0 6 3.46 42.0 6 2.78 24.0 6 2.47 33.67 6 2.02 20.33 6 3.21 19.0 6 3.12 95.0 6 3.50 80.0 6 1.32 90.0 6 2.50 80.0 6 5.89 85.0 6 4.92 70.0 6 3.50 65.0 6 0.29 60.0 6 5.06 62.0 6 3.50 80.0 6 5.29 82.0 6 4.33 75.0 6 0.50 70.0 6 4.75 71.0 6 3.91 80.0 6 4.09 7.29

30.17 6 3.71 31.17 6 4.08 34.17 6 4.62 35.00 6 5.77 35.83 6 5.35 35.83 6 3.33 31.33 6 5.55 31.17 6 6.57 30.00 6 3.80 32.17 6 3.08 31.17 6 4.06 26.17 6 5.28 27.00 6 6.32 24.17 6 5.33 25.00 6 2.23 34.33 6 6.25 33.33 6 4.73 34.00 6 4.18 30.17 6 6.38 24.00 6 3.05 24.00 6 2.32 23.00 6 2.00 26.33 6 7.25 27.17 6 2.12 39.17 6 6.36 33.17 6 2.75 28.00 6 3.13 30.17 6 3.33 31.17 6 4.25 30.33 6 3.84 32.33 6 2.85 29.17 6 2.31 30.00 6 4.58 25.33 6 5.12 28.17 6 4.69 27.00 6 3.91 31.00 6 3.31 29.00 6 2.30 33.00 6 4.18 5.27

longest shoots (237.6 and 230.3, respectively). The northern clone KO-MA had the shortest shoot (157.0 cm) followed by KO-ME with the value of 161.0 cm (Table 3). The basal shoot diameter varied from 0.32 (KO-ME) to 0.60 cm (KZS1, KZ-S2, and MA-S1). The highest and lowest number of shoot internodes were observed in KZ-S2 (23.67) and T-DO2 (13.5), respectively. The leaf length and leaf width ranged from 34.05 to 55.45 cm and from 1.83 to 4.21 cm, respectively. Moreover, the highest and lowest leaf lengths were observed in G-GR1 and Q-BE1,and Q-BE3 respectively. MA-S1 had the highest and Q-BE2 had the lowest leaf widths (Table 3). The leaf area per shoot ranged from 664 cm2 (KZD2) to 321 cm2 (KO-ME and KE-S1) (Table 4). The highest aboveground dry weight per shoot was observed in KZ-OJ (12.83 g) whereas KE-S1 had the lowest aboveground dry weight per shoot (7.86 g). The results obtained showed that the plants of clones from the southern areas were taller and thicker with more nodes, and they also were more vigorous than plants from more northern areas. The percentage of flowering shoots at the end of growing season was ranged from 15.0 (KE-S2) to 95.0 (MA-S1). In all clones, the time of panicle appearance was negatively correlated with latitude of origin (Figure 2). The range of panicle length was from 23.00 cm (KZ-OJ) to 39.17 cm (MA-S1). The panicles were 370

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Number of days until panicle appearance 144.77 6 140.38 6 139.22 6 142.05 6 146.48 6 143.58 6 162.57 6 161.37 6 169.33 6 165.28 6 162.13 6 165.22 6 163.52 6 170.37 6 172.18 6 150.32 6 153.50 6 155.27 6 190.35 6 192.37 6 197.58 6 196.53 6 200.28 6 203.93 6 159.38 6 158.23 6 172.32 6 177.58 6 179.40 6 168.57 6 169.37 6 168.33 6 167.37 6 170.83 6 171.60 6 173.33 6 172.75 6 170.25 6 173.45 6 5.46

6.23 3.99 5.08 4.97 3.79 4.08 5.60 5.02 3.85 5.09 4.39 3.46 3.89 4.32 1.83 2.63 2.88 7.52 5.80 3.59 2.65 5.61 2.81 4.54 3.07 7.12 3.34 4.19 3.20 5.09 3.24 3.42 7.28 4.83 5.63 2.78 4.35 4.36 5.18

Number of days until senescence appearance 160.00 6 156.00 6 155.00 6 158.00 6 162.00 6 159.00 6 179.17 6 178.00 6 186.17 6 177.33 6 180.17 6 182.17 6 180.17 6 185.50 6 189.17 6 172.17 6 176.67 6 166.60 6 208.17 6 209.83 6 215.17 6 214.00 6 218.33 6 222.50 6 176.17 6 175.00 6 189.00 6 193.00 6 197.00 6 185.00 6 186.00 6 185.17 6 184.17 6 187.00 6 188.17 6 190.00 6 189.17 6 187.17 6 190.00 6 5.97

3.14 3.29 3.25 3.80 4.32 4.00 4.36 6.87 3.99 6.37 2.47 2.60 7.75 2.64 2.93 7.46 4.01 5.42 3.34 5.76 5.22 3.62 6.11 3.51 5.62 5.50 6.25 4.77 4.17 8.67 3.92 4.52 4.42 5.33 7.91 4.41 3.40 5.79 4.47

larger in southern clones than those of northern clones. The senescence time varied among clones, with the most striking difference being A-MO3 that started senescing in July, and KZ-S2 which started senescing in October (Table 4). The phenotypic correlation between traits was shown in Table 5. Shoot length was significantly and positively correlated with all traits except percentage of flowering shoots. The strongest relationship was between shoot length and aboveground dry weight (r2 5 0.88). Leaf length also was

Figure 2. Relationship between the latitude of origin and time of panicle appearance for clones of common reed.

Table 5. Simple correlation coefficients among morphological traits in 39 clones of common reed.

Shoot length

Leaf length

Shoot length 1.00 Leaf length 0.45** 1.00 Leaf width 0.40* 0.25 Shoot diameter 0.69** 0.41** Number of internodes 0.45** 20.16 Leaf area 0.67** 0.39* Aboveground dry weight 0.88** 0.44** Percentage of flowering shoots 20.84** 20.41** Panicle length 0.32* 0.35* Number of days until panicle appearance 0.41** 0.39* Number of days until senescence appearance 0.65** 0.46**

Leaf width

1.00 0.24

Shoot Number of diameter internodes

Leaf area

Percentage of Aboveground flowering dry weight shoots

Panicle length

Number of days until panicle appearance

Number of days until senescence appearance

1.00

0.57** 0.30

20.06 0.01

1.00 0.64**

1.00

0.41**

20.22

0.82**

0.42**

0.42** 0.37*

0.22 0.23

0.79** 0.38*

20.39* 0.32*

0.47**

0.23

0.47**

0.34*

0.01

0.54**

1.00 0.69** 0.31*

1.00 0.24

1.00

0.36*

0.35*

0.34*

0.37*

1.00

0.40*

0.22

0.57**

0.60**

0.69**

1.00

**’* Significant at 1% and 5%, respectively.

Figure 4. Principal components analysis (PCA) scores of clones of common reed calculated from morphological data.

positively correlated with shoot diameter, leaf area, aboveground dry weight, panicle length, time of panicle appearance, and senescence time, but negatively correlated with the percentage of flowering shoots and number of internodes. The percentage of flowering shoots and leaf area also exhibited negative correlation with each other. Clustering of common reed clones on the basis of distance values for morphological traits produced a dendrogram with four major groups (Figure 3). The first group was comprised of six clones form Ardebil (A-MO1, A-MO2, A-MO3, A-MO4, A-MO5, and A-MO6), one population from Mazandaran (MA-S1), and five clones from Golestan (G-GR1, G-GR2, G-GR3, G-GO1, and G-GO2). The r Figure 3. Dendrogram generated for clones of common reed on the basis of unweighted pair group method with arithmetic averages (UPGMA) using morphological traits data from 2 yr. Scale represents Euclidean distance value (d ).


N

371

Table 6. Similarity matrix among common reed clones using Nei and Li’s coefficient based on RAPD bands. KZ- KZA-MO1 A-MO2 A-MO3 A-MO4 A-MO5 A-MO6 KZ-D1 KZ-D2 KZ-OJ MO S1 KZ-S2 MA-S1 MA-S2 KE-S1 KE-S2 KE-BI KE-MA A-MO1 A-MO2 A-MO3 A-MO4 A-MO5 A-MO6 KZ-D1 KZ-D2 KZ-OJ KZ-MO KZ-S1 KZ-S2 MA-S1 MA-S2 KE-S1 KE-S2 KE-BI KE-MA Q-BE1 Q-BE2 Q-BE3 T-SHR T-SHS T-VGO T-VG1 T-VG2 T-VG3 T-DO1 T-DO2 T-MO1 T-MO2 KO-ME KO-MA KO-NA G-GR1 G-GO1 G-GO2 G-GR2 G-GR3

1.00 0.78 0.76 0.72 0.73 0.73 0.61 0.62 0.62 0.70 0.62 0.65 0.49 0.57 0.64 0.69 0.59 0.66 0.64 0.67 0.67 0.62 0.59 0.57 0.61 0.61 0.54 0.66 0.63 0.64 0.66 0.65 0.64 0.65 0.64 0.59 0.54 0.63 0.56

1.00 0.81 0.69 0.69 0.74 0.56 0.55 0.61 0.65 0.61 0.64 0.48 0.55 0.69 0.68 0.64 0.70 0.60 0.64 0.62 0.57 0.54 0.54 0.62 0.60 0.53 0.71 0.60 0.61 0.63 0.63 0.59 0.64 0.61 0.61 0.55 0.65 0.61

1.00 0.67 0.64 0.67 0.61 0.61 0.55 0.58 0.61 0.59 0.52 0.51 0.57 0.61 0.57 0.58 0.56 0.58 0.63 0.60 0.54 0.54 0.56 0.58 0.48 0.64 0.61 0.58 0.58 0.54 0.52 0.57 0.61 0.58 0.53 0.60 0.55

1.00 0.74 0.76 0.68 0.68 0.67 0.74 0.56 0.66 0.58 0.63 0.62 0.67 0.61 0.69 0.53 0.63 0.55 0.64 0.66 0.66 0.59 0.64 0.61 0.59 0.67 0.64 0.66 0.64 0.64 0.69 0.65 0.66 0.61 0.64 0.58

1.00 0.75 0.60 0.61 0.65 0.71 0.55 0.64 0.50 0.52 0.61 0.65 0.58 0.63 0.63 0.58 0.64 0.59 0.64 0.58 0.56 0.54 0.57 0.70 0.63 0.60 0.60 0.62 0.59 0.59 0.57 0.55 0.55 0.60 0.59

1.00 0.56 0.60 0.62 0.68 0.55 0.60 0.47 0.61 0.62 0.69 0.53 0.62 0.59 0.61 0.59 0.57 0.57 0.57 0.59 0.61 0.56 0.62 0.65 0.62 0.64 0.59 0.60 0.63 0.60 0.67 0.61 0.65 0.62

1.00 0.84 0.70 0.72 0.72 0.69 0.66 0.59 0.56 0.62 0.63 0.62 0.55 0.59 0.65 0.69 0.68 0.70 0.59 0.63 0.58 0.60 0.67 0.61 0.66 0.63 0.62 0.58 0.67 0.62 0.51 0.61 0.55

1.00 0.72 0.71 0.71 0.72 0.57 0.62 0.53 0.62 0.64 0.57 0.58 0.63 0.61 0.71 0.70 0.68 0.59 0.65 0.53 0.56 0.64 0.63 0.64 0.58 0.56 0.55 0.70 0.65 0.50 0.58 0.55

second group included ten clones from Tehran (T-SHR, TSHS, T-VGO, T-VG1, T-VG2, T-VG3, T-DO1, T-DO2, T-MO1, and T-MO2), three clones from Qom (Q-BE1, Q-BE2, and Q-BE3), and one clone from Mazandaran (MA-S2). The third group contained six clones from Khozestan (KZ-SH, KZ-OJ, KZ-D1, KZ-D2, KZ-A1, and KZ-A2). The fourth group comprised three clones from Khorasan (KO-MA, KO-ME, and KO-NA) and four clones from Kermanshah (KE-S1, KE-S2, KE-BI, and KE-MA). What we can conclude from this dendrogram is the clear separation of the clones based on location (province), with the exception of the clones from Mazandaran. In the PCA analysis, the morphological traits of clones did not discriminate well between clones (Figure 4) in agreement with Curn et al (2007). The first and second PCA axes of morphological traits accounted for 49.29% and 18.57% of total variation, respectively (Figure 4). The traits containing aboveground dry weight, number of internodes, leaf area, and shoot length explained variability associated with the first axis. The panicle length, number of days until panicle appearance, and senescence explained variability associated with the second axis. RAPD Analysis. A total of 149 bands were screened, among which 123 were polymorphic. The number of bands per 372

N


1.00 0.83 0.69 0.70 0.59 0.60 0.68 0.69 0.62 0.63 0.60 0.65 0.65 0.68 0.67 0.68 0.61 0.58 0.55 0.68 0.73 0.66 0.69 0.63 0.61 0.62 0.68 0.72 0.71 0.72 0.72

1.00 0.66 0.75 0.55 0.64 0.72 0.68 0.64 0.67 0.63 0.65 0.66 0.67 0.69 0.65 0.64 0.64 0.59 0.75 0.72 0.70 0.75 0.67 0.68 0.66 0.70 0.69 0.66 0.73 0.70

1.00 0.71 0.59 0.58 0.66 0.59 0.71 0.66 0.58 0.63 0.63 0.71 0.61 0.65 0.70 0.67 0.66 0.60 0.65 0.63 0.69 0.56 0.63 0.57 0.66 0.58 0.56 0.58 0.63

1.00 0.62 0.63 0.62 0.63 0.68 0.67 0.58 0.63 0.65 0.72 0.66 0.68 0.71 0.69 0.67 0.65 0.63 0.70 0.70 0.64 0.69 0.69 0.65 0.61 0.54 0.63 0.62

1.00 0.45 0.55 0.57 0.54 0.53 0.49 0.56 0.57 0.63 0.54 0.54 0.58 0.56 0.53 0.54 0.60 0.52 0.54 0.56 0.57 0.53 0.43 0.47 0.51 0.49 0.51

1.00 0.62 0.55 0.61 0.60 0.48 0.54 0.51 0.62 0.64 0.61 0.61 0.61 0.63 0.56 0.65 0.69 0.69 0.61 0.60 0.65 0.58 0.64 0.53 0.55 0.53

1.00 0.66 0.58 0.65 0.65 0.65 0.64 0.59 0.62 0.60 0.56 0.60 0.58 0.69 0.62 0.59 0.61 0.69 0.67 0.66 0.57 0.67 0.68 0.65 0.67

1.00 0.56 0.68 0.66 0.65 0.65 0.63 0.59 0.65 0.61 0.61 0.65 0.64 0.60 0.58 0.64 0.77 0.68 0.70 0.58 0.65 0.63 0.68 0.64

1.00 0.83 0.53 0.59 0.63 0.61 0.55 0.55 0.67 0.59 0.67 0.58 0.60 0.66 0.68 0.63 0.70 0.65 0.58 0.51 0.57 0.53 0.56

1.00 0.51 0.60 0.60 0.61 0.58 0.58 0.65 0.58 0.68 0.61 0.64 0.63 0.68 0.69 0.70 0.69 0.59 0.58 0.58 0.58 0.63

primer varied from 8 to 15 with an average of 9.31. The average proportion of polymorphic markers across primers was 83.1%, ranging between 73.3% (UBC13) and 92% (UBC1) (Table 2). Estimates of genetic similarity of RAPD based on 123.1 polymorphic markers among 39 clones of common reed ranged from 0.43 to 0.85 with an average of 0.60 (Table 6). Maximum similarity was with T-MO1 and T-MO2 (0.85); minimum similarity was with MA-S1 and G-GR1 (0.43). Cluster analysis resulted in grouping of the 39 clones of common reed into four main clusters in 0.62 distance unit (Figure 5). The first cluster had two subdivisions. The first subdivision contained six clones form Ardebil (A-MO1, A-MO2, A-MO3, A-MO4, A-MO5, and A-MO6), three clones from Khorasan (KO-MA, KO-ME, and KO-NA), and four clones form Kermanshah (KE-S1, KE-S2, KE-BI, and KE-MA). The second subdivision contained six clones from Khozestan (KZ-SH, KZ-OJ, KZ-D1, KZ-D2, KZ-A1, and KZ-A2), one clone from Mazandaran (MA-S2), and 10 clones from Tehran (T-SHR, T-SHS, T-VGO, T-VG1, T-VG2, T-VG3, T-DO1, T-DO2, T-MO1, and T-MO2). The second cluster included clones from Qom (Q-BE1, Q-BE2, and Q-BE3). Two clones from Golestan (G-GO1 and G-GO2) form the third cluster, and three clones from the Golestan (G-GR1, G-GR2, and G-GR3) formed the fourth

Table 6. Extended. Q- QBE1 BE2

QBE3

TSHR

TSHS

TVGO

TVG1

TVG2

TVG3

TDO1

TDO2

TMO1

TMO2

KOME

KOMA

KONA

GGR1

GGGGGO1 GO2 GR2 GR3

1.00 0.76 0.80 0.57 0.53 0.59 0.55 0.55 0.56 0.64 0.57 0.59 0.63 0.57 0.61 0.52 0.51 0.57 0.52 0.57 0.58

1.00 0.68 0.63 0.54 0.60 0.53 0.59 0.70 0.63 0.62 0.64 0.60 0.66 0.57 0.62 0.58 0.60 0.58 0.64

1.00 0.80 0.64 0.64 0.65 0.61 0.57 0.66 0.58 0.65 0.62 0.63 0.59 0.61 0.64 0.56 0.64 0.52

1.00 0.60 0.64 0.66 0.61 0.61 0.65 0.58 0.62 0.58 0.62 0.51 0.55 0.56 0.56 0.58 0.52

1.00 0.64 0.64 0.63 0.65 0.74 0.65 0.69 0.57 0.53 0.59 0.55 0.56 0.58 0.68 0.60

1.00 0.79 0.76 0.65 0.65 0.64 0.71 0.62 0.65 0.64 0.53 0.52 0.54 0.57 0.62

1.00 0.70 0.59 0.67 0.64 0.67 0.62 0.64 0.60 0.50 0.51 0.53 0.59 0.58

1.00 0.56 0.62 0.61 0.66 0.69 0.68 0.62 0.47 0.51 0.55 0.56 0.60

1.00 0.78 0.72 0.70 0.59 0.59 0.60 0.58 0.57 0.59 0.65 0.67

1.00 0.73 0.73 0.57 0.62 0.56 0.58 0.57 0.59 0.65 0.64

1.00 0.85 0.55 0.60 0.64 0.59 0.55 0.58 0.62 0.67

1.00 0.62 0.68 0.64 0.59 0.60 0.62 0.64 0.63

1.00 0.84 0.76 0.55 0.60 0.58 0.58 0.58

1.00 0.70 0.59 0.58 0.60 0.56 0.57

1.00 0.62 0.66 0.65 0.63 0.59

1.00 0.71 0.57 0.59 0.55

1.00 0.62 0.68 0.59

1.00 0.71 0.60 0.55 0.62 0.62 0.63 0.59 0.67 0.63 0.67 0.69 0.61 0.62 0.64 0.58 0.58 0.55 0.62 0.64

1.00 0.73 1.00 0.70 0.75 1.00

cluster. The fifth cluster contained only one clone from Mazandaran (MA-S1). Another interesting attempt was the comparison of genetic similarities within provinces. The geographic distance between KZ-D1 and KZ-D2 was less than 12 km and genetic similarity between these clones was 0.84. All clones from Qom (Q-BE1, Q-BE2, and Q-BE3) were placed in the same cluster. Genetic distances among these clones were less because Qom is a small province with a low variability in climatic conditions. Similarly, genetic distances among clones from Ardrbil (A-MO1, A-MO2, A-MO3, A-MO4, A-MO5, and A-MO6) were small. Genetic distances among clones from Tehran were large; this might be because of variable climatic conditions in Tehran. T-SHR and T-SHS clones were from the south but T-DO1, T-DO2, T-MO1, and T-MO2 clones were from northwest, and T-VGO, T-VG1, T-VG2, and T-VG3 clones were from southeastern of Tehran. Discussion Figure 5. Dendrogram of individual clones of common reed constructed on the basis of RAPD data, by unweighted pair group method with arithmetic averages (UPGMA) method, resulting from similarity matrix calculated with the metric of Nei and Li (1979).

The genetic diversity in clonal plant population has been studied by some researchers, and they found substantial amounts of diversity in most plant species (Diggle et al. 1998; Ellstrand and Roose 1987; Khudamrongsawart et al. Diyanat et al.: Genetic diversity of common reed

N

373

2004; Widen et al. 1994). European studies of common reed, examining genetic variation and population genetic structure, have shown high levels of genetic divergence among populations (Koppitz et al. 1997; Ku¨hl and Neuhaus 1993; Zeidler et al. 1994), both homogenous (Koppitz et al. 1997) or heterogenous populations (McKee and Richards 1996), or a combination of the two (Ku¨hl and Neuhaus 1993; Zeidler et al. 1994). The high degree of genetic diversity detected among reed populations in this study agrees with European studies. McLellan et al. (1997) stated that gene flow by means of dispersal of vegetative propagules plays an important role in providing variation among or within clonal populations. Our results are in agreement with Bastlova´ et al. (2006), who showed that the clones from the southern areas grew taller and more vigorously than plants from more northern areas. The clones from southern areas allocated the reduced dry mass proportion to generative reproduction. The results are in agreement with Clevering et al. (2001), who showed that differences in length of growing season, time of flowering, morphology, and biomass allocation were sustained when clones of common reed from different geographic origins were transplanted to common environments. Karunaratne and Asaeda (2002) have shown that rhizome biomass and rhizome standing stocks of nonstructural carbohydrates and mineral nutrients decrease in the early growing season and increase later in the year in common reed. Storage is especially important in perennial plants living in regions with cold winters, because the nonstructural carbohydrates are involved in the tolerance of cold climatic conditions, including the danger of frost damage and prolonged winters followed by vegetative periods with low temperatures, which limits carbon assimilation (Klimesˇ et al. 1999). Therefore, the common reed clones in northern regions are not as productive as those in warmer lower latitudes. Weber and Schimd (1998) also showed populations of two goldenrod (Solidago) species originating from northern locations flowered earlier and reached a smaller size at maturity than plants from southern locations. Photoperiod, a factor of great importance, is highly variable across latitude gradient and influences plant lifehistory traits. Variability in phenology found in our study might be strongly correlated with physiological requirement for floral initiation and probably is genetically based. RAPD is an effective method to detect intra- and interpopulation variation and still is used widely for these purposes in many plants (Bussell et al. 2005; Curn et al. 2007; Keller 2000; Koppitz 1999; Koppitz et al. 1997; Ku¨hl et al. 1999). Our results also show that RAPD is suitable for genetic diversity assessment in reed clones. In common reed research, the existence of ecotypes has been mentioned frequently. However, in most instances, common reed clones have not been grown under similar (controlled) conditions. Therefore, it is hard to tell whether differences found between populations or clones are due to environmentally induced variation rather than to genetic differences. In this study we investigated genetic diversity in 39 Iranian clones of common reed based on RAPD markers and morphological traits. For this study, plants were grown under similar environmental conditions in terms of soil, water, nutrients, and climate. It is, therefore, fair to believe that any differences in morphology traits are genetically determined. Clevering et al. (2001) also showed that differences in length of growing season, time of flowering, morphology, and biomass allocation were 374

N


sustained when genotypes of common reed from different geographic origins were transplanted to common environments. Genetics and environments both play a role in the expression of common reed characteristics. Cluster analysis based on morphological traits grouped clones better than RAPD markers, this possibly could be explained by the fact that morphological traits provided a better genome-wide coverage than RAPD markers. Possibly, these variations among clones will assist in successful management of common reed using chemical or other methods of control.

Sources of Materials 1

Leaf area meter, Li-Cor, Inc., Lincoln, NE. Sixteen random primers, University of British Colombia, Vancouver, Canada. 3 MgCl2 (50 Mm), 103 PCR Buffer, dNTPs Mix (10 mM), Taq DNA polymerase (100U [5u/ml]), CinnaGen, Co., Tehran, Iran. 4 Statistical software, Statistical Analysis Systems (version 9), SAS Institute, Inc., 100 SAS Campus Drive, Cary, NC 27513. 2

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Received October 30, 2010, and approved March 29, 2011.


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