Variation in Populations of the Cutgrass Leersia Hexandra

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Aquatic Botany 84 (2006) 359–362 www.elsevier.com/locate/aquabot Short communication Inter-simple sequence repeat (ISSR) variation in populations of the cutgrass Leersia hexandra Zhiping Song, Yun Guan, Jun Rong, Xian Xu, Bao-Rong Lu * Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, Fudan University, Shanghai 200433, China Received 14 November 2004; received in revised form 22 November 2005; accepted 30 November 2005

Abstract Genetic variability of a perennial clonal aquatic weed Leersia hexandra was estimated by the ISSR assay. A total of 225 L. hexandra individuals collected from 10 populations in China were analyzed. The 12 used ISSR primers generated 175 bands, with an average of 14.6 per primer. The average values of percentage of polymorphic bands (PPB), effective number of alleles (Ae), and gene diversity (He) in these populations were 49.7%, 1.258, and 0.154, respectively, indicating a considerable genetic diversity in this species. Signiﬁcant genetic differentiation was found among populations with the major genetic variability within populations based on Nei’s genetic diversity analysis (Gst = 0.417) and AMOVA (40.1%). Genetic variation is independent of geographical distance. This pattern of genetic variation may be associated with reproductive mode of its mixed breeding systems although mainly through vegetative propagation. # 2005 Elsevier B.V. All rights reserved.

Keywords: Leersia hexandra; Genetic diversity; ISSR; Differentiation; Clonal plant

1. Introduction It is difficult to identify genets in investigating genetic variation of clonal species. Inter-simple sequence repeats Genetic study of clonal plant species provides reliable (ISSRs) are regions lying within the microsatellite repeats that information on their population dynamics and detailed are extensively used to estimate genetic variation of clonal demographic data (Tolvanen et al., 2004; Ren et al., 2005). plants due to their high polymorphism (Li and Ge, 2001). They A low level of genetic diversity is expected in clonal plants offer a great potential to determine intra- and inter-genomic (Loveless and Hamrick, 1984; Eckert, 1999; Carter and Sytsma, diversity compared with other arbitrary primers, since they 2001). In contrast, clonal populations can also maintain reveal variations within unique regions of the genome at considerable genetic diversity, comparable to sexually repro- multiple loci simultaneously (Zietkiewicz et al., 1994). ISSRs ducing species (e.g., Li and Ge, 2001; Alfonso-Corrado et al., exhibit specificity of sequence-tagged-site markers, but need no 2004; Li et al., 2004; Pluess and Stocklin, 2004; Ren et al., sequence information for primer synthesis, thus having 2005). The cutgrass Leersia hexandra Swartz. (Poaceae) is an advantages of random markers. ISSRs have become popular aquatic clonal species and widely distributed in the tropical and and effective markers for measuring genetic diversity in plants warm temperate regions of the northern and southern hemi- (e.g., Jin et al., 2003; Pluess and Stocklin, 2004). The objective spheres (Gould, 1968). L. hexandra usually grows along the of this study is to investigate the genetic diversity within and margins of marshes, streams, ponds, lakes, swamps, ditches, among populations of L. hexandra to obtain baseline data for canals, and in water up to 1.8 m deep, and can sometimes form this aquatic clonal species. floating islands (Godfrey and Wooten, 1979; Hoyer et al., 1996). Although it can reproduce by seed, it exhibits mainly clonal growth with rhizomes or leafy stolons (Villafan˜e, 1989). 2. Materials and methods However, little is known about its genetic variation. 2.1. Population sampling and DNA extraction

* Corresponding author. Tel.: +86 21 65643668; fax: +86 21 65643668. A total of 225 L. hexandra individuals were sampled E-mail address: [email protected] (B.-R. Lu). representing 10 natural populations from Hunan, Fujian, and

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Guangdong Provinces and Shanghai city of China (Table 1). tions were performed using POPGENE program version 1.31 About 1 g fresh leaf samples from each individual were (Yeh et al., 1999). The nonparametric analysis of molecular collected and placed in a plastic bag containing silica gel for variance (AMOVA) program version 1.5 was also used to fast drying and brought back to a laboratory for DNA isolation. describe genetic structure and variability among populations, as Sampled individuals were 5 m apart to minimize the possibility described by Excoffier et al. (1992). The number of permutations of sampling connected ramets. Total genomic DNA was for significant testing was set at 5000 for AMOVA. The effect of extracted using a protocol described by Doyle and Doyle spatial separation on genetic structure was tested by the Mantel (1987). test (Mantel and Valand, 1970) on matrices of genetic (Nei, 1978) and geographic distances between populations. Mantel test was 2.2. PCR assay and electrophoresis performed with 1000 random permutations using the software developed by Bonnet and Van De Peer (2002). The UPGMA tree To determine appropriate primers suitable for L. hexandra, (Nei, 1978) was constructed using NTsys program version 1.8 86 ISSR primers purchased from The University of British (Rohlf, 1994) to examine relationship of the populations. Columbia (UBC SSR Primer (RAPD) Synthesis Project Oligonucletide Set 100/9) were screened. Twelve ISSR primers 3. Results produced clear and reproducible bands (>3 replications) were selected for amplifying all DNA samples after amplification 3.1. Genetic diversity conditions were optimized. PCR reactions were performed in a total volume of 20 mL, with 1 buffer, 2–3.25 mM MgCl2, A total of 175 ISSR bands were generated with the 12 0.25 mM dNTPs, 0.2 mM primer, 0.5 unit of Taq DNA primers (Table 2) (14.6 bands/primer on average), and 165 of polymerase (TakaRa Biotchnology Co., Ltd.), and 20 ng of these were polymorphic in L. hexandra populations (Table 1). template DNA. After an initial denaturation of 4 min at 94 8C, The parameters of genetic diversity showed that the percen- 40 cycles for 45 s at 94 8C, 45 s at 50–54 8C (variable for tages of polymorphic bands (PPB) of L. hexandra populations different primers), and 45 s at 72 8C were performed, followed varied from 29.1 to 72.0% (49.7% on average). The mean by the final 7 min extension at 72 8C. The amplifications were number of effective alleles (Ae) was 1.234 at the population carried out in a DNA thermal cycler (Eppendorf Mastercycler level. The gene diversity values (He)ofL. hexandra populations Gradient). PCR products were electrophoresed in the 1.5% ranged from 0.092 to 0.209 (0.156 on average). Among the 10 horizontal agarose gel with the voltage of 5 V cm1 and populations, Gaozhou population (LG) had the lowest genetic visualized under ultraviolet light after staining in 1 mgmL1 diversity levels, while Chenxi population (LC) showed the ethidium bromide. Photodocumentation was taken for each gel. highest genetic diversity (Table 1).

2.3. Data analysis 3.2. Genetic differentiation

The amplified DNA polymorphic fragments (bands) were The coefficient of genetic differentiation between popula- scored as presence (1) or absence (0), and the data matrix of the tions (Gst) was 0.417, indicating over 40% genetic variation ISSR phenotypes was assembled for further analysis. The between L. hexandra populations. AMOVA analysis further percentage of polymorphic bands (PPB), effective allele number revealed highly significant ( p < 0.001) genetic differences (Ae), and gene diversity (He) were calculated to estimate genetic among the 10 populations. Of the total genetic diversity, 40.1% variation. Population differentiation was analyzed for poly- was attributable to among-populations diversity and the rest morphism between populations by Gst.Geneflow(Nm)was (59.9%) to differences within populations. The level of gene estimated from Nm = (1/4)(1 Gst)/Gst (Nei, 1987). All calcula- flow (Nm) was 0.699 individuals per generation. Population

Table 1 The parameters of genetic diversity of Leersia hexandra populations included in the study

Population Sample site Sample size No. of polymorphic band PPB (%) Ae He CL Chaling, Hunan Province 59 126 72.0 1.310 0.188 LB Boluo, Guangdong Province 7 66 37.7 1.201 0.122 LC Chenxi, Guangdong Province 24 121 69.1 1.345 0.209 LF Fugang, Guangdong Province 19 71 40.6 1.242 0.141 LD Gaozhou, Guangdong Province 12 88 50.3 1.298 0.171 LG Gaozhou, Guangdong Province 17 51 29.1 1.154 0.092 LH Huilai, Guangdong Province 22 81 46.3 1.244 0.145 LZ Zengcheng, Guangdong Province 12 90 51.4 1.286 0.167 LP Zhangpu, Fujian Province 47 118 67.4 1.324 0.197 LQ Qingpu, Shanghai 6 58 33.1 1.177 0.106 Mean (S.E.) 87 (26.9) 49.7 (15.4) 1.258 (0.065) 0.154 (0.039) Species 225 165 94.3 1.432 0.261

PPB: percentage of polymorphic bands; Ae: effective number of alleles; He: gene diversity. 中国科技论文在线 http://www.paper.edu.cn

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Table 2 The list of ISSR primers used in the study Primer Sequence (50–30) Anneal temperature (8C) No. of bands No. of polymorphic bands 814 (CT)8A 50 14 12 816 (CA)8T 52 13 11 829 (TG)8C 51 13 13 836 (AG)8(CT)A 52 15 14 840 (GA)8(CT)A 52 11 11 851 (Gt)8(CT)G 52 12 10 855 (AC)8(CT)T 54 17 16 857 (AC)8(CT)G 50 13 12 886 (ACG)(AGT)(ACG) (CT)7 50 17 17 887 (AGT)(ACG)(AGT) (CT)7 50 18 18 889 (AGT)(CGT)(AGT) (AC)7 51 16 16 890 (ACG)(AGT)(ACG) (GT)7 53 16 16 Total 175 165

genetic differentiation was not significantly correlated with set was detected in some L. hexandra populations (Liu et al., physical distance of the populations (Mantel test, r = 0.508, 2005). The consequence of certain features, such as wide- p = 0.073). This pattern was further confirmed by the UPGMA distribution and perennial life form might also contribute to tree based on Nei’s (1978) unbiased genetic similarity (Fig. 1), high genetic diversity of L. hexandra, as in other perennial in which geographically close populations LD and LG showed herbs (Hamrick and Godt, 1990). relatively distant genetic relationship, as well as, the popula- Genetic structure is an important feature reflecting gene tions LB, LZ, and LF (Fig. 1). flow, mating system in a population and the extent of population diversity (Li and Ge, 2001; Ren et al., 2005). In L. hexandra, the 4. Discussion over 40% genetic variability between populations may be due to its reproductive mode. L. hexandra shows both clonal growth To the best of our knowledge, this is the first report of genetic and sexual reproduction (Song, personal observations). The diversity in Leersia species. In general, a relatively high genetic clonal propagation theoretically has similar effects for variability (He = 0.154 0.039) was detected compared with population genetic structure as strict selfing (inbreeding). other clonal plants, suggesting that some clonal plants can have Inbreeding mode usually reduces gene exchange (gene flow) high levels of genetic diversity. The high level of genetic both between different individuals and populations, leading to diversity in L. hexandra may be interpreted as occasional sexual significant differentiation between populations (Slatkin, 1987). recruitments in some habitats. Studies show that low rate of Based on the general understanding of association between seedling recruitment is sufficient to maintain relatively high genetic structure and breeding systems, we predict that L. levels of genotypic variation within plant populations (Pluess hexandra most likely reproduce by selfing, because the value of and Stocklin, 2004; Ren et al., 2005). In fact, a low rate of seed Gst is near to the mean of selfing species (0.41 versus 0.51

Fig. 1. UPGMA dendrogram based on Nei’s (1978) genetic distance, combined with geographical locations illustrating genetic and geographical relationships among the 10 populations of Leersia hexandra from China. CL population sampled from Hunan Province, LQ population sampled from Shanghai City, LP population sampled from Fujian Province, the others sampled from Guangdong Province. Sample sizes are listed in Table 1. 中国科技论文在线 http://www.paper.edu.cn

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