Bot. Bull. Acad. Sin. (1999) 40: 319-327

Chou and Tsai — IGS variation of Cogongrass in Taiwan

Genetic variation in the intergenic spacer of ribosomal DNA of Imperata cylindrica (L.) Beauv. var. major (Cogongrass)

populations in Taiwan

Chang-Hung Chou^1,2,4, Chi-Chu Tsai^2,3

¹Institute of Botany, Academia Sinica, Taipei 115, Taiwan

²Graduate Institute of Botany, National Taiwan University, Taipei 107, Taiwan

(Received October 12, 1998; Accepted April 13, 1999)

Abstract. Genetic variation in the intergenic spacer (IGS) of ribosomal DNA (rDNA) repetitive sequences in inter- and intra-specific populations of Cogongrass [Imperata cylindrica (L.) Beauv. var. major (Nees) Hubb] in Taiwan, was studied by employing PCR-amplified RFLP analysis. Each IGS region of 45 individuals from 15 populations was amplified using a pair of complementary primers to conserved regions in the 5`end of 17S and 3'end of 25S rRNA genes. Five IGS length variants were found at 2,730 bp, 2,830 bp, 2,930 bp, 3,030 bp and 3,130 bp. Of them, 2930 bp was the main type of IGS length of Cogongrass in Taiwan. Within an individual Cogongrass, one to two IGS length types were distinguished. Those samples amplified by PCR were digested with 13 restriction enzymes, and 283 bands were revealed. Of them, 248 bands were polymorphic. Clustering analysis was conducted based on data obtained from bands distributed in all samples mentioned, and two major clusters were found. The Chuwei population was distinctly different from the remaining populations. Moreover, the findings of the PCR-amplified RFLP analysis also indicated that the IGS region of rDNA provides a good genetic marker and a potential tool for the study of microevolutionary process.

Keywords: Cogongrass; IGS; Imperata cylindrica; PCR-amplified RFLP; rDNA.

Introduction

Imperata cylindrica (Cogongrass), an aggressive rhizomatous grass, is distributed widely in many parts of the world from tropical to subtropical areas (Al-Juboory and Hassawy, 1980). It can be spread both by seeds and rhizomes (Wilcut et al., 1988). The light, thistle-like seeds of Cogongrass are capable of being transported over great distances, and this has probably contributed to increased distribution. The seed production is rapid and well adapted to new environments (Al-Juboory and Hassawy, 1980; Dickens, 1974). The plant exhibits a wide adaptability to different climatic and environmental regimes but is sensitive to salinity, leading to many ecotypes. Basic information on the taxonomy, physiological ecology, and morphology of this grass is fairly extensive. Little was known regarding the molecular ecology of the grass until Cheng and Chou (1997) initiated a series of molecular studies by selecting six Cogongrass populations in Taiwan. They (1997) concluded that the Chuwei (CW) population located at the mouth of the Tamshui River has formed an ecotype distinct from the remaining five populations in other parts of Taiwan.

In higher eukaryotes, ribosomal RNA genes (rDNAs) are organized as families with repeated genes in tandem arrays at the nucleolar organizer regions of chromosomes. The copy number of repeated genes in rDNAs usually reveals from 100 to 1,000 copies per diploid cell in animals, and from 500 to 40,000 copies per diploid cell in plants. Each repeating unit usually consists of the transcribed region coding for 17S, 5.8S and 25S rRNAs, which are highly conserved among various organisms, and the intergenic spacer (IGS) which is highly variable in length and primary structure among organisms and individuals (Appels et al., 1980; Waldron et al., 1983; Rogers and Bendich, 1987; Taira et al., 1988; Kato et al., 1990; Perry and Palukaitis, 1990;Beech and Strobeck, 1993; Borisjuk et al., 1994). The primary source of rDNA length variation is the number of subrepeats within the IGS (Bhatia et al., 1996; Kaufman et al., 1996). Therefore, sequence comparisons of rRNAs genic regions were used to study phylogenetic relationships among organisms (Schaal and Learn, 1988; Molina et al., 1993) while the IGS region provided a good genetic marker for the study of the microevolutionary process in inter- and intra-specific populations (Schaal and Learn, 1988).

Recently, techniques such as restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) have been widely used for genetic diversity analysis (Torres et al., 1993). However, conventional RFLP techniques involving southern blotting are

³Present address: Taichung District Agricultural Improvement Station, Changhua 515, Taiwan

⁴Corresponding author. E-mail: chou@gate.sinica.edu.tw

Botanical Bulletin of Academia Sinica, Vol. 40, 1999

laborious, time-consuming, and inadequate for large-scale analysis (Halward et al., 1992; Williams et al., 1990). On the other hand, RAPD techniques provide a powerful tool to detect genetic variation in natural and man-made populations. The technique requires only small amounts of DNA, is simple, and costs less than other techniques (Huff et al., 1993). Nevertheless, it has certain limitations, including its sensitivity to reaction conditions and difficulties surrounding the reproducibility of amplification products (Hadrys et al., 1992; Schierwater and Ender, 1993). Recently, the PCR technique has been increasingly applied to problems in genetics, biomedical sciences, evolutionary biology, and ecology (Arnheim and Erlich, 1992). The technique of PCR-amplified RFLP was initially introduced to detect variations in the chloroplast gene rpoC₁, C₂ region among 14 species of Astragalus (Fabaceae) (Liston, 1992). After that, several reports attempted to detect genetic variation by PCR-amplified RFLP, but most of them focused on detecting genetic variations of the chloroplast DNA region in plants (Ghareyazie et al., 1995; Tsumura et al., 1995, 1996; Lashermes et al., 1996; Ziegenhagen and Fladung, 1997). In the present study, the technique was employed to detect the variation in IGS of rDNA among 15 populations of Cogongrass in Taiwan.

Materials and Methods

Sites of Sampling

Fifteen sites—located at Fengshan (FS), Potzu (PZ), Fengyuan (FY), Chuangwei (TW), Hualien (HL), Chengkung (CK), Yehliu (YL), Sarlum (SL), Hoshe (HS), Kengting (KT), Penghu (PH), Lutao (LT), Lanyu (LY), Chuwei (CW), and Nankang (NK)—were selected for this study (Figure 1). Characteristics of the habitats, including soil texture, altitude, and weather conditions, were described by Tsai (1994). The sites were chosen to be representative of different habitats and climates in Taiwan.

Plant Materials

Rhizomes of Cogongrass collected from the 15 habitats of Taiwan (Figure 1) were transplanted into pots which were set in the greenhouse of the Institute of Botany, Academia Sinica at Taipei, Taiwan. Environmental conditions—such as the amount of water, soil type, humidity, temperature, and light intensity—were under control.

Preparation of Total Cellular DNA

Total cellular DNA from the leaves of transplanted Cogongrass was prepared by using an extraction technique modified from that of Shure et al. (1983). Three grams of fresh leaves were harvested and ground to powder with liquid nitrogen in a mortar and pestle, then transferred into a 30 ml centrifuge tube containing 10 ml of urea buffer (8.0 M urea, 0.05 M NaCl, 0.05 M Tris-HCl pH 7.5, 0.02 M EDTA, 1% sarcosyl), preheated in 60°C water. We mixed the sample thoroughly and incubated it in a water bath at 60°C, inverting the tube constantly. We then, add

Figure 1. The sampling locations of I. cylindrica in Taiwan. The alphabes indicate that the samples were analyzed in the present study. The abbreviations of sampling sites are: FS (Fengshan), PZ (Potzu), FY (Fengyuan), TW (Chuangwei), HL (Hualien), CK (Chengkung), YL (Yehliu), SL (Sarlum), HS (Hoshe), KT (Kengting), PH (Penghu), LT (Lutao), LY (Lanyu), CW (Chuwei) and NK (Nankang).

ed 10 ml phenol: chloroform (1:1, v/v) [Tris pH 8.0 saturated], inverting many times gently, and centrifuged the tube at 10,000 rpm (Sigma 2K15; Nr12139) for 10 min at 4°C. We transferred the supernatant to a new centrifuge tube by filtering through two layers of miracloth, after adding 0.7 volume of 2-propanol and 1/10 volume 4.4 M NH₄OAc. The tube was centrifuged at 10,000 rpm for 10 min at 4°C to collect precipitated DNA. The DNA pellet was resuspended with 5 ml TE (10 mM, Tris-HCl pH 8.0, 1 mM EDTA) and incubated with 50 µg DNase-free RNase (Sigma) for 10 min at 65°C. The RNase and remaining protein were extracted with an equal volume of phenol:chloroform (1:1, v/v) [Tris pH 8.0 saturated] and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was transferred to a new tube. Then, the DNA was precipitated by the addition of a 1/10 volume 4.4 M NH₄OAc and three volumes of 95% ethanol. Precipitated DNA was collected by centrifugation at 10,000 rpm for 10 min at 4°C, washed with 70% ethanol twice, and dried before being redissolved in 100-150 µl of TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Approximate yield amounts were calculated by a spectrophotometer (Beckman DU-20), and the DNA samples were stored at -20°C.

Chou and Tsai — IGS variation of Cogongrass in Taiwan

PCR Primers

Oligonucleotides used for PCR priming were designed from conserved regions of the 5'end of the 17S rDNA sequence and the complementary sequence of the 3'end of 25S rDNA from previously described sequences (Takaiwa et al., 1990; Kiss et al., 1989a; Kiss et al., 1989b; Lakshmikumaran and Negi, 1994 ). Two primer sets for amplifying IGS of rDNA were designated as IG1: 5'-CTACTGGCAGGATCAACCAGG-3' and IG2: 5'-TTGCTGCCACGATCCACTGAG-3'.

PCR Amplification

PCR reaction was performed by using a 50 µl mixture, containing 30 mM Tricine pH 8.4, 2 mM MgCl₂, 0.01% BSA, 5 mM 2-mercaptoethanol and 0.1% Thesit, with four dNTPs (0.8 mM each), primers (0.5 µM each), 2.5 units of Taq DNA polymerase (Promega) and 32 ng genomic DNA, and 50 µl mineral oil (Ponce and Micol, 1992). Amplification reactions were done in dry-block, two-step thermal cycles. In the first step, the mixture was incubated at 94°C for 5 min, followed by 10 cycles of denaturation at 94°C for 1 min, annealing at 61°C for 1 min and extension at 72°C for 3 min. The second step was conducted as follows: 30 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 3 min, followed by a final extension for 10 min at 72°C. Those reaction samples were detected by agarose gel electrophoresis of 10 µl of the PCR products (0.8%, w/v in TAE), staining by 0.5 µg/ml of ethidium bromide (EtBr), and photographing under the exposure of UV light.

Restriction Enzyme Digestion

DNA fragments amplified by PCR were digested with 13 restriction enzymes, namely HaeIII, TaqI, AluI, RsaI, BanI, CfoI, MspI, BstOI, Eco0109I, HinfI, StyI, EcoRV and SmaI. All restriction enzymes digested were carried out under the conditions recommended by manufacturers (BRL or Promega), using 5-10 units of enzyme per microgram (µg) of DNA, and incubating the reaction for 3 h to overnight.

Gel Electrophoresis

About 5-10 µg of digested DNA mixed with tracking dye [0.25% bromophenol blue, 40% (w/v) sucrose in water] were electrophoresed overnight at 50 V-80 V in 2.5%-5.0% NuSieve 3:1 agarose gels (FMC Bioproducts) with 0.5 µg/ml EtBr. The pattern of bands was examined under UV light.

DNA Labeling and Southern Hybridization

The DNA band on the gels was recovered by glassmilk (BIO 101, Geneclean Kit II). Probes were labeled with digoxigenin. Prehybridization and hybridization were performed with a non-radioactive DNA labeling and detection Kit (Boehringer Mannheim). Detection of hybrid DNAs was achieved by chemiluminescent reaction using AMPPD on Kodac x-omat film. Immunological detection

was also done using color solution (containing NBT solution and x-phosphate) on a membrane (Boehringer Mannheim). The aforementioned reaction protocol was recommended by the manufacturers.

DNA Cloning and Sequencing

PCR product of CW₃ DNA samples in Cogongrass was recovered by glassmilk, cloned into T-vector (Promega), and transformed into the E. coli strain `JM109' competent cell (Promega). After bacteria culture and plasmid extraction (by Boehringer Mannheim, Qiagen-tip 20), the DNA sample was sequenced by the dideoxy chain-termination method using the Auto Read Sequencing Kit (Pharmacia). The aforementioned reaction protocol was recommended by the manufacturers.

Data Analysis

Data of bands obtained from PCR-amplified RFLP of Cogongrass populations were analyzed statistically using similarity coefficients (S), where S=2N_AB/N_A+N_B, and N_AB represents the number of co-migrating fragments (in considering all enzymes). N_A and N_B are the total number of bands for sample A and sample B, respectively (Chapco et al., 1992; Wilde et al., 1992). A dendrogram was constructed based on data of the similarity matrix, using the unweighted pair-group method analysis (UPGMA) (Rohlf et al., 1982).

Results

PCR Amplification

DNA fragments amplified from PCR using primers IG1 and IG2 were completed from 45 samples of Cogongrass. Five PCR products of different lengths were found, 2,800 bp, 2,900 bp, 3,000 bp, 3,100 bp and 3,200 bp, respectively (Figure 2). Both the 5`end and 3'end of the PCR product in the CW₃ sample was partly sequenced to identify the PCR product that was IGS of rDNA (data not shown). Using the PCR product of the CW₃ sample and hybridizing it with the others indicated that the PCR products of all samples fell into IGS regions (data not shown). The PCR product of the CW₃ sample was identified and contained both 48 bp of 25S rRNA gene and 24 bp of 17S rRNA gene. Thus, five different IGS length variants of rDNA of Cogongrass in Taiwan were detected, namely 2,730 bp, 2,830 bp, 2,930 bp, 3,030 bp and 3,130 bp. Among the five IGS lengths, type 2,930 bp was the most abundant with a 75.5% presence among 45 samples of Cogongrass; type 2,730 bp was the second most abundant with 24.4%; type 3,030 bp was third with 22.2%, and types 2,830 bp and 3,130 bp were as low as 4.4%. Furthermore, populations FS, PZ, TW, CK, YL, HS, KT, LY and NK analyzed by PCR possess two types of IGS length with a 31.1% presence among 45 samples of Cogongrass, one of them was minor, and the remaining populations revealed only one type with 68.8% (Table 1). Restriction enzyme EcoRI was chosen for rDNA length variation analysis because it cleaved both

Botanical Bulletin of Academia Sinica, Vol. 40, 1999

25S rRNA gene and 17S rRNA gene once (Taira et al., 1988). Total genomic DNAs of all samples were digested by EcoRI. After electrophoresing by 0.8% agarose gel and staining with EtBr, a band of about 8.8 kb appeared (data not shown). Those bands can be hybridized with the IGS of CW₃ sample (Figure 3). The findings confirmed that the rDNA of Cogongrass exhibited many copy numbers in tandem repeat and only one main type of rDNA length within individuals of Cogongrass.

PCR-Amplified RFLP

Each PCR product of 45 samples of Cogongrass was digested with 13 restriction enzymes, namely HaeIII, TaqI, AluI, RsaI, BanI, CfoI, MspI, BstOI, Eco0109I, HinfI, StyI, EcoRV and SmaI, revealing 283 bands in total. Of them, 248 bands were polymorphic, reaching 87.6% (Table 2). Of these restriction enzymes, TaqI, AluI, RsaI, MspI, Eco0109I, StyI, and SmaI revealed powerful polymorphic bands above 90% in the IGS region of rDNA among the 15 Cogongrass populations, while EcoRV exhibited rather low polymorphism (Figure 4).

Clustering Analysis

Based on PCR-amplified RFLP of IGS, the similarity was computed between pairs of samples (data not shown). A phylogenetic tree was constructed from similarity by UPGMA. Based on this tree, the genetic variation within the population was greater than that between populations in some individuals of aforementioned populations, namely FS, CK, YL, HS, KT, LY and NK. Furthermore, minor population differentiation was revealed, namely in PZ, FY, TW, HL, SL, PH and LT. Obviously, the aforementioned populations of Cogongrass in Taiwan could be divided into two clusters. The CW population forms one cluster, and the remaining 14 populations revealed another cluster (Figure 5).

Discussion

Since the 17S-5.8S-25S structural gene region is very conserved in sequence and is approximately 6.0 kb in most grasses, the differences in rDNA repeat sizes are due to variation in the IGS length (Pillay, 1996). However, in some plants, there are two or more types of IGS length among individual members of a plant population while in others the length heterogeneity of IGS cannot be detected (Rogers and Bendich, 1987; Lakshmikumaran and Negi, 1994). Generally, the variability of IGS length is smaller in cultivated species than in wild type species. A reduction in the variability of rDNA spacer may be due to domestication (Cordesse et al., 1990). However, the results of PCR showed the variability of IGS length in Cogongrass, which is wild, to be rather low, most individuals being of only

Figure 3. The result of southern hybridization from six populations of I. cylindricain Taiwan hybridized by PCR product of CW₃ sample. The abbreviations of sampling sites see Figure 1. The arabic numbers indicate different sample of each population.

Figure 2. The IGS region of rDNA in 15 populations of I. cylindrica in Taiwan. The IGS region was obtained by the PCR amplification of primers IG1 and IG2. The abbreviations of sampling sites see Figure 1. The arabic numbers indicate different samples of each population.

Figure 4. The band pattern of IGS region of rDNA of I. cylindrica obtained by using PCR, digested with RsaI restriction enzyme, and separated by 2.5% NuSieve 3:1 agarose gel. The abbreviations of sampling sites see Figure 1. The arabic numbers indicate different samples of each population.

Chou and Tsai — IGS variation of Cogongrass in Taiwan

Table 1. A summary of IGS length and number of IGS length variation from 15 populations of I. cylindrica based on PCR.

Location of population Length of IGS (bp)

and (abbreviation) 2730 2830 2930 3030 3130

Fengshan (FS) 1 + +^* 2 +^* + 3 + +^*

Potzu (PZ) 1 + +^* 2 + +^* 3 + +^*

Fengyuan (FY) 1 + 2 + 3 +

Chuangwei (TW) 1 + +^* 2 + +^* 3 + +^*

Hualien (HL) 1 +

2 +

3 +

Chengkung (CK) 1 +

2 +

3 +

Yehliu (YL) 1 +

2 +

3 +

Sarlum (SL) 1 +

2 +

3 +

Hoshe (HS) 1 +

2 +

3 +

Kengting (KT) 1 + +^*

2 +

3 +

Penghu (PH) 1 +

2 +

3 +

Lutao (LT) 1 +

2 +

3 +

Lanyu (LY) 1 + +^*

2 + +^*

3 + +^*

Chuwei (CW) 1 +

2 +

3 +

Nankang (NK) 1 + +^*

2 +

3 +

Total 45 11 2 34 10 2

% of total sample 24.4 4.4 75.5 22.2 4.4

+ : Presence of major band.

+^*: Presence of minor band.

Botanical Bulletin of Academia Sinica, Vol. 40, 1999

Table 2. The polymorphic fragment of IGS of rDNA from 15 populations of I. cylindrica digested by 13 restriction enzymes.

Enzyme Recognition site Restriction fragment number (A) Polymorphic band

Number % of (A)

HaeIII GG¯CC 38 32 84.2

TaqI T¯CGA 11 10 90.9

AluI AG¯CT 9 9 100

RsaI GT¯AC 15 15 100

BanI G¯G(T/C)(A/G)CC 13 11 84.6

CfoI GCG¯C 32 25 78.1

MspI C¯CGG 46 42 91.3

BstOI CC¯(A/T)GG 32 27 84.4

Eco0109I Pu^bG¯GN^aCCPy^c 12 11 91.7

HinfI G¯ANTC 23 19 82.6

StyI C¯C(A/T)(A/T)GG 25 24 96.0

EcoRV GAT¯ATC 5 2 40.0

SmaI CCC¯GGG 22 21 95.5

Total 283 248 87.6

^aN, represent A, G, C or T.

^bPu, represent purine.

^cPy, represent pyrimidine.

one type (68.9%) (Figure 2 and Table 1). The variability was also as low as one type when hybridization techniques were used (Figure 3). The variation in rDNA spacer length can reflect both a phylogenetic relationship and a limited ecological introgression (Cordesse et al., 1990). Cogongrass in Taiwan contained five types of IGS length variants, namely 2,730 bp, 2,830 bp, 2,930 bp, 3,030 bp and 3,130 bp. However, no positive correlation appeared between rDNA length variation and geographical distribution. For example, samples collected from YL, SL, NK and CW were located at the coastal area of Taipei county, but the IGS length patterns of the YL, SL and NK populations were different from that of the CW population (Table 1 and Figure 1). The report that the heterogeneous length of IGS varied with the copy number is primarily based on small subrepeats of several plants (Cordesse et al., 1990; Kaufman et al., 1996). The variability of IGS length ranged from 1 kb to 12 kb or higher for different plant species (Rogers and Bendich, 1987), and small subrepeat length varied from 30 bp to 350 bp (Beech and Strobeck, 1993). Therefore, we infer a 100 bp small subrepeat sequence among IGS of rDNA of Cogongrass in Taiwan. A phylogenetic tree of IGS based on PCR-amplified RFLP analysis suggested that the CW population was unique and distinguished from the remaining 14 populations (Figure 5). Among these populations no obvious geographical relationship was exhibited (Figure 1 and Figure 5).

The variability of IGS length that appeared within populations, namely TW, CK, YL, HS, KT, LY and NK, was greater (Table 1). Furthermore, the results of the cluster analysis of PCR-amplified RFLP in IGS of rDNA also showed that the genetic variation within the population was greater than that between population in some individuals of the aforementioned populations, namely FS, CK, YL, HS, KT, LY and NK (Figure 5). In view of the two

Figure 5. A dendrogram of cluster analysis based on PCR-amplified RFLP of IGS region from 15 populations of I. cylindrica in Taiwan. The abbreviations of sampling sites see Figure 1. The arabic numbers indicate different sample of each population.

Chou and Tsai — IGS variation of Cogongrass in Taiwan

rDNA may spread among the structure of tandem repeat rDNAs by the processes of unequal recombination and gene conversion (Beech and Strobeck, 1993; Bhatia et al., 1996). This study has suggested that this variant plays some role in selecting the variants of the IGS sequence in rDNAs. Furthermore, we suggested that the IGS of rDNA family in the CW population of Cogongrass might maintain a few other types of IGS sequence to adapt to the changing environment.

Morphological characters such as leaf length, leaf width, and plant height were measured based on the transplanting of rhizome of Cogongrass from the field to the greenhouse. The Cogongrass growing at the CW site had waxy crystals on its surface and a rib with a hollow structure without ground tissue (Cheng and Chou, 1997). The CW population is located at the mouth of the Tamshui River, where severe environmental pressures are present, such as frequent submergence in high saline water and oxygen deficiency at the roots (Chang, 1996). Therefore, we infer that the development of waxy crystals on the leaf surface is for the purpose of reducing evaporation, and the hollow structure on the rib without the ground tissue has evolved to help the plant adapt to oxygen deficiency.

In conclusion, the present study has shown the genetic variation of IGS length and sequence of rDNA among intra- and inter-specific populations of Cogongrass based on PCR-amplified RFLP. It supported the previous findings of RAPD and morphological characters that the CW population of Cogongrass was distinctly different from the remaining populations and that microevolution was running in the CW population. Moreover, the analysis of IGS of rDNA based on PCR-amplified RFLP seems to be an easy and powerful tool to elucidate population structure and microevolution.

Acknowledgements. The study was financially supported by grants to C. H. Chou from the Five-year Research Plan of the Institute of Botany, Academia Sinica, Taipei. We are grateful to Drs. T.Y. Chou, C.Y. Huang, C. Tu, S. Huang, and Y. F. Chen for their valuable comments and helpful discussion in the course of the study.

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