Assessment of Genetic Variation and Distribution Pattern of Thalictrum petaloideum Detected by RAPDs

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1 Acta Botanica Sinica 2004, 46 (2): Assessment of Genetic Variation and Distribution Pattern of Thalictrum petaloideum Detected by RAPDs XIE Lei, LI Liang-Qian *, ZHANG Da-Ming (Laboratory of Systematic and Evolutionary Botany, Institute of Botany, The Chinese Academy of Sciences, Beijing , China) Abstract: Random amplified polymorphic DNA (RAPD) method was applied to assess genetic variation and population structure of Thalictrum petaloideum L. (Ranunculaceae). Two hundred and forty-six individuals from 11 populations of the species were investigated by RAPD profiles. Twenty selected RAPD primers generated 125 bands, in which 120 were polymorphic. The results revealed a high level of genetic variation (percentage of polymorphic bands (PPB) was 96%, Nei s gene diversity (h) was and shannon s information index (I ) was at the species level). The differentiation among the populations was high (G ST = ) in this species. Result of analyzing of molecular variance (AMOVA) showed that 38.88% of genetic variance was found among the populations. Positive correlation with r = (P = ) was found between genetic distance and geographic distance among populations. Two populations distributed in the drainage basin of Yangtz River affined genetically and formed one clade and the rest nine populations formed the other clade in both unweighted pair-group method using arithmetic average (UPGMA) trees made by two different methods. It was very clear that these two populations were very special, and must be closely related in history, despite the fact that they now share quite weak link to the rest populations through gene communication. Key words: Thalictrum petaloideum ; random amplified polymorphic DNA (RAPD); genetic diversity; distribution pattern The genus Thalictrum is one of the largest and most diverse genera in buttercup family (Ranunculaceae). Thalictrum petaloideum is a species of the genus, which is a diploid and widely distributed in northern China with several populations reaching the drainage basin of Yangtz River (Wu and Raven, 2001) (Fig.1). Plants in the drainage basin of Yangtz River have some characters different from the ones in northern China, marked by bigger leaflets, stronger stems, shadowy and lower altitude inhabited environment. Wang (1984) considered that its modern distribution pattern might be affected by the glacial events and its final geographical structure was most likely formed through recolonizing after the last glacial events and in Pleistocene Ice Age. In this study, The random amplified polymorphic DNA (RAPD) (Williams et al., 1990) method was used to analyze the population genetic structure of T. petaloideum and provided valuable molecular evidence for its genetic variation. Population genetic structure is considered as spatiotemporal distribution of genetic variation (Ge, 1997). It can reflect evolutionary processes of populations (Hamrick and Loveless, 1989). In order to elucidate these processes, patterns and mechanisms of evolution and to explain phylogenetic relationships among taxa, we should reveal the range of genetic variation, genetic structure, diversification trend and other factors affecting genetic structure of the populations. Presently, many biochemical and DNA molecular markers have been used in the studying of population structure. Fig.1. Distributional pattern of Thalictrum petaloideum. Received 5 Aug Accepted 29 Aug Supported by the National Natural Science Foundation of China ( ). * Author for correspondence. <lilq@ns.ibcas.ac.cn>

2 RAPD is frequently applied to reveal population genetic variation, divergence and biogeography (Schaal and Leverich, 2001). A large number of studies have successfully applied this method to study genetic variation in plants despite its shortcomings, such as unnecessity of prior DNA sequence information, relatively unbiased portion of the genome sampling, simplicity to use, lower cost, and smaller amount of plant material used (Qian et al., 2000). 1 Materials and Methods 1.1 Materials Leaves of Thalictrum petaloideum L. collected from 246 individuals of 11 populations and dried by silica gel were stored at 20 (Table 1). Individuals of nine populations that covered most of northern distribution area of T. petaloideum were involved and two populations from the drainage basin of Yangtz River were also carefully gathered for the study. 1.2 Methods DNA isolation Total genomic DNA of a single leaf was extracted from silica-gel-dried leaves with a modified CTAB method (Zou et al., 2001) RAPD-PCR amplification Twenty of 100 primers provided by Operon, Shanghai, which produced clear and polymorphic banding patterns among different populations in the prior screening were selected (A-15, B-5, D-20, G-8, G-10, G-19, L-18, S-18, T-6, V-6, Y-11, Y-14, Y-20, Z-17, Z-18, K-9, K-10, O-15, P-2, R-1). PCR reactions were carried out in a volume of 10 µl containing 10 ng template DNA, 50 mmol/l Tris-HCL ph 8.3, 500 µg/ml BSA 10% Ficoll, 1 mmol/l Tartrazine, 2 mmol/l MgCl µmol/l dntp, 1 µmol/l primer and 0.5 U Taq polymerase. DNA amplification was performed in a Rapidcycler 1818 (Idaho Tech.), programmed for an initial s, s, s for 2 cycles, followed by 40 cycles of 94 0 s, 36 0 s, and s, and ended with 72 7 min. Amplification products were analyzed by electrophoresis on 1.5% agarose gel stained with ethidium bromide, and photographed under ultraviolet light. Molecular weights were estimated using a 100 bp DNA ladder Data analysis Amplified fragments were scored as binominal data, i.e., the presence as 1 and absence as 0 for the homologous bands. The matrix of RAPD phenotype was analyzed based on several indices of population genetics, such as observed number of alleles (na), effective number of alleles (ne), Nei s gene diversity (h), shannon s information index (I) and G ST through POPGENE program (Yeh et al., 1997). DCFA1.0 (Zhang, 2001) program was also used to calculate the similarity coefficients for all pairs of samples. We then analyzed the distribution of genetic variance by Analysis of Molecular Variance AMOVA Excoffier 1993 for combining the similarity coefficients. Two dendrograms using unweighted pair group method (UPGMA) (Sneath and Sokal, 1973) were constructed for estimating the genetic similarity based on Nei s coefficients and average taxonomic distance of band frequency. NTSYSpc2.02a software Rohlf 1997 was used to calculate the correlation between genetic distance and geographic distance among populations. 2 Results 2.1 Level of polymorphism Twenty decamer primers produced 125 reproducible and clear amplification bands, which were employed for analyzing (Fig. 2). We got 245 RAPD phenotypes from 246 individuals of T. petaloideum. The percentage of polymorphic bands (PPB) at species level was 96.00% (Table 2). 2.2 Population genetic structure Four indices of population genetics: observed number of alleles (na), effective number of alleles (ne), Nei s gene diversity (h), shannon s information index (I), were shown Table 1 Origin and sampling size of Thalictrum petaloideum used in this study Population Origin Date Voucher Habitat Altitude Sample m size YKS Yakeshi, Nei Mongol Jun., 2002 XL Mountaintop; near cropland QS Qianshan, Liaoning Jul., 2002 XL Sunny mountaintop BHS Baihuashan, Beijing May, 2002 XL Meadow of the mountaintop X W Xiaowutaishan, Hebei Jun., 2002 XL Sunny and dry place W T Wutaishan, Shanxi Jun., 2002 W Sunny slope of the mountain ZX Zheyangshan, Gansu Jul., 2002 XL Sunny and dry meadow TS Tianshui, Gansu Jul., 2002 XL Sunny and dry XJ Tianshan, Xinjiang Jun., 2002 XL Mountain slope; meadow BQ Baoku, Qinghai Sept.2002 XL Sunny mountain slope HB Zaoyang, Hubei Jun., 2002 XL Forest shade LY Langyashan, Anhui May,2002 XL Forest shade

3 XIE Lei et al.: Assessment of Genetic Variation and Distribution Pattern of Thalictrum petaloideum Detected by RAPDs Table 2 Percentage of polymorphic bands (PPB) in different populations of Thalictrum petaloideum Population Sample size Number of polymorphic bands PPB (%) YKS QS BHS X W W T ZX TS BQ XJ HB LY Average Species Abbrevations are the same as in Table 1. Fig.2. Amplified RAPD electrophorestic pattern of Thalictrum petaloideum. a. Primer p178. b. Primer p90. c. Primer p291. in Table 3. G ST computed by POPGENE is , and after excluding the two Yangtz River populations, G ST is Results of AMOVA analyses Results of AMOVA analyses indicated that majority of variation are distributed within populations (Table 4). 2.4 UPGMA results Two UPGMA dendrograms, both illustrating the genetic relationships among populations, were identical in topology (Figs.3, 4). We used NTSYSpc2.02a software Rohlf 1997 to test the correlation between genetic distance and geographic distance among populations by its MXCOMP function. The correlation between two similarity matrices showed a positive correlation with r = with high Table 3 Indices of population genetics in Thalictrum petaloideum Population Population size Observed number of Effective number of Nei s gene Shannon s information alleles (na) alleles (ne) diversity (h) index (I) YKS QS BHS X W W T ZX TS XJ BQ HB LY Average Variance Species Abbreviations are the same as in Table 1. Table 4 Results of Analysis of Molecular Variance (AMOVA) analysis in Thalictrum petaloideum Source of variation Variance expectation Percentage P value Among populations % <0.01 Within population % <0.01 Significance tests were performed using permutations.

4 Fig.3. Diagram of UPGMA dendrograms made by average taxonomic distance of band frequency illustrating the genetic relationships among populations in Thalictrum petaloideum. Abbreviatioins are the same as in Table 1. Fig.4. Diagram of UPGMA dendrograms made by Nei s coefficients illustrating genetic relationships among populations in Thalictrum petaloideum. Abbreviations are the same as in Table 1. statistic significance (P = ). 3 Discussion 3.1 Genetic diversity and population structure We examined the level of genetic polymorphism in T. petaloideum by RAPD analysis. At species level, the percentage of polymorphic bands (PPB) were 96%, it was higher than that of other species reported before in the Ranunculaceae (Markus et al., 2000; Cole and Kuchenreuther 2001). Shannon s information index (I) showed the same result, and its value of was much higher than the other results of population-level studies. Nei s gene diversity h gotten in our research was According to Nybom and Bartish (2000), average value of dicotyledons was It also indicated that high level of genetic polymorphism indwelled this species. This may attribute to its outcrossing reproductive behavior and lacking of clonal vegetation. The G ST value of the species was , meaning that per cent of the total variation was among populations. This value was rather high (Hamrick and Godt, 1990). It indicated that distinctive divergence happened among populations. Theoretically, higher G ST values would exist in annual, incrossing and spreading seeds by weight, herbaceous species than that in perennial, outcrossing and spreading seeds by wind species. As for T. petaloideum, this result seemed to be conflicting and its story must be different. It was perennial herb, but the life span of individuals was relatively short. Moreover, although it was a widely distributed species in northern China, the distribution area was rather discontinuous. Based on our fieldwork, individuals of T. petaloideum distributed in northern area were restricted in higher altitude, sunny and dry places. So discontinuity of environment resulted in isolating of populations. Furthermore, distance of the populations, as represented by our sampling, was long enough to avoid intercommunication of gene among populations. These were the main causes of the high value of the G ST. AMOVA analyses also showed much inter-population variation. From what was discussed above we could find that T. petaloideum was a species that possessed abundant variation and is under the process to form new subspecies. 3.2 Geographical inferences Two UPGMA dendrograms showed identical topology. Two populations from the drainage basin of Yangtz River were clustered together and separated from the other nine populations from the root. The other nine northern populations formed two clades, which represented the plants in eastern and western areas respectively. We tested the correlation between genetic and geographic distances ( r = ). It suggested that those two factors were positively related although the correlation was weak. This was because the two Yangtz River populations had greater genetic distance and smaller geographic distance from the other nine populations. When these two populations were removed, the value from the rest nine populations was rather high ( ). From these results, we knew that apart from Yangtz River populations, distinctive relationship between the genetic distance and the geographical distance could be found. Whereas, the two Yangtz River populations exhibited high genetic similarity but they were distinctive from the northern populations. Although to draw a conclusion might not be safe on how these two populations had Zhang F-M A preliminary study on speciation of Aconitum delavayvi complex (Ranunculaceae) in Hengduan Mountains. PhD thesis.

5 XIE Lei et al.: Assessment of Genetic Variation and Distribution Pattern of Thalictrum petaloideum Detected by RAPDs proceeded a evolutionary process in developing and diversifying from the rest northern populations yet from the RAPD data, it was very clear that these two populations were very special, and these two ones must be closely related in history, despite the fact that they now share quite weak link to the rest populations through gene communication. Acknowledgements: Thanks go to Dr. LIU Jian-Quan, Dr. WANG Ying-Wei and Mr. MAO Jian-Feng for sampling from Qinghai, Shanxi and Xinjiang. We would also like to thank Prof. GE Song for his excellent comments on the manuscript. We were indebted to Dr. ZHANG Fu-Min for his kindly instruction on data processing. References: Cole C T, Kuchenreuther M A Molecular markers reveal little genetic differentiation among Aconitum noveboracense and A. columbianum (Ranunculaceae) populations. Am J Bot, 88: Excoffier L Analysis of molecular variance (AMOVA) version Genetics and Biometry Laboratory, University of Geneva, The Switzerland. Ge S Review and prospect of the study on plant population genetic structure. Li C-S. Advances in Plant Science. Vol. 1. Beijing: Higher Education Press (in Chinese) Hamrick J L, Godt M J W Allozyme diversity in plant species. Brown A H D, Clegg M T. Plant Population Genetics, Breeding and Genetic Resources. Sunderland: Sinauer Hamrick J L, Loveless M D Associations between the breeding system and the genetic structure of tropical tree populations. Bock J, Linhart Y. Evolutionary Ecology of Plants. Boulder: Westview Press Markus F, RenéH, Daniel P, Markus P, Mark van K, Bernhard S RAPD variation among and within small and large populations of the rare clonal plant Ranunculus reptans (Ranunculaceae). Am J Bot, 87: Nybom H, Bartish I V Effect of life history traits and sampling strategies on genetic diversity estimates obtained with RAPD markers in plants. Perspect Plant Ecol Evol Syst, 3: Qian W, Ge S, Hong D-Y A study of genetic diversity of wild rice Oryza granulata from China using RAPD and ISSR markers. Acta Bot Sin, (in Chinese with English abstract) Rohlf F J NTSYS: numerical taxonomy and multivariate analysis system, version 2.02a. Exeter Software. Setauket, New York, USA. Schaal B A, Leverich W J Plant population biology and systematics. Taxon, 50: Sneath P H A, Sokal R R Numerical Taxonomy. San Francisco: Freeman. Wang W-T Notulae de Ranunculaceis Sinensibus ( ). Acta Bot Yunnan, 6: (in Chinese with English abstract) Williams J G K, Kubelik A R, Livak K J, Raflski J A, Tingey S V DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res, 18: Wu Z Y, Raven P H Flora of China. Tomus 6. Beijing: Science Press Yeh F C, Yang R C, Boyle T B J, Ye Z H, Mao J X POGENE, the user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Edmonton, Alberta, Canada. Zhang F-M DCFA 1.0, a program companied with AMOVA to compute the matrix of distance. Laboratory of Systematics and Evolutionary Botany, Institute of Botany, The Chinese Academy of Sciences, Beijing. (in Chinese) Zou Y-P, Ge S, Wang X-D Molecular markers used in systematics and development botany. Beijing: Science Press , (in Chinese) (Managing editor: ZHAO Li-Hui)