Published online 30 July 2007
Published in Crop Sci 47:1737-1743 (2007)
© 2007 Crop Science Society of America
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PLANT GENETIC RESOURCES
Genetic Diversity and Population Structure of Caragana korshinskii Revealed by AFLP
Z. Wanga,
H. W. Gaoa,*,
Y. Q. Wub and
J. G. Hanc
a Institute of Animal Science, Chinese Academy of Agricultural Science, China
b Dep. of Plant and Soil Science, Oklahoma State Univ., Stillwater, OK
c Institute of Grassland Science, China Agricultural University, China. This study was supported by the Hi-Tech Research and Development Program (No. 2002AA241091) from the Ministry of Science and Technology of China
* Corresponding author (gaohongwen{at}263.net).
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ABSTRACT
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Caragana korshinskii Kom. is a perennial sandy grassland and desert deciduous shrub species, indigenous to and distributed in the northwest of China and Mongolia, and important in vegetation rehabilitation of widely degraded and degrading semiarid and arid regions because of its high ecological and economic values. To assess the genetic diversity and population structure of C. korshinskii, 10 natural populations from the Loess plateau and Inner Mongolia plateau in China were analyzed for amplified fragment length polymorphisms. Four primer combinations produced 358 bands across a total of 98 individuals. A high percentage of polymorphic loci was observed at species level (p = 93.9%). Multivariate analyses separated accessions into two regional groups corresponding to the Loess and Inner Mongolia plateaus. Based on analysis of molecular variance, 77.8% of the genetic variation of C. korshinskii was within population, 7% difference between regions, and 15.2% among collection sites within regions. An indirect estimate of the number of migrants per generation (Nm = 1.7) indicated that gene flow was high among populations of the species. A significant correlation (r = 0.58) between genetic and geographic distance was detected. Results of this study suggested that C. korshinskii has a high genetic variability and potential as a source of variation for breeding programs.
Abbreviations: AFLP, amplified fragment length polymorphism • AMOVA, analysis of molecular variance • ISSR, inter–simple sequence repeat • ITS, internal transcribed spacer • RAPD, random amplified polymorphic DNA
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INTRODUCTION
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CARAGANA BELONG to the Papilionoideae (Leguminosae) and comprise about 70 species (Polhill, 1981), mainly distributed from Northern Europe and central Asian, east toward Russian Siberia, Korea, and Japan, and southward to Nepal, Bhutan, Sikkim, and northern Indian (Fu, 1989). Species of this genus have distinctive morphological variations and ecological adaptations, especially to drought and cold.
Korshinsk peashrub (C. korshinskii Kom.), a perennial sandy grassland and desert deciduous shrub species, is indigenous to and distributed in half-fixed and fixed sandy regions in the northwest of China and Mongolia (Fu, 1989). Caragana korshinskii is a diploid (2n = 2x = 16) and is an allogamous insect-pollinated species (Xu and Hao, 1989). Generally, C. korshinskii turns green in April, blooms in May, and sets seed and seneces in July. This species is prized for its drought resistance and has a valuable role in vegetation succession from shifting dunes to sandy grasslands, in restoring degraded land by fixing atmospheric nitrogen, and in forming shrub belts for crops or artificial grassland (Zhang, 1994). In the northwest of China, it is also used as a critical livestock forage for sheep, goat, deer, and camel. Especially during wintertime, over 20% of shoots of korshinsk peashrub still can be used as fodder (Fu, 1989). Caragana korshinskii is also used as green fertilizer, fuel, and honey and wood-based panel production, as well (Li et al., 2000; Wang and Gao, 2003).
Pod and seed traits are the most variable characteristics within and among C. korshinskii and related species (Wang et al., 1994a; Wang et al., 2005b). Geographical and ecologica1 factors have a remarkable influence on its phenotypic traits (Song et al., 2005; Wang et al., 2005b). Wang et al. (1994b) has used total seed proteins for the study of genetic structure of Caragana populations and showed that more than 90% of the gene diversity existed within populations, indicating a high level of gene flow (Zhou et al., 2001). Allozyme diversity of C. korshinskii was assessed by Wang et al. (2006), demonstrating high genetic variation at both species and population levels. Approximately 84.8% of the total genetic variation occurred within populations. Wei et al. (1999) have reported that 82.4% of the molecular variation existed within the Caragana populations as revealed by random amplified polymorphic DNA (RAPD) analysis. Liu et al. (2005) used inter–simple sequence repeat (ISSR) markers to detect genetic variation of C. polourensis populations (79.07%) and C. turfanensis (41.86%) from Xinjiang of China. Yang et al. (2006) have reported a high level of gene flow (Nm = 1.9936) in C. davazamcii. Hou et al. (2006) has examined the interspecific relationship of C. korshinskii, C.microphylla, and C. davazamcii based on internal transcribed spacer (ITS) and noncoding region of chloroplast trnL-F, combined with morphological data and geographical distribution. The trnL-F sequence of C. davazamcii is identical to that of C. microphylla but differs distinctly from that of C. korshinskii. The high congruence of ITS copies through both direct and cloning sequencing rejects the recent hybridization hypothesis of C. davazamcii between C. microphylla and C. korshinskii.
The exploration of plant genetic resources and the design of plant improvement programs require a detailed knowledge of the amount and distribution of genetic diversity within species. Molecular markers can provide a relatively unbiased method of quantifying such genetic diversity. Dominant DNA marker amplified fragment length polymorphisms (AFLPs) (Vos et al., 1995) offer a lot of advantages, such as low quantities of template DNA required, no need of sequence data for primer designs, random distribution throughout the genome, and stability and reproducibility (Vos et al., 1995). The AFLP technique (Vos et al., 1995) has been proved to be effective in the analyses of genetic variation below the species level, particularly in investigations of population structure and the differentiation of subpopulations (Karola and Jensen, 2000; Man and Ohnish, 2002; Larson et al., 2004). In the present study, we used AFLPs (i) to quantify genetic diversity within and among collection sites, (ii) to quantify genetic diversity within and among geographical regions (Loess plateau versus Inner Mongolia plateau), and (iii) to estimate number of migrants per generations between sites and regions.
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MATERIALS AND METHODS
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Plant Materials and DNA Extraction
Ten korshinsk peashrub populations were collected from seven sites on the Loess plateau (LP-PG, LP-HQ1, LP-HQ2, LP-SM, LP-YL, LP-JB, and LP-DB) and three sites on the Inner Mongolia plateau (IMP-ETK, IMP-HJ, and IMP-DS) in the summer of 2003. These collection sites included nine counties and three provinces of China, which covered almost all the geographical range of C. korshinskii (Fig. 1
, Table 1). About 100 seeds were collected from each of 8 to 10 individuals (mother plants) at each collection site. A total of 98 individuals were used in the study. Five to 10 randomly selected seeds from each individual were sown into greenhouse pots, and leaves of 3-wk seedlings grown from them were harvested as one sample and stored at –70°C until used in DNA preparation. Total genomic DNA was isolated following a modification of the SDS "miniextraction" protocol developed by Edwards et al. (1991), and involving an additional phenol-chloroform extraction, alcohol precipitations, and acetate salt rinse. The DNA sample was resuspended in 1x TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and diluted to 200 ng/µL and stored at 4°C or –20°C for future use.
AFLP Analysis
AFLP analysis was performed according to Vos et al. (1995) with some modifications (Wang et al., 2005a). The extracted genomic DNA was digested with 0.15 µL EcoRI (20 U/µL) and 0.3 µL MseI (10 U/µL) restriction enzymes and ligated to 50 pmol/µL EcoRI and 50 pmol/µL MseI adaptors. The EcoRI adaptor consisted of the combination of two primers: 5'-CTCGTAGACTGCGTACC-3' and 3'-CATCTGACGCATGGTTAA-5'. Similarly, the MseI adaptor contained two primers: 5'-GACGATGAGTCCTGAG-3' and 3'-TACTCAGGACTCAT-5'. The procedures of digestion and ligation were performed together for 12 h at 37°C.
The digested and ligated template was preamplified using EcoRI + 1(5'-GACTGCGTACCAATTCA-3') and MseI + 1(5'-GATGAGTCCTGAGTAAC-3') primers. A total volume of 20 µL reaction mixture containing 2 µL of the digestion/ligation mixture, 0.6 µL each of the preamplification primers (50 ng/µL), 2 µL 10x PCR buffer, 1.2 µL Mg2+ (25 mM), 0.16 µL dNTPs (25 mM), 0.12 µL Taq polymerase (5 U/µL) was prepared and subjected to 5 min at 94°C; 30 cycles of 35 s at 94°C, 35 s at 56°C, 60 s at 72°C; 5 min at 72°C.The products of preamplification was checked in a 1% agarose gel and diluted 15 times in 1x TE buffer.
Four pairs of selective AFLP primers were used for selective amplification. Selective amplification reaction mixture (20 µL) was prepared with 2 µL diluted preamplification products, 0.8 µL each EcoRI and MseI selective primer (50 ng/µL), 2µL 10x PCR buffer, 1.2 µL Mg2+ (25 mM), 0.18 µL dNTPs (25 mM), 0.2 µL Taq polymerase (5 U/µL). This step was performed for 5 min at 95°C; 13 touchdown cycles of 35 s at 94°C, 35 s at 65°C (–0.7°C per cycle), 60 s at 72°C; 35 cycles of 35 s at 94°C, 35 s at 56°C, 60 s at 72°C. Amplified products were mixed with 7.5 µL of formamide loading buffer (98% formamide, 10 mM EDTA, 0.1% bromophonol blue, 0.1% xylene cyanol), denatured at 95°C for 5 min, and resolved on 6% denaturing polyacrylamide gels (acrylamide-bis-acrylamide [20:1], 7.5 M urea–1x TBE buffer) at a constant 2400 V, until the forward-running dye (bromophenol blue) reached the end of the gel. Gel was stained with silver following the method of Van Toai et al. (1996) with some modifications (Wang et al., 2005a). A PBR322/HindIII DNA ladder was used to determine the size of the AFLP fragments.
Data Analysis
Polymorphic DNA bands were scored as present (1), absent (0) for each DNA sample, excluding the smeared and weak ones, by visual inspection. The percentage of polymorphic loci (P), and Shannon's information index of diversity (I) were analyzed using POPGENE (Yeh et al., 1999), assuming Hardy–Weinberg equilibrium. The amount of gene flow among these populations was estimated as Nm = (1/GST – 1)/4.
The analysis of molecular variance (AMOVA) was conducted to calculate variance components and their significance levels for variation between plants from the Inner Mongolia plateau and the Loess plateau, among populations within a region, and within populations using AMOVA version 1.55 (Excoffier et al., 1992). The input files for AMOVA were prepared by the aid of DCFA 1.1 program written by Zhang (2001).
Nei's genetic distances (D) were calculated for each pairwise combination among populations (Nei, 1978). Cluster analysis using the Neighbor–Joining method was performed using NTSYS-pc, version 2.0 (Rohlf, 1997). A principle coordinate analysis was also performed for display the population differentiation using the NTSYS-pc software (Rohlf, 1997). To test for a correlation between genetic distances and geographical distances among populations, a Mantel test was performed (Smouse et al., 1986).
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RESULTS
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AFLP Profile
Among the 98 individuals, AFLP analysis with four primer combinations generated a total of 358 scorable DNA bands and 336 (93.9%) polymorphic bands (Table 2). The number of bands per primer combination produced ranged from 72 to 116, with an average of 89.5 (Table 2). Each of the 98 individuals presented a unique AFLP genotype, indicating that no two wild plants were genetically identical. The AFLP fragments scored across individuals ranged from 50 to 800 bp, with most fragments in the 200- to 600-bp range.
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Table 2. Numbers of amplified fragment length polymorphisms (AFLPs) generated with four selective AFLP primer pairs of Caragana korshinskii.
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Genetic Diversity
The percentage of polymorphic loci (P) within populations ranged from 62% (LP-DB) to 80.5% (IMP-DS), with an average of 69.5% (Table 1). The Shannon's diversity index (I) ranged from 0.284 (LP-DB) to 0.426 (IMP-DS), with an average of 0.365 at the population level and 0.477 at the species level (Table 1). In particular, when populations were grouped according to geographic region, genetic diversity of the Loess plateau group (P = 83.8%, I = 0.441) was lower than that of the Inner Mongolia plateau group (P = 92.5%, I = 0.468), as revealed by both the percentage of polymorphic loci and the Shannon's diversity index (Table 1). A significant correlation was found between intrapopulation genetic diversity of korshinsk peashrub with latitude (r = 0.785, p < 0.01 for I; r = 0.656, p < 0.05 for P, data not show).
Population Structure
Hierarchical AMOVA analysis indicated that most of the variation was accounted for by differentiation among individuals within populations (77.8%), with the least differentiation found between Loess plateau and Inner Mongolia plateau (7%), and the remainder (15.2%) partitioned among populations within regions (Table 3). There were significant differences between populations from the Loess plateau and the Inner Mongolia plateau (p < 0.001), and among all 10 populations (p < 0.001), and within populations (p < 0.001). Based on GST values, the estimated number of migrants per generation (Nm) between populations in the Loess plateau and Inner Mongolia plateaus was 1.84 and 3.65, respectively, with a level of 1.7 at the species level (Table 1). Nei's genetic distance is shown in Table 4. The value of the gene distance between populations ranged from 0.028 to 0.132 with a mean of 0.094.
Cluster Analysis
The Neighbor–Joining cluster analysis (Fig. 2
) and principle coordinate analysis (Fig. 3
) separated populations into two clusters: Loess plateau and Inner Mongolia plateau (Fig. 2). The Mantel test revealed that there was a statistically significant correlation between pairwise genetic distance and corresponding geographic distance among all 10 populations (r = 0.58, p < 0.05), and 33.6% of the genetic variation was explained by geographic distance.
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Figure 3. Dispersion graph from principal coordinate (PC) analysis of amplified fragment length polymorphism data of 98 individuals of Caragana korshinskii. 1, LP-PG; 2, LP-HQ1; 3, LP-HQ2; 4, LP-SM; 5, LP-YL; 6, LP-JB; 7, LP-DB; 8, IMP-ETK; 9, IMP-HJ; and 10, IMP-DS.
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DISCUSSION
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In the present study, high genetic diversity was observed at species level (P = 93.9%, I = 0.477) and the population level (P = 69.5%, I = 0.365), a result consistent with its morphological and allozyme diversity (Wang et al., 2005a, 2006). When the genetic diversity of korshinsk peashrub was compared to that of other Caragana species, it was slightly lower than that of the C. davazamcii (P = 98.01%) reported by Yang et al. (2006) and was higher than that of the C. polourensis (P = 79.07%) and C. turf (P = 41.86%) reported by Liu et al. (2005), although the use of different methods (e.g., the number of loci, populations sampled, and the molecular markers studied) may preclude meaningful direct comparisons. On the basis of its life history characteristics, korshinsk peashrub is a long-lived, out-crossing, insect-pollinated species (Xu and Hao, 1989), and the combination of these life history traits enable the species to maintain a high level of genetic diversity (Hamrick and Godt, 1989; Hamrick et al., 1992).
Fahima et al. (2002) reported that there were some correlations between genetic diversity with ecogeographic variation. In this study, we found a significant correlation between intrapopulation genetic diversity of korshinsk peashrub with latitude. Of course, the limited number of sampling sites and other ecological factors may influence the results. Many studies have also shown strong relationships between levels of genetic polymorphism and degree of environmental stress (Hedric, 1986; New, 2001). Yang et al. (2006) have reported Shannon's information index of C. davazamcii increased along with changes of the habitats from east to west and with lack of average annual rainfall; genetic diversity increased as well. The arid environment of korshinsk peashrub is characterized by low precipitation and high annual precipitation fluctuations (Geng, 1986). Accordingly, we hypothesize that such a stressful environment may lead to the maintaining of high levels of intrapopulation genetic variation. Of course, we had to admit the impact of aerial seeding, which has been employed since the 1970s in China. This movement must have contributed to the high gene flow among the Chinese korshinsk peashrub populations, although we have no ability to estimate its magnitude.
Based on AMOVA analysis, 77.8% of genetic variation of C. korshinskii was within population. Similar results were obtained for C. davazamcii (79.95%) (Yang et al., 2006). An ISSR analysis revealed approximately 91.7% genetic variation for C. polourensis among populations (Liu et al., 2005).
The population genetic structure of a species is affected by a number of evolutionary factors including mating system, gene flow, seed dispersal, and mode of reproduction, as well as natural selection (Hamrick and Godt, 1989). Moreover, reproductive biology is the most important factor in determining the genetic structure of plant populations. Nybom and Bartish (2000), who estimated the genetic diversity using RAPD markers, compiled mean GST values of 0.59, 0.19, and 0.23 for selfing, mixed mating, and out-crossing plant species, respectively. Compared with these values, the populations of korshinsk peashrub are close to that with an out-crossing breeding system.
In addition, the high intrapopulation variability and genetic homogeneity across populations could also have arisen by high levels of gene flow. A migration rate of 0.5 was considered sufficient to overcome the diversifying effects of random drift (Ellstrand and Elam, 1993). In this study, the estimated gene flow of korshinsk peashrub (Nm = 1.70) was higher than the average value reported for out-crossed animal-pollinated species (Nm = 1.154) and higher than that of mixed-mating species (Nm = 0.727) (Hamrick and Godt, 1989).
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ACKNOWLEDGMENTS
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The authors gratefully thank Profs. TieLiang ShangGuan, Jun Chen, DaWei Zhang, XiaoHai Shu, and JingXun Qin for help locating and sampling populations; Drs. Yongliang Zhao and Lifang Zhang for help in the lab; and Dr. Fuming Zhang and Mr. Xi Yang for data analysis.
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NOTES
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All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication September 5, 2006.
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ABSTRACT
INTRODUCTION
