C 2006) Biochemical Genetics, Vol. 44, Nos. 1/2, February 2006 ( DOI: 10.1007/s10528-006-9011-8
A Study of Conservation Genetics in Cupressus chengiana, an Endangered Endemic of China, Using ISSR Markers Bingqing Hao,1,2 Wang Li,2,3 Mu Linchun,2 Yao Li,2 Zhang Rui,2 Tang Mingxia,2 and Bao Weikai1 Received 25 January 2005—Final 27 July 2005 Published online: 3 May 2006
ISSR markers were used to analyze the genetic diversity and genetic structure of eight natural populations of Cupressus chengiana in China. ISSR analysis using 10 primers was carried out on 92 different samples. At the species level, 136 polymorphic loci were detected. The percentage of polymorphic bands (PPB) was 99%. Genetic diversity (He ) was 0.3120, effective number of alleles (Ae ) was 1.5236, and Shannon’s information index (I) was 0.4740. At the population level, PPB = 48%, Ae = 1.2774, He = 0.1631, and I = 0.2452. Genetic differentiation (Gst ) detected by Nei’s genetic diversity analysis suggested 48% occurred among populations. The partitioning of molecular variance by AMOVA analysis indicated significant genetic differentiation within populations (54%) and among populations (46%; P < 0.0003). The average number of individuals exchanged between populations per generation (Nm ) was 0.5436. Samples from the same population clustered in the same population-specific cluster, and two groups of Sichuan and Gansu populations were distinguishable. A significantly positive correlation between genetic and geographic distance was detected (r = 0.6701). Human impacts were considered one of the main factors to cause the rarity of C. chengiana, and conservation strategies are suggested based on the genetic characters and field investigation, e.g., protection of wild populations, reestablishment of germplasm bank, and reintroduction of more genetic diversity. KEY WORDS: Cupressus chengiana; ISSR; genetic diversity; genetic structure; differentiation.
1 Chengdu
Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, P. R. China. of Life Sciences, Sichuan University, Chengdu 610064, P. R. China. 3 To whom correspondence should be addressed; e-mail:
[email protected]. 2 School
31 C 2006 Springer Science+Business Media, Inc. 0006-2928/06/0200-0031/1
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Hao, Wang, Mu, Yao, Zhang, Tang, and Bao
INTRODUCTION As described by the Chinese common name “Minjiang bai mu” Cupressus chengiana S. Y. Hu, a long-lived and monoecious conifer of the Cupressaceae, was found originally in the Minjiang basin of Sichuan Province in China. Being an endemic species, it is restricted to arid-dry valleys of the upper reaches of the three rivers of Minjiang and Daduhe in Sichuan Province and Bailongjiang in Gansu Province, all of which have the characteristics of severe drought, an essentially rainless state (500–700 mm), high evaporation (1400–2000 mm), and windiness (Liu et al., 2001). C. chengiana shows significant tolerance for less fertile soils, for dry and cold habitats, and can even grow on rocky cliffs and hillsides. It is a priority for forestation and soil and water preservation in these dry valleys. On the other hand, it has extensive histories of use in furniture making, temple construction, and boat building because of its fine timber and special aroma. Most of the natural populations of C. chengiana are distributed at an elevation of 1100– 2600 m, close to villages, so that they usually suffer from human impacts such as logging and grazing. Today, C. chengiana has declined sharply in its distribution areas and populations because of demographic and environmental stochasticity. It has been listed as a second-class protection plant in the Red Book of China: Rare and Threatened Plants (Fu, 1992). Therefore, it is necessary to protect this endangered plant for its high economic and ecological values, and for the conservation of biodiversity. Genetic diversity plays an important role in the conservation of rare and endangered species. Researchers generally consider that loss of genetic variation is usually accompanied by reduction of the species’ ability to cope with changes in the environment, which causes the species to become rare (Frankham et al., 2002; Ge et al., 1998; Hogbin et al., 2000; Maki and Horie, 1999). The genus Cupressus consists of about 20 species and varieties distributed discontinuously in North America, eastern Asia, and the Mediterranean (Zheng and Fu, 1978), and most of them are rare and endangered. Only a few researchers have examined the conservation genetics of this genus using isozyme or RAPD markers (Bartel et al., 2003; Rushforth et al., 2003; Sabrina and Sabri, 1999). In studies of C. chengiana, information about its genetic diversity and the genetic structure of populations remains cryptic, except for some reports about its taxonomic status, chemical components, and karyomorphology (Rushforth et al., 2003; Laurence et al., 1998; Li and Fu, 1996; Shi and Wang, 1994). Consequently, the comprehensive analysis of genetic variation within and among populations of C. chengiana will be the key to reveal the cause of rarity and to adopt suitable conservation recommendations. Recently, molecular markers have been frequently applied to ecological genetic and conservation genetic studies (Kjolner et al., 2004; Schonswetter et al., 2004). ISSR (intersimple sequence repeat; Weising et al., 1995; Zietkiewicz et al., 1994) is a modification of the SSR approach (simple sequence repeat) that uses
Conservation Genetics in Cupressus chengiana
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a single primer based on SSR (microsatellites) that are common in the genome. Compared with the RAPD method (random amplified polymorphic DNA), the longer primer (16–20 bp) can precisely target the template DNA and improve reliability and reproducibility (Weising et al., 1995). In addition, the ISSR approach does not need highly purified DNA, experienced technique, and significant expense in comparison with AFLP (amplified fragment length polymorphism). Thus, ISSR is widely used to reveal the genetic variation of plants (Fern´andez et al., 2002; Hodkinson et al., 2002; Mattioni et al., 2002). In this study, ISSR analysis was employed to investigate the genetic diversity of C. chengiana, which was sampled throughout the current geographical range, with the following objectives: (1) to assess the extent of genetic variation and structure of eight natural populations; (2) to clarify the relationships among the populations and subpopulations by statistical and cluster analysis; (3) to discuss the conservation implications based on the genetic characteristics of C. chengiana. MATERIALS AND METHODS Sample Collection For this study, 92 samples from eight natural populations of C. chengiana were collected from different geographic sites along the uprivers of Minjiang and Daduhe in Sichuan Province and Bailongjiang in Gansu Province (Table I, Fig. 1). Fresh leaf samples were collected and preserved in silica gel until required for DNA isolation. From each population, 10–14 individual samples were used for the ISSR analysis. Herbarium voucher specimens from each population are housed at the Herbarium of Botany (SZ), Sichuan University, and researchers can access the vouchers by the herbarium numbers 0305001-0305008.
Table I. Description of Eight Natural Populations of C. chengiana Population
Location
Sample size
Latitude (N)
BW
Baiwan of maerkang, Sichuan Kangding, Sichuan Rezu of maerkang, Sichuan Xiaojin, Sichuan Jinchuan, Sichuan Mao, Sichuan Wen, Gansu Zhouqu, Gansu
14
31◦ 54
102◦ 02
2400
11 11
30◦ 34
102◦ 00 101◦ 90
1700 2600
12 10 11 13 10
31◦ 03 31◦ 46 31◦ 67 32◦ 95 33◦ 81
102◦ 24 101◦ 98 103◦ 89 104◦ 70 104◦ 38
2420 2280 1690 1100 1850
KD RZ XJ JC MO WE ZQ
31◦ 63
Longitude (E) Altitude (m)
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Hao, Wang, Mu, Yao, Zhang, Tang, and Bao
Fig. 1. Distribution of eight natural populations of Cupressus chengiana in China.
DNA Extraction DNA was extracted from the young leaves using the CTAB method with some modifications (Zou et al., 2001). Desiccated leaf tissues were ground in liquid nitrogen, transferred to a 1.5-mL tube holding 800 µL of preheated 2 × CTAB extraction buffer containing 1% 2-mercaptoethanol and incubated at 65◦ C for 60 min. Subsequently, samples were extracted with equal volumes of chloroform/isoamylalcohol (24:1, v/v) twice, and the aqueous phase was mixed with two third volume of chilled isopropanol. Precipitated DNA was collected by centrifugation and washed with 70% ethanol. After air-drying and resuspension in 1 mL of sterile distilled water, it was treated with RNase (1 mg/mL) for 30 min at 37◦ C and then purified with equilibrated phenol and 750 mL of chloroform/isoamylalcohol (24:1, v/v). The purified DNA was reprecipitated from the aqueous phase using chilled ethanol, air-dried, and resuspended in sterile water. The quality and quantity of DNA were checked on 1.0% agarose gel electrophoresis with a 15,000 bp DNA Marker kit (Takara Biotech).
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Table II. Polymorphism of ISSR Bands Amplified by the 10 ISSR Primers UBC primer code 814 825 834 835 840 845 847 848 851 895 Average Total
Primer sequence CTC TCT CTC TCT CTC TA ACA CAC ACA CAC ACA CT AGAGAGAGA GAG AGA GYT AGAGAGAGA GAG AGA GYC GAG AGA GAG AGA GAG AYT CTC TCT CTC TCT CTC TRG CAC ACA CAC ACA CAC ARC CAC ACA CAC ACA CAC ARG GTG TGT GTG TGT GTG TYG AGA GTT GGT AGC TCT TGA TC
Number of bands scored 8 10 13 21 11 16 12 14 18 15 13.8 138
Note. Single-letter abbreviations for mixed base positions: N (A,G,C,T), R (A,G), Y (C,T), B (C,G,T; i.e., not A), D (A,G,T; i.e., not C), H (A,C,T; i.e., not G), V (A,C,G; i.e., not T), K (G,T; Keto in large groove), M (A,C; Amino in large groove), S (G,C; Strong, 3 H-bonds), W(A,T; Weak, 2 H-bonds).
ISSR Analysis Ten ISSR primers showing clear and reproducible band patterns were selected from 100 ISSR primers obtained from the University of British Columbia Biotechnology Laboratory (Table II). The PCR amplification was performed in a 20-µL reaction volume, containing 2 µL 10 × reaction buffer, 1.5 mM MgCl2 , 250 µM dNTPs, 0.8 µM primer, 10 ng DNA template, and 1 U Taq DNA polymerase (Takara Biotech). The mixture was overlaid with mineral oil and subjected to PCR on a PTC-100 Programmable Thermal Controller (MJ Research) programmed for an initial step of 5 min at 94◦ C, followed by 48 cycles of 30 s at 94◦ C, 45 s at 48◦ C, 2 min at 72◦ C, and a 7 min final extension step at 72◦ C. PCR products were analyzed on 2% agarose gels in 0.5 × TBE buffer. Gels were stained with ethidium bromide and visualized and photographed with ultraviolet light. Molecular weights were estimated using a 100 bp DNA ladder (Takara Biotech). Data Analysis Because of the dominance of ISSR markers, it is assured that each band represented the phenotype at a single biallelic locus. Amplified bands were scored as present (1) or absent (0). Popgene version 1.31 (Yeh et al., 1997), AMOVA (Excoffier et al., 1992), and NTSYSpc version 2.02c (Rohlf, 1998) were used to calculate parameters in genetic diversity as follows: (1) Np (number of polymorphic loci); (2) PPB; (3) Ao (observed number of alleles per locus) and Ae (effective number of alleles per locus); (4) He (Nei’s gene diversity) and I (Shannon’s information
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Hao, Wang, Mu, Yao, Zhang, Tang, and Bao
index); (5) Nei’s genetic distances (D) and genetic identity (IN); (6) cluster analysis with unweighted pair group with arithmetic average (UPGMA; Sun et al., 1998); (7) coefficient of gene differentiation among populations within species (Gst ; Nei, 1972); (8) gene flow (Nm ); (9) Mantel test between geographic and Nei’s genetic distance. RESULTS ISSR Polymorphism Amplified bands ranging from 100 to 2100 bp were scored as present (1) or absent (0). Statistical analysis was based on 138 amplified ISSR markers, and 13.8 bands were amplified on average by each primer (Table II). Of the 138 bands, 99% (136) were polymorphic, with an average genetic diversity of 0.3120. The average genetic diversity and percentage of polymorphic loci for each population are summarized in Table III. Some difference was observed among populations with regard to the genetic variation indices, such as Ao , He , and PPB. The WE population had the highest PPB at 64%, and JC had the lowest at 34%. The range of Ae was from 1.1883 to 1.3666, somewhat lower than the Ao . Shannon’s information index (I) ranged from 0.1705 to 0.3181, with the same trend line as PPB and He . The genetic variation indices at the species level were PPB = 99%, Ao = 1.9855, Ae = 1.5236, He = 0.3120, and I = 0.4740, much higher than the mean values of eight populations, especially the PPB (Table III). Based on the values of Nei’s total gene diversity (Ht = 0.3132) and Nei’s gene diversity within populations (Hs = 0.1631), Nei’s genetic differentiation (Gst ) was calculated to be 0.4791 using Popgene software; that is, 48% of gene Table III. Genetic Variation of Eight Populations of C. chengiana Population
Np
PPB (%)
Ao
Ae
He
I
BW KD RZ XJ JC MO WE ZQ Mean Species level SD
65 65 63 61 47 56 88 83 66 136
47.10 47.10 45.65 44.20 34.06 40.58 63.77 60.14 47.83 98.55
1.4710 1.4710 1.4565 1.4420 1.3406 1.4058 1.6377 1.6014 1.4783 1.9855 0.1199
1.2782 1.2846 1.2786 1.2600 1.1883 1.2335 1.3297 1.3666 1.2774 1.5236 0.3249
0.1626 0.1660 0.1622 0.1510 0.1125 0.1376 0.2001 0.2130 0.1631 0.3120 0.1522
0.2435 0.2479 0.2421 0.2263 0.1705 0.2071 0.3064 0.3181 0.2452 0.4740 0.1926
Note. Np : Number of polymorphic loci; PPB: Percentage of polymorphic loci; Ao : Observed number of alleles per locus; Ae : Effective number of alleles per locus; He : Nei’s gene diversity; I: Shannon’s information index.
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Table IV. Nei’s Analysis of Gene Differentiation Among Populations Nei’s total gene diversity (Ht ) Mean value of loci SD
0.3132 0.0229
Nei’s gene diversity Nei’s genetic in population (Hs ) differentiation (Gst ) 0.1631 0.0112
0.4791
Gene flow (Nm ) 0.5436
differentiation occurred among populations and 52% within populations, which showed a relatively higher level of genetic differentiation within populations. The average number of individuals exchanged between populations per generation (Nm ) was 0.5436 (Table IV). In addition, for the purpose of detecting the relationship among and within populations, molecular variance was examined using AMOVA based on ISSR banding patterns. The variance component found within populations was 54%, and 46% was found among populations (Table V). Nei’s Genetic Identity and Genetic Distances Nei’s genetic identity (IN) and genetic distance (D) matrices are shown in Table VI, based on analyzing the ISSR fragment patterns of all individuals in eight populations. The IN value varied from 0.6996 to 0.9307, and the pairwise genetic distance was between 0.0718 and 0.3573. Among the eight populations, the genetic identity between XJ and BW was the highest; that between ZQ and RZ was the lowest. Furthermore, relationships of all 92 individuals were revealed by cluster analysis based on Jaccard’s similarity coefficient using the UPGMA method (Fig. 2). All individuals of the same population were arranged in the same populationspecific cluster, except one individual of WE (shown by the arrow), and the two major groups were distinguishable. Six populations of Sichuan Province were clustered into one group (BW, XJ, RZ, KD, MO, and JC), and the WE and ZQ populations of Gansu Province clustered on the other branch. To clarify the relationships between populations further, the Mantel test was carried out between Table V. AMOVA Among and Within Populationsa
Variance among populations Variance within populations a Significance
df
Variance component
Percentage of variance component
p-value
7
11.4300
46.25
