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Genetic Diversity of Natural Myrica rubra Sieb.et Zucc Populations in Guangxi Revealed by ISSR Markers
Agricultural Sciences in China
May 2010
2010, 9(5): 626-632
Genetic Diversity of Natural Myrica rubra Sieb.et Zucc Populations in Guangxi Revealed by ISSR Markers HE Xin-hua1, PAN Hong1, DENG Li-bao1, PAN Jie-chun1, LI Feng1 and LI Yang-rui2 1 2
College of Agriculture, Guangxi University, Nanning 530004, P.R.China Guangxi Crop Genetic Improvement and Biotechnology Lab, Guangxi Academy of Agricultural Sciences, Nanning 530007, P.R.China
Abstract The present study was conducted to assess the molecular characterization and genetic diversity amongst natural populations of Myrica rubra in Guangxi Zhuang Autonomous Region, China, thus to provide scientific evidence for germplasm conservation and exploitation. Using ISSR (inter-simple sequence repeats) markers, the level of genetic variation and the molecular characterization of 10 natural populations of M. rubra, originated from Guangxi Zhuang Autonomous Region in China, were performed. Based on 11 primers, 123 clear and reproducible DNA fragments were generated, of which 95 (77.24%) were polymorphic. The average value of Nei’s gene diversity (He) was 0.268. The coefficient of genetic differentiation (Gst) was 0.341, revealing that 34.1% of the total molecular variance existed among populations. The Mantel statistical testing showed that the genetic distance was correlated to the geographic distance, but the correlation was not significant. Ten populations were divided into two big clusters according to unweighted pair group method with arithmetic mean (UPGMA) analysis. One consisted of populations of Rongxian (RX), Hepu (HP), Liangqing (LQ), Marshan (MS), Lingshan (LS) and Shansi (SS), which originated from the southern Guangxi, while the other was composed of Guanyang (GY) and Lingui (LG) populations of northern Guangxi, Huanjiang (HJ) populations of northwestern Guangxi and Shanglin (SL) populations of southern Guangxi. The level of genetic variation in wild M. rubra population distributed in Guangxi is high. Gene drift within the population was responsible for genetic variation in wild M. rubra in Guangxi, and the effect of the genetic flow among inter-populations was not significant. Classification of wild M. rubra populations was correlated to climate and environment. The molecular characterization and diversity assessment of M. rubra is of immense value for planning conservation of its genetic resources and their exploitation for further studies. Key words: Myrica rubra, population, genetic diversity, ISSR
INTRODUCTION Myrica rubra Sieb.et Zucc (also named as Chinese tree berry, red bayberry and yangmei) is an important subtropical fruit native to China. In China, M. rubra is distributed from eastern coastal area of Taiwan to Ruili County of Yunan Province in the west and from
Sanya City of Hainan Province in the south to Hanzhong district of Shanxi Province in the north at the latitude of 18°-33° N. The main economic planting zone is located in Zhejiang, Jiangsu, Fujian, Guangdong, Jiangxi, Anhui, Hunan and Guizhou provinces in China (Li et al. 1999; He et al. 2004a, b, 2006). Guangxi is also an important distribution zone and planting region of M. rubra.
Received 24 April, 2009 Accepted 20 September, 2009 Correspondence HE Xin-hua, Professor, Tel: +86-771-3270184, Fax: +86-771-3235612, E-mail: [email protected]
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60137-1
Genetic Diversity of Natural Myrica rubra Sieb.et Zucc Populations in Guangxi Revealed by ISSR Markers
Guangxi is located in southern China between 20°54´ and 26°20´ N latitude and covers an area of 236 661 km2. The three climate zones of North tropic, Sub-tropic and Mid Sub-tropic distributed in this area from south to north. The intricate but superior natural environmental conditions led to the development of extremely rich biological species and complex ecological combinations in this area. These conditions have also made the M. rubra resources in Guangxi much abundant. Most of them naturally exist and distributed in sporadic islands or bands. In recent years, the wild M. rubra resources in Guangxi have been seriously damaged due to the unreasonable and artificial afforestation through burning-slash, uprooting activities, etc. Therefore, it has become very important to protect and utilize the M. rubra resources. The studies on wild Myrica populations have not yet been initiated in China or abroad. In order to genetically characterize M. rubra populations as well as to facilitate programs aimed at its genetic improvement, restoration conservation and sustainable management, suitable molecular markers are required to reliably assess the genetic diversity. Some taxonomical and morpho-physiological characteristics of M. rubra in Guangxi have been reported in previous studies (Li et al. 1999; He et al. 2004a, b, 2006), but its genetic diversity has not been studied yet. Inter-simple sequence repeat (ISSR) marker is a microsatellite-derived genetic fingerprinting method based on the amplification of DNA fragments occurring in the genome in regions where a particular SSR (short sequence repeat) motif occurs on opposite strands within a short and amplifiable distance (Zietkiewicz et al. 1994). It has been broadly and successfully used in studies on genetic diversity, phylogenetics in a wide range of plant species or plant populations (Fang et al. 1998; Ajibade et al. 2000; Martin and Sánchez-Yélamo 2000; Qian
627
et al. 2001; Sankar and Moore 2001; Qiu et al. 2002; Pradeep et al. 2002; He et al. 2005; Yu and Ai 2007). The present study has been conducted to analyze molecular characterization and assess genetic diversity amongst natural populations of M. rubra in Guangxi, and to provide scientific evidence for germplasm conservation and exploitation using ISSR Markers. The genetic construction, genetic variance degree and the patterns of gene flow within and between different populations were studied by using ISSR-PCR to understand genetic diversity of natural M. rubra populations in Guangxi.
MATERIALS AND METHODS Plant materials A total of 105 individuals of M. rubra corresponding to 10 natural populations, distributed in 10 natural habitats with varying climate and topography, were collected from 10 counties of Guangxi Zhuang Autonomous Region, China (Table 1). The samples were collected at random and the geographical distance between the populations was more than 30 km. The fresh matured leaves were sampled and stored in a refrigerator at -30°C.
DNA extraction The genomic DNA of M. rubra was extracted by the optimized CTAB method (He et al. 2005). The concentration and quality of DNA were determined by the spectrophotometer (Eppendorf, Germany) and 0.8% agarose gel electrophoresis. The purified DNA was preserved in the refrigerator at -30°C.
Table 1 The ecological and geographical parameters of the M. rubra populations samples Population 1) LQ SL MS LS SS HP RX LG GY HJ 1)
Geographic origin in Guangxi
Longitude (E)/Latitude (N)
Annual rainfall (mm)
Annual mean temperature (°C)
Sample sizes
Liangqing district Shanglin County Mashan County Lingshan County Shangsi County Hepu County Rongxian County Lingui County Guanyang County Huanjiang County
107°55´/22°45´ 108°43´/23°18´ 107°46´/23°35´ 109°03´/21°52´ 107°52´/21°53´ 109°16´/21°33´ 110°25´/22°27´ 110°02´/24°31´ 110°30´/25°21´ 108°13´/24°55´
1 300.0 1 789.2 1 667.6 1 649.0 1 217.3 1 667.0 1 698.9 1 869.0 1 538.4 1 569.5
21.7 20.9 21.3 21.7 21.7 22.3 21.3 19.1 17.9 17.8
5 14 4 8 5 5 9 22 14 19
LQ, Liangqing; SL, Shanglin, MS, Marshan; LS, Lingshan; SS, Shansi (SS); HP, Hepu; RX, Rongxian; LG, Lingui; GY, Guanyang; HJ, Huanjiang.
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
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HE Xin-hua et al.
ISSR analysis 116 ISSR primers were designed according to Qiu et al. (2005) and public sequences of University of British Columbia, Canada, and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, China). 11 primers (Table 2) were selected from 116 primers tested based on the clarity and reproducibility of the band patterns and were used to conduct the genetic analysis of M. rubra populations. Table 2 Sequences of 11 primers used in ISSR analysis Primer UBC808 UBC811 UBC812 UBC835 UBC840 UBC888 UBC889 ym4 ym5 ym6 a919
Sequence 5´ primer
3´
(AG)8C (GA)8C (GA)8 A (AG)8YC HBH(AG)7 BDB(CA)7 DBD(AC)7 GSG(GT)6 GGA(GTG)4 CCA(GTG)4 DBD(CA)6
Annealing temperature (°C)
Number of bands recorded
52.0 51.0 49.0 53.7 53.0 52.5 52.5 52.3 52.3 50.5 50.0
11 11 12 10 12 10 12 12 10 12 11
Each 20 μL amplification reaction consisted of 10 mmol L-1 Tris-HCl (pH 8.3), 50 mmol L-1 KCl, 1.5 mmol L-1 MgCl2, 0.25 mmol L -1 mixed dNTP, 0.3 μmol L-1 primer, 1 U Taq polymerase (Bioer Technology, China), and approximately 60-90 ng genomic DNA. Amplification was performed in a GeneAmpTM PCR System 9700 (Perkin-Elmer Corp., Norwalk, CT, USA) under the following conditions: initial denaturation for 5 min at 94°C, followed by 45 cycles of 1 min at 94°C, 1 min at annealing temperature (49-54°C) and 2 min at 72°C followed by final extension of 10 min at 72°C. The PCR products were separated on 2.0% agarose gels and stained by 0.1% ethidium bromide. The molecular weights were estimated with reference to GeneRulerTM 100 bp DNA ladder plus (MBI, USA). The gel images were recorded and the band sizes were quantified using a Gel Doc 2 000TM system (Bio-Rad, USA).
files were scored for each individual band as present (1) or absent (0) on the basis of size comparison with standard (GeneRulerTM 100 bp DNA Ladder Plus). The following parameters were generated using the program POPGENE 1.31 to describe intra and inter-population genetic variation: Nei’s gene diversity index (He), Shannon’s information index (Ho), the observed number of alleles (Na) and the effective number of alleles (Ne). Genetic divergence between populations was investigated using Nei’s unbiased genetic distances (D) and genetic identities (I). Nei’s unbiased genetic distances were calculated for all population pairs and used to construct a phylogenetic tree (UPGMA) (Nei 1978). The genetic construction was further investigated using Nei’s gene diversity statistics, including the total genetic diversity (HT), genetic diversity within populations (HS), and the relative magnitude of genetic differentiation among populations Gst = (HT - HS)/HT (Nei 1973). An estimate of gene flow among populations (Nm) was computed using the formula of McDermott and McDonald (1993) Nm = (1 - Gst)/2Gst. The distribution of genetic variation among the populations was analyzed by AMOVA 1.55 software and a Mantel test (Mantel 1967) in the NTSYS-pc 2.10e software was used to test whether the matrix of genetic distances correlated with the matrix of geographical distances.
RESULTS ISSR characteristics in M. rubra 105 individuals from 10 natural populations of M. rubra were amplified with 11 selected primers. The 11 selected primers generated altogether 123 unambiguous and reproducible bands, of which 95 (77.24%) were polymorphic, the sizes ranged from 300 to 1800 bp (Fig.1). The numbers of bands varied from 10 to 12, with an average of 11.18 bands per primer (Table 2). The results showed that wild M. rubra populations distributed in various areas of Guangxi possess rich polymorphism.
Data analysis Genetic diversity within populations Only bands that were unambiguously scored across all samples were included in the analysis. The ISSR pro-
In individual populations, the percentage of polymor-
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Genetic Diversity of Natural Myrica rubra Sieb.et Zucc Populations in Guangxi Revealed by ISSR Markers
629
Fig. 1 Electrophoresis pattern amplified from some Myrica cultivars in Guangxi with primer UBC840. Lane M, GeneRulerTM 100 bp DNA ladder; other lanes represent cultivars listed in Table 1.
phic loci (PPB) ranged from 31.7 to 62.6%, with an average of 49.0 % (Table 3). Nei’s gene diversities (He) varied from 0.121 to 0.222, with an average of 0.175, and Shannon’s indices (Ho) ranged from 0.180 to 0.331, with an average of 0.261. In this study, the genetic diversity amongst GY populations was the highest with He and Ho values of 0.222 and 0.331, respectively, while the genetic diversity amongst Hepu populations was found to be the lowest with He and Ho values of 0.1210 and 0.180, respectively. The genetic diversity of populations from high to low ranked as follows: GY > LS > HJ > LG > SS> RX > MS > SL > LQ > PH. The same results were also obtained by observing the number of alleles (Na) and effective number of alleles (Ne) (Table 3). When calculated across the populations, the He and Ho values equaled 0.268 and 0.402, respectively, and the Na and Ne values equaled 1.772 and 1.454, respectively.
Genetic construction of populations The analysis of the genetic construction revealed a considerable level of genetic differentiation among various populations of M. rubra investigated. The total gene diversity (HT) and gene diversity within populations (HS) were 0.265 and 0.175, respectively. The coefficient of genetic differentiation (Gst) amongst interpopulations of wild M. rubra was 0.341 which indicated 34.1% variation in inter-populations while 65.9% variation within the populations. Data from AMOVA molecular detection proved that there was 22.4% genetic variation in inter-populations (Table 4), this further strengthened the results and illustrated that genetic differentiation of M. rubra was affected more in interpopulation groups. Gene flow estimated by Gst (Nm = 0.5(1 - Gst)/Gst) was 0.9682, it indicated that there was some genetic differentiation between the populations
Table 3 Genetic variability parameters of wild M. rubra populations in Guangxi based on ISSR markers Population RX HP LS LQ SS SL MS HJ LG GY Mean Species
Observed number of alleles (Na)
effective number of alleles (Ne)
Nei’s genetic diversity index (He)
Shannon’s information index (Ho)
Percentage of polymorphic loci (%, PPB)
1.447 1.317 1.553 1.366 1.480 1.480 1.398 1.626 1.626 1.610 1.490 1.772
1.274 1.204 1.361 1.249 1.318 1.242 1.265 1.353 1.308 1.380 1.295 1.454
0.161 0.121 0.208 0.144 0.186 0.149 0.158 0.207 0.190 0.222 0.175 0.268
0.240 0.180 0.309 0.213 0.275 0.229 0.233 0.313 0.293 0.331 0.261 0.402
44.72 31.71 55.28 36.59 47.97 47.97 39.84 62.60 62.60 60.98 49.03 77.24
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Table 4 Analysis of molecular variance (AMOVA) of wild M. rubra populations Variance source Inter-populations (AP) Within the population (WP) Total
df
Variance component
9 95 104
4.096 14.175 18.271
Percentage (%)
P*
22.4 77.76
< 0.001 < 0.001
Mantel test analysis, the result showed that the genetic distance had positive correlation with the geographical distance (r = 0.37082, P= 0.9906) (Table 5).
Genetic relationship
, significant test of 1 000 exchange haplotypes.
*
of wild Myrica, and variation of species was effected within the population also.
Relationship between geographic distance and genetic distance The geographic distance and genetic distance between the wild M. rubra populations were calculated with
Based on results of Nei’s genetic distance, a dendrogram was constructed by the UPGMA method (Fig.2). Ten wild M. rubra populations in Guangxi were gathered into two clusters, one included populations of RX, PH, LQ, MS, LS and SS, and the other included populations of SL, HJ, LG and GY. Among them, the genetic distance between RX and PH populations was 0.039, their genetic relationship was the nearest. However, the genetic distance between populations of RX and GY was 0.1748 showing the farthest genetic distance among them.
Table 5 Genetic distance and geographical distances between wild M. rubra populations in Guangxi 1) Population RX HP LS LQ SS SL MS HJ LG GY 1)
RX ***
0.039 0.075 0.087 0.163 0.140 0.120 0.163 0.157 0.175
HP 181.9 ***
0.069 0.050 0.155 0.101 0.080 0.142 0.146 0.164
LS 134.3 77.3 ***
0.061 0.057 0.139 0.075 0.104 0.129 0.085
LQ 224.2 145.9 104.9 ***
0.130 0.122 0.088 0.139 0.124 0.153
SS
SL
MS
266.1 130.4 134.3 73.5
194 191.6 121.9 83.2 156.6
265.4 224.2 171.6 81.5 140.4 74.8
***
0.178 0.131 0.121 0.127 0.092
***
0.126 0.110 0.083 0.134
***
0.114 0.122 0.138
HJ
LG
GY
335.9 374.8 303.5 246.9 313.3 182.8 172.8
240.1 371 295 308.1 380.5 227.2 278.1 199.7
269.7 429.3 357.7 390 459.5 311.3 370.8 295.1 97.9
***
0.084 0.115
***
0.076
***
Geographical and genetic distances are given above and below the diagonal.
DISCUSSION Genetic diversity The level of population genetic diversity is related to the size of the population, the bigger populations generally have higher level of genetic diversity (Sankar and Moore 2001). The results indicated that the genetic diversity of wild M. rubra in Guangxi at the level of population (PPB = 49.03%, He = 0.175, Ho = 0.261) was lower than that at the level of species (PPB = 73.70%, He = 0.268, Ho = 0.402). Among them, the genetic diversity of PH population was the lowest, and the GY was the highest. The investigations showed that PH population was destroyed greatly by human and only distributed dispersedly. Their trees were propagated by air layering and might have been originated from a
Fig. 2 UPGMA dendrogram of wild M. rubra populations in Guangxi based on Nei’s genetic distance.
few old M. rubra trees. As a result, the genetic diversity within populations is relatively lower. HJ and LG populations still have wild M. rubra gene pool with clump distribution, and the individual quantities in the
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Genetic Diversity of Natural Myrica rubra Sieb.et Zucc Populations in Guangxi Revealed by ISSR Markers
population are large, so their genetic diversity within populations is higher. Ten populations were gathered separately into two big clusters. One consisted of populations of RX, PH, LQ, MS, LS and SS, which originated from the southern Guangxi, while the other was composed of GY and LG populations of northern Guangxi, HJ populations of northwestern Guangxi and SL populations of southern Guangxi. The climates at southern, northwestern and northern Guangxi vary greatly but various populations from different regions still clustered together. The possible reason for this might be the habitat of SL populations, collected from Daming Mountain scenic area. This population grows at the altitude of 1 200-1 400 m where the local climate is similar to that of climate in north and northwest areas of Guangxi. M. rubra mainly pollinates through wind or insects and belongs to cross-pollinating plants; its seed setting rate is relatively high. However, the seed-coat of M. rubra is very hard and seed sprouting is difficult under normal circumstances. Some physiological factors such as vernalization before seed germination greatly limit survival quantity and rate of M. rubra. This special breeding system also limits the natural distribution of M. rubra. The similar research on population structure and life habits of plants has been done by Hamrick and Godt (1990). They were of the opinion that there are many factors influencing the changes in plant populations. Breeding system, distribution and habits of plant life may play a leading role in the population genetic evolution.
Effect of genetic differentiation on population structure The results showed that the genetic differentiation coefficient (Gst) amongst inter-populations of wild M. rubra was 0.341, which indicated 34.1% variation in inter-populations while 65.9% variation within the populations. The same result was also obtained by the AMOVA analysis. When Nm < l, the level of genetic diversity maintained within a population is more susceptible to genetic drift (Wright 1951). The gene flow (Nm) in the study was 0.968. Therefore, genetic variation of wild M. rubra in Guangxi is mainly due to gene drift within the population, and the effect of the gene
631
flow among inter-populations is not significant. The result from the similar studies on population structure of wild mango by Yu and Ai (2007) showed that the genetic variation is mainly related to the limit of gene flowing among the populations, and the far geographical distance is one of the main factors that make the exchange of gene inconvenience. SL population possesses more individuals, but its genetic differentiation coefficient is not very high, it may be related to its geographical altitude which located between 1 200 to 1 400 m altitude. The geographical distance hinders the pollens movement from one place to another, thus increases the frequency of crossing within a population resulted in low genetic differentiation coefficient.
Protection strategy of wild M. rubra in Guangxi On the basis of results obtained in the present study and the land surveys, some active protection measurements must be recommended to ensure appropriate levels of genetic variation of the wild M. rubra resources in Guangxi. The protection measurements are as follows: (1) Actions of cutting or destroying wild M. rubra must be strictly prohibited; (2) the in situ conservation of M. rubra should be established in natural protection zone; (3) the gene pool and germplasm resources of M. rubra should be secured for exploitation in various breeding programs.
Acknowledgements The research was supported by the National Natural Science Foundation of China (30560007). The authors appreciate Dr Manoj Kumar Srivastava from Indian Institute of Sugercane Reaearch for his critical review of the manuscript.
References Ajibade S R, Weeden N F, Chite S M. 2000. Inter simple sequence repeat analysis of genetic relationships in the genus Vigna. Euphytica, 111, 47-55. Fang D Q, Krueger R R, Roose M L.1998. Phylogenetic relationships among selected citrus germplasm accessions revealed by inter-simple sequence repeat (ISSR) markers. Journal of the American Society for Horticultural Science, 123, 612-617. Hamrick J L, Godt M J W. 1990. Allozyme Diversity in Plant Species, Plant Population Genetics, Breeding, and Genetic
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
632
HE Xin-hua et al.
Resources. Sunderland Mass, Sinauer, Sip-Associates. pp. 43-63.
of the USA, 70, 3321-3323. Nei M. 1978. Estimation of average heterozygosity and genetic
He X H, Chen L G, Asghar S, Chen Y. 2004a. Red bayberry (Myrica rubra), a promising fruit and forest tree in China.
distance from a small number of individuals. Genetics, 89, 583-590.
Journal of American Pomological Society, 58, 163-168. He X H, Chen L G, Chen Y, Guo C L. 2004b. China Myrica
Pradeep R M, Sarla N, Siddiq E A. 2002. Inter simple sequence repeat (ISSR) polymorphism and its application in plant
resources and its utilization and research evaluation. Journal of Fruit Trees, 21, 467-471. (in Chinese)
breeding. Euphytica, 128, 9-17. Qian W, Ge S, Hong D Y. 2001. Genetic variation within and
He X H, Li Y R, Guo Y Z, Tang Z P, Li R B. 2005. Genetic analysis of 23 mango cultivar collection in Guangxi Province
among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers. Theoretical and
revealed by ISSR. Molecular Plant Breeding, 3, 829-834. He X H, Pan H, She J C, Guo Y Z. 2006. Progress on Myrica.
Applied Genetics, 102, 440-449. Qiu Y X, Fu C X, Kong H H. 2002. ISSR analysis of different
Fujian Fruit Trees, 139, 16-23. (in Chinese) Li X J, Lv J L, Li S Y. 1999. Progress on research of Myrica in
Myrica species. Journal of Agricultural Biotechnology, 10, 343-346. (in Chinese)
China. Journal of Sichuan University, 17, 224-228. (in Chinese)
Sankar A A, Moore G A. 2001. Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the
Mantel N. 1967. The detection of disease clustering and a generalized regression approach. Cancer Research, 27, 209-
genetic linkage map. Theoretical and Applied Genetics, 102, 206-214.
212. Martin J P, Sánchez-Yélamo M D. 2000. Genetic relationships
Wright S. 1951. The genetical structure of populations. Annals of Engenics, 15, 323-354.
among species of the genus Diplotaxis (Brassicaceae) using inter-simple sequence repeat markers. Theoretical and Applied
Yu X M, Ai C X. 2007. ISSR analysis of genetic diversity of wild mango populations. Journal of Fruit Trees, 24, 329-333. (in
Genetics, 101, 1234-1241. McDermott J M, McDonald B A. 1993. Gene flow in plant
Chinese) Zietkiewicz E, Rafalski A, Labuda D. 1994. Genome
pathosystems. Annual Review of Phytopathology, 31, 353373.
fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics, 20, 176-
Nei M. 1973. Analysis of gene diversity in subdivided populations. Proceedings of National Academy of Sciences
183. (Managing editor ZHANG Yi-min)
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
May 2010
2010, 9(5): 626-632
Genetic Diversity of Natural Myrica rubra Sieb.et Zucc Populations in Guangxi Revealed by ISSR Markers HE Xin-hua1, PAN Hong1, DENG Li-bao1, PAN Jie-chun1, LI Feng1 and LI Yang-rui2 1 2
College of Agriculture, Guangxi University, Nanning 530004, P.R.China Guangxi Crop Genetic Improvement and Biotechnology Lab, Guangxi Academy of Agricultural Sciences, Nanning 530007, P.R.China
Abstract The present study was conducted to assess the molecular characterization and genetic diversity amongst natural populations of Myrica rubra in Guangxi Zhuang Autonomous Region, China, thus to provide scientific evidence for germplasm conservation and exploitation. Using ISSR (inter-simple sequence repeats) markers, the level of genetic variation and the molecular characterization of 10 natural populations of M. rubra, originated from Guangxi Zhuang Autonomous Region in China, were performed. Based on 11 primers, 123 clear and reproducible DNA fragments were generated, of which 95 (77.24%) were polymorphic. The average value of Nei’s gene diversity (He) was 0.268. The coefficient of genetic differentiation (Gst) was 0.341, revealing that 34.1% of the total molecular variance existed among populations. The Mantel statistical testing showed that the genetic distance was correlated to the geographic distance, but the correlation was not significant. Ten populations were divided into two big clusters according to unweighted pair group method with arithmetic mean (UPGMA) analysis. One consisted of populations of Rongxian (RX), Hepu (HP), Liangqing (LQ), Marshan (MS), Lingshan (LS) and Shansi (SS), which originated from the southern Guangxi, while the other was composed of Guanyang (GY) and Lingui (LG) populations of northern Guangxi, Huanjiang (HJ) populations of northwestern Guangxi and Shanglin (SL) populations of southern Guangxi. The level of genetic variation in wild M. rubra population distributed in Guangxi is high. Gene drift within the population was responsible for genetic variation in wild M. rubra in Guangxi, and the effect of the genetic flow among inter-populations was not significant. Classification of wild M. rubra populations was correlated to climate and environment. The molecular characterization and diversity assessment of M. rubra is of immense value for planning conservation of its genetic resources and their exploitation for further studies. Key words: Myrica rubra, population, genetic diversity, ISSR
INTRODUCTION Myrica rubra Sieb.et Zucc (also named as Chinese tree berry, red bayberry and yangmei) is an important subtropical fruit native to China. In China, M. rubra is distributed from eastern coastal area of Taiwan to Ruili County of Yunan Province in the west and from
Sanya City of Hainan Province in the south to Hanzhong district of Shanxi Province in the north at the latitude of 18°-33° N. The main economic planting zone is located in Zhejiang, Jiangsu, Fujian, Guangdong, Jiangxi, Anhui, Hunan and Guizhou provinces in China (Li et al. 1999; He et al. 2004a, b, 2006). Guangxi is also an important distribution zone and planting region of M. rubra.
Received 24 April, 2009 Accepted 20 September, 2009 Correspondence HE Xin-hua, Professor, Tel: +86-771-3270184, Fax: +86-771-3235612, E-mail: [email protected]
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60137-1
Genetic Diversity of Natural Myrica rubra Sieb.et Zucc Populations in Guangxi Revealed by ISSR Markers
Guangxi is located in southern China between 20°54´ and 26°20´ N latitude and covers an area of 236 661 km2. The three climate zones of North tropic, Sub-tropic and Mid Sub-tropic distributed in this area from south to north. The intricate but superior natural environmental conditions led to the development of extremely rich biological species and complex ecological combinations in this area. These conditions have also made the M. rubra resources in Guangxi much abundant. Most of them naturally exist and distributed in sporadic islands or bands. In recent years, the wild M. rubra resources in Guangxi have been seriously damaged due to the unreasonable and artificial afforestation through burning-slash, uprooting activities, etc. Therefore, it has become very important to protect and utilize the M. rubra resources. The studies on wild Myrica populations have not yet been initiated in China or abroad. In order to genetically characterize M. rubra populations as well as to facilitate programs aimed at its genetic improvement, restoration conservation and sustainable management, suitable molecular markers are required to reliably assess the genetic diversity. Some taxonomical and morpho-physiological characteristics of M. rubra in Guangxi have been reported in previous studies (Li et al. 1999; He et al. 2004a, b, 2006), but its genetic diversity has not been studied yet. Inter-simple sequence repeat (ISSR) marker is a microsatellite-derived genetic fingerprinting method based on the amplification of DNA fragments occurring in the genome in regions where a particular SSR (short sequence repeat) motif occurs on opposite strands within a short and amplifiable distance (Zietkiewicz et al. 1994). It has been broadly and successfully used in studies on genetic diversity, phylogenetics in a wide range of plant species or plant populations (Fang et al. 1998; Ajibade et al. 2000; Martin and Sánchez-Yélamo 2000; Qian
627
et al. 2001; Sankar and Moore 2001; Qiu et al. 2002; Pradeep et al. 2002; He et al. 2005; Yu and Ai 2007). The present study has been conducted to analyze molecular characterization and assess genetic diversity amongst natural populations of M. rubra in Guangxi, and to provide scientific evidence for germplasm conservation and exploitation using ISSR Markers. The genetic construction, genetic variance degree and the patterns of gene flow within and between different populations were studied by using ISSR-PCR to understand genetic diversity of natural M. rubra populations in Guangxi.
MATERIALS AND METHODS Plant materials A total of 105 individuals of M. rubra corresponding to 10 natural populations, distributed in 10 natural habitats with varying climate and topography, were collected from 10 counties of Guangxi Zhuang Autonomous Region, China (Table 1). The samples were collected at random and the geographical distance between the populations was more than 30 km. The fresh matured leaves were sampled and stored in a refrigerator at -30°C.
DNA extraction The genomic DNA of M. rubra was extracted by the optimized CTAB method (He et al. 2005). The concentration and quality of DNA were determined by the spectrophotometer (Eppendorf, Germany) and 0.8% agarose gel electrophoresis. The purified DNA was preserved in the refrigerator at -30°C.
Table 1 The ecological and geographical parameters of the M. rubra populations samples Population 1) LQ SL MS LS SS HP RX LG GY HJ 1)
Geographic origin in Guangxi
Longitude (E)/Latitude (N)
Annual rainfall (mm)
Annual mean temperature (°C)
Sample sizes
Liangqing district Shanglin County Mashan County Lingshan County Shangsi County Hepu County Rongxian County Lingui County Guanyang County Huanjiang County
107°55´/22°45´ 108°43´/23°18´ 107°46´/23°35´ 109°03´/21°52´ 107°52´/21°53´ 109°16´/21°33´ 110°25´/22°27´ 110°02´/24°31´ 110°30´/25°21´ 108°13´/24°55´
1 300.0 1 789.2 1 667.6 1 649.0 1 217.3 1 667.0 1 698.9 1 869.0 1 538.4 1 569.5
21.7 20.9 21.3 21.7 21.7 22.3 21.3 19.1 17.9 17.8
5 14 4 8 5 5 9 22 14 19
LQ, Liangqing; SL, Shanglin, MS, Marshan; LS, Lingshan; SS, Shansi (SS); HP, Hepu; RX, Rongxian; LG, Lingui; GY, Guanyang; HJ, Huanjiang.
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ISSR analysis 116 ISSR primers were designed according to Qiu et al. (2005) and public sequences of University of British Columbia, Canada, and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, China). 11 primers (Table 2) were selected from 116 primers tested based on the clarity and reproducibility of the band patterns and were used to conduct the genetic analysis of M. rubra populations. Table 2 Sequences of 11 primers used in ISSR analysis Primer UBC808 UBC811 UBC812 UBC835 UBC840 UBC888 UBC889 ym4 ym5 ym6 a919
Sequence 5´ primer
3´
(AG)8C (GA)8C (GA)8 A (AG)8YC HBH(AG)7 BDB(CA)7 DBD(AC)7 GSG(GT)6 GGA(GTG)4 CCA(GTG)4 DBD(CA)6
Annealing temperature (°C)
Number of bands recorded
52.0 51.0 49.0 53.7 53.0 52.5 52.5 52.3 52.3 50.5 50.0
11 11 12 10 12 10 12 12 10 12 11
Each 20 μL amplification reaction consisted of 10 mmol L-1 Tris-HCl (pH 8.3), 50 mmol L-1 KCl, 1.5 mmol L-1 MgCl2, 0.25 mmol L -1 mixed dNTP, 0.3 μmol L-1 primer, 1 U Taq polymerase (Bioer Technology, China), and approximately 60-90 ng genomic DNA. Amplification was performed in a GeneAmpTM PCR System 9700 (Perkin-Elmer Corp., Norwalk, CT, USA) under the following conditions: initial denaturation for 5 min at 94°C, followed by 45 cycles of 1 min at 94°C, 1 min at annealing temperature (49-54°C) and 2 min at 72°C followed by final extension of 10 min at 72°C. The PCR products were separated on 2.0% agarose gels and stained by 0.1% ethidium bromide. The molecular weights were estimated with reference to GeneRulerTM 100 bp DNA ladder plus (MBI, USA). The gel images were recorded and the band sizes were quantified using a Gel Doc 2 000TM system (Bio-Rad, USA).
files were scored for each individual band as present (1) or absent (0) on the basis of size comparison with standard (GeneRulerTM 100 bp DNA Ladder Plus). The following parameters were generated using the program POPGENE 1.31 to describe intra and inter-population genetic variation: Nei’s gene diversity index (He), Shannon’s information index (Ho), the observed number of alleles (Na) and the effective number of alleles (Ne). Genetic divergence between populations was investigated using Nei’s unbiased genetic distances (D) and genetic identities (I). Nei’s unbiased genetic distances were calculated for all population pairs and used to construct a phylogenetic tree (UPGMA) (Nei 1978). The genetic construction was further investigated using Nei’s gene diversity statistics, including the total genetic diversity (HT), genetic diversity within populations (HS), and the relative magnitude of genetic differentiation among populations Gst = (HT - HS)/HT (Nei 1973). An estimate of gene flow among populations (Nm) was computed using the formula of McDermott and McDonald (1993) Nm = (1 - Gst)/2Gst. The distribution of genetic variation among the populations was analyzed by AMOVA 1.55 software and a Mantel test (Mantel 1967) in the NTSYS-pc 2.10e software was used to test whether the matrix of genetic distances correlated with the matrix of geographical distances.
RESULTS ISSR characteristics in M. rubra 105 individuals from 10 natural populations of M. rubra were amplified with 11 selected primers. The 11 selected primers generated altogether 123 unambiguous and reproducible bands, of which 95 (77.24%) were polymorphic, the sizes ranged from 300 to 1800 bp (Fig.1). The numbers of bands varied from 10 to 12, with an average of 11.18 bands per primer (Table 2). The results showed that wild M. rubra populations distributed in various areas of Guangxi possess rich polymorphism.
Data analysis Genetic diversity within populations Only bands that were unambiguously scored across all samples were included in the analysis. The ISSR pro-
In individual populations, the percentage of polymor-
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Genetic Diversity of Natural Myrica rubra Sieb.et Zucc Populations in Guangxi Revealed by ISSR Markers
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Fig. 1 Electrophoresis pattern amplified from some Myrica cultivars in Guangxi with primer UBC840. Lane M, GeneRulerTM 100 bp DNA ladder; other lanes represent cultivars listed in Table 1.
phic loci (PPB) ranged from 31.7 to 62.6%, with an average of 49.0 % (Table 3). Nei’s gene diversities (He) varied from 0.121 to 0.222, with an average of 0.175, and Shannon’s indices (Ho) ranged from 0.180 to 0.331, with an average of 0.261. In this study, the genetic diversity amongst GY populations was the highest with He and Ho values of 0.222 and 0.331, respectively, while the genetic diversity amongst Hepu populations was found to be the lowest with He and Ho values of 0.1210 and 0.180, respectively. The genetic diversity of populations from high to low ranked as follows: GY > LS > HJ > LG > SS> RX > MS > SL > LQ > PH. The same results were also obtained by observing the number of alleles (Na) and effective number of alleles (Ne) (Table 3). When calculated across the populations, the He and Ho values equaled 0.268 and 0.402, respectively, and the Na and Ne values equaled 1.772 and 1.454, respectively.
Genetic construction of populations The analysis of the genetic construction revealed a considerable level of genetic differentiation among various populations of M. rubra investigated. The total gene diversity (HT) and gene diversity within populations (HS) were 0.265 and 0.175, respectively. The coefficient of genetic differentiation (Gst) amongst interpopulations of wild M. rubra was 0.341 which indicated 34.1% variation in inter-populations while 65.9% variation within the populations. Data from AMOVA molecular detection proved that there was 22.4% genetic variation in inter-populations (Table 4), this further strengthened the results and illustrated that genetic differentiation of M. rubra was affected more in interpopulation groups. Gene flow estimated by Gst (Nm = 0.5(1 - Gst)/Gst) was 0.9682, it indicated that there was some genetic differentiation between the populations
Table 3 Genetic variability parameters of wild M. rubra populations in Guangxi based on ISSR markers Population RX HP LS LQ SS SL MS HJ LG GY Mean Species
Observed number of alleles (Na)
effective number of alleles (Ne)
Nei’s genetic diversity index (He)
Shannon’s information index (Ho)
Percentage of polymorphic loci (%, PPB)
1.447 1.317 1.553 1.366 1.480 1.480 1.398 1.626 1.626 1.610 1.490 1.772
1.274 1.204 1.361 1.249 1.318 1.242 1.265 1.353 1.308 1.380 1.295 1.454
0.161 0.121 0.208 0.144 0.186 0.149 0.158 0.207 0.190 0.222 0.175 0.268
0.240 0.180 0.309 0.213 0.275 0.229 0.233 0.313 0.293 0.331 0.261 0.402
44.72 31.71 55.28 36.59 47.97 47.97 39.84 62.60 62.60 60.98 49.03 77.24
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Table 4 Analysis of molecular variance (AMOVA) of wild M. rubra populations Variance source Inter-populations (AP) Within the population (WP) Total
df
Variance component
9 95 104
4.096 14.175 18.271
Percentage (%)
P*
22.4 77.76
< 0.001 < 0.001
Mantel test analysis, the result showed that the genetic distance had positive correlation with the geographical distance (r = 0.37082, P= 0.9906) (Table 5).
Genetic relationship
, significant test of 1 000 exchange haplotypes.
*
of wild Myrica, and variation of species was effected within the population also.
Relationship between geographic distance and genetic distance The geographic distance and genetic distance between the wild M. rubra populations were calculated with
Based on results of Nei’s genetic distance, a dendrogram was constructed by the UPGMA method (Fig.2). Ten wild M. rubra populations in Guangxi were gathered into two clusters, one included populations of RX, PH, LQ, MS, LS and SS, and the other included populations of SL, HJ, LG and GY. Among them, the genetic distance between RX and PH populations was 0.039, their genetic relationship was the nearest. However, the genetic distance between populations of RX and GY was 0.1748 showing the farthest genetic distance among them.
Table 5 Genetic distance and geographical distances between wild M. rubra populations in Guangxi 1) Population RX HP LS LQ SS SL MS HJ LG GY 1)
RX ***
0.039 0.075 0.087 0.163 0.140 0.120 0.163 0.157 0.175
HP 181.9 ***
0.069 0.050 0.155 0.101 0.080 0.142 0.146 0.164
LS 134.3 77.3 ***
0.061 0.057 0.139 0.075 0.104 0.129 0.085
LQ 224.2 145.9 104.9 ***
0.130 0.122 0.088 0.139 0.124 0.153
SS
SL
MS
266.1 130.4 134.3 73.5
194 191.6 121.9 83.2 156.6
265.4 224.2 171.6 81.5 140.4 74.8
***
0.178 0.131 0.121 0.127 0.092
***
0.126 0.110 0.083 0.134
***
0.114 0.122 0.138
HJ
LG
GY
335.9 374.8 303.5 246.9 313.3 182.8 172.8
240.1 371 295 308.1 380.5 227.2 278.1 199.7
269.7 429.3 357.7 390 459.5 311.3 370.8 295.1 97.9
***
0.084 0.115
***
0.076
***
Geographical and genetic distances are given above and below the diagonal.
DISCUSSION Genetic diversity The level of population genetic diversity is related to the size of the population, the bigger populations generally have higher level of genetic diversity (Sankar and Moore 2001). The results indicated that the genetic diversity of wild M. rubra in Guangxi at the level of population (PPB = 49.03%, He = 0.175, Ho = 0.261) was lower than that at the level of species (PPB = 73.70%, He = 0.268, Ho = 0.402). Among them, the genetic diversity of PH population was the lowest, and the GY was the highest. The investigations showed that PH population was destroyed greatly by human and only distributed dispersedly. Their trees were propagated by air layering and might have been originated from a
Fig. 2 UPGMA dendrogram of wild M. rubra populations in Guangxi based on Nei’s genetic distance.
few old M. rubra trees. As a result, the genetic diversity within populations is relatively lower. HJ and LG populations still have wild M. rubra gene pool with clump distribution, and the individual quantities in the
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Genetic Diversity of Natural Myrica rubra Sieb.et Zucc Populations in Guangxi Revealed by ISSR Markers
population are large, so their genetic diversity within populations is higher. Ten populations were gathered separately into two big clusters. One consisted of populations of RX, PH, LQ, MS, LS and SS, which originated from the southern Guangxi, while the other was composed of GY and LG populations of northern Guangxi, HJ populations of northwestern Guangxi and SL populations of southern Guangxi. The climates at southern, northwestern and northern Guangxi vary greatly but various populations from different regions still clustered together. The possible reason for this might be the habitat of SL populations, collected from Daming Mountain scenic area. This population grows at the altitude of 1 200-1 400 m where the local climate is similar to that of climate in north and northwest areas of Guangxi. M. rubra mainly pollinates through wind or insects and belongs to cross-pollinating plants; its seed setting rate is relatively high. However, the seed-coat of M. rubra is very hard and seed sprouting is difficult under normal circumstances. Some physiological factors such as vernalization before seed germination greatly limit survival quantity and rate of M. rubra. This special breeding system also limits the natural distribution of M. rubra. The similar research on population structure and life habits of plants has been done by Hamrick and Godt (1990). They were of the opinion that there are many factors influencing the changes in plant populations. Breeding system, distribution and habits of plant life may play a leading role in the population genetic evolution.
Effect of genetic differentiation on population structure The results showed that the genetic differentiation coefficient (Gst) amongst inter-populations of wild M. rubra was 0.341, which indicated 34.1% variation in inter-populations while 65.9% variation within the populations. The same result was also obtained by the AMOVA analysis. When Nm < l, the level of genetic diversity maintained within a population is more susceptible to genetic drift (Wright 1951). The gene flow (Nm) in the study was 0.968. Therefore, genetic variation of wild M. rubra in Guangxi is mainly due to gene drift within the population, and the effect of the gene
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flow among inter-populations is not significant. The result from the similar studies on population structure of wild mango by Yu and Ai (2007) showed that the genetic variation is mainly related to the limit of gene flowing among the populations, and the far geographical distance is one of the main factors that make the exchange of gene inconvenience. SL population possesses more individuals, but its genetic differentiation coefficient is not very high, it may be related to its geographical altitude which located between 1 200 to 1 400 m altitude. The geographical distance hinders the pollens movement from one place to another, thus increases the frequency of crossing within a population resulted in low genetic differentiation coefficient.
Protection strategy of wild M. rubra in Guangxi On the basis of results obtained in the present study and the land surveys, some active protection measurements must be recommended to ensure appropriate levels of genetic variation of the wild M. rubra resources in Guangxi. The protection measurements are as follows: (1) Actions of cutting or destroying wild M. rubra must be strictly prohibited; (2) the in situ conservation of M. rubra should be established in natural protection zone; (3) the gene pool and germplasm resources of M. rubra should be secured for exploitation in various breeding programs.
Acknowledgements The research was supported by the National Natural Science Foundation of China (30560007). The authors appreciate Dr Manoj Kumar Srivastava from Indian Institute of Sugercane Reaearch for his critical review of the manuscript.
References Ajibade S R, Weeden N F, Chite S M. 2000. Inter simple sequence repeat analysis of genetic relationships in the genus Vigna. Euphytica, 111, 47-55. Fang D Q, Krueger R R, Roose M L.1998. Phylogenetic relationships among selected citrus germplasm accessions revealed by inter-simple sequence repeat (ISSR) markers. Journal of the American Society for Horticultural Science, 123, 612-617. Hamrick J L, Godt M J W. 1990. Allozyme Diversity in Plant Species, Plant Population Genetics, Breeding, and Genetic
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
632
HE Xin-hua et al.
Resources. Sunderland Mass, Sinauer, Sip-Associates. pp. 43-63.
of the USA, 70, 3321-3323. Nei M. 1978. Estimation of average heterozygosity and genetic
He X H, Chen L G, Asghar S, Chen Y. 2004a. Red bayberry (Myrica rubra), a promising fruit and forest tree in China.
distance from a small number of individuals. Genetics, 89, 583-590.
Journal of American Pomological Society, 58, 163-168. He X H, Chen L G, Chen Y, Guo C L. 2004b. China Myrica
Pradeep R M, Sarla N, Siddiq E A. 2002. Inter simple sequence repeat (ISSR) polymorphism and its application in plant
resources and its utilization and research evaluation. Journal of Fruit Trees, 21, 467-471. (in Chinese)
breeding. Euphytica, 128, 9-17. Qian W, Ge S, Hong D Y. 2001. Genetic variation within and
He X H, Li Y R, Guo Y Z, Tang Z P, Li R B. 2005. Genetic analysis of 23 mango cultivar collection in Guangxi Province
among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers. Theoretical and
revealed by ISSR. Molecular Plant Breeding, 3, 829-834. He X H, Pan H, She J C, Guo Y Z. 2006. Progress on Myrica.
Applied Genetics, 102, 440-449. Qiu Y X, Fu C X, Kong H H. 2002. ISSR analysis of different
Fujian Fruit Trees, 139, 16-23. (in Chinese) Li X J, Lv J L, Li S Y. 1999. Progress on research of Myrica in
Myrica species. Journal of Agricultural Biotechnology, 10, 343-346. (in Chinese)
China. Journal of Sichuan University, 17, 224-228. (in Chinese)
Sankar A A, Moore G A. 2001. Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the
Mantel N. 1967. The detection of disease clustering and a generalized regression approach. Cancer Research, 27, 209-
genetic linkage map. Theoretical and Applied Genetics, 102, 206-214.
212. Martin J P, Sánchez-Yélamo M D. 2000. Genetic relationships
Wright S. 1951. The genetical structure of populations. Annals of Engenics, 15, 323-354.
among species of the genus Diplotaxis (Brassicaceae) using inter-simple sequence repeat markers. Theoretical and Applied
Yu X M, Ai C X. 2007. ISSR analysis of genetic diversity of wild mango populations. Journal of Fruit Trees, 24, 329-333. (in
Genetics, 101, 1234-1241. McDermott J M, McDonald B A. 1993. Gene flow in plant
Chinese) Zietkiewicz E, Rafalski A, Labuda D. 1994. Genome
pathosystems. Annual Review of Phytopathology, 31, 353373.
fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics, 20, 176-
Nei M. 1973. Analysis of gene diversity in subdivided populations. Proceedings of National Academy of Sciences
183. (Managing editor ZHANG Yi-min)
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.