Hu, Y

Li, Y

Peng, M

Wang, L

Xie, X

Yang, J

English

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African Journal of Biotechnology Vol. 8 (18), pp. 4490-4494, 15 September, 2009     Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2009 Academic Journals     Full Length Research Paper  Genetic diversity in three morphological types of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao as revealed by inter simple sequence repeat markers  Xiaolong Xie1,2,3, Yanping Hu1,2, Li Wang1,2, Jian Yang1,2, Yi Li1* and Min Peng1*  1Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, Qinghai, P.R. China. 2Graduate University of Chinese Academy of Sciences, Beijing 100049, P.R. China. 3Henan University of Traditional Chinese Medicine, Zhengzhou 450008, P.R. China.  Accepted 20 July, 2009  Inter simple sequence repeat (ISSR) markers were used to detect the genetic diversity in three morphological types of cultivated Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao discovered in Gansu province of China. Eight primers used for analysis generated 165 scorable bands of which 162 (98.18%) were polymorphic. The percentage polymorphic bands (PPB), Shannon’s information index (I) and Nei’s gene diversity (h) for 3 types ranged from 59.39%, 0.2912, 0.1901 to 78.18%, 0.3411, 0.2238 respectively, indicating each type had a high genetic diversity. Analysis of molecular variance (AMOVA) showed 90.41% of the total genetic diversity existed within the 3 morphological types, whereas only 9.59% occurred among them. The UPGMA tree based on SM similarity coefficients implied that the 3 types had genetically differentiated though not obviously. The high genetic diversity suggested that each type had a potential to develop new strains or cultivars and ISSR was a potentially useful tool in the process of selective breeding of A. membranaceus var. mongholicus.   Key words: Astragalus membranaceus var. mongholicus, morphological type, genetic diversity, ISSR, Radix astragali.   INTRODUCTION  Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao, a perennial herbaceous plant, is defined as one of the genuine botanical sources of Radix astragali (“Huangqi” in Chinese) in the Chinese Pharmacopoeia. As a commonly used traditional Chinese medicine, Radix astragali has the functions of reinforcing “qi” (vital energy), of  strengthening  the  superficial  resistance,  of    *Corresponding author. E-mail: liyi@nwipb.ac.cn (Yi Li), pengm@nwipb.ac.cn (Min Peng). Tel.: +86971-6101454. Fax: +86971-6143282.  Abbreviations: AMOVA, analysis of molecular variance; CTAB, cetyltrimethylammonium bromide; ISSR, inter simple sequence repeat; PCR, polymerase chain reaction; UPGMA, unweighed pair-group method using arithmetic average. inducing diuresis and of promoting the discharge of pus and the growth of new tissue (Pharmacopoeia Com-mission of People’s Republic of China, 2005). In China, the wild resources of Radix astragali are gradually decreasing due to its exhaustive exploitation and hence the commercial supply of it is mainly taken from farming sources, especially from cultured A. membranaceus var. mongholicus (Wang and Xie, 2004; Ma et al., 2000; Feng and Xiao, 2002). According to our investigation into A. membranaceus var. mongholicus in Longxi county, Gansu province, there exist 3 morphological types (R type, RG type and G type) in the cultivated population (Xie et al., unpublished data). R type possesses red dapples on both stems and pods more or less, while G type has completely green stems and pods without any flammulation. For RG type, the stems are same as the G type ones  while  the  pods  are  Xie et al.        4491    Table 1. ISSR primers used for PCR amplification of A. membranaceus var. mongholicus, together with optimum Mg2+ concentration and annealing temperature for each primer.  Primer Sequence of the primer(5’ - 3’)* Mg2+ (mM) Annealing temperature (°C) UBC809 AGA GAG AGA GAG AGA GG 2.10 56.2 UBC836 AGA GAG AGA GAG AGA GYA 1.95 50.0 UBC841 GAG AGA GAG AGA GAG AYC 2.10 51.0 UBC842 GAG AGA GAG AGA GAG AYG 1.95 53.4 UBC856 ACA CAC ACA CAC ACA CYA 1.80 48.3 UBC861 ACC ACC ACC ACC ACC ACC 1.80 59.4 UBC868 GAA GAA GAA GAA GAA GAA 1.95 47.4 UBC891 HVH TGT GTG TGT GTG TG 2.10 54.2  *Y = (C,T); H = (A,C,T), that is, not G; V = (A,C,G), that is, not T.    same as the R type ones. The morphological diversity as well as the results of our previous studies (Xie et al., 2004, 2007) showed the cultivated population is very heterogeneous, which has resulted in a rapid decline of the yield and quality of Radix astragali in this region. On the other hand, the different morphological types may be useful genetic resources for the breeding program of A. membranaceus var. mongholicus. The objective of the present study was to provide genetic references for Radix astragali selection and breeding by analyzing the genetic diversity in the 3 morphological types using ISSR mar-kers.   MATERIALS AND METHODS  Plant material and DNA extraction  A total of 32 individuals comprised of 16 plants of R type, 6 plants of RG type and 10 plants of G type were sampled from Longxi county for this study. Fresh leaves of per plant were desiccated with silica gel in a plastic zip-lock bag and stored at -20°C until DNA isolation. Total DNA was extracted by a cetyltrimethylammonium bromide (CTAB) protocol of Tian et al. (2004) with minor modifications. Dried leaf materials were rapidly ground with about 0.025 g PVP powder and a little quartz sand and mixed with 900 ul preheated 2 × CTAB extraction buffer [100 mM Tris-HCl (pH 8.0), 1.4 M NaCl, 20 mM EDTA, 2%(w/v) CTAB] containing 0.1% (v/v) -mercaptoethanol in a 2.0 ml centrifuge tube. The mixture was extracted twice with an equal volume of chloroform:isoamyl alcohol (24:1,v/v) followed by centrifugation at 10,000 g for 10 min. The resulting supernatant was mixed with 2/3 volumes of ice-cold isopropanol and kept at -20°C for 2 h. The total DNA, recovered by centrifugation at 10,000 g for 6 min, was washed twice with 70% ethanol, air dried and finally re-suspended in 150 ul sterilized double distilled water. The extracted DNA was quantified with a DNA/HindIII marker in 0.8% agarose gel.   ISSR-PCR amplification  38 ISSR primers of the UBC set #9 (Sangon, Shanghai, China) were screened with three DNA samples. Reaction volumes were 20 ul and consisted of 2.8 ul  10 × PCR  buffer  (100 mM  Tris-HCl,  pH  8.3 and 500 mM KCl), 1.8 - 2.1 mM MgCl2 (Table 1), 325 uM dNTPs (Sangon, Shanghai, China), 1U Taq DNA polymerase (TaKaRa Inc., Dalian, China), 0.3 uM primer and 0.84 ng DNA template. All the amplifications were carried out in a Dyad DiscipleTM Peltier Thermal Cycler (Bio-RAD, USA) with the following program: an initial denaturation step at 94°C for 5 min; followed by 40 cycles of 45 s at 94°C, 60 s at a specific annealing temperature depending on a specific primer (Table 1), 90 s extension at 72°C and a final 7 min at 72°C. After resolved by electrophoresis in 1.5% agarose gel in 1 × TAE buffer, amplification products were stained with ethidium bromide (0.5 ug/ml) and photographed under UV light. Molecular weights were estimated with a 200 bp DNA ladder marker (TaKaRa Inc., Dalian, China).   Data analysis   The ISSR amplification products were scored as “1” for presence and “0” for absence across the analyzed samples. The binary data matrix, thus obtained was used for further analyses. Genetic diver-sity parameters including the percentage of polymorphic bands (PPB), Shannon’s information index (I), Nei’s (1973) gene diversity (h) and effective number of alleles (ne) were calculated with POPGENE version 1.31 (Yeh et al., 1999). Analysis of molecular variance (AMOVA) was made as described by Excoffier et al. (1992) to examine variability among and within types and the signi-ficance of variance components was tested by 1000 random permutations. Cluster analysis of the individuals was performed with the unweighed pair-group method using arithmetic average (UPG MA) method based on SM similarity coefficients using NTSYSpc version 2.0 (Rohlf, 1998).   RESULTS AND DISCUSSION  Eight primes that produced clear, reproducible and poly-morphic bands were selected for PCR analysis (Table 1). A total of 165 bands ranging from 270 to 1650 bp in size, were generated with an average of 20.63 bands per primer, of which 162 (98.18%) were polymorphic among 32 plants with a mean of 20.25 bands. The percentage of polymorphic bands of each primer varied from 94.74% (primers UBC841 and UBC861) to100% (UBC809, UBC836, UBC842 and UBC856) (Table 2).  Of the 3 morphological types studied, R type  produced  4492         Afr. J. Biotechnol.    Table 2. Number of amplification products generated with 8 ISSR primers from 32 individuals of three morphological types of A. membranaceus var. mongholicus.  Primer Total number of bands Number of polymorphic bands Percentage of polymorphic bands (%) Bands size (bp) UBC809 24 24 100 380-1600 UBC836 25 25 100 350-1250 UBC841 19 18 94.74 270-1600 UBC842 20 20 100 300-1500 UBC856 16 16 100 520-1550 UBC861 19 18 94.74 420-1600 UBC868 22 22 100 370-1650 UBC891 20 19 95 380-1400 Total 165 162   Average 20.63 20.25      Table 3. Genetic diversity parameters for three morphological types (R type, RG type and G type) of A. membranaceus var. mongholicus.   Type ne*(±SD) h**(±SD) I***(±SD) Number of polymorphic bands Percentage of polymorphic bands (%) R 1.3514 ± 0.3428 0.2155 ± 0.1793 0.3358 ± 0.2485 129 78.18 RG 1.3138 ± 0.3503 0.1901 ± 0.1872 0.2912 ± 0.2690 98 59.39 G 1.3742 ± 0.3605 0.2238 ± 0.1890 0.3411 ± 0.2657 116 70.30 Total 1.3875 ± 0.3174 0.2432 ± 0.1608 0.3847 ± 0.2109 162 98.18  *ne = Effective number of alleles, **h = Nei’s gene diversity, ***I = Shannon’s information index.    the highest number of polymorphic bands (129, 78.18%) while RG type gave the least polymorphism (98, 59.39%). Although the minimums of Nei’s (1973) gene diversity (0.1901), Shannon’s information index (0.2912) and effective number of alleles (1.3138) still existed in RG type, the maximums (h = 0.2238, I = 0.3411, ne = 1.3742) occurred in G type instead of R type (Table 3). However, these parameters showed that each morphological type of A. membranaceus var. mongholicus had a high level of genetic diversity comparing with the long-lived perennial herbs and widespread distributed species (Yao et al., 2008), indicating each type had a potential to develop strains or cultivars. The high genetic diversity detected may be mostly attributed to 2 reasons:   1) A. membranaceus var. mongholicus is a cross-pollinating species, thus it can maintain higher heterozygosity as compared to self-pollinating plant species (Joshi and Dhawan, 2007; Xie et al., 2004).  2) Being informative and polymorphic at intraspecies level, ISSR markers can reveal more variability (Joshi and Dhawan, 2007; Qian et al., 2001; Goulo et al., 2001).  The results of AMOVA with a high significance (P < 0.001) showed that most of the whole genetic diversity (90.41%) resided within the 3 morphological types, whereas only 9.59% existed among types.  An UPGMA dendrogram based on SM coefficients between all studied individuals is shown in Figure 1. In this dendrogram, except few individuals including RG1, R1, R2, R13, R14, G1 and G2, most from the same morphological type formed a separate group without any other types’ individuals involved. This indicated:   1) 3 morphological types have genetically differentiated to some degree, which could be corroborated by the results of AMOVA and hence it is feasible to classify plants by the forenamed morphological characteristics.  2) If RG1, R1, R2, R13, R14, G1 and G2 are excluded, each type can be further purified.  3) The excluded individuals are genetic materials as important as those remained in each type.  Generally, in the process of breeding by selection, tradi-tional approach of genotype identification depends on morphological differences. However, morphological iden-tification has several disadvantages such as large scale and long term field evaluation trials, environmental and developmental plasticity of the traits  and  limited  number  Xie et al.        4493      Figure 1. Dendrogram illustrating genetic relationships among 32 individuals of three morphological types of A. membranaceus var. mongholicus generated by UPGMA method based on SM similarity coefficients calculated on ISSR analysis (Individuals with R, RG and G belong to R, RG and G type, respectively).    of phenotypic makers available (Goulo et al., 2001; Manimekalai and Nagarajan, 2006). Furthermore, A. membranaceus var. mongholicus being a perennial herb, some morphological markers such as pod color are available for use until the sowed seeds grow at least for 2 years. On the contrary, molecular markers are available in unlimited numbers, irrespective of environment and the development stage of the plant and can be accomplished in a relatively shorter period (Goulo et al., 2001; Manimekalai and Nagarajan, 2006). Therefore, if conven-tional approach of selective breeding is assisted with appropriate molecular markers, reasonable and efficient strategies will be employed and consequently the period of artificial selection will be shortened. In this study, ISSR effectively revealed the levels of genetic diversity and differentiation in the 3 morphological types, which breed-ers would benefit from, suggesting ISSR is a potentially powerful and helpful tool for breeding program of A. membranaceus var. mongholicus.   ACKNOWLEDGMENTS   We thank associate professor Ying Yu of  Northwest A&F University in China for the English version of the manuscript. This study was supported by National Key Technology R&D Program of China (2007BAD64B04).   REFERENCES   Excoffier L, Smouse PE, Quattro JM (1992). Analysis of molecular variance inferred from metric distance among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics, 131: 479-491. Feng YX, Xiao PG (2002). Radix astragali. In: Xiao PG (ed) Modern Chinese Materia Medica, vol. 1. Chemical Industry Press, Beijing, pp. 876-893. Goulo L, Valdiviesso T, Santana C, Oliveria CM (2001). Comparison between phenetic characterization using RAPD and ISSR markers and phenotypic data of cultivated chestnut (Castanea sativa Mill.). Genet. Resour. Crop Evol. 48: 329-338. Joshi P, Dhawan V (2007). Analysis of genetic diversity among Swertia chirayita genotypes. Biol. Plant. 51: 764-768. Ma XQ, Duan JA, Zhu DY, Dong TTX, Tsim KWK (2000). Species identification of Radix Astragali (Huangqi) by DNA sequence of its 5S-rRNA spacer domain. Phytochemistry, 54: 363-368. Manimekalai R, Nagarajan P (2006). Assessing genetic relationships among coconut (Cocos nucifera L.) accessions using inter simple sequence repeat markers. Sci. Hortic. 108: 49-54. Nei M (1973). Analysis of gene diversity in subdivided population. Proc. Natl. Acad. Sci. USA, 70: 3321-3323. Pharmacopoeia   Commission   of  People’s Republic  of  China  (2005).  4494         Afr. J. Biotechnol.    Pharmacopoeia of the People’s Republic of China (vol. 1). Chemical Industry Press, Beijing, pp. 212-213. Qian W, Ge S, Hong DY (2001). Genetic variation within and among populations of a wild rice Oryza granulata from China detected by RAPD and ISSR markers. Theor. Appl. Genet. 102: 440-449. Rohlf FJ (1998). NTSYSpc Numerical Taxonomy and Multivariate Analysis System version 2.0 User Guide. Exeter Software, Setauket, New York. Tian CJ, Lei YD, Shi SH, Nan P, Chen JK, Zhong Y (2004). Genetic diversity of sea buckthorn (Hippophae rhamnoides) populations in northeastern and northewestern China as revealed by ISSR markers. New Forest, 27: 229-237. Wang S, Xie Y (2004). China Species Red List (vol. 1 Red List). Higher Education Press, Beijing, p. 343. Xie XL, Hu YP, Zhao XD, Wang L, Li Y (2007). Numerical analysis of esterase isozymes in seeds of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao cultivated in Longxi county. J. Grad. Sch. Chin. Acad. Sci. 24: 525-529.                                                           Xie XL, Wang XS, Zhao L, Wang L, Li Y (2004). Morphotype diversity of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao. J. Anhui Agric. Sci. 32: 1203-1204.  Yao H, Zhao Y, Chen DF, Chen JK, Zhou TS (2008). ISSR primer screening and preliminary evaluation of genetic diversity in wild populations of Glycyrrhiza uralensis. Biol. Plant, 52: 117-120. Yeh FC, Yang RC, Boyle T (1999). Microsoft Window-based Freeware for Population Genetic Analysis (POPGENE version 1.31). University of Alberta and Centre for International Forestry Research.  

Genetic diversity in three morphological types of Astragalus membranaceus (Fisch.) Bge. var.mongholicus (Bge.) Hsiao as revealed by inter simple

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Genetic diversity in three morphological types of Astragalus membranaceus (Fisch.) Bge. var.mongholicus (Bge.) Hsiao as revealed by inter simple

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