Biochemical Systematics and Ecology 51 (2013) 301307

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Biochemical Systematics and Ecology

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Genetic diversity of castor bean (Ricinus communis L.) in Northeast China revealed by ISSR markers
Chao Wang a, d, Guo-rui Li c, d,1, Zhi-yong Zhang b, d, Mu Peng c, d, Yu-si Shang a, d, Rui Luo a, d, Yong-sheng Chen c, d, *
a

College of Agriculture, Inner Mongolia University for the Nationalities, Tongliao 028043, China Tongliao Academy of Agricultural Science, Inner Mongolia, Tongliao 028015, China c College of Life Science, Inner Mongolia University for Nationalities, Tongliao 028043, China d Inner Mongolia Industrial Engineering Research Center of Universities for Castor, Tongliao 028043, China
b

a r t i c l e i n f o
Article history: Received 22 June 2013 Accepted 28 September 2013 Available online 22 October 2013 Keywords: Ricinus communis L. Genetic diversity ISSR Gene ow

a b s t r a c t
Inter-Simple Sequence Repeat (ISSR) markers were employed to analyze the genetic diversity of Ricinus communis L. in northeastern China plants. We selected ten primers that produced clear, reproducible and multiple bands for these experiments and 179 bands were obtained across 39 genotypes. Polymorphic band ratios ranged from 100% to a minimum of 78.9% with an average of 96.4% while band numbers were comprised between 13 (UBC823) and 23 (UBC856). The results obtained from UPGMA clustering dendrogram and PCoA lead to 39 distinct castor bean accessions belonging to four major groups. We found that all groups shared a common node with 66% similarity while Jaccards similarity coefcient ranged from 0.58 to 0.92. Compatible inference was also observed from the high values of heterozygosity (Ht 0.3378 0.0218), Neis genetic diversity (H 0.1765 0.2090), and Shannons information index (I 0.4942 0.1872). In addition, our data reveal a Neis genetic differentiation index (GST) of 0.3452 and estimated the gene ow (Nm) at 0.9482. These ndings clearly suggest a genetic diversity in castor bean germplasms from various geographic origins and contribute to our understanding of breeding and conservation of castor beans. 2013 Elsevier Ltd. All rights reserved.

1. Introduction Ricinus communis L., commonly known as castor bean, is an industrial oil crop widely distributed in arid and semi-arid regions of the world (Govaerts et al., 2000). Castor bean oil constitutes more than 45% of seed weight and is used in a variety of industrial processes, including the manufacture of lubricants, pharmaceutical products, avoring ingredients, and paints. Castor bean plants are grown in both northern and southern regions of China, the second largest castor bean producer after India. According to their different growth requirements, castor beans are divided into annual (northeast China) and perennial (south of China) varieties (Chen and Huang, 2012). Wild castor beans are tall perennial tropical shrubs above 10 m

* Corresponding author. College of Life Science, Inner Mongolia University for Nationalities, Tongliao 028043, China. Tel.: 86 0475 8314624; fax: 86 0475 8314668. E-mail addresses: chenys_2012@hotmail.com, wujing630951374@163.com (Y.-s. Chen). 1 The author contributed equally to this work. 0305-1978/$ see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2013.09.017

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found in the southern provinces. In northern China, castor beans are annual as a result of cold weather, succumbing to frost in winter and growing to only 24 m high in summer. Northeast R. communis L. is a cluster type plant with multiple shoot-buds and slender lateral roots (Huang et al., 2012) presenting green or purple stems and branches with wax powder and small leaves. The raceme of the central stem has a wide cylindrical shape, is short and thinly scattered, with a low growing position. Most of the capsules have thorns growing on the long carpopodium and either dehiscing naturally or when they are not mature. The northeastern castor bean plant is classied as an early maturing variety, forming its rst mature raceme 7590 days after seedling. Recently, studies have been designed for assessment of genetic variation in castor bean idioplasms using molecular marker techniques (Allan et al., 2008; Gajera et al., 2010; Pecina-Quintero et al., 2013). Inter Simple Sequence Repeat (ISSR) marker technology is based on the high variation of short repeats observed in plant populations (Sarwat, 2012; Xie et al., 2011). ISSRs show high Genetic polymorphism, providing valuable site information and revealing the various microsatellite variations between individuals. ISSR markers are dominant traits, following Mendelian patterns. ISSR marker technology combines the advantages of Random Amplied Polymorphic DNA (RAPD) and simple sequence repeat (SSR), resulting in lower cost as well as required DNA amounts (Liu et al., 2008; Wang et al., 2009, 2013). Using RAPD and ISSR markers, Gajera et al. (2010) detected high genetic diversity mainly within castor bean accessions of genetic improvement programs. Amplied fragment length polymorphism (AFLP) and simple sequence repeat (SSR) were recently used for genetic diversity analysis of 200 samples comprising 41 castor bean accessions from 35 countries (PecinaQuintero et al., 2013). These authors speculated that SSR markers yield higher percentages of polymorphic loci, higher heterozygosity and a wider range of genetic distances among accessions than does AFLP markers. The present study aimed to examine the genetic variability within and among R. communis L. cultivated in the northeastern region of China, using ISSR markers. The data collected will contribute to identication, rational exploitation and conservation of germplasms of castor beans in northeastern China. 2. Materials and methods 2.1. Plant material A total of 39 populations were sampled throughout the distribution areas of R. communis L. in northeast China, including Hei Longjiang, Jilin, Liaoning and Inner Mongolia provinces (Fig. 1). Fresh leaves in squaring period were randomly collected in each population and frozen in liquid nitrogen for molecular analyses. The detailed locations of the studied populations are summarized in Table 1. 2.2. DNA extraction Genomic DNA was extracted by the CTAB method (Doyle and Doyle, 1987) with minor modications. DNA concentrations and purity were evaluated by electrophoresis on 0.8% agarose gels based on relative band intensities in comparison to the DL 15000 Marker (TaKaRa, Japan). Finally, the DNA samples were diluted to 60 ng/ml and stored at 20  C for ISSR analysis. 2.3. PCR amplication A total of 100 ISSR primers were synthesized by BGI (China), according to the public biotechnology website of University of British Columbia. These primers were used initially for amplication to optimize the PCR conditions, Ten (10) of them yielded clear, reproducible and relatively high polymorphism bands (Table 2). ISSR-PCR amplications were performed in 20 ml reactions containing 60 ng genomic DNA templates, 1 PCR Buffer, 2.0 mM MgCl2, 0.25 mM of each dNTP, 10 pmol primers, and 2 U of Taq DNA polymerase. Amplication reactions were performed on a PCR Thermal Cycler Dice (TaKaRa, Japan) under the following conditions: initial denaturation at 94  C for 5 min followed by 35 cycles of 94  C for 1 min, annealing at optimal temperature for 1 min and 72  C for 3 min, and a nal 10 min elongation step at 72  C. The PCR products were analyzed electrophoretically on 3.0% (w/v) agarose gels in 1 TAE Buffer at 90 V for 1.5 h. A total of 1 ml 6 loading buffer (TaKaRa, Japan) was added to each reaction before electrophoresis. Gels with amplication fragments were visualized and photographed under UV light using a Gel Imaging & Analysis System (Beijing Beony Instrument, China) and molecular weights were estimated based on the DL 2000 Marker (TaKaRa, Japan). 2.4. Data analysis Each primer was used to amplify all samples twice and the resulting band patterns showed good reproducibility. The amplied fragments were scored as 1 for presence and 0 for absence of the band for each primer genotype combination for the ISSR analysis. Genetic parameters including percentage of polymorphic bands (PPB), observed number of alleles (Na), effective number of alleles (Ne), Neis gene diversity (H), Shannons index (I) (Nei, 1987), Neis genetic differentiation index among populations (GST) and gene ow (Nm) were calculated using POPGENE version 1.32 (Yeh et al., 1999). An estimate of Nm among populations was computed using the equation Nm 0.5(1 GST)/GST (Sork et al., 1999). A dendrogram was created using the unweighted pair group method with arithmetic average (UPGMA) with the SAHN module of NTSYS-pc version 2.10s

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Fig. 1. A distribution map showing the 39 castor beans accessions (black dots) collected from northeast China in the present study.

to determine the Jaccard similarity coefcients (Rohlf, 1992; Strimmer and Von Haeseler, 1996). Principle coordinate analysis (PCoA) and the NTSYS-pc version 2.10s statistical package were used to generate 3D scatter plots and for the evaluation of the genetic distribution of individual accessions, respectively. Finally, genetic relationships were compared by visual examination of the dendrogram (Waterman and Smith, 1978). 3. Results The ten selected ISSR primers produced 179 bands, including 172 polymorphic, across 39 genotypes. This corresponds to an average of 17.2 polymorphic bands per primer. Percentages of polymorphic bands ranged from 100 to a minimum of 78.9 with an average of 96.4 (Table 2). The fragments sizes varied from 200 to 2500 bp and the number of bands ranged from 13 (UBC823) to 23 (UBC856). The ISSR bands were scored for presence (1) or absence (0) among the genotypes and used for UPGMA analysis. A dendrogram was generated from the ISSR data using the SAHN through UPGMA clustering method (Fig. 2). As shown in Fig. 2, Jaccards similarity coefcients ranged from 0.58 to 0.92. In addition, we found a mean number of alleles (Na) of 1.9609 in the 39 castor bean accessions from northeastern China (Table 3). Interestingly, Na was highest (1.7989) for castor bean populations from the Inner Mongolia province and lowest for plants from Jilin province (1.4413). The mean effective number of alleles (Ne) for the 39 accessions was 1.5570. Highest and lowest Ne values were found in Inner Mongolia province (1.4250) and Jilin province (1.3113) populations, respectively. Similarly, Neis gene diversity (H) and Shannons Information Index (I) found for Inner Mongolia province populations were the highest of the values determined in the four populations (H 0.2540; I 0.3854).

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Table 1 List of the castor beans (Ricinus communis L.) included in the work. No. of accession HL-1 HL-2 HL-3 HL-4 HL-5 HL-6 IM-1 IM-2 IM-3 IM-4 IM-5 IM-6 IM-7 IM-8 IM-9 IM-10 IM-11 IM-12 IM-13 IM-14 IM-15 IM-16 IM-17 JL-1 JL-2 JL-3 JL-4 LN-1 LN-2 LN-3 LN-4 LN-5 LN-6 LN-7 LN-8 LN-9 LN-10 LN-11 LN-12 Location Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Town Zhaodong, Province Hei Longjiang Zhaodong, Province Hei Longjiang Hulen, Province Hei Longjiang Hulen, Province Hei Longjiang Hulen, Province Hei Longjiang Hulen, Province Hei Longjiang Chifeng, Province Inner Mongolia Chifeng, Province Inner Mongolia Kulen, Province Inner Mongolia Kulen, Province Inner Mongolia Tongliao, Province Inner Mongolia Horqin, Province Inner Mongolia Horqin, Province Inner Mongolia Alu Horqin, Province Inner Mongolia Kailu, Province Inner Mongolia Nimen, Province Inner Mongolia Nimen, Province Inner Mongolia Tongliao, Province Inner Mongolia Oliabe, Province Inner Mongolia Horqin, Province Inner Mongolia Horqin, Province Inner Mongolia Horqin, Province Inner Mongolia Horqin, Province Inner Mongolia Sanling, Province Jilin Suanglu, Province Jilin sipen, Province Jilin Zhenlie, Province Jilin Janpin, Province Liaoning Janpin, Province Liaoning Keiyun, Province Liaoning Keiyun, Province Liaoning Keiyun, Province Liaoning Keiyun, Province Liaoning Panshen, Province Liaoning Seamain, Province Liaoning Seamain, Province Liaoning Seamain, Province Liaoning Xingcheng, Province Liaoning Zhouyin, Province Liaoning Altitude (m) 146 144 152 143 161 152 601 612 263 264 179 180 177 526 239 366 401 179 168 175 188 148 180 187 133 164 138 578 556 90 85 91 90 4 29 43 40 5 18 Latitude (North) 46.05 46.10 46.15 46.13 46.16 46.11 42.25 42.30 42.73 43.00 43.65 44.05 43.78 43.87 43.60 42.86 42.80 43.65 43.78 43.62 44.71 44.02 44.14 44.27 43.51 43.16 43.78 41.40 41.40 42.54 42.50 42.40 42.36 41.24 42.00 42.15 42.00 40.61 41.02 Longitude (East) 125.96 126.03 126.84 126.88 126.77 126.80 118.88 118.40 121.77 122.03 122.24 122.02 122.18 120.06 121.31 120.65 120.50 122.24 122.41 122.25 122.65 121.95 121.88 123.96 123.50 124.35 127.49 119.64 119.66 124.03 123.89 124.10 124.00 121.99 122.81 123.00 122.59 120.72 120.08 Color of plant stem Green Green Green Green Green Green Green Green Green Green Green Green Green Green Purple Lavender Purple Purple Purple Green Purple Lavender Red Green Green Purple Lavender Purple Green Green Green Green Purple Lavender Green Green Green Green Lavender Fruit type Smooth Lappa Lappa Lappa Lappa Lappa Lappa Lappa Lappa Lappa Lappa Lappa Smooth Lappa Lappa Lappa Smooth Lappa Lappa Lappa Smooth Lappa Lappa Lappa Lappa Lappa Lappa Lappa Lappa Lappa Lappa Lappa Lappa Lappa Smooth Lappa Lappa Lappa Lappa Fruit Hundred kernel weight (g) 35.0 31.9 40.3 29.7 33.4 32.6 27.1 37.5 36.1 31.7 29.8 32.7 30.5 31.7 31.0 30.7 34.5 30.9 30.8 31.3 31.0 32.2 29.6 28.8 31.3 30.4 27.4 34.5 36.3 37.9 27.4 33.7 33.2 31.2 35.0 32.8 32.0 35.0 31.9

Fruit

Fruit

Fruit

Fruit

In the dendrogram, the 39 castor bean accessions were distinctly separated into four major groups. These four groups shared a common node at 66% similarity (Fig. 2). Group I included four samples from Liaoning province (LN-7, LN-4, LN-6 and LN-11). Group II was further subdivided into 2 with 8 samples in subgroup II1 and 4 samples in subgroup II2. The subgroup II1 included JL-4, HL-1, HL-3, HL-5, IM-13, HL-2, HL4 and IM-3, while HL-6, IM-8, IM-9 and IM-14 formed subgroup II2. The 17 genotypes within Group III were further divided into two subgroups. The subgroup III1 comprised JL-1, IM-7, LN-3, JL-3, JL-2, IM-4, IM-5 and IM-12. Within the subgroup III1, JL2 and IM-4 were closely related, displaying a similarity coefcient of 0.92. The subgroup III2 consisted of LN-8, IM-17, IM-6, LN5, LN-12, LN-9, LN-10, IM-16 and IM-15. The last group IV included the remaining 6 genotypes including LN-1, LN-2, IM-10, IM11, IM-1 and IM-2. Importantly, the PCoA clearly separated the 39 castor bean individuals into 4 distinct groups, using data from the ISSR markers (Fig. 3), in accordance with the UPGMA cluster analysis. 4. Discussion ISSR is a quite efcient tool in exploring genetic variations and assessing diversity and has been widely used for identication of germplasms in many plant species (Abdul Kareem et al., 2012; Hassanpour et al., 2013; Lv et al., 2012). To date, few studies have applied this technology to study the genetic diversity of the castor bean plants (Allan et al., 2008; Gajera et al., 2010; Pecina-Quintero et al., 2013). Using ISSR marker analyses, highest percentage of polymorphic loci (up to 79.9%), largest number of polymorphic loci (up to 143), and the highest Shannon information index (0.3854) were found in castor bean accessions from Inner Mongolia province (Table 3). Meanwhile, the lowest values were recorded in castor bean populations from Jilin province (PPB 44.1%, PB 79, I 0.2582). These data suggest that castor bean samples from Inner Mongolia province possess relatively higher

C. Wang et al. / Biochemical Systematics and Ecology 51 (2013) 301307 Table 2 List of 10 primers selected from UBC (University of British Columbia) used for ISSR amplication. Primer name UBC812 UBC823 UBC826 UBC846 UBC855 UBC856 UBC864 UBC881 UBC885 UBC891 Total Mean Sequence 50 30 GAG AGA GAG AGA GAG AA TCT CTC TCT CTC TCT CC ACA CAC ACA CAC ACA CC CAC ACA CAC ACA CAC ART ACA CAC ACA CAC ACA CYT ACA CAC ACA CAC ACA CYA ATG ATG ATG ATG ATG ATG GGG TGG GGT GGG GTG BHB GAG AGA GAG AGA GA HVH TGT GTG TGT GTG TG Tm ( C) 50.00 52.00 52.00 53.00 53.00 53.00 48.00 54.00 51.33 50.67 No. of bands scored 17 13 20 19 16 23 18 18 16 19 179 17.9 No. of polymorphic bands 17 13 20 18 16 22 18 17 16 15 172 17.2 Percentage of polymorphic markers (%) 100 100 100 94.7 100 95.6 100 94.4 100 78.9 96.36

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Range of the bands size (bp) 4501500 4802500 5002500 2502000 2502000 2502000 4502000 4502000 5002500 2502000 2002500

Note: N (A, G, C, T), R (A, G), Y (C, T), B (C, G, T) (I. e. not A), H (A, C, T) (I. e. not G), V (A, C, G) (I. e. not T).

genetic variation. The high genetic diversity could be attributed to a wide geographic distribution, which has been shown to strongly correlate with the variation levels within a population (Hamrick and Godt, 1996). Indeed, similar data have been reported for Piperia yadonii (George et al., 2009), Viola pubescens (Culley et al., 2007), Sapindaceae (Mahar et al., 2011). The castor bean plants IM-1, IM-2, IM-10 and IM-11 grew in the hills of the Yanshan Mountain, in complex topographic conditions and relatively high altitudes (Tian, 2005) while castor bean samples from Inner Mongolia province mainly grew in the plain upstream of the West Liaohe River (Zhengkai et al., 2000). This geographical isolation might have promoted a high genetic diversity in castor bean plants. When the 39 castor bean accessions were analyzed for their ISSR markers, we found a Jaccards similarity coefcient ranging from 0.58 to 0.92, demonstrating a relatively high diversity in these samples. A compatible inference could also be derived from the high values obtained for heterozygosity (Ht 0.3378 0.0218), Neis genetic diversity (H 0.1765 0.2090) and Shannons information index (I 0.4942 0.1872). Neis genetic differentiation index (GST) for ISSR was 0.3452, indicating that 34.5% of the total genetic variability was among populations and 65.5% within populations (Table 3). The estimate of gene ow (Nm, 0.9482) was less than 1, stating clearly that gene migration was limited among widely distributed populations (Slatkin, 1985, 1987). For castor bean germplasms from northeastern China, molecular differentiation might be very pronounced. Indeed gene exchange is limited by the geographical isolation among the castor bean plants, and their form of pollination (pollen distributed by wind). On the other hand, human activities have also strongly contributed to the genetic differentiation of the castor bean accessions. Consequently, most types were geographically clustered (Kadmon and Pulliam,

Fig. 2. Dendrogram produced by Jaccards coefcient and UPGMA clustering based on ISSR in the 39 castor beans plants.

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Table 3 Summary of genetic variation statistics for all loci of ISSR. Population code POP 1 POP 2 POP 3 POP 4 Mean Observed number of alleles (Na) (mean SD) 1.4413 1.7095 1.7989 1.5642 1.9609 0.4979 0.4553 0.4020 0.4972 0.1944 Effective number of alleles (Ne) (mean SD) 1.3113 1.3997 1.4250 1.3846 1.5570 0.3874 0.3721 0.3502 0.3917 0.3168 Neis (1973) gene diversity (H) (mean SD) 0.1765 0.2347 0.2540 0.2196 0.3285 0.2090 0.1949 0.1838 0.2092 0.1468 Shannons information index (I) (mean SD) 0.2582 0.3538 0.3854 0.3228 0.4942 0.2999 0.2728 0.2552 0.2987 0.1872 Total gene diversity (Ht) (mean SD) 0.1927 0.2429 0.2665 0.2365 0.3378 0.0490 0.0360 0.0345 0.0473 0.0218 Number of polymorphic loci (PB) 79 127 143 101 172 Percentage of polymorphic loci (PPB) 44.13 70.95 79.89 56.42 96.09

Note: POP1, POP 2, POP 3, POP 4 represented the accessions collected from Jilin, Liaoning, Inner Mongolia and Hei Longjiang provinces respectively.

Fig. 3. Principal coordinates analysis (PCoA) for the ISSR evaluation of the castor beans germplasm.

1993). However, there were some varieties in the population structure where mutual penetration and interspersed distribution were more evident. UPGMA of Jaccards similarity values and the PCoA clustering data separated the 39 castor bean samples from northeastern China into four major distinct groups (Figs. 2 and 3), reecting the geographic distribution patterns of these populations (Iwatsuki, 1972). Group I represented the accessions cultivated on the Liaodong Peninsula in relatively abundant rainfall. Group II mainly included the accessions from the Songnen Plain with fertile soil and extremely low mean annual temperatures. Group III accessions were mainly found in the plain region of the West Liaohe River, with barren gravel soil and scarce rainfall. The castor bean samples grown in the hills of Yanshan Mountain constituted Group IV (Fig. 1). These ndings clearly indicate a distinct differentiation between castor bean germplasms from various geographic origins. Grouping by the two clustering methods revealed geographical afliations (Nino-Vega et al., 2000). Therefore, differentiation of castor bean gene pools from different regions might have resulted from reproductive isolation and divergent natural selection arising from wide geographic separation. Overall, the Inter Simple Sequence Repeats (ISSR) marker technology was an effective tool for studying gene polymorphism in R. communis L. grown in northeastern China and our data suggest a high genetic diversity of castor bean germplasms. These ndings could contribute to breeding and conservation of castor beans. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31060194) and the Ministry of Education new century excellent talent support plan (NCET-08-0870). References
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