Biochemical Systematics and Ecology 51 (2013) 301–307

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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,*

aCollege of Agriculture, Inner Mongolia University for the Nationalities, Tongliao 028043, Chinab Tongliao Academy of Agricultural Science, Inner Mongolia, Tongliao 028015, ChinacCollege of Life Science, Inner Mongolia University for Nationalities, Tongliao 028043, Chinad Inner Mongolia Industrial Engineering Research Center of Universities for Castor, Tongliao 028043, China

a r t i c l e i n f o

Article history:Received 22 June 2013Accepted 28 September 2013Available online 22 October 2013

Keywords:Ricinus communis L.Genetic diversityISSRGene flow

* Corresponding author. College of Life Science, In0475 8314668.

E-mail addresses: chenys_2012@hotmail.com, w1 The author contributed equally to this work.

0305-1978/$ – see front matter � 2013 Elsevier Ltdhttp://dx.doi.org/10.1016/j.bse.2013.09.017

a b s t r a c t

Inter-Simple Sequence Repeat (ISSR) markers were employed to analyze the genetic di-versity of Ricinus communis L. in northeastern China plants. We selected ten primers thatproduced clear, reproducible and multiple bands for these experiments and 179 bandswere obtained across 39 genotypes. Polymorphic band ratios ranged from 100% to aminimum of 78.9% with an average of 96.4% while band numbers were comprised between13 (UBC823) and 23 (UBC856). The results obtained from UPGMA clustering dendrogramand PCoA lead to 39 distinct castor bean accessions belonging to four major groups. Wefound that all groups shared a common node with 66% similarity while Jaccard’s similaritycoefficient ranged from 0.58 to 0.92. Compatible inference was also observed from thehigh values of heterozygosity (Ht ¼ 0.3378 � 0.0218), Nei’s genetic diversity(H ¼ 0.1765 � 0.2090), and Shannon’s information index (I ¼ 0.4942 � 0.1872). In addition,our data reveal a Nei’s genetic differentiation index (GST) of 0.3452 and estimated the geneflow (Nm) at 0.9482. These findings clearly suggest a genetic diversity in castor beangermplasms from various geographic origins and contribute to our understanding ofbreeding 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-aridregions of the world (Govaerts et al., 2000). Castor bean oil constitutes more than 45% of seed weight and is used in a va-riety of industrial processes, including the manufacture of lubricants, pharmaceutical products, flavoring ingredients, andpaints.

Castor bean plants are grown in both northern and southern regions of China, the second largest castor bean producerafter India. According to their different growth requirements, castor beans are divided into annual (northeast China) andperennial (south of China) varieties (Chen and Huang, 2012). Wild castor beans are tall perennial tropical shrubs above 10 m

ner Mongolia University for Nationalities, Tongliao 028043, China. Tel.: þ86 0475 8314624; fax: þ86

ujing630951374@163.com (Y.-s. Chen).

. All rights reserved.

C. Wang et al. / Biochemical Systematics and Ecology 51 (2013) 301–307302

found in the southern provinces. In northern China, castor beans are annual as a result of cold weather, succumbing to frost inwinter and growing to only 2–4 m high in summer. Northeast R. communis L. is a cluster type plant with multiple shoot-budsand slender lateral roots (Huang et al., 2012) presenting green or purple stems and branches with wax powder and smallleaves. The raceme of the central stem has awide 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 notmature. The northeastern castor bean plant is classified as an early maturing variety, forming its first mature raceme 75–90days after seedling.

Recently, studies have been designed for assessment of genetic variation in castor bean idioplasms usingmolecular markertechniques (Allan et al., 2008; Gajera et al., 2010; Pecina-Quintero et al., 2013). Inter Simple Sequence Repeat (ISSR) markertechnology is based on the high variation of short repeats observed in plant populations (Sarwat, 2012; Xie et al., 2011). ISSRsshow high Genetic polymorphism, providing valuable site information and revealing the various microsatellite variationsbetween individuals. ISSR markers are dominant traits, following Mendelian patterns. ISSR marker technology combines theadvantages of Random Amplified Polymorphic DNA (RAPD) and simple sequence repeat (SSR), resulting in lower cost as wellas 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 ofgenetic improvement programs. Amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) wererecently used for genetic diversity analysis of 200 samples comprising 41 castor bean accessions from 35 countries (Pecina-Quintero et al., 2013). These authors speculated that SSR markers yield higher percentages of polymorphic loci, higherheterozygosity 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 north-eastern region of China, using ISSR markers. The data collected will contribute to identification, rational exploitation andconservation 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, includingHei Longjiang, Jilin, Liaoning and InnerMongolia provinces (Fig.1). Fresh leaves in squaring periodwere randomly collected ineach population and frozen in liquid nitrogen for molecular analyses. The detailed locations of the studied populations aresummarized in Table 1.

2.2. DNA extraction

Genomic DNAwas extracted by the CTAB method (Doyle and Doyle, 1987) with minor modifications. DNA concentrationsand purity were evaluated by electrophoresis on 0.8% agarose gels based on relative band intensities in comparison to the DL15000 Marker (TaKaRa, Japan). Finally, the DNA samples were diluted to 60 ng/ml and stored at �20 �C for ISSR analysis.

2.3. PCR amplification

A total of 100 ISSR primers were synthesized by BGI (China), according to the public biotechnology website of University ofBritish Columbia. These primers were used initially for amplification to optimize the PCR conditions, Ten (10) of them yieldedclear, reproducible and relatively high polymorphism bands (Table 2). ISSR-PCR amplifications were performed in 20 ml re-actions containing 60 ng genomic DNA templates, 1� PCR Buffer, 2.0 mMMgCl2, 0.25 mM of each dNTP, 10 pmol primers, and2 U of Taq DNA polymerase. Amplification reactions were performed on a PCR Thermal Cycler Dice (TaKaRa, Japan) under thefollowing conditions: initial denaturation at 94 �C for 5 min followed by 35 cycles of 94 �C for 1 min, annealing at optimaltemperature for 1 min and 72 �C for 3 min, and a final 10 min elongation step at 72 �C. The PCR products were analyzedelectrophoretically 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 amplification fragments were visualized and photo-graphed under UV light using a Gel Imaging & Analysis System (Beijing Beony Instrument, China) andmolecular weights wereestimated 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. Theamplified fragments were scored as ‘1’ for presence and ‘0’ for absence of the band for each primer genotype combination forthe ISSR analysis. Genetic parameters including percentage of polymorphic bands (PPB), observed number of alleles (Na),effective number of alleles (Ne), Nei’s gene diversity (H), Shannon’s index (I) (Nei, 1987), Nei’s genetic differentiation indexamong populations (GST) and gene flow (Nm) were calculated using POPGENE version 1.32 (Yeh et al., 1999). An estimate ofNm among populations was computed using the equationNm¼ 0.5(1� GST)/GST (Sork et al., 1999). A dendrogramwas createdusing the unweighted pair groupmethod with arithmetic average (UPGMA) with the SAHNmodule of NTSYS-pc version 2.10s

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

C. Wang et al. / Biochemical Systematics and Ecology 51 (2013) 301–307 303

to determine the Jaccard similarity coefficients (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 thegenetic distribution of individual accessions, respectively. Finally, genetic relationships were compared by visual examinationof the dendrogram (Waterman and Smith, 1978).

3. Results

The ten selected ISSR primers produced 179 bands, including 172 polymorphic, across 39 genotypes. This corresponds toan average of 17.2 polymorphic bands per primer. Percentages of polymorphic bands ranged from 100 to a minimum of 78.9with 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 forUPGMA analysis. A dendrogramwas generated from the ISSR data using the SAHN through UPGMA clusteringmethod (Fig. 2).As shown in Fig. 2, Jaccard’s similarity coefficients 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) forcastor bean populations from the Inner Mongolia province and lowest for plants from Jilin province (1.4413). The meaneffective number of alleles (Ne) for the 39 accessions was 1.5570. Highest and lowest Ne values were found in Inner Mongoliaprovince (1.4250) and Jilin province (1.3113) populations, respectively. Similarly, Nei’s gene diversity (H) and Shannon’s In-formation Index (I) found for Inner Mongolia province populations were the highest of the values determined in the fourpopulations (H ¼ 0.2540; I ¼ 0.3854).

Table 1List of the castor beans (Ricinus communis L.) included in the work.

No. ofaccession

Location Altitude(m)

Latitude(North)

Longitude(East)

Color ofplant stem

Fruit type Hundred kernelweight (g)

HL-1 Town Zhaodong, Province Hei Longjiang 146 46.05 125.96 Green Smooth Fruit 35.0HL-2 Town Zhaodong, Province Hei Longjiang 144 46.10 126.03 Green Lappa 31.9HL-3 Town Hulen, Province Hei Longjiang 152 46.15 126.84 Green Lappa 40.3HL-4 Town Hulen, Province Hei Longjiang 143 46.13 126.88 Green Lappa 29.7HL-5 Town Hulen, Province Hei Longjiang 161 46.16 126.77 Green Lappa 33.4HL-6 Town Hulen, Province Hei Longjiang 152 46.11 126.80 Green Lappa 32.6IM-1 Town Chifeng, Province Inner Mongolia 601 42.25 118.88 Green Lappa 27.1IM-2 Town Chifeng, Province Inner Mongolia 612 42.30 118.40 Green Lappa 37.5IM-3 Town Kulen, Province Inner Mongolia 263 42.73 121.77 Green Lappa 36.1IM-4 Town Kulen, Province Inner Mongolia 264 43.00 122.03 Green Lappa 31.7IM-5 Town Tongliao, Province Inner Mongolia 179 43.65 122.24 Green Lappa 29.8IM-6 Town Horqin, Province Inner Mongolia 180 44.05 122.02 Green Lappa 32.7IM-7 Town Horqin, Province Inner Mongolia 177 43.78 122.18 Green Smooth Fruit 30.5IM-8 Town Alu Horqin, Province Inner Mongolia 526 43.87 120.06 Green Lappa 31.7IM-9 Town Kailu, Province Inner Mongolia 239 43.60 121.31 Purple Lappa 31.0IM-10 Town Nimen, Province Inner Mongolia 366 42.86 120.65 Lavender Lappa 30.7IM-11 Town Nimen, Province Inner Mongolia 401 42.80 120.50 Purple Smooth Fruit 34.5IM-12 Town Tongliao, Province Inner Mongolia 179 43.65 122.24 Purple Lappa 30.9IM-13 Town Oliabe, Province Inner Mongolia 168 43.78 122.41 Purple Lappa 30.8IM-14 Town Horqin, Province Inner Mongolia 175 43.62 122.25 Green Lappa 31.3IM-15 Town Horqin, Province Inner Mongolia 188 44.71 122.65 Purple Smooth Fruit 31.0IM-16 Town Horqin, Province Inner Mongolia 148 44.02 121.95 Lavender Lappa 32.2IM-17 Town Horqin, Province Inner Mongolia 180 44.14 121.88 Red Lappa 29.6JL-1 Town Sanling, Province Jilin 187 44.27 123.96 Green Lappa 28.8JL-2 Town Suanglu, Province Jilin 133 43.51 123.50 Green Lappa 31.3JL-3 Town sipen, Province Jilin 164 43.16 124.35 Purple Lappa 30.4JL-4 Town Zhenlie, Province Jilin 138 43.78 127.49 Lavender Lappa 27.4LN-1 Town Janpin, Province Liaoning 578 41.40 119.64 Purple Lappa 34.5LN-2 Town Janpin, Province Liaoning 556 41.40 119.66 Green Lappa 36.3LN-3 Town Keiyun, Province Liaoning 90 42.54 124.03 Green Lappa 37.9LN-4 Town Keiyun, Province Liaoning 85 42.50 123.89 Green Lappa 27.4LN-5 Town Keiyun, Province Liaoning 91 42.40 124.10 Green Lappa 33.7LN-6 Town Keiyun, Province Liaoning 90 42.36 124.00 Purple Lappa 33.2LN-7 Town Panshen, Province Liaoning 4 41.24 121.99 Lavender Lappa 31.2LN-8 Town Seamain, Province Liaoning 29 42.00 122.81 Green Smooth Fruit 35.0LN-9 Town Seamain, Province Liaoning 43 42.15 123.00 Green Lappa 32.8LN-10 Town Seamain, Province Liaoning 40 42.00 122.59 Green Lappa 32.0LN-11 Town Xingcheng, Province Liaoning 5 40.61 120.72 Green Lappa 35.0LN-12 Town Zhouyin, Province Liaoning 18 41.02 120.08 Lavender Lappa 31.9

C. Wang et al. / Biochemical Systematics and Ecology 51 (2013) 301–307304

In the dendrogram, the 39 castor bean accessions were distinctly separated into four major groups. These four groupsshared 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 2with 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,HL-4 and IM-3, while HL-6, IM-8, IM-9 and IM-14 formed subgroup II2. The 17 genotypes within Group III were further dividedinto 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, JL-2 and IM-4were closely related, displaying a similarity coefficient of 0.92. The subgroup III2 consisted of LN-8, IM-17, IM-6, LN-5, 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, IM-11, IM-1 and IM-2.

Importantly, the PCoA clearly separated the 39 castor bean individuals into 4 distinct groups, using data from the ISSRmarkers (Fig. 3), in accordance with the UPGMA cluster analysis.

4. Discussion

ISSR is a quite efficient tool in exploring genetic variations and assessing diversity and has been widely used for identi-fication of germplasms in many plant species (Abdul Kareem et al., 2012; Hassanpour et al., 2013; Lv et al., 2012). To date, fewstudies 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 (upto 143), and the highest Shannon information index (0.3854) were found in castor bean accessions from Inner Mongoliaprovince (Table 3). Meanwhile, the lowest values were recorded in castor bean populations from Jilin province (PPB ¼ 44.1%,PB ¼ 79, I ¼ 0.2582). These data suggest that castor bean samples from Inner Mongolia province possess relatively higher

Table 2List of 10 primers selected from UBC (University of British Columbia) used for ISSR amplification.

Primer name Sequence 50–30 Tm (�C) No. of bandsscored

No. ofpolymorphic bands

Percentage ofpolymorphic markers (%)

Range of thebands size (bp)

UBC812 GAG AGA GAG AGA GAG AA 50.00 17 17 100 450–1500UBC823 TCT CTC TCT CTC TCT CC 52.00 13 13 100 480–2500UBC826 ACA CAC ACA CAC ACA CC 52.00 20 20 100 500–2500UBC846 CAC ACA CAC ACA CAC ART 53.00 19 18 94.7 250–2000UBC855 ACA CAC ACA CAC ACA CYT 53.00 16 16 100 250–2000UBC856 ACA CAC ACA CAC ACA CYA 53.00 23 22 95.6 250–2000UBC864 ATG ATG ATG ATG ATG ATG 48.00 18 18 100 450–2000UBC881 GGG TGG GGT GGG GTG 54.00 18 17 94.4 450–2000UBC885 BHB GAG AGA GAG AGA GA 51.33 16 16 100 500–2500UBC891 HVH TGT GTG TGT GTG TG 50.67 19 15 78.9 250–2000Total – – 179 172 – 200–2500Mean – – 17.9 17.2 96.36 –

Note: N ¼ (A, G, C, T), R ¼ (A, G), Y ¼ (C, T), B ¼ (C, G, T) (I. e. not A), H ¼ (A, C, T) (I. e. not G), V ¼ (A, C, G) (I. e. not T).

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genetic variation. The high genetic diversity could be attributed to a wide geographic distribution, which has been shown tostrongly correlate with the variation levels within a population (Hamrick and Godt, 1996). Indeed, similar data have beenreported for Piperia yadonii (George et al., 2009), Viola pubescens (Culley et al., 2007), Sapindaceae (Mahar et al., 2011). Thecastor bean plants IM-1, IM-2, IM-10 and IM-11 grew in the hills of the Yanshan Mountain, in complex topographic conditionsand relatively high altitudes (Tian, 2005) while castor bean samples from Inner Mongolia province mainly grew in the plainupstream of the West Liaohe River (Zhengkai et al., 2000). This geographical isolation might have promoted a high geneticdiversity in castor bean plants.

When the 39 castor bean accessions were analyzed for their ISSR markers, we found a Jaccard’s similarity coefficientranging from 0.58 to 0.92, demonstrating a relatively high diversity in these samples. A compatible inference could also bederived from the high values obtained for heterozygosity (Ht¼ 0.3378� 0.0218), Nei’s genetic diversity (H¼ 0.1765� 0.2090)and Shannon’s information index (I ¼ 0.4942 � 0.1872). Nei’s genetic differentiation index (GST) for ISSR was 0.3452, indi-cating that 34.5% of the total genetic variability was among populations and 65.5% within populations (Table 3). The estimateof gene flow (Nm, 0.9482) was less than 1, stating clearly that gene migration was limited among widely distributed pop-ulations (Slatkin, 1985, 1987). For castor bean germplasms from northeastern China, molecular differentiation might be verypronounced. Indeed gene exchange is limited by the geographical isolation among the castor bean plants, and their form ofpollination (pollen distributed by wind). On the other hand, human activities have also strongly contributed to the geneticdifferentiation of the castor bean accessions. Consequently, most types were geographically clustered (Kadmon and Pulliam,

Fig. 2. Dendrogram produced by Jaccard’s coefficient and UPGMA clustering based on ISSR in the 39 castor beans plants.

Table 3Summary of genetic variation statistics for all loci of ISSR.

Populationcode

Observed numberof alleles (Na)(mean � SD)

Effective numberof alleles (Ne)(mean � SD)

Nei’s (1973) genediversity (H)(mean � SD)

Shannon’sinformationindex (I) (mean � SD)

Total genediversity (Ht)(mean � SD)

Number ofpolymorphicloci (PB)

Percentage ofpolymorphicloci (PPB)

POP 1 1.4413 � 0.4979 1.3113 � 0.3874 0.1765 � 0.2090 0.2582 � 0.2999 0.1927 � 0.0490 79 44.13POP 2 1.7095 � 0.4553 1.3997 � 0.3721 0.2347 � 0.1949 0.3538 � 0.2728 0.2429 � 0.0360 127 70.95POP 3 1.7989 � 0.4020 1.4250 � 0.3502 0.2540 � 0.1838 0.3854 � 0.2552 0.2665 � 0.0345 143 79.89POP 4 1.5642 � 0.4972 1.3846 � 0.3917 0.2196 � 0.2092 0.3228 � 0.2987 0.2365 � 0.0473 101 56.42Mean 1.9609 � 0.1944 1.5570 � 0.3168 0.3285 � 0.1468 0.4942 � 0.1872 0.3378 � 0.0218 172 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.

C. Wang et al. / Biochemical Systematics and Ecology 51 (2013) 301–307306

1993). However, there were some varieties in the population structure where mutual penetration and interspersed distri-bution were more evident.

UPGMAof Jaccard’s similarity values and the PCoA clustering data separated the 39 castor bean samples fromnortheasternChina into four major distinct groups (Figs. 2 and 3), reflecting 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 temper-atures. Group III accessions were mainly found in the plain region of theWest Liaohe River, with barren gravel soil and scarcerainfall. The castor bean samples grown in the hills of Yanshan Mountain constituted Group IV (Fig. 1). These findings clearlyindicate a distinct differentiation between castor bean germplasms from various geographic origins. Grouping by the twoclustering methods revealed geographical affiliations (Nino-Vega et al., 2000). Therefore, differentiation of castor bean genepools from different regions might have resulted from reproductive isolation and divergent natural selection arising fromwide geographic separation.

Overall, the Inter Simple Sequence Repeats (ISSR) marker technology was an effective tool for studying gene poly-morphism in R. communis L. grown in northeastern China and our data suggest a high genetic diversity of castor beangermplasms. These findings 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 Edu-cation “new century excellent talent support plan” (NCET-08-0870).

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