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Genetic variations within a collection of anthuriums unraveled by morphological traits and AFLP markers
Biochemical Systematics and Ecology 45 (2012) 34–40
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Genetic variations within a collection of anthuriums unraveled by morphological traits and AFLP markers Yaying Ge 1, Fei Zhang 1, Xiaolan Shen, Yongming Yu, Xiaoyun Pan, Xiaojing Liu, Jianxin Liu, Gangmin Pan, Danqing Tian* Flower Research and Development Centre, Zhejiang Academy of Agricultural Sciences, No. 79 Tangwan, Xiaoshan District, Hangzhou 311202, Zhejiang Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 May 2012 Accepted 7 July 2012 Available online 4 August 2012
In this study, genetic variations among a collection of anthuriums were comparatively investigated using morphological traits and amplified fragment length polymorphism (AFLP) markers. Both morphological and AFLP-based data clustered the accessions from Anthurium andraeanum, Anthurium scherzerianum, and Anthurium clarinervium into three separate groups, with a significant though low correlation (r ¼ 0.3) observed between the morphological and AFLP-based similarity matrices. The AFLP-based principal coordinate analysis (PCoA) divided the entire accessions into three parts, reinforcing the AFLP-based clustering. Moreover, the Bayesian-based structuring assigned the anthurium accessions to three subgroups but with 27 accessions retained in the admixed groups, probably implying the highly heterozygous genome of the investigated anthuriums. The findings of this study would add new knowledge of genetic diversity among anthuriums as well as embark on a useful beginning for a rational hybridization breeding for anthuriums in future. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Anthurium Genetic diversity Population structure Morphology AFLP
1. Introduction Anthurium (Areaceae), native to Central and South America, is one of the most popular ornamental crops. The attractive characteristics including vibrant inflorescence with straight spathe, candle-like spadix, exotic foliage, and particularly the long lasting ‘flower’ of anthurium have ensured its great commercial importance, and therefore anthuriums have currently occupied a large part in flower industry, especially in terms of cut-flower and potted ornamentals. Now, demand for new good quality varieties of anthuriums is increasing. Many attempts have been made to increase the diversity of anthuriums to meet the preferences of different groups of people (Elibox and Umaharan, 2008; Avila-Rostant et al., 2010). Complete knowledge of genetic diversity is indispensable for efficient utilization of genetic resources and effective breeding. By far many studies have attempted to address the genetic diversity of anthurium accessions (Ranamukhaarachchi et al., 2001; Acosta-Mercao et al., 2002; Devanand et al., 2004; Nowbuth et al., 2005; Andrade et al., 2009; Yasin and Mayadevi, 2010; Gantait and Sinniah, 2011). However, anthurium germpalsms could by no means be thoroughly characterized due to the localized and limited samples in the previous published articles, and therefore necessary to assess the genetic variability within a specific collection of anthuriums.
* Corresponding author. Tel.: þ86 571 82713881; fax: þ86 571 82721053. E-mail address: [email protected] (D. Tian). 1 The authors contributed equally to this work. 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.07.023
Y. Ge et al. / Biochemical Systematics and Ecology 45 (2012) 34–40
35
In the present study, we aimed to address the genetic variability of the commercial varieties currently cultivated in China and recently selected hybrids of anthurium based on morphological traits and AFLP markers. The findings of this study will add a new knowledge with respect to genetic diversity of anthuriums and therefore facilitate a rational breeding design in future. 2. Materials and methods 2.1. Plant materials and phenotyping In this study, 60 accessions of anthuriums, a representative of the commercially cultivated gene pools in China, were selected. Of the 60 accessions, 57 accessions were from Anthurium andraeanum, two from Anthurium scherzerianum, and one from Anthurium clarinervium. In addition, the cultivated varieties were encoded with a suffix An, and the recently selected hybrids with a suffix S (Table 1). An4 and An44 are A. scherzerianum and An6 is A. clarinervium. For the S-coded accessions, S1–S3 were selected hybrids of An27 An32, and S4 and S5 hybrids of An36 An37, S6 and S7 hybrids of An36 An17, S8 hybrids of An38 An45, S9 hybrids of An32 An15, and An10 hybrids of An3 An15. The anthurium samples were collected from various breeders or production companies, and cultivated as potted flowers in the semi-controlled greenhouse at Flower Research and Development Centre, Zhejiang Academy of Agricultural Sciences, China. A total of nine phenotypic traits were recorded for the flowering plants (Table 2). A summary of the investigated traits is depicted in Table S1. 2.2. DNA isolation and AFLP profile Young leaves were collected from each accession, frozen with liquid nitrogen and grinded into powder. Genomic DNA was isolated following a CTAB-based procedure (Murray and Thompson, 1980). DNA concentration was estimated in comparison with known concentrations of Lambda DNA in 0.8% agarose gel. AFLP analyses were performed as described by Vos et al. (1995) with some modifications. The pre-amplification products were diluted 20-fold and were used as a template for the selective amplification. Selective amplification was conducted by using fluorescently labeled EcoRI or Pstl primers. A total of 48 selective primers of EcoRI þ 3/Pstl þ 3 were initially applied to screen polymorphisms using the DNA of four randomly chosen accessions; and consequently eight polymorphic primer combinations were chosen to genotype the whole accessions. Amplification was conducted on a Gene Amp PCR System 9600 (Perkin Elmer, USA). PCR products were mixed with loading buffer and loaded on 4% polyacrylamide gels after heat
Table 1 The investigated accessions of anthuriums in this study. Code
Accession
Botanical classification
Code
Accession
Botanical classification
An1 An2 An3 An4 An5 An6 An7 An8 An9 An10 An11 An12 An13 An14 An15 An16 An17 An18 An19 An20 An21 An22 An23 An24 An25 An26 An27 An28 An29 An30
Alabamb Pink Champion Dakota Artus Altimo Species Baleno Fantasy Love Red Love Butterfly Dream Amalia Red Fiesta Red Queen White Angel Big Beauty Beauty Silence Ping Love Robino Adios Kiss True Love Secret Love Classie Red Red Victory Nathalive Yukon Paradi Feroza Season Latino
A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A.
An31 An32 An33 An34 An35 An36 An37 An38 An39 An40 An41 An42 An43 An44 An45 An46 An47 An48 An49 An50 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10
Coattails Red Sempre Poncho Burning Love Tricolore Happy Purple Flag Mystral Sierra Tender Love Manbu Music Red Fire Lybra Beijing Success Red Crown Fiorino Micra Chico Green Tucano Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid
A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A.
andraeanum andraeanum andraeanum scherzerianum andraeanum clarinervium andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum
andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum scherzerianum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum
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Y. Ge et al. / Biochemical Systematics and Ecology 45 (2012) 34–40 Table 2 The morphological traits recorded for the investigated anthurium accessions in this study. Trait
Value
Leaf type Leaf color Peduncle color Spathe size Spathe type Spathe glossiness Spathe color Inflorescence type Tillering capacity
Heart shaped; long-heart shaped; triangle; oval; pointed oval; lanceolate; stripe Dark green; green; light green Dark green; moderate green; light green; green-red; dark red; red; light red Large; medium; small Oval; heart shaped; long-heart shaped; round; willow-leaf shaped Bright; moderate; dark Pink; red; orange; green; white; dark red; purplish red; orange red Up-right; inclined; twisty; arc-shaped Good; moderate; poor
denaturation. The products were fractionated on PRISM 377 sequencer (ABI) using electrophoresis. The internal 70–500 size standard ROX 500 (ABI) was run with the samples to estimate the size of fragments. All AFLP fragments were scored dominantly and recorded in a 0/1-matrix for the peak absence/presence along with their sizes. The binary scores were manually compared with the electropherograms to re-confirm the presence or absence of peaks. 2.3. Statistical analysis Two dendrograms were separately constructed based on the phenotype- and AFLP-based genetic similarity using unweighted pair group method with arithmetic average (UPGMA) method with the SAHN module of NTSYS-pc 2.2 (Rohlf, 2005), and the normalized Mantel statistic Z test (Mantel, 1967) was used to determine the level of association between the two similarity matrices. Then based on the AFLP data, the principal coordinated analysis (PCoA) was performed to show the differentiation of the bromeliad accessions in a two-dimensional array of eigenvectors using the DCENTER and EIGEN modules of NTSYS-pc. Lastly, a Bayesian-based clustering implemented in STRUCTURE software version 2.3.3 with admixture (Pritchard et al., 2000) was performed on the AFLP data to better delineate population substructures of genetically similar accessions. STURCUTURE was run 5 times independently with the number of subgroups (K) ranging from 1 to 15. Each run implemented with a burn-in period of 100,000 steps followed by 100,000 Monte Carlo Markov Chain replicates (Hubisz et al., 2009). That run with the maximum likelihood was applied to subdivide the accessions into different subgroups with the membership probabilities threshold 0.80 as well as the maximum membership probability among subgroups. Those accessions <0.80 membership probabilities were retained in the admixed group. An ad hoc measure DK proposed by Evanno et al. (2005) was used to detect the true K present in the SRAP marker data. 3. Results 3.1. Morphological clustering Based on the nine investigated morphological traits, pair-wise similarity coefficient varied from 0.65 to 1.0. The sixty anthurium accessions were classified into three main groups (I, II, III) at similarity coefficient of 0.70 (Fig. 1). Group I consisted of all accessions from A. andraeanum, group II the two accessions from A. scherzerianum, and group III the accession from A. clarinervium, well concordant with their botanical classification as depicted in Table 1. The group I could be further divided into six sub-clusters, whereas no correlation was found between sub-clusters and morphology. 3.2. Molecular characterization Out of the 48 primer combinations, eight selective primers generating a clear and informative pattern were chosen for genotyping. As a result, a total of 1467 fragments were produced, of which 1459 (99.45%) were polymorphic (Table 3). The number of polymorphic bands for each primer combination varied between 164 and 199 and averaged w182. Based on the AFLP marker data, the similarity coefficient ranged from 0.68 to 0.88. A dendrogram based on this similarity matrix is shown in Fig. 2. In this dendrogram, the whole accessions were grouped into three main clusters (I, II, III) at similarity coefficient of 0.73, well in concordance with their botanical classification as well. Cluster I could be also divided into several sub-clusters, whereas with no significant correlation between sub-clusters and morphology either. Results from the principal coordinate analysis (PCoA) revealed that the first three coordinates accounted for 16.83% of the molecular variations. According to the two-dimensional plots, the 60 anthurium accessions generally dispersed in threecentered parts (Fig. 3), reinforcing the clustering of the accessions from A. andraeanum. The pattern of genetic diversity and population structure of the investigated anthuriums was further characterized with a Bayesian-based approach. The peak value for the ad hoc quantity with respect to DK was observed for K ¼ 3 (Fig. 4); as a result, the 60 anthurium accessions were assigned to three subgroups (Fig. 5). Within the membership probability 0.80,
Y. Ge et al. / Biochemical Systematics and Ecology 45 (2012) 34–40
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Fig. 1. Dendrogram of the 60 anthurium accessions, derived from the UPGMA cluster analysis based on phenotype-based similarity coefficients. The accession codes An4 and An44 are A. scherzerianum, An6 is A. clarinervium, and the others are A. andraeanum. For details please see Table 1.
sub-group A included nine accessions, sub-group B 18 accessions and sub-group C six accessions, with the remained 27 accessions retained in the admixed groups (Fig. 5, Table S2). 3.3. Mantel test between similarity matrices To compare the morphological and AFLP marker based similarity matrices, the Mantel test was performed. The result indicated a significant but low correlation (r ¼ 0.30) between the two matrices. 4. Discussion A complete knowledge of genetic variability among crop germplasm is a prerequisite for rational management of genetic resources as well as for successful crop improvement (Gowda et al., 2011). Recently, some efforts have been focused on the genetic diversity and relationship among anthuriums. Ranamukhaarachchi et al. (2001) identified the genetic relationship of nine potted anthuriums with RAPD markers. Yasin and Mayadevi (2010) reported a high genetic diversity among 12 commercial varieties of A. andraeanum using RAPD markers. Nowbuth et al. (2005) assessed the genetic variations among 24 cut-flower cultivars of A. andraeanum with RAPD markers. Devanand et al. (2004) fingerprinted a relatively large sample of 58 accessions of container-grown anthuriums with AFLP markers. In addition, the genetic variations in natural populations of Anthurium species were also characterized (Acosta-Mercao et al., 2002; Andrade et al., 2009). The findings of these reports add a knowledge regarding the genetic variation of anthuriums. Morphological traits are of great importance in selecting rational parents for hybridization breeding and have also been applied to assess genetic diversity of many crops (Beyene et al., 2005; Finger et al., 2010). In this study, the nine investigated Table 3 Amplified result of AFLP analysis for the 60 accessions of cultivated anthuriums. Primers
Total bands
Polymorphic bands
% of polymorphic bands
E-AAC/M-CAA E-AAC/M-CTA E-AAC/M-CTG E-AAG/M-CAA E-ACA/M-CTT E-ACC/M-CAC E-ACC/M-CAG E-ACC/M-CTC Total Average
184 186 180 200 182 165 185 185 1467 183.38
183 184 179 199 180 164 185 185 1459 182.38
99.45 98.92 99.44 99.50 98.90 99.39 100 100 – 99.45
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Y. Ge et al. / Biochemical Systematics and Ecology 45 (2012) 34–40
Fig. 2. Dendrogram of the 60 anthurium accessions, derived from the UPGMA cluster analysis based on AFLP-based similarity coefficients. The accession codes An4 and An44 are A. scherzerianum, An6 is A. clarinervium, and the others are A. andraeanum. For details please see Table 1.
morphological traits grouped the sixty anthuriums into three main clusters consonant with their botanical classifications. However, the morphological traits hardly separated the accessions of A. andraeanum grouped in cluster I (Table 1). Probably this implies the phenotypic similarity of accessions from A. andraeanum and difficulty in identifying their genetic diversity via morphology, thereby consistent with the earlier study by Nowbuth et al. (2005).
Fig. 3. Matrix plot of the first two coordinates of principal coordinate analysis (PCoA) based on the AFLP data, showing associations among the investigated anthuriums accessions. The accession codes An4 and An44 are A. scherzerianum, An6 is A. clarinervium, and the others are A. andraeanum. For details please see Table 1.
Y. Ge et al. / Biochemical Systematics and Ecology 45 (2012) 34–40
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Fig. 4. Magnitude of DK from structure analysis as a function of K, calculated following the DK methods as proposed by Evanno et al. (2005), for the investigated anthuriums based on the AFLP data. The modal value of these distributions indicates the true K or the uppermost level of structure, here three subgroups.
AFLP markers were proven to be an effective approach to characterizing genetic variation of many crops including anthuriums (Devanand et al., 2004; Andrade et al., 2009). In this study, the eight primers produced a total of 1459 (99.45%) polymorphic markers, indicative of a high level of genotypic variations among the investigated anthurium accessions. Comparatively, lower similarity coefficients (0.68–0.88) were observed in this study rather than in the former reports (Ranamukhaarachchi et al., 2001; Devanand et al., 2004; Nowbuth et al., 2005). This also suggests the high genetic diversity within anthuriums in this study. The AFLP-based clustering grouped the whole accessions herein into three main groups, reinforcing the morphological clustering and the botanical classifications. Several previous literatures have reported the lack of correlation between the two clustering based on morphological traits and molecular markers (Beyene et al., 2005; Solouki et al., 2008; Finger et al., 2010; Pham et al., 2011). In this study, the morphology and AFLP-based clustering analyses unequivocally differentiated the sixty anthuriums into three main groups, both well corresponding to their botanical classifications. However, a significant but low correlation was observed between the similarity matrices. This is probably due to the discordance of the clustering of the accessions from A. andraeanum that are largely resulted from the difference in neutrality between morphological traits and AFLP markers. Additionally, the ten selected hybrids (S1–S10) included in this study were not grouped together with their parents. Recently, Gawenda et al. (2011) reported that the new selected hybrids of Phalaenopsis orchids were inclined to cluster together; Zhang et al. (2012) confirmed that AFLP marker rather than pedigree information is more effective in quantifying the genetic relatedness for Aechmea species or hybrids. In this work, the failure to distinctly identify the relationship between the hybrids and parents for anthuriums suggested to some extent their complex genetic backgrounds with several clones or species caused by intense hybridization. To further unravel the genetic diversity and population structure of cultivated anthuriums, a Bayesian-based approach implemented in STURCTURE was adopted. The structuring assigned 33 accessions to three subgroups, whereas with the other 27 accessions retained in the admixed groups. Obviously this indicates the presence of highly heterozygous genome of the investigated anthuriums here. This cause should be attributed to the intense hybridization breeding works for anthuriums during the last decades. Nowadays, the conventional hybridization is one of the most effective approaches to breed new varieties for anthuriums though the advents of some new molecular breeding methods. Crossing between the genetically diverse parents helps
Fig. 5. Bayesian admixture proportion of individual plants of the investigated anthuriums for a K ¼ 3 population model. The K ¼ 3 ‘subgroups’ were identified by the program STRUCTURE. The red, green, and blue indicate subgroups A, B, and C, respectively. The length of the colored sections of each vertical bar (the admixtures) is proportional to the probabilities of assignment to the colored groups represented in that bar (i.e. accession). The Y-axis represents the probability of assignment, and the X-axis is the series of accessions arranged in some arbitrary order. For the detailed probabilities of assignment for the investigated accessions, please refer to Table S2 Electronic Supplementary Materials. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Y. Ge et al. / Biochemical Systematics and Ecology 45 (2012) 34–40
broadening variations in segregating generations and then to increase the opportunities of selecting for excellent hybrid plants. In this study, high genetic variations and some main clusters were comparatively observed using morphological traits and AFLP markers. The findings of this study would add new knowledge of genetic diversity and population structure of anthuriums as well as permit a sound hybridization breeding for anthuriums in future. Acknowledgments This research was financially supported by the Key Science Technology Specific Projects of Zhejiang Province (2009C12095), the Key Scientific and Technological Innovation Team Project of Zhejiang Province (2011R50034-14), and Hangzhou Special Project of Seeds and Seedlings (20110332H14). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bse.2012. 07.023. References Acosta-Mercao, D., Bird-Pico, F.J., Kolterman, D.A., 2002. Genetic variability of Anthurium crenatum (L.) Kunth (Araceae) in Puerto Rico. Caribb. J. Sci. 38, 111–124. Andrade, I.M., Mayo, S.J., van den Berg, C., Fay, M.F., Cherster, M., Lexer, C., Kirkup, D., 2009. Genetic variation in natural population of Anthurium sinuatum and A. pentaphyllum var. pentaphyllum (Araceae) from north-east Brazil using AFLP molecular markers. Bot. J. Linn. Soc. 159, 88–105. Avila-Rostant, O., Lennon, A.M., Umaharan, P., 2010. Spathe color variation in Anthurium andraeanum Hort. and its relationship to vacuolar PH. HortScience 45, 1768–1772. Beyene, Y., Botha, A.M., Myburg, A.A., 2005. Genetic diversity in traditional Ethiopian highland maize accessions assessed by AFLP markers and morphological traits. Biodivers. Conserv. 15, 2655–2671. Devanand, P.S., Chao, C.T., Chen, J., Henny, R.J., 2004. AFLP analysis of genetic relationships among container-grown Anthurium cultivars. HortScience 39, 861. Elibox, W., Umaharan, P., 2008. Inheritance of major spathe colors in Anthurium andraeanum Hort. is determined by three major genes. HortScience 43, 787–791. Evanno, G., Regnaut, S., Goudet, J., 2005. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14, 2611–2620. Finger, F.L., Lannes, S.D., Schuelter, A.R., Doege, J., Comerlata, A.P., Goncalves, L.S.A., Ferreira, F.R.A., Clovis, L.R., Scapim, C.A., 2010. Genetic diversity of Capsicum chinensis (Solanaceae) accessions based on molecular markers and morphological and agronomical traits. Genet. Mol. Res. 9, 1852–1964. Gantait, S., Sinniah, U.R., 2011. Morphology, flow cytometry and molecular assessment of ex-vitro grown micropropagated anthurium in comparison with seed germinated plants. Afr. J. Biotechnol. 10, 13991–13998. Gawenda, I., Schröder-Lorenz, A., Debener, T., 2011. Markers for ornamental traits in Phalaenopsis orchids: population structure, linkage disequilibrium and association mapping. Mol. Breed. http://dx.doi.org/10.1007/s11032-011-9620-8. Gowda, S.J.M., Randhawa, G.J., Bisht, I.S., Fire, P.K., Singh, A.K., Abraham, Z., Dhillon, B.S., 2011. Morpho-agronomic and simple sequence repeat-based diversity in colored rice (Oryza sativa L.) germplasm from peninsular India. Genet. Resour. Crop Evol.. http://dx.doi.org/10.1007/s10722-01109674-9 Hubisz, M.J., Falush, D., Stephens, M., Pritchard, J.K., 2009. Inferring weak population structure with the assistance of sample group information. Mol. Ecol. Resour. 9, 1322–1332. Mantel, N., 1967. The detection of disease clustering and a generalized regression approach. Cancer Res. 27, 209–220. Murray, M.G., Thompson, W.F., 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8, 4321–4325. Nowbuth, P., Khittoo, G., Bahorun, T., Venkatasamy, S., 2005. Assessing genetic diversity of some Anthurium andraeanum Hort. cut-flower cultivars using RAPD markers. Afr. J. Biotechnol. 4, 1189–1194. Pham, T.D., Geleta, M., Bui, T.M., Bui, T.C., Merker, A., Carlsson, A.S., 2011. Comparative analysis of genetic diversity of sesame (Sesamum indicum L.) from Vietnam and Cambodia using agro-morphological and molecular markers. Hereditas 148, 28–35. Pritchard, J., Stephens, M., Donnelly, P., 2000. Inference of population structure using multilocus genotype data. Genetics 155, 945–959. Ranamukhaarachchi, D.G., Henny, R.J., Guy, C.L., Li, Q.B., 2001. DNA fingerprinting to identify nine Anthurium pot plant cultivars and examine their genetic relationship. HortScience 36, 758–760. Rohlf, F.J., 2005. NTSYSpc Numerical Taxonomy and Multivariate Analysis System. Version 2.2. Exeter Software, New York. Solouki, M., Mehdikhani, H., Zeinali, H., Emamjomeh, A.A., 2008. Study of genetic diversity in chamomile (Matricaria chamomilla) based on morphological traits and molecular markers. Sci. Hortic. 117, 281–287. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Fritjers, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407–4414. Yasin, J.K., Mayadevi, P., 2010. Genetic diversity among commercial varieties of Anthurium andreanum Linden using RAPD markers. J. Genet. Transgenet. 1, 11–15. Zhang, F., Wang, W., Ge, Y., Shen, X., Tian, D., Liu, J., Liu, X., Yu, X., Zhang, Z., 2012. Genetic relatedness among Aechmea species and hybrids inferred from AFLP markers and pedigree data. Sci. Hortic. 139, 39–45.
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Genetic variations within a collection of anthuriums unraveled by morphological traits and AFLP markers Yaying Ge 1, Fei Zhang 1, Xiaolan Shen, Yongming Yu, Xiaoyun Pan, Xiaojing Liu, Jianxin Liu, Gangmin Pan, Danqing Tian* Flower Research and Development Centre, Zhejiang Academy of Agricultural Sciences, No. 79 Tangwan, Xiaoshan District, Hangzhou 311202, Zhejiang Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 May 2012 Accepted 7 July 2012 Available online 4 August 2012
In this study, genetic variations among a collection of anthuriums were comparatively investigated using morphological traits and amplified fragment length polymorphism (AFLP) markers. Both morphological and AFLP-based data clustered the accessions from Anthurium andraeanum, Anthurium scherzerianum, and Anthurium clarinervium into three separate groups, with a significant though low correlation (r ¼ 0.3) observed between the morphological and AFLP-based similarity matrices. The AFLP-based principal coordinate analysis (PCoA) divided the entire accessions into three parts, reinforcing the AFLP-based clustering. Moreover, the Bayesian-based structuring assigned the anthurium accessions to three subgroups but with 27 accessions retained in the admixed groups, probably implying the highly heterozygous genome of the investigated anthuriums. The findings of this study would add new knowledge of genetic diversity among anthuriums as well as embark on a useful beginning for a rational hybridization breeding for anthuriums in future. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Anthurium Genetic diversity Population structure Morphology AFLP
1. Introduction Anthurium (Areaceae), native to Central and South America, is one of the most popular ornamental crops. The attractive characteristics including vibrant inflorescence with straight spathe, candle-like spadix, exotic foliage, and particularly the long lasting ‘flower’ of anthurium have ensured its great commercial importance, and therefore anthuriums have currently occupied a large part in flower industry, especially in terms of cut-flower and potted ornamentals. Now, demand for new good quality varieties of anthuriums is increasing. Many attempts have been made to increase the diversity of anthuriums to meet the preferences of different groups of people (Elibox and Umaharan, 2008; Avila-Rostant et al., 2010). Complete knowledge of genetic diversity is indispensable for efficient utilization of genetic resources and effective breeding. By far many studies have attempted to address the genetic diversity of anthurium accessions (Ranamukhaarachchi et al., 2001; Acosta-Mercao et al., 2002; Devanand et al., 2004; Nowbuth et al., 2005; Andrade et al., 2009; Yasin and Mayadevi, 2010; Gantait and Sinniah, 2011). However, anthurium germpalsms could by no means be thoroughly characterized due to the localized and limited samples in the previous published articles, and therefore necessary to assess the genetic variability within a specific collection of anthuriums.
* Corresponding author. Tel.: þ86 571 82713881; fax: þ86 571 82721053. E-mail address: [email protected] (D. Tian). 1 The authors contributed equally to this work. 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.07.023
Y. Ge et al. / Biochemical Systematics and Ecology 45 (2012) 34–40
35
In the present study, we aimed to address the genetic variability of the commercial varieties currently cultivated in China and recently selected hybrids of anthurium based on morphological traits and AFLP markers. The findings of this study will add a new knowledge with respect to genetic diversity of anthuriums and therefore facilitate a rational breeding design in future. 2. Materials and methods 2.1. Plant materials and phenotyping In this study, 60 accessions of anthuriums, a representative of the commercially cultivated gene pools in China, were selected. Of the 60 accessions, 57 accessions were from Anthurium andraeanum, two from Anthurium scherzerianum, and one from Anthurium clarinervium. In addition, the cultivated varieties were encoded with a suffix An, and the recently selected hybrids with a suffix S (Table 1). An4 and An44 are A. scherzerianum and An6 is A. clarinervium. For the S-coded accessions, S1–S3 were selected hybrids of An27 An32, and S4 and S5 hybrids of An36 An37, S6 and S7 hybrids of An36 An17, S8 hybrids of An38 An45, S9 hybrids of An32 An15, and An10 hybrids of An3 An15. The anthurium samples were collected from various breeders or production companies, and cultivated as potted flowers in the semi-controlled greenhouse at Flower Research and Development Centre, Zhejiang Academy of Agricultural Sciences, China. A total of nine phenotypic traits were recorded for the flowering plants (Table 2). A summary of the investigated traits is depicted in Table S1. 2.2. DNA isolation and AFLP profile Young leaves were collected from each accession, frozen with liquid nitrogen and grinded into powder. Genomic DNA was isolated following a CTAB-based procedure (Murray and Thompson, 1980). DNA concentration was estimated in comparison with known concentrations of Lambda DNA in 0.8% agarose gel. AFLP analyses were performed as described by Vos et al. (1995) with some modifications. The pre-amplification products were diluted 20-fold and were used as a template for the selective amplification. Selective amplification was conducted by using fluorescently labeled EcoRI or Pstl primers. A total of 48 selective primers of EcoRI þ 3/Pstl þ 3 were initially applied to screen polymorphisms using the DNA of four randomly chosen accessions; and consequently eight polymorphic primer combinations were chosen to genotype the whole accessions. Amplification was conducted on a Gene Amp PCR System 9600 (Perkin Elmer, USA). PCR products were mixed with loading buffer and loaded on 4% polyacrylamide gels after heat
Table 1 The investigated accessions of anthuriums in this study. Code
Accession
Botanical classification
Code
Accession
Botanical classification
An1 An2 An3 An4 An5 An6 An7 An8 An9 An10 An11 An12 An13 An14 An15 An16 An17 An18 An19 An20 An21 An22 An23 An24 An25 An26 An27 An28 An29 An30
Alabamb Pink Champion Dakota Artus Altimo Species Baleno Fantasy Love Red Love Butterfly Dream Amalia Red Fiesta Red Queen White Angel Big Beauty Beauty Silence Ping Love Robino Adios Kiss True Love Secret Love Classie Red Red Victory Nathalive Yukon Paradi Feroza Season Latino
A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A.
An31 An32 An33 An34 An35 An36 An37 An38 An39 An40 An41 An42 An43 An44 An45 An46 An47 An48 An49 An50 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10
Coattails Red Sempre Poncho Burning Love Tricolore Happy Purple Flag Mystral Sierra Tender Love Manbu Music Red Fire Lybra Beijing Success Red Crown Fiorino Micra Chico Green Tucano Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid
A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A.
andraeanum andraeanum andraeanum scherzerianum andraeanum clarinervium andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum
andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum scherzerianum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum andraeanum
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Y. Ge et al. / Biochemical Systematics and Ecology 45 (2012) 34–40 Table 2 The morphological traits recorded for the investigated anthurium accessions in this study. Trait
Value
Leaf type Leaf color Peduncle color Spathe size Spathe type Spathe glossiness Spathe color Inflorescence type Tillering capacity
Heart shaped; long-heart shaped; triangle; oval; pointed oval; lanceolate; stripe Dark green; green; light green Dark green; moderate green; light green; green-red; dark red; red; light red Large; medium; small Oval; heart shaped; long-heart shaped; round; willow-leaf shaped Bright; moderate; dark Pink; red; orange; green; white; dark red; purplish red; orange red Up-right; inclined; twisty; arc-shaped Good; moderate; poor
denaturation. The products were fractionated on PRISM 377 sequencer (ABI) using electrophoresis. The internal 70–500 size standard ROX 500 (ABI) was run with the samples to estimate the size of fragments. All AFLP fragments were scored dominantly and recorded in a 0/1-matrix for the peak absence/presence along with their sizes. The binary scores were manually compared with the electropherograms to re-confirm the presence or absence of peaks. 2.3. Statistical analysis Two dendrograms were separately constructed based on the phenotype- and AFLP-based genetic similarity using unweighted pair group method with arithmetic average (UPGMA) method with the SAHN module of NTSYS-pc 2.2 (Rohlf, 2005), and the normalized Mantel statistic Z test (Mantel, 1967) was used to determine the level of association between the two similarity matrices. Then based on the AFLP data, the principal coordinated analysis (PCoA) was performed to show the differentiation of the bromeliad accessions in a two-dimensional array of eigenvectors using the DCENTER and EIGEN modules of NTSYS-pc. Lastly, a Bayesian-based clustering implemented in STRUCTURE software version 2.3.3 with admixture (Pritchard et al., 2000) was performed on the AFLP data to better delineate population substructures of genetically similar accessions. STURCUTURE was run 5 times independently with the number of subgroups (K) ranging from 1 to 15. Each run implemented with a burn-in period of 100,000 steps followed by 100,000 Monte Carlo Markov Chain replicates (Hubisz et al., 2009). That run with the maximum likelihood was applied to subdivide the accessions into different subgroups with the membership probabilities threshold 0.80 as well as the maximum membership probability among subgroups. Those accessions <0.80 membership probabilities were retained in the admixed group. An ad hoc measure DK proposed by Evanno et al. (2005) was used to detect the true K present in the SRAP marker data. 3. Results 3.1. Morphological clustering Based on the nine investigated morphological traits, pair-wise similarity coefficient varied from 0.65 to 1.0. The sixty anthurium accessions were classified into three main groups (I, II, III) at similarity coefficient of 0.70 (Fig. 1). Group I consisted of all accessions from A. andraeanum, group II the two accessions from A. scherzerianum, and group III the accession from A. clarinervium, well concordant with their botanical classification as depicted in Table 1. The group I could be further divided into six sub-clusters, whereas no correlation was found between sub-clusters and morphology. 3.2. Molecular characterization Out of the 48 primer combinations, eight selective primers generating a clear and informative pattern were chosen for genotyping. As a result, a total of 1467 fragments were produced, of which 1459 (99.45%) were polymorphic (Table 3). The number of polymorphic bands for each primer combination varied between 164 and 199 and averaged w182. Based on the AFLP marker data, the similarity coefficient ranged from 0.68 to 0.88. A dendrogram based on this similarity matrix is shown in Fig. 2. In this dendrogram, the whole accessions were grouped into three main clusters (I, II, III) at similarity coefficient of 0.73, well in concordance with their botanical classification as well. Cluster I could be also divided into several sub-clusters, whereas with no significant correlation between sub-clusters and morphology either. Results from the principal coordinate analysis (PCoA) revealed that the first three coordinates accounted for 16.83% of the molecular variations. According to the two-dimensional plots, the 60 anthurium accessions generally dispersed in threecentered parts (Fig. 3), reinforcing the clustering of the accessions from A. andraeanum. The pattern of genetic diversity and population structure of the investigated anthuriums was further characterized with a Bayesian-based approach. The peak value for the ad hoc quantity with respect to DK was observed for K ¼ 3 (Fig. 4); as a result, the 60 anthurium accessions were assigned to three subgroups (Fig. 5). Within the membership probability 0.80,
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Fig. 1. Dendrogram of the 60 anthurium accessions, derived from the UPGMA cluster analysis based on phenotype-based similarity coefficients. The accession codes An4 and An44 are A. scherzerianum, An6 is A. clarinervium, and the others are A. andraeanum. For details please see Table 1.
sub-group A included nine accessions, sub-group B 18 accessions and sub-group C six accessions, with the remained 27 accessions retained in the admixed groups (Fig. 5, Table S2). 3.3. Mantel test between similarity matrices To compare the morphological and AFLP marker based similarity matrices, the Mantel test was performed. The result indicated a significant but low correlation (r ¼ 0.30) between the two matrices. 4. Discussion A complete knowledge of genetic variability among crop germplasm is a prerequisite for rational management of genetic resources as well as for successful crop improvement (Gowda et al., 2011). Recently, some efforts have been focused on the genetic diversity and relationship among anthuriums. Ranamukhaarachchi et al. (2001) identified the genetic relationship of nine potted anthuriums with RAPD markers. Yasin and Mayadevi (2010) reported a high genetic diversity among 12 commercial varieties of A. andraeanum using RAPD markers. Nowbuth et al. (2005) assessed the genetic variations among 24 cut-flower cultivars of A. andraeanum with RAPD markers. Devanand et al. (2004) fingerprinted a relatively large sample of 58 accessions of container-grown anthuriums with AFLP markers. In addition, the genetic variations in natural populations of Anthurium species were also characterized (Acosta-Mercao et al., 2002; Andrade et al., 2009). The findings of these reports add a knowledge regarding the genetic variation of anthuriums. Morphological traits are of great importance in selecting rational parents for hybridization breeding and have also been applied to assess genetic diversity of many crops (Beyene et al., 2005; Finger et al., 2010). In this study, the nine investigated Table 3 Amplified result of AFLP analysis for the 60 accessions of cultivated anthuriums. Primers
Total bands
Polymorphic bands
% of polymorphic bands
E-AAC/M-CAA E-AAC/M-CTA E-AAC/M-CTG E-AAG/M-CAA E-ACA/M-CTT E-ACC/M-CAC E-ACC/M-CAG E-ACC/M-CTC Total Average
184 186 180 200 182 165 185 185 1467 183.38
183 184 179 199 180 164 185 185 1459 182.38
99.45 98.92 99.44 99.50 98.90 99.39 100 100 – 99.45
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Fig. 2. Dendrogram of the 60 anthurium accessions, derived from the UPGMA cluster analysis based on AFLP-based similarity coefficients. The accession codes An4 and An44 are A. scherzerianum, An6 is A. clarinervium, and the others are A. andraeanum. For details please see Table 1.
morphological traits grouped the sixty anthuriums into three main clusters consonant with their botanical classifications. However, the morphological traits hardly separated the accessions of A. andraeanum grouped in cluster I (Table 1). Probably this implies the phenotypic similarity of accessions from A. andraeanum and difficulty in identifying their genetic diversity via morphology, thereby consistent with the earlier study by Nowbuth et al. (2005).
Fig. 3. Matrix plot of the first two coordinates of principal coordinate analysis (PCoA) based on the AFLP data, showing associations among the investigated anthuriums accessions. The accession codes An4 and An44 are A. scherzerianum, An6 is A. clarinervium, and the others are A. andraeanum. For details please see Table 1.
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Fig. 4. Magnitude of DK from structure analysis as a function of K, calculated following the DK methods as proposed by Evanno et al. (2005), for the investigated anthuriums based on the AFLP data. The modal value of these distributions indicates the true K or the uppermost level of structure, here three subgroups.
AFLP markers were proven to be an effective approach to characterizing genetic variation of many crops including anthuriums (Devanand et al., 2004; Andrade et al., 2009). In this study, the eight primers produced a total of 1459 (99.45%) polymorphic markers, indicative of a high level of genotypic variations among the investigated anthurium accessions. Comparatively, lower similarity coefficients (0.68–0.88) were observed in this study rather than in the former reports (Ranamukhaarachchi et al., 2001; Devanand et al., 2004; Nowbuth et al., 2005). This also suggests the high genetic diversity within anthuriums in this study. The AFLP-based clustering grouped the whole accessions herein into three main groups, reinforcing the morphological clustering and the botanical classifications. Several previous literatures have reported the lack of correlation between the two clustering based on morphological traits and molecular markers (Beyene et al., 2005; Solouki et al., 2008; Finger et al., 2010; Pham et al., 2011). In this study, the morphology and AFLP-based clustering analyses unequivocally differentiated the sixty anthuriums into three main groups, both well corresponding to their botanical classifications. However, a significant but low correlation was observed between the similarity matrices. This is probably due to the discordance of the clustering of the accessions from A. andraeanum that are largely resulted from the difference in neutrality between morphological traits and AFLP markers. Additionally, the ten selected hybrids (S1–S10) included in this study were not grouped together with their parents. Recently, Gawenda et al. (2011) reported that the new selected hybrids of Phalaenopsis orchids were inclined to cluster together; Zhang et al. (2012) confirmed that AFLP marker rather than pedigree information is more effective in quantifying the genetic relatedness for Aechmea species or hybrids. In this work, the failure to distinctly identify the relationship between the hybrids and parents for anthuriums suggested to some extent their complex genetic backgrounds with several clones or species caused by intense hybridization. To further unravel the genetic diversity and population structure of cultivated anthuriums, a Bayesian-based approach implemented in STURCTURE was adopted. The structuring assigned 33 accessions to three subgroups, whereas with the other 27 accessions retained in the admixed groups. Obviously this indicates the presence of highly heterozygous genome of the investigated anthuriums here. This cause should be attributed to the intense hybridization breeding works for anthuriums during the last decades. Nowadays, the conventional hybridization is one of the most effective approaches to breed new varieties for anthuriums though the advents of some new molecular breeding methods. Crossing between the genetically diverse parents helps
Fig. 5. Bayesian admixture proportion of individual plants of the investigated anthuriums for a K ¼ 3 population model. The K ¼ 3 ‘subgroups’ were identified by the program STRUCTURE. The red, green, and blue indicate subgroups A, B, and C, respectively. The length of the colored sections of each vertical bar (the admixtures) is proportional to the probabilities of assignment to the colored groups represented in that bar (i.e. accession). The Y-axis represents the probability of assignment, and the X-axis is the series of accessions arranged in some arbitrary order. For the detailed probabilities of assignment for the investigated accessions, please refer to Table S2 Electronic Supplementary Materials. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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broadening variations in segregating generations and then to increase the opportunities of selecting for excellent hybrid plants. In this study, high genetic variations and some main clusters were comparatively observed using morphological traits and AFLP markers. The findings of this study would add new knowledge of genetic diversity and population structure of anthuriums as well as permit a sound hybridization breeding for anthuriums in future. Acknowledgments This research was financially supported by the Key Science Technology Specific Projects of Zhejiang Province (2009C12095), the Key Scientific and Technological Innovation Team Project of Zhejiang Province (2011R50034-14), and Hangzhou Special Project of Seeds and Seedlings (20110332H14). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bse.2012. 07.023. References Acosta-Mercao, D., Bird-Pico, F.J., Kolterman, D.A., 2002. Genetic variability of Anthurium crenatum (L.) Kunth (Araceae) in Puerto Rico. Caribb. J. Sci. 38, 111–124. Andrade, I.M., Mayo, S.J., van den Berg, C., Fay, M.F., Cherster, M., Lexer, C., Kirkup, D., 2009. Genetic variation in natural population of Anthurium sinuatum and A. pentaphyllum var. pentaphyllum (Araceae) from north-east Brazil using AFLP molecular markers. Bot. J. Linn. Soc. 159, 88–105. Avila-Rostant, O., Lennon, A.M., Umaharan, P., 2010. Spathe color variation in Anthurium andraeanum Hort. and its relationship to vacuolar PH. HortScience 45, 1768–1772. Beyene, Y., Botha, A.M., Myburg, A.A., 2005. Genetic diversity in traditional Ethiopian highland maize accessions assessed by AFLP markers and morphological traits. Biodivers. Conserv. 15, 2655–2671. Devanand, P.S., Chao, C.T., Chen, J., Henny, R.J., 2004. AFLP analysis of genetic relationships among container-grown Anthurium cultivars. HortScience 39, 861. Elibox, W., Umaharan, P., 2008. Inheritance of major spathe colors in Anthurium andraeanum Hort. is determined by three major genes. HortScience 43, 787–791. Evanno, G., Regnaut, S., Goudet, J., 2005. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14, 2611–2620. Finger, F.L., Lannes, S.D., Schuelter, A.R., Doege, J., Comerlata, A.P., Goncalves, L.S.A., Ferreira, F.R.A., Clovis, L.R., Scapim, C.A., 2010. Genetic diversity of Capsicum chinensis (Solanaceae) accessions based on molecular markers and morphological and agronomical traits. Genet. Mol. Res. 9, 1852–1964. Gantait, S., Sinniah, U.R., 2011. Morphology, flow cytometry and molecular assessment of ex-vitro grown micropropagated anthurium in comparison with seed germinated plants. Afr. J. Biotechnol. 10, 13991–13998. Gawenda, I., Schröder-Lorenz, A., Debener, T., 2011. Markers for ornamental traits in Phalaenopsis orchids: population structure, linkage disequilibrium and association mapping. Mol. Breed. http://dx.doi.org/10.1007/s11032-011-9620-8. Gowda, S.J.M., Randhawa, G.J., Bisht, I.S., Fire, P.K., Singh, A.K., Abraham, Z., Dhillon, B.S., 2011. Morpho-agronomic and simple sequence repeat-based diversity in colored rice (Oryza sativa L.) germplasm from peninsular India. Genet. Resour. Crop Evol.. http://dx.doi.org/10.1007/s10722-01109674-9 Hubisz, M.J., Falush, D., Stephens, M., Pritchard, J.K., 2009. Inferring weak population structure with the assistance of sample group information. Mol. Ecol. Resour. 9, 1322–1332. Mantel, N., 1967. The detection of disease clustering and a generalized regression approach. Cancer Res. 27, 209–220. Murray, M.G., Thompson, W.F., 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8, 4321–4325. Nowbuth, P., Khittoo, G., Bahorun, T., Venkatasamy, S., 2005. Assessing genetic diversity of some Anthurium andraeanum Hort. cut-flower cultivars using RAPD markers. Afr. J. Biotechnol. 4, 1189–1194. Pham, T.D., Geleta, M., Bui, T.M., Bui, T.C., Merker, A., Carlsson, A.S., 2011. Comparative analysis of genetic diversity of sesame (Sesamum indicum L.) from Vietnam and Cambodia using agro-morphological and molecular markers. Hereditas 148, 28–35. Pritchard, J., Stephens, M., Donnelly, P., 2000. Inference of population structure using multilocus genotype data. Genetics 155, 945–959. Ranamukhaarachchi, D.G., Henny, R.J., Guy, C.L., Li, Q.B., 2001. DNA fingerprinting to identify nine Anthurium pot plant cultivars and examine their genetic relationship. HortScience 36, 758–760. Rohlf, F.J., 2005. NTSYSpc Numerical Taxonomy and Multivariate Analysis System. Version 2.2. Exeter Software, New York. Solouki, M., Mehdikhani, H., Zeinali, H., Emamjomeh, A.A., 2008. Study of genetic diversity in chamomile (Matricaria chamomilla) based on morphological traits and molecular markers. Sci. Hortic. 117, 281–287. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Fritjers, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407–4414. Yasin, J.K., Mayadevi, P., 2010. Genetic diversity among commercial varieties of Anthurium andreanum Linden using RAPD markers. J. Genet. Transgenet. 1, 11–15. Zhang, F., Wang, W., Ge, Y., Shen, X., Tian, D., Liu, J., Liu, X., Yu, X., Zhang, Z., 2012. Genetic relatedness among Aechmea species and hybrids inferred from AFLP markers and pedigree data. Sci. Hortic. 139, 39–45.