Native to eastern regions in North America, the genus Aronia is a group of deciduous shrubs in the Rosaceae family, subtribe Pyrinae. The Pyrinae subtribe has a base chromosome count of n = 17 (Postman, 2011), and Aronia species are commonly found as diploids (2n = 2x = 34) and tetraploids (2n = 4x = 68) with some occurrence of triploids (Brand, 2010; Hovmalm et al., 2004; Leonard et al., 2013). Polyploidization events in Aronia likely resulted from the fusion of unreduced gametes from the same or different species to produce autopolyploids and allopolyploids, respectively. The three commonly accepted Aronia species include A. arbutifolia (L.) Pers., red chokeberry (tetraploid); A. melanocarpa (Michx.) Elliott, black chokeberry (diploid and tetraploid); and A. prunifolia (Marshall) Reheder, purple chokeberry (triploid and tetraploid). A fourth species of Aronia has been recognized as A. mitschurinii [(A.K. Skvortsov & Maitul) tetraploid], and it is used in commercial fruit production, usually as the cultivars Viking or Nero (Leonard et al., 2013). However, genetic marker and phenotypic data suggest that nearly all A. mitschurinii cultivars, an intergeneric hybrid involving A. melanocarpa × Sorbus aucuparia L. (Leonard et al., 2013; Skvortsov and Mautulina, 1982), are genetically identical and likely renames of a single genotype (J.D. Mahoney and M.H. Brand, unpublished data). Interest in Aronia is high because their fruits contain high levels of antioxidants and polyphenols (Brand et al., 2017; Wu et al., 2004; Zheng and Wang, 2003), they are valuable as replacements for invasive exotic ornamental plants (Brand, 2010), and they are widely adapted to various geographic regions (Dirr, 2009; McKay, 2001).
Aronia flowers are thought to be protogynous and self-compatible (Connolly, 2014). Polyploid Aronia species have been reported to reproduce apomictically, via gametophytic apomixis, resulting in embryos that are identical or nearly identical to maternal plants (Brand, 2010). Hovmalm et al. (2004) reported that diploid A. melanocarpa produced highly heterogeneous offspring and tetraploid plants produced homogeneous offspring, suggesting that polyploid Aronia reproduce apomictically. Gametophytic apomixis occurs when a progenitor cell in the megasporangium forms a megagametophyte (Grossniklaus et al., 2001; Richards, 2003). Gametophytic apomixis is further classified into two categories: diplospory and apospory. In diplosporous apomictic plants, the megagametophyte forms from an unreduced or partially reduced megaspore. When a partially reduced megaspore is involved, meiosis is initiated but fails before completion and cell division continues mitotically (Bicknell and Koltunow, 2004). The result is an unreduced megagametophyte derived from a megaspore in which homologous recombination and one round of segregation may have occurred. Apospory refers to an unreduced megagametophyte arising from nucellar or integument tissue (Koltunow and Grossniklaus, 2003). Talent (2009) mentions that pseudogamous gametophytic apospory is common in the Maloid Rosaceae (Pyrinae), where seed development requires pollination, but the embryo has no paternal inheritance and only the endosperm is fertilized. Both diplospory and apospory have been reported to occur in the same species, including the Pyrinae genera Crataegus (Muniyamma and Phipps, 1979, 1984a, 1984b) and Sorbus (Jankun and Kovanda, 1988).
In normal sexual reproduction, genetic uniformity and hybrid vigor are lost after the F1 generation, but with apomixis these traits can be maintained through many generations due to a fixed heterozygosity (Koltunow et al., 1995; Ortiz et al., 2013; Richards 2003). For this reason, seed propagation of apomictic selections is possible and allows growers to achieve high yields while avoiding more expensive vegetative propagation methods (Barcaccia and Albertini 2013). In apomictic temperate fruit crops, it is advantageous to use vegetative propagation from mature phase plants rather than regenerate from juvenile seed. Aronia requires 3 to 5 years before reaching the mature phase for flowering to occur, so use of apomictic seeds delays fruit production. However, Mahoney et al. (2018) report that cotyledons have a greater shoot regeneration rate than mature phase leaf explants; therefore, it may be advantageous to use apomictic seed tissue (i.e., cotyledons) for genetic transformation of Aronia. Although apomixis can be an advantage, it also can present challenges to controlled breeding and genetic exchange. For example, genetic improvement of polyploid Aronia genotypes has been hindered by the occurrence of apomixis during attempted crosses.
A number of molecular marker techniques such as random amplified polymorphic DNA (RAPD), intersimple sequence repeat, and cpDNA marker analysis have been popular in identifying apomixis in plants (Arnholdt-Schmitt, 2000; Hovmalm et al., 2004; Ludwig et al., 2013; Robertson et al., 2010; Smolik et al., 2011). AFLP analysis often has been preferred over other molecular methods for its efficiency (Leonard et al., 2013; Lubell et al., 2008; Obae and Brand, 2013). The AFLP technique has proven to be a more cost-effective way of producing a large number of markers (Mueller and Wolfenbarger, 1999). In this study, we use AFLP, in conjunction with ploidy analysis and plant phenotype, to elucidate the reproductive mechanisms of Aronia species and reveal the occurrence of apomixis within the genus and among ploidy levels.
Anonymous, 2007 AFLP plant mapping protocol. Applied Biosystems, Foster City, CA. 2 July 2018. <https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_040959.pdf>
Arnholdt-Schmitt, B. 2000 RAPD analysis: A method to investigate aspects of the reproductive biology of Hypericum perforatum L Theor. Appl. Genet. 100 906 911
Baarlen, P.V., van Dijk, P.J., Hoekstra, R.F. & Jong, J.H.D. 2000 Meiotic recombination in sexual diploid and apomictic triploid dandelions (Taraxacum officinale L.) Genome 43 827 835
Bartish, I.V., Hylmö, B. & Nybom, H. 2001 RAPD analysis of interspecific relationships in presumably apomictic Cotoneaster species Euphytica 120 273 280
Brand, M.H., Connolly, B.A., Levine, L.H., Richards, J.T., Shine, S.M. & Spencer, L.E. 2017 Anthocyanins, total phenolics, ORAC and moisture content of wild and cultivated dark-fruited Aronia species Scientia Hort. 224 332 342
Campbell, C.S. & Dickinson, T.A. 1990 Apomixis, patterns of morphological variation, and species concepts in subfam. Maloideae (Rosaceae) Syst. Bot. 15 124 135
Connolly, B.A. 2014 Collection, description, taxonomic relationships, fruit biochemistry, and utilization of Aronia melanocarpa, A. arbutifolia, A. prunifolia, and A. mitschurinii. Framingham State University, Framingham, Doctoral Dissertations, 342
Dirr, M. 2009 Manual of woody landscape plants. 6th ed. Stipes Publishing, Champaign, IL
Grossniklaus, U., Nogler, G.A. & van Dijk, P.G. 2001 How to avoid sex: The genetic control of gametophytic apomixis Plant Cell 13 1491 1498
Hovmalm, H.A., Jeppsson, N., Bartish, I.V. & Nybom, H. 2004 RAPD analysis of diploid and tetraploid populations of Aronia points to different reproductive strategies within the genus Hereditas 141 301 312
Koltunow, A.M., Bicknell, R.A. & Chaudhury, A.M. 1995 Apomixis: Molecular strategies for the generation of genetically identical seeds without fertilization Plant Physiol. 108 1345 1352
Lehrer, J.M., Brand, M.H. & Lubell, J.D. 2008 Induction of tetraploidy in meristematically active seeds of Japanese barberry (Berberis thunbergii var. atropurpurea) through exposure to colchicine and oryzalin Scientia Hort. 119 67 71
Leonard, P.J., Brand, M.H., Connolly, B.A. & Obae, S.G. 2013 Investigation of the origin of Aronia mitschurinii using amplified fragment length polymorphism analysis HortScience 48 520 524
Lubell, J.D., Brand, M.H. & Lehrer, J.M. 2008 AFLP identification of Berberis thunbergii cultivars, inter-specific hybrids, and their parental species J. Hort. Sci. Biotechnol. 83 55 63
Ludwig, S., Robertson, A., Rich, T., Djordjevic, M., Cerovic, R., Houston, L., Harris, S. & Hiscock, S. 2013 Breeding systems, hybridization and continuing evolution in Avon Gorge Sorbus Ann. Bot. 111 563 575
Mahoney, J.D., Apicella, P.V. & Brand, M.H. 2018 Adventitious shoot regeneration from in vitro leaves of Aronia mitschurinii and cotyledons of closely related Pyrinae taxa Scientia Hort. 237 135 141
Muniyamma, M. & Phipps, J.B. 1984a Studies in Crataegus. X. A note on the occurrence of diplospory in Crataegus dissona Sarg. (Maloideae, Rosaceae) Can. J. Genet. Cytol. 26 249 252
Muniyamma, M. & Phipps, J.B. 1984b Studies in Crataegus. XI. Further cytological evidence for the occurrence of apomixes in North American hawthorns Can. J. Bot. 62 2316 2324
Obae, S. & Brand, M.H. 2013 Using amplified fragment length polymorphism markers to confirm identity and correct labeling of Japanese Barberry (Berberis thunbergii) cultivars in the market HortScience 48 150 157
Oksanen, J., Blanchet, F.G., Friendly, M., Kindt, R., Legendre, P., McGlinn, D., Minchin, P.R., O’Hara, R.B., Simpson, G.L., Solymos, P., Stevens, M.H.M., Szoecs, E. & Wagner, H. 2017 vegan: Community Ecology Package. R package version 2.5-2. 2 July 2018. <https://CRAN.R-project.org/package=vegan>
Ortiz, J.P.A., Quarin, C.L., Pessino, S.C., Acuña, C., Martínez, E.J., Espinoza, F., Hojsgaard, D., Sartor, M.E., Cáceres, M.E. & Pupilli, F. 2013 Harnessing apomictic reproduction in grasses: What we have learned from Paspalum Ann. Bot. 112 767 787
Robertson, A., Rich, T., Allen, A., Houston, L., Roberts, C., Bridle, J., Harris, S. & Hiscock, S. 2010 Hybridization and polyploidy as drivers of continuing evolution and speciation in Sorbus Mol. Ecol. 19 1675– 1690
Schmidt, A., Schmid, M.W. & Grossniklaus, U. 2015 Plant germline formation: Common concepts and developmental flexibility in sexual and asexual reproduction Development 142 229 241
Skvortsov, A.K. & Mautulina, Y.K. 1982 On the differences between the cultivated chokeberry and its wild progenitors Bull. Central Botanical Garden 126 35 40
Smolik, M., Ochimian, I. & Smolik, B. 2011 RAPD and ISSR methods used for fingerprinting selected, closely related cultivars of Aronia melanocarpa Not. Bot. Horti Agrobo. 39 276 284
Talent, N. 2009 Evolution of gametophytic apomixis in flowering plants: An alternative model from Maloid Rosaceae Theory Biosci. 128 121 138
Wu, X., Gu, L., Prior, R.L. & McKay, S. 2004 Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucus and their antioxidant capacity J. Agr. Food Chem. 52 7846 7856
Zheng, W. & Wang, S.Y. 2003 Oxygen radical absorbing capacity of phenolics in blueberries, cranberries, chokeberries, and lingonberries J. Agr. Food Chem. 51 502 509