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Exploring the Genetic Diversity of Wild Cranberry Populations in the Upper Midwestern United States

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Exploring the Genetic Diversity of Wild Cranberry Populations in the Upper Midwestern United States Published October 24, 2019 RESEARCH Exploring the Genetic Diversity of Wild Cranberry Populations in the Upper Midwestern United States Lorraine Rodríguez-Bonilla,* Fabian Rodríguez Bonilla, Daniel Matusinec, Eric Wiesman, Sean D. Schoville, Amaya Atucha, and Juan Zalapa* L. Rodríguez-Bonilla, A. Atucha, and J. Zalapa, Dep. of Horticulture, ABSTRACT Univ. of Wisconsin, Madison, WI 53706; F.R. Bonilla, Dep. of Biology, Plant breeding continuously evolves to satisfy Univ. of Puerto Rico Mayaguez, Mayaguez, Puerto Rico 00680; D. the needs of the growing population, but in Matusinec, Dep. of Atmospheric and Oceanic Sciences and Political many crops, it has resulted in a decline in the Science, Univ. of Wisconsin, Madison, WI 53706; E. Wiesman and genetic diversity available. Therefore, increasing J. Zalapa, USDA-ARS, Vegetable Crops Research Unit, Univ. of knowledge of the range, genetic relationships, Wisconsin, Madison, WI 53706; S.D. Schoville, Dep. of Entomology, and diversity among crop wild relatives (CWR) Univ. of Wisconsin, Madison, WI 53706. Received 5 June 2019. Accepted is essential for the efficient use of available 30 Sept. 2019. *Corresponding authors ([email protected]; germplasm in breeding programs. In cran- [email protected]). Assigned to Associate Editor Joseph Robins. berries (Vaccinium macrocarpon Ait.), most Abbreviations: CTAB, cetyltrimethylammonium bromide; CWR, cultivars share the same genetic background crop wild relative(s); EDTA, ethylenediaminetetraacetic acid; PCA, based on only a few wild selections. This limits principal component analysis; PCoA, principal coordinate analysis; the breeding pool for selection to support the PC, principal component; PCR, polymerase chain reaction; RAPD, cranberry industry. Therefore, we studied 36 random amplified polymorphic DNA; SSR, simple sequence repeat. wild populations of V. macrocarpon and V. oxycoccos L. across Wisconsin and Minne- lant breeding is crucial for food production and food sota using 32 simple sequence repeat (SSR) security; however, multiple cycles of selection can result in markers. We found high levels of heterozygosity P in both species, despite previous molecular the loss of genetic diversity (Cheema, 2018). This genetic diver- markers studies revealing low genetic varia- sity loss leads to crop homogeneity, which makes food systems tion. In V. macrocarpon, a total of 294 alleles vulnerable to biotic and abiotic stresses, as has been the case of and moderate to high levels of heterozygosity many crops, such as bananas (Musa acuminata Colla, M. balbisiana (observed [HO] = 0.51, total [HT] = 0.66) were Colla) and potatoes (Solanum tuberosum L.) (Ullstrup, 1972; Rowe, found. As expected for outcrossed polyploid 1984; National Research Council, 1993; Dempewolf et al., 2017). species (4x), higher levels of heterozygosity (HO A way to reduce homogeneity is to find, characterize, and use = 0.81, HT = 0.83) were found in V. oxycoccos wild plant genetic resources as a source of traits for crop improve- than in V. macrocarpon. A comparison between ment (Brozynska et al., 2016). Crop wild relatives (CWR) have wild V. macrocarpon with a group of cultivated provided wild germplasm with many valuable traits for breeding and experimental hybrids found a distinct sepa- cultivated crops, such as resistance to pests, as is the case of resis- ration between both groups, suggesting there is tance to rice blast (Pyricularia grisea) (Liu et al., 2002) and resistance diversity in the wild that needs to be explored and incorporated into breeding programs. The to wheat blast (Magnaporthe oryzae Triticum pathotype) (Anh et results of our studies are the first to explore in al., 2018). Crop wild relatives also possess traits for tolerance to depth the genetic diversity of wild cranberry abiotic stress, such as salt tolerance from soybean [Glycine max (L.) populations in the upper midwestern United Merr.] to tobacco (Nicotiana tabacum L.) (Ji et al., 2010) and cold States and provide novel information to support in situ conservation efforts to protect CWR of Published in Crop Sci. 59:2413–2428 (2019). one of the few North American native crops. doi: 10.2135/cropsci2019.06.0367 © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). CROP SCIENCE, VOL. 59, NOVEMBER–DECEMBER 2019 WWW.CROPS.ORG 2413 sweetening resistance in potato (Hamernik et al., 2009), MATERIALS AND METHODS and traits influencing yield and quality such as in tomato Plant Materials (Solanum lycopersicum L.) (Tanksley et al., 1996; Brozynska Plant material of 572 samples of wild cranberries (putatively et al., 2016; Zhang et al., 2017). Therefore, exploring and collected as V. macrocarpon or V. oxycoccos) was obtained from 36 understanding the genetic diversity of CWR of plants locations across Wisconsin (22) and Minnesota (14) (Table 1). native to North America can help us prepare to face future The locations visited were public lands, Department of agricultural challenges and help boost local production. Natural Resources State Natural Areas, State Scientific Areas, In the United States, very few species used for food or Forest Service lands. Permits were obtained to sample and production are native to North America, and even fewer collect in locations in both states. Using satellite imagery in Google Earth Pro and data provided by the Department of have CWR in nature (Khoury et al., 2013). The American Natural Resources of both states, we located potential collec- cranberry (Vaccinium macrocarpon Ait.) is a diploid, woody tion sites. Prior to visiting the populations, satellite imagery perennial species native to North America with vast amounts was surveyed to define the area of collection to design the of unexplored wild populations and wild relatives sharing best strategy to sample the overall genetic diversity at each site overlapping ranges and environments (Darrow and Camp, while avoiding clones. In the sites, coordinates were logged per 1945; Vander Kloet, 1988). Cranberry domestication and sample, cuttings of a single vine ?20 cm long were obtained, cultivation began in the 1800s with several documented and samples were collected at least 5 m apart to ensure that wild selections (Chandler and Demoranville, 1958; Dana, plant tissue collected for each sample came from a single plant 1983; Eck, 1990). Most cultivated cranberries share a similar and/or genotype. Plant tissue was stored in a bag with wet genetic background; in fact, all hybrid cultivars released in towels, labeled, and kept in a cold bag. A total of 20 plants were the history of cranberry breeding have been derived from collected by location when possible based on the geography ‘Early Black’, ‘Howes’, ‘McFarlin’, and ‘Searles’ (the “Big and environmental conditions. Leaf material was then stored at −4°C for DNA extractions. Plant material from cultivated Four”) and a few other native selections (Eck 1990). V. macrocarpon was obtained from the USDA-ARS, Vegetable Vaccinium oxycoccos L. is mostly a tetraploid species, Crop Research Unit, Cranberry Genetics and Genomics which grows as a small evergreen perennial vine that Laboratory at the University of Wisconsin–Madison. The produces small overwintering berries that have a similar germplasm collected in this study will be kept ex situ and flavor profile to cranberries and exhibit a superior anti- housed in the USDA-ARS, Vegetable Crop Research Unit oxidant profile (Vander Kloet, 1988; Jacquemart, 1997; in Madison, WI, and selected accessions will be added to the Česonienė et al., 2011; Brown et al., 2012). As one of the National Clonal Germplasm Repository collection. V. macrocarpon wild relatives in the continental United States, V. oxycoccos provides great potential for developing DNA Extraction interspecific hybrids to transfer desirable traits into cran- Genomic DNA extractions were performed using leaf tissue per berry, such as tolerance or resistance to abiotic stressors. a cetyltrimethylammonium bromide (CTAB) method (Doyle Because V. oxycoccos has a circumboreal distribution and is and Doyle 1987) modified in our laboratory to isolate high- quality, clean DNA. A total of 10–20 mg of frozen tissue was adapted to high latitudes and harsh winters (Areškevičiūtė et al., 2006), the species could be an excellent source of ground by hand using liquid N and transferred to a 2.0-ml tube, and 700 ml of 2% CTAB extraction buffer (20 mM ethylenedi- cold hardiness genes, as is the case of other wild relatives aminetetraacetic acid [EDTA], 0.1 M Tris-HCl pH 8.0, 1.4 M to crops such as rice (Oryza sativa L.) (Shakiba et al., 2017) NaCl, 2% CTAB) was added and mixed by inversion. The and potato (Hamernik et al., 2009). solution was then incubated at 65°C for 45 min. After incu- Therefore, exploring, studying, and using wild cran- bation, 400 ml of a chloroform-isoamyl alcohol (24:1) solution berry diversity in breeding is crucial to the future of was added to the tubes and gently mixed by inversion. Samples cranberry breeding and production. To meet emerging were then centrifuged for 5 min at 14,000 rpm, 500 ml of the challenges, there is a need to identify and transfer needed supernatant was transferred to a fresh 1.5-ml tube containing traits into cultivated cranberries, as well as develop hybrids 50 ml of 10% CTAB buffer, and the sample was mixed by inver- with novel traits (Vorsa et al., 2008). In the United States, sion. A total of 750 ml of cold isopropanol (100%) was added, little has been done to analyze and understand the wild and then samples were incubated from 2–48 h at a tempera- genetic diversity of native crops such as V. macrocarpon and ture of −20°C. Samples were gently mixed by inversion and V. oxycoccos. This study represents the most comprehensive centrifuged at 14,000 rpm for 20 min. After centrifugation, the supernatant was discarded, and the resulting pellet was air dried analysis of wild populations of cranberries in Wisconsin for 5 min.
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