Quantifying apple diversity: A phenomic characterization of Canada’s Apple Biodiversity Collection

A future with a secure and safe food supply requires humanity to preserve and exploit the vast variation available across agricultural plant species. Apples are one of the most widely consumed fruits and provide significant nutritional value worldwide. Here, we characterize key agricultural traits in a diverse collection of apples to provide a foundation for future apple improvement. We show that commercially successful apple varieties capture only a small fraction of apple diversity, and demonstrate that significant improvement is possible by tapping into existing genetic diversity. The domesticated apple (Malus domestica) is thought to have been cultivated for over 3,000 years (Zohary et al. 2012). Genetic evidence suggests that the main progenitor species of the domesticated apple is Malus sieversii from Central Asia (Velasco et al. 2010); however, significant gene flow from Malus sylvestris has also been detected (Sun et al. 2020; Duan et al., 2017; Cornille et al. 2012). Red color, reduced acidity, larger fruit, and firmness appear to have been under selection during apple domestication and improvement (Ma et al., 2015; Migicovsky et al., 2021). The apple's widespread geographic distribution and long-standing popularity have resulted in over 10,000 named apple cultivars with a fascinating diversity of phenotypes (Liang et al., 2015). Despite this tremendous diversity, a small number of cultivars make up a significant proportion of production. For example, in 2018, only four cultivars accounted for over 50% of apple production in the USA (“US Apple Noiton Nybom et al., 2013). In addition, breeding apples that retain their firmness after long-term storage have been a key breeding target (Kouassi et al., 2009). Not only is it important for breeders to select for firmness and firmness retention, but it is also crucial to breed for the phenological traits associated with firmness and firmness retention. This is especially important given that the optimal phenological breeding targets are expected to shift over time due to climate change. Characterizing how fruit texture and phenological traits are associated with each other can enable the development of new cultivars that adapt to climate change and meet consumer preferences. Germplasm collections serve as important reservoirs of genetic diversity for crop improvement. They contain the high levels of diversity that are essential for identifying valuable phenotypes that can be leveraged to develop improved cultivars (Bramel Jackson, 1997; Migicovsky Ward, 1978). Most recently, Canada's Apple Biodiversity Collection (ABC) was established in Nova Scotia, Canada, with over 1,000 accessions that includes trees primarily belonging to the domesticated apple, M. domestica, and its primary wild ancestor, M. sieversii. The ABC was established as a dual-purpose orchard. The first purpose of the ABC is to preserve and maintain potentially valuable apple genetic and phenotypic diversity. Second, the ABC is specifically designed to enable accurate measurements of phenological and fruit quality traits primarily for the purposes of genetic mapping. The trees in the ABC were grafted at the same time to the same rootstock and planted in duplicate in a randomized block design to control for positional effects in the orchard. The result is an apple population that is ideally suited to accomplish a phenome-wide characterization of apples. Here we present a comprehensive evaluation of Canada's ABC through phenotyping of phenological traits and fruit quality traits both at harvest and after 3 months of cold storage. The Apple Biodiversity Collection (ABC) is located at the Agriculture and Agri-Food Canada (AAFC) Kentville Research and Development Centre in Nova Scotia, Canada (45.071767, −64.480466). The ABC contains 1,119 apple accessions that were grafted to M.9 rootstock in August 2011 and allowed to grow outdoors until November 2012 when they were removed from the orchard and stored in moist sawdust at 2°C until planting. On May 31, 2013, we planted each of the 1,119 accessions in duplicate in a 5-acre orchard that was tile drained and fumigated with Telone® soil fumigant. The trees were spaced 1.5 m within rows and 5 m between rows. The trees were trained to a trellis system with wires at 1.5 and 2.4 meters above the ground. Soil amendments, training, thinning, and pruning were performed to industry standards. The ABC consists of apple accessions from the United States Department of Agriculture (USDA) Plant Genetic Resources Unit apple germplasm collection in Geneva, New York, USA; commercial cultivars from the Nova Scotia Fruit Growers’ Association Cultivar Evaluation Trial; and advanced breeding material from the AAFC Kentville breeding program. The collection contains mostly M. domestica accessions including cider, dessert, processing, heritage, and elite cultivars. The orchard also contains 78 accessions of the wild progenitor species M. sieversii from Central Asia. It is possible that pairs of accessions within the ABC may be clonally related in some cases. Here we treat each accession as a unique sample in downstream analyses and future genetic investigation will reveal the degree of clonal relatedness in the collection. The trees in the ABC are not available for propagation as most of the material was imported from the USDA under a section 43 import permit from the Canadian Food Inspection Agency (Permit #P-2011–00222) which prohibits the sale or distribution of the germplasm. This mixed-model accounts for fixed effects of an accession and the random effects of position depending on Block (north/south), north-to-south position within the block (rGrid), east-to-west position within the block (cGrid), and the interactions between these random effects. The measurements resulted in 39 phenotypic variables collected from 2014 to 2018, which are summarized in Data S1. All phenology traits that were recorded as dates (e.g., harvest date and flowering date) were converted into Julian days. Harvest date was recorded for the 2016 and 2017 harvest seasons. During both seasons, 20 apples were picked randomly from each tree. Trees ready for harvest were flagged at the beginning of the week and harvested over the subsequent days of that week. Due to the diversity of the accessions and the variation in ripening time, a variety of methods were used to determine when to harvest. Dropped apples or changes in background skin color were indicators of harvestable trees (Watkins, 2003). In addition, a sample apple was taken from each tree and touched to assess firmness, tasted to assess starch and sweetness, cut in half to check browning of seeds, and sprayed with iodine solution to evaluate starch content (Blanpied et al., of apple was in of weight of an and were strongly 1 we only present in the main of the Apple accessions were into on species domestica M. geographic and using primarily from the USDA Germplasm as as as M. in the were M. domestica in analyses these are by as M. sylvestris or were from of between species. All accessions that were as dessert, or were as only as were as as wild and rootstock cultivars were from the of between and For geographic accessions that in Europe or Asia were as that in and New Zealand were as The year of was also from the or from for accessions that were as named cultivars. All and were performed in accession was planted in but one of an trees or not fruit, the from only a tree. The phenology and fruit measurements storage were for their in the orchard by the using the et al., in which resulted in one and were not most only a tree was Fruit quality measurements taken after storage were also not for in the orchard for each the fruit from the duplicate trees was being in cold storage. measurements are the value across apples within an evaluate how fruit quality during the between the measurements storage and after storage was calculated for each between phenotypes were assessed using the in assess how the most commercially successful cultivars from the of the we phenotypes from of the top cultivars in the USA (“US Apple et al., 2019). 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